Alpha-helical protein nanofibrils

Abstract

The present invention relates to the field of microbial self-assembling protein fibrils as novel bionanomaterials. More specifically, the present invention relates to protein nanofibril materials comprising Bacillus endospore appendage (ENA) proteins, which are spontaneously folding helix hairpin subunits assembling into alpha-helical multimeric (A-ENA) fibrils with a hydrophobic core and covalently connected by one or more isopeptidic bonds, providing for a nanofibril of high stability and tensile strength. The invention further relates to said protein nanofibrils which are engineered or modified to provide for functionalized bionanomaterials. In particular, the invention relates to methods for recombinant production of said self-assembling protein nanofibrils, and their use in modifying bacterial endospores and endospore activity or pathogenicity.

Description

[0001] ALPHA-HELICAL PROTEIN NANOFIBRILS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of microbial self-assembling protein fibrils as novel bionanomaterials. More specifically, the present invention relates to protein nanofibril materials comprising Bacillus endospore appendage (ENA) proteins, which are spontaneously folding helix hairpin subunits assembling into alpha-helical multimeric (A-ENA) fibrils with a hydrophobic core and covalently connected by one or more isopeptidic bonds, providing for a nanofibril of high stability and tensile strength. The invention further relates to said protein nanofibrils which are engineered or modified to provide for functionalized bionanomaterials. In particular, the invention relates to methods for recombinant production of said self-assembling protein nanofibrils, and their use in modifying bacterial endospores and endospore activity or pathogenicity.

[0004] BACKGROUND

[0005] Nanotechnology facilitates to control events at the nanoscale and is applied in designing novel functionalized nanomaterials including nanofibers, nanowires, nanofibrils, and nanoparticles, among others. Nanofibrils are typically built from polysaccharides, such as cellulose and chitin, and / or proteins, such as silk fibroin, thereby not only providing exquisite mechanical characteristics, but also taking into account the need for biosustainable and biodegradable alternatives. The most abundant starting material to date is cellulose, which as a polysaccharide in nanometric form can be isolated and modified for applications as biomaterials. Second, protein nanofibrils, such as silk, spidersilk and collagen, were characterized in animals and plants and described as promising nanostructures for the development of bionanomaterials and biomedical applications, amongst others. Moreover, starting from biological production, the modification and exploitation of these bionanomaterials enhances the potential to provide materials with advantageous biological properties. The applications are numerous, and in an economy that is turning into a more ecological and biosustainable society, their value is growing and the demand for commercially viable solutions is enormous.

[0006] Bacterial spores are a remarkable example of biological resilience, representing a dormant survival form able to withstand extended periods (decades) of adverse conditions, including physical and chemical hazards such as high salinity, acidity, temperature, and drought. Accordingly, the proteinaceous structures that make up the endospore coating and its appendages require material properties compatible with these harsh conditions. Previously, a radically new class of biological nanofibers was structurally unravelled and identified on the endospores of Bacillus cereus sensu lato (s.l.), named ENdospore Appendages or ENAs (Pradhan et al. 2021, the EMBO Journal el06887; Remaut et al. WO2022 / 029325). Based on the self-assembling protein fibrillar structures present in endospore samples, the proteins constituting these protein assemblies were classified as S-ENA and L-ENA proteins. These nanofibrils clearly form a novel family of building blocks for future bionanomaterials, and further functionalization, because of their high flexibility and elasticity, with interesting properties such as remarkable chemical and physical strength, withstanding low pH, proteinase treatment, pasteurization, desiccation and high vacuum. Moreover, the S- and L-ENA fibrils' self-assembling properties facilitate high yield recombinant microbial production and the extreme stability of the fibrils aids in easy isolation from cellular debris, proteins, nucleic acids and membranes, while retaining the native structure and properties. At least for S-ENA-based fibrils, functionalization by insertion of a peptide in two distinct surface loops present on the protein was demonstrated (Remaut et al. WO2022 / 029325). Though to maintain the self-assembling nature of the proteins and the mechanical properties of the resulting fibrils, heterologous insertions are inherently limited in length and types of possible folds for engineering, and N- or C- terminal functionalization is not desired in this S-or L-Ena type of fibrils. The S-ENA fibrils were reported to contain intermolecular disulfide covalent linkages to increase their tensile strength, stability and rigidity. Such rigidifying covalent interactions are known from other types of protein-materials as for instance the formation of intermolecular isopeptide bonds, which occurs for instance also in the ubiquitously known ubiquitination reactions.

[0007] Bacteria, in general Gram-positive bacteria, have pilins which make use of enzyme-mediated intermolecular isopeptide bonds to covalently connect subunits and build long thin fibrillar structures onto to the cell surface (Hendrickx et al. 2011, Nat. Rev. Micro. 9, 166-176). In addition to these enzymedependent processes, intramolecular isopeptide bonds embedded in the hydrophobic environment of the p-strand based Immunoglobulin can form autocatalytically and serve to increase the proteaseresistance, thermal and mechanical stability of the pili forming protein domains (e. g. collagen adhesin domains A (CnaA), such as FimA, and CnaB , such as Spy; Kang et al. 2007, Science, 318(5856):1625-8; Echelman et al. 2016 PNAS;113(9):2490-5). Such autocatalytic isopeptide bonds form through a proximity-induced reaction of which the self-assembling nature depends primarily on the geometry of the participating amino acids and the hydrophobic moiety in which the isopeptide unit is formed (Kang and Baker, 2011 Trends Biochem Sci. 36(4):229-37). The autocatalytic isopeptide bond formation geometries identified in the immunoglobulin-like domains of bacterial pili have successfully been employed biotechnologically to covalently couple engineered derivatives such as in the split domain architectures found in the SpyCatcher - SpyTag or SnoopCatcher - SnoopTag technologies (Zakeri et al. 2012, Proc. Nat. Acad. Sci. USA 109:E690-97; Hatlem et al. 2019, Int J Mol Sci 20(9): 2129). To date, equivalent isopeptide bond formation geometries that allow the covalent linkage of alpha-helical elements are lacking, limiting the stability and application area of natural and synthetic alphahelical protein materials.

[0008] In view of improving and further customizing protein-based materials and nanofibril innovations, novel types of protein fibrils with superior characteristics in terms of chemical stability, mechanical resistance, bio-engineering potential and sustainable manufacturing and biodegradability options are required.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention discloses a novel type of protein-based nanofibrils derived from Bacillus endospores, in particular from Bacillus thuringiensis, where said fibrils appear on the surface of the endospore, typically in connection with the parasporal bodies. The protein nanofibrils of the present invention were characterized herein for the first time as to be fabricated from alpha-helical hairpin proteins, and -in line with previously annotated Endospore-Appendage protein families (see Pradhan et al., 2021),- these newly annotated fibril-assembling proteins were called Alpha-helical ENdospore Appendage proteins, or A-ENA proteins. This novel protein family is unique by showing an ingeniously simplistic monomer or subunit structure of two antiparallel alpha helices connected by a short turn of amino acids and ending in free N- and C-termini, wherein the subunits are self-organizing into two helically wound protofilaments or protofibrils (e.g. see Figure 3), with as a result a nanofibril providing for an environment in which the side chains of the A-ENA monomers are positioned as a helical wheel allowing cross-linking by an incredible pattern of isopeptide bonds (e.g. see Figure 7). The presence of intermolecular covalent interactions of the A-ENA subunits within and between the protofibrils provides for an irreversibly organized structural component to form an A-ENA fibril of high tensile strength. The invention relates to these protein nanofibrils constituting A-ENA or A-ENA-based protein variants defined by a rational pattern of residues with nucleophilic, electrophilic, and acid / base properties for the design of isopeptide bond forming units to covalently stabilize helix-helix contacts. Furthermore, through recombinant production in (microbial) hosts, the nanofibrils can as well be manufactured starting from the expression of recombinant A-ENA or A-ENA-like or A-ENA-based variant proteins from a chimeric gene construct. Self-assembly is conserved thanks to the structural conservation of the protein family. Finally, the simple topology of the A-ENA-based variant or engineered A-ENA fibrils allows for A-ENA nanofibrils to carry N- and C-terminal in addition to internal Loop or turn-inserted (poly)peptide constructs resulting in a tuneable, high-density functionalization of the fibrils.

[0011] So a first aspect of the invention relates to a protein-based fibril, called herein protein nanofibril, composed or consisting of two protofibrils, wherein each protofibril comprises two or more monomer protein subunits, hence said fibril comprising at least a tetramer, wherein each monomer protein subunit comprises covalently connected amino acid sequence fragments, wherein said covalent connection is a HaRe / A-Ena / 819 peptide bond, according to the formula: N-terminal lock (NTL) – helix 1– Linker (L) – helix 2 – C-terminal tail (CT), wherein said monomer protein subunit self-assembles when expressed or present in aqueous solution into two helices, helix 1 and helix 2, as an alpha-helical antiparallel coiled coil protein structure, and wherein helix 1 and helix 2 each comprise a continuous sequence of at least 5 heptad (H) elements according to the formula H1-1 - H1-2 - H1-3 - H1-4 - H1-5 for helix 1, and H2-1 – H2-2 – H2-3 – H2-4 – H2- 5 for helix 2, wherein said heptad elements each comprise seven amino acid residues designated ‘abcdefg’ with the following consensus sequences: - Heptad1-1: X-X-X- Φ-X-X- Φ, - Heptad1-2: Φ-X-ѱ- Φ-ѱ- Φ-ɣ, - Heptad1-3: Φ-X-ѱ- Φ-ѱ-X- Φ, - Heptad1-4: Φ-N-ѱ-ɣ-ѱ-ɣ- δ, - Heptad1-5: Φ- Φ-X- Φ- Φ-X-X, - Heptad2-1: Φ-X-X- Φ- ΦΦX- Φ, - Heptad2-2: Φ-X-X- Φ-X-X-X, - Heptad2-3: Φ-X-X- Φ- Φ-δ-δ, - Heptad2-4: Φ- Φ- Φ- Φ- Φ-X- δ, - Heptad2-5: Φ-ɣ-X- Φ- Φ-X-X, wherein: - ^ is a hydrophobic amino acid selected form the list of M, V, I, L, A, G, H, W, Y, F for at least 70 % of said ^ positions indicated in said heptad elements; preferably a bulky amino acid selected from L, M, I, V, F, or W; - Ѱ is a short side chain residue, selected from the list of V, C, G, A, P, S, T, N, D; preferably an amino acid selected from A, G, S, T; - ɣ is a residue that serves as acid / base catalyst selected from Glu (E) or Asp (D), for at least one or more of said ɣ positions indicated in said heptad elements; - δ is a residue capable to serve as isopeptide bond ‘donor’ or nucleophile selected from Lysine, for at least one or more of said δ positions indicated in said heptad elements; - ^ is a residue capable to serve as isopeptide bond ‘acceptor’ or electrophile selected from Glu (E) , or Asp (D), Gln (Q) or Asn (N), for at least one or more of said ^ positions indicated in said heptad elements; - and ɣ , δ, and ^ ^may be any amino acid at the one or more remaining positions not involved in IPB formation, X can be any type of amino acid, and wherein the linker (L) fragment comprises at least 4 amino acids, and N-terminal lock (NTL) and C- terminal tail (CT) fragments comprise at least one amino acid, and wherein said monomeric protein subunits are interconnected through at least one or more isopeptidic bonds (IPBs).

[0012] Specifically, said protein nanofibril forms at least one IPB to covalently interconnect the two protofibrils (f) and (f'). More specifically, said at least one IPB interconnecting said two protofibrils (f) and (f') is present between the monomeric protein subunits (i) of protofibril (f) and the monomer subunits (i' and / or i' + 1) of protofibril (f') between the side chain of the amino acid acting as a nucleophile and the side chain of the amino acid acting as electrophile, respectively, at positions:

[0013] H2-3f of (i) and H2-4e of (i'); and / or

[0014] H2-3f of (i'+l) and H2-4e of (i).

[0015] Alternatively, said protein nanofibril forms at least one IPB to covalently interconnect the two protofibrils (f') and (f), wherein said at least one IPB interconnecting said two protofibrils (f') and (f) is present between the monomeric protein subunits (i') of protofibril (f') and the monomer subunits (i and / or i + 1) of protofibril (f) between the side chain of the amino acid acting as a nucleophile and the side chain of the amino acid acting as electrophile, respectively, at positions:

[0016] H2-3f of (i') and H2-4e of (i); and / or

[0017] H2-3f of (i+1) and H2-4e of (i').

[0018] In a further embodiment, the protein nanofibril has a tertiary structure with a minimal tetrameric entity as shown in Figure 3c, and / or wherein the monomeric protein subunits (i + / - n) of protofibril (f) and the monomer subunit (i'+ / - n) of protofibril (f') are covalently connected through at least one or more IPBs, preferably 10 IPBs, wherein the IPBs are formed, as for example shown in Figure 9, between the side chain of the amino acid acting as a nucleophile and the side chain of the amino acid acting as electrophile, respectively, at positions:

[0019] NTL of (i) and Hl-5b of (i-5), or (i-4), (i-3) or (i-2);

[0020] Hl-4g of (i) and H2-2a of (i-1);

[0021] H2-3g of (i) and H2-4a of (i-1);

[0022] H2-3f of (i) and H2-4e of (i');

[0023] H2-4g of (i) and Hl-3a of (i-1);

[0024] H2-4g of (i+1) and Hl-3a of (i);

[0025] NTL of (i+5) or (i+4), (i+3) or (i+2) and Hl-5b of (i);

[0026] Hl-4g of (i+1) and H2-2a of (i);

[0027] H2-3g of (i+1) and H2-4a of (i); and / or H2-3f of (i'+l) and H2-4e of (i).

[0028] Alternatively, protein nanofibril has a tertiary structure with a minimal tetrameric entity as shown in Figure 3c, and / or wherein the monomeric protein subunits (i + / - n) of protofibril (f) and the monomer subunit (i'+ / - n) of protofibril (f') are covalently connected through at least one or more IPBs, preferably 10 IPBs, wherein the IPBs are formed, between the side chain of the amino acid acting as a nucleophile and the side chain of the amino acid acting as electrophile, respectively, at positions:

[0029] NTL of (i) and Hl-5b of (i-5), (i-4), (i-3), or (i-2);

[0030] Hl-4g of (i) and H2-2a of (i-1);

[0031] H2-3g of (i) and H2-4a of (i-1);

[0032] H2-3f of (i) and H2-4e of (i'-l);

[0033] H2-4g of (i) and Hl-3a of (i-1);

[0034] H2-4g of (i+l)and Hl-3a of (i);

[0035] NTL of (i+5), (i+4), (i+3), or (i+2) and Hl-5b of (i);

[0036] Hl-4g of (i+1) and H2-2a of (i);

[0037] H2-3g of (i+1) and H2-4a of (i); and / or

[0038] H2-3f of (i') and H2-4e of (i).

[0039] In a further embodiment, said protein nanofibril comprises Heptad elements of the helices al and a2 with the following consensus sequences:

[0040] - Hl-1: X-X-X-Φ-X-X-Φ,

[0041] - Hl-2: Φ -X-(S / T)- Φ-(A / G)- Φ-(E / Q)

[0042] - Hl-3: E-X- X-Φ - X-(H / N)-Φ

[0043] - Hl-4: Φ -N-(A / G / S)-E-(G / A)-E-K

[0044] - Hl-5: Φ-X-X-Φ-Φ-X-X

[0045] - H2-1: Φ-X-X-Φ-Φ-X-Φ,

[0046] - H2-2: (N / Q / D)-X-X-Φ -X-X-X

[0047] - H2-3: Φ -X-X-Φ Φ-(K / X)-(K / X)

[0048] - H2-4: (E / Q)-Φ Φ -(L)-(Q / X)-X-K,

[0049] - H2-5: Φ-y-X-Φ Φ-X-X, wherein the one-letter amino acid codes are applied for specific residues and ' / ' is read as 'or', and wherein <t> is a hydrophobic amino acid selected form the list of M, V, I, L, A, G, H, W, Y, F; preferably a bulky amino acid selected from L, M, I, V, F, or W; y is a residue that serves as acid / base catalyst, selected from Glu (E) , Gin (Q) or Asp (D);

[0050] X can be any type of amino acid.

[0051] A further specific embodiment relates to said protein nanofibril wherein said monomeric protein subunits are based on, derived from, or originating from Bacillus proteins, in particular, Bacillus Endospore appendage (ENA) proteins, even more specific Alphahelical (A-)ENA proteins, preferably from Bacillus thuringiensis.

[0052] Further embodiments provide for said protein nanofibril with an N-terminal Lock (NTL) amino acid sequence corresponding to M-fJ-O-Z-X-O-P, wherein: tp= short side chain residue, selected from the list of V, C, G, A, P, S, T, N, D; preferably A, G, S or T;

[0053] <t> = a hydrophobic amino acid, selected from the list of M, V, I, L, A, G, H, W, Y, F; preferably from the list of L, M, I, V, F, or W;

[0054] X = any amino acid, Z is Ala or Proline, and M and P are one letter code for the respective amino acids Methionine and Proline.

[0055] More specifically, said protein nanofibril may comprise an NTL with an amino acid sequence corresponding to M- tp -O-Z-X-O-P, wherein tp is selected from S, G, or T, <t> is a hydrophobic amino acid, selected from the list of M, V, I, L, A, G, H, W, Y, F, preferably from the list of L, M, I, V, F, or W; and X is any amino acid, Z is Ala or Proline, and M and P are one letter code for the respective amino acids Methionine and Proline.

[0056] A further specific embodiment relates to said protein fibrils wherein the NTL-helixl-L-helix2-CT sequence of the monomer consists of an A-ENA protein sequence, wherein said A-ENA protein sequence is selected from the list of proteins depicted by their access codes provided in Table 3. In a specific embodiment, said A-ENA protein sequence is selected from the list of SEQ ID NOs: 1-6, or a modified or engineered or functionalized fibril consisting of A-ENA proteins selected from SEQ ID NO: 1-6, wherein the NTL, and / or Linker, and / or CT are modified.

[0057] A further specific embodiment relates to said protein fibrils wherein the NTL-helixl-L-helix2-CT sequence of the monomer consists of an A-ENA (like) protein as defined by the Hidden Markov Model (hmm) profiles for Helix 1 and Helix 2 as shown in Figure 11, and as depicted in Tables 1 and 2, respectively (obtained from 593 A-ENA homologues identified in this work), or a modified or engineered or functionalized fibril consisting of said A-ENA (like) proteins, wherein the NTL, and / or Linker, and / or CT are modified. The skilled person may apply (online) tools and databases to verify whether a protein sequence complies to the HMM profile representing a protein class or family (e.g. https: / / www.ebi.ac.uk / Tools / hmmer / ), which thus allows to use the information discloses herein in Table 1-3 and Figure 11 to determine whether a protein belongs to the A-Ena family.

[0058] A further specific embodiment relates to any one of said protein fibrils, wherein the NTL is truncated in that it is at least 1, but less than 4 amino acids.

[0059] Another embodiment relates to said protein-based fibril, wherein the monomeric protein subunit comprises an NTL of at least 4 amino acids and the monomeric protein is modified on its surface, or is a mutant variant as compared to the consensus motif of the fragments NTL - helix 1- L- helix 2 - CT, as defined herein, with at least one or more amino acid substitutions in the heptads of the helix 1 or helix 2 regions at the following positions: Hl-la, Hl-lb, Hl-lc, Hl-3f, Hl-4f, Hl-5b, Hl-5f, H2-lb, H2-lc, and / or H2-5b, with an amino acid that is different from the list of amino acids provided in the consensus sequences described herein for said heptad position.

[0060] In an alternative embodiment, said protein-based fibril comprises a monomeric protein subunit comprising an NTL of at least 1, but less than 4 amino acids, and the monomeric protein is modified on its surface, or is a mutant variant as compared to the consensus motif of the fragments NTL - helix 1- L- helix 2 - CT, as defined herein, with at least one or more amino acid substitutions in the heptads of the helix 1 or helix 2 regions at the following positions: Hl-la, Hl-lb, Hl-lc, Hl-lf, Hl-2f, Hl-3b, Hl-3f, Hl- 4b, Hl-4f, Hl-5b, Hl-5f, H2-lb, H2-lc, H2-2b, H2-2f, H2-3f, H2-4f, H2-5b, H2-5c and / or H2-5f, with an amino acid that is different from the list of amino acids provided in the consensus sequences described herein for said heptad position.

[0061] Further embodiments relate to said protein nanofibril, wherein the monomeric protein comprising the consensus sequence corresponding to the NTL-helixl-L-helix2-CT formula as defined herein is further linked to a tag or protein domain or is conjugated at the N-terminus, C-terminus or within the Linker region of the monomer for forming a functionalized fibril. Said fusion or linking may be performed directly or with a (flexible and / or optimized) linker sequence in between. Said self-assembling protein fibril thus comprises one or more monomers wherein a functional fusion to a further moiety is present, wherein said further moiety may be a folded protein, a tag, a label, or a functional moiety, fused or conjugated directly or via a linker to said A-ENA-based monomer. In a specific embodiment, the monomeric protein of said protein fibril which is linked to a further moiety to form a functionalized protein nanofibril is obtained from (recombinant) expression from a genetic fusion of the monomer operably linked to said further functional moiety. More specifically said genetic fusion of the monomer may be present as at its N-terminal or C-terminal end, or as an insertion in the linker region. In another specific embodiment, said monomeric protein of said protein nanofibril is functionalized or modified post-fibril formation, and the modified or functionalized protein nanofibril is obtained from orthogonal conjugation of one or more monomeric protein subunits of said protein nanofibril after self-assembly of the fibril. In a preferred embodiment, said further orthogonal conjugation comprises a covalent linkage, more preferably an isopeptide binding, even more preferably the functionalization comprises a modification comprising a peptide tag / binding partner pair derived from an isopeptide bond forming strand - pilus subunit fold complementation pair, more specifically a (Spyjtag / Collagen adhesin domain B (CnaB2) Catcher type (such as SpyCatcher) or a (Snoop)Tag / D4 domain (RrgA) catcher type (such as SnoopCatcher) binding partner type of protein or fusion protein pair (Hatlem et al. 2019, Int J Mol Sci 20(9): 2129).

[0062] Further embodiments provide for said protein nanofibril composed of monomeric proteins as described herein, wherein at least one monomer, preferably an A-ENA or A-ENA-based variant monomer, further comprises a fusion with a heterologous protein domain for surface display on the protein nanofibril. Particular embodiments relate to said protein nanofibrils comprising at least one monomer as described herein further fused to a heterologous protein domain, wherein said fusion is made at the N-terminal or C -terminal end of the monomer, or inserted at the L or Linker region of the monomer. In specific further embodiments, said heterologous protein domain fused to said at least one protein monomer comprises a VHH, for nanobody surface display, or comprises a counterpart of a peptide tag / binding partner pair, preferably comprising covalent interacting bonds such as an isopeptide bond, more specifically for instance a tag / catcher binding pair such as the SpyTag / SpyCatcher or SnoopTag / SnoopCatcher, or comprises a further protein which upon presence on the surface of said protein nanofibril is used for surface display. In a further specific embodiment, said heterologous protein comprises a sequence selected from SEQ. ID Nos: 7 -11, 13- 15, or 67.

[0063] In one specific embodiment, said protein nanofibril comprises said monomers fused to a heterologous protein domain, wherein each monomer is identical forming a homopolymeric fibril upon self-assembly. In a further alternative embodiment said protein nanofibril comprising one or more monomeric protein subunits as described herein wherein each of the monomers further comprises a heterologous protein domain, which comprises a flexible (optimized) linker between the monomer as described herein and the heterologous protein domain, wherein the protein domain has a hydrodynamic radius below 11 nm, which is suitable for surface display when the heterologous protein domain is present on homopolymeric fibrils.

[0064] Another specific embodiment relates to said nanofibril wherein the monomer subunit as described herein is fused to a heterologous protein domain in at least one monomer, resulting in a protein nanofibril wherein after self-assembly of the monomers into protofibrils and intertwining to a nanofibril at least two different sequences of monomeric proteins appear as part of a heteropolymeric fibril.

[0065] Further specific embodiments provide for said protein nanofibril, which was isolated from a bacterial or endospore environment 'as such', and / or is a protein nanofibril consisting of said protein material without further biological material such as endospore or parasporal attaching components. In a further specific embodiment said protein nanofibril as described herein is isolated from a recombinantly produced bacterial cell culture, preferably of Bacillus or from Bacillus cell cultures or sporulating cultures, preferably from Bacillus thuringiensis, wherein said bacterial cell culture comprises a heterologous A- ENA or A-ENA-based or modified A-ENA monomeric protein subunit for self-assembly into the protein nanofibril as described herein. Alternatively, said protein nanofibril as described herein is a recombinantly produced protein nanofibril, such as a protein fibril expressed in a host cell cytoplasm and isolated from said host cell, wherein said host cell may be a prokaryotic or eukaryotic host cell.

[0066] In a second aspect, the invention relates to a bacterial endospore, preferably a Bacillus endospore, which is modified as compared to a wild type bacterial endospore, preferably a Bacillus endospore, in that the endospore comprises and / or displays the modified A-ENA monomeric protein subunits as described herein, or the functionalized protein nanofibril as described herein.

[0067] More specifically, said modified bacterial endospore, preferably Bacillus endospore, may lack an endogenous A-ENA monomeric protein complying to the consensus sequence of the NTL-helixl-L-helix2- CT formula as described herein, and said bacterial stain, preferably Bacillus strain comprises an exogenously introduced A-ENA monomeric protein comprising said consensus sequence in the format of the NTL-helixl-L-helix2-CT formula as described herein, or comprises said protein nanofibril displaying said heterologously or exogenously introduced modified A-ENA monomeric protein or monomeric mutant variant or monomeric fusion protein as previously described herein.

[0068] Further embodiments relate to the use of said modified bacterial endospore, preferably Bacillus endospore, for increasing bacterial endospore activity, more preferably for improving or increasing Bt pathogenic activity.

[0069] An alternative embodiment relates to the application of said protein nanofibril described herein in combination with a bacterial endospore or bacterial sporulating suspension, preferably a Bacillus endospore or Bacillus suspension, to enhance virulence of said bacteria or endospores, preferably of said Bacillus or Bt spores, as to enhance the pesticidal activity, preferably the insecticidal activity, of said endospore or suspension. Further aspects of the invention relate to nucleic acid molecules, vectors, host cells, or compositions for providing the A-ENA monomer in modified, mutant variant or fusion subunit for spontaneous assembly of the protein nanofibril.

[0070] A final aspect of the invention relates to a method to produce the A-ENA protein nanofibril or A-ENA- based variant protein nanofibril as described herein, comprising the steps of:

[0071] Introducing a nucleic acid molecule encoding an A-ENA monomer, or A-ENA based monomer or modified A-ENA monomer, as described herein in a cell, or recombinant expression of said monomeric protein as described herein in a host cell, and incubating the cells allowing self-assembly of said monomeric protein into protofibrils and consequently protein nanofibril assembly; or alternatively culturing the host cell comprising said monomeric protein subunits or assembling said protein nanofibril described herein, obtaining the self-assembled monomers and / or protein nanofibrils from the host cell and / or cell suspension, preferably through cell lysis, and

[0072] Purifying the self-assembled protein nanofibrils, preferably through resuspension from the insoluble fraction and / or further purification from the cell lysate.

[0073] Specific embodiments further relate to a method to produce said recombinant A-ENA protein nanofibrils wherein one or more protein monomers are modified, mutant variants, or fusion proteins as described herein, also called functionalized A-ENA protein nanofibrils herein. Alternatively, isolation and / or purification of the protein fibril in the final step is performed in an alternative manner as known by the skilled person, for removing cell debris, DNA, or host cell proteins or compounds which are undesired for further applying a composition of the protein nanofibril or functionalized protein nanofibril obtained from said method.

[0074] DESCRIPTION OF THE FIGURES

[0075] The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

[0076] Figure 1. Identification of A-type Endospore Appendages (A-ENA) as a novel family of protein fibrils produced on the endospores of Bacillus thuringiensis Sv. Israelensis (Bti). Transmission electron microscopy (TEM) characterization of a Bti spore biofilm shows the presence of a network of nanoscale fibrils, (a) Stain-free TEM micrograph of a resuspended Bti spore biofilm, composed of endospores and parasporal bodies (PSBs), grown on LB agar and deposited onto a Formvar / Carbon grid; (b) Higher magnification image of the same grid used in (a) revealing the A-ENA filaments that permeate the biofilm; (c) Negative stain TEM (nsTEM) micrograph of a Bti endospore revealing A-ENA filaments that emanate from the spore surface; (d-f) Examples of A-ENA mediated coupling between spore-PSB, spore- spore-PSB and spore-spore, respectively; (g) nsTEM micrograph of a cluster of parasporal bodies decorated with A-ENA filaments that emanate from the PSB surface; (h) nsTEM micrograph of wild-type A-ENA fibrils with corresponding 2D class average (box size 384x384A); (i) cryoEM micrograph of vitrified wild-type A-ENA fibrils with corresponding 2D class average (box size 230x230A).

[0077] Figure 2. Recombinant production of self-assembling A-ENA fibrils, (a) nsTEM micrograph of recombinant A-ENA (UniprotKB Q8KNV8; SEQ. ID NO: 1) expressed in the cytoplasm of E. coli. A-ENA self assembles into protein nanofibrils that are isolated from the culture by resuspension and incubation of cell pellets into EDTA - lysozyme, incubation in warm 1% SDS, followed by centrifugation, and consecutive wash steps (resuspension & centrifugation) in water to remove dissolved lysate (full procedure in Example 2). The resulting suspension of A-ENA fibrils was deposited on a Formvar / Carbon Supported Copper Grid, and stained with a 2% (w / v) uranyl acetate solution for observation by nsTEM (scale bar: 100 nm; inset: corresponding 2D class average with box size 384x384A); (b) Reconstructed cryoEM volume (resolution: 2.59A according to the 0.143 FSC criterion) derived from recombinant A- ENA fibrils vitrified in deionized water; (c) Corresponding Fourrier shell correlation function of the volume shown in panel b; (d) Cartoon representation of the atomic model built based on the electrostatic potential map shown in (b).

[0078] Figure 3. Architecture and structural visualization of A-type Endospore Appendages (A-ENA). (a, b) Ribbon and space-filling models of A-ENA fibrils viewed (a) perpendicular to and (b) along the fibril long axis. A-ENA fibrils are composed of two protofibrils (colored white and dark grey, and labelled f and f') that intertwine into a two-start helical superstructure (i.e. a parallel double helix) with a twist and rise of approximately 12° and 10.8 A, respectively, (c) Top; ribbon and surface representation of a pair of juxtaposed A-ENA monomers, labelled i and i'. A-ENA subunits consist of an alpha-helical hairpin with an N-terminal extension projecting downwards along the fibril axis. The A-ENA helix hairpin makes an approximately 68° angle to the fibril long axis. Within a single vertical plane of the fibril, two A-ENA monomers, one of each protofibril, interact laterally to form a dimeric entity. Middle; A-ENA pairs stack axially, thereby extending each protofibril, with a relative twist of approximately 12° and rise of 10.8 A, to form a tetrameric complex, which can be considered a minimalistic fibril entity, or fibril nucleus. Bottom; fibril extension is consequently achieved via axial stacking of additional dimeric units at either poles of an A-ENA tetramer, each a relative twist and rise of approximately ± 12° and 10.8 A.

[0079] A-ENA fibrils are drawn with the subunits' C-termini pointing upwards (referred to as C-terminal or pointed fibril end, i.e. C-pole), and the subunits' N-terminal extensions pointing down (referred to as N- terminal or barbed fibril end, i.e. N-pole). A-ENA monomers are called i and i' in the f and f' protofibril respectively, with incremental (i.e. i+1; i+2, i+3, etc.) or decremental (i.e. i-1, i-2, i-3, etc.) numbering for every stacked protomer towards the C- and N-pole, respectively.

[0080] Figure 4. Structure and protomer interactions in A-ENA fibrils of Bacillus thuringiensis Sv. israelensis (Bti). (a) Cartoon representation of a single Bti A-ENA monomer (UniprotKB Q8KNV8; SEQ ID NO: 1) as identified in the cryoEM structure of recombinant A-ENA fibrils. A-ENA monomers as shown here are composed of an alpha-helical hairpin (al and a2) with a 13 residue N-terminal extension (NTL; i.e. res 2 to 14 of SEQ ID NO: 1). Residues shown in stick representation (residue number shown in bold, underscored) partake in intermolecular isopeptide bond (IPB) formation with neighbouring A-ENA monomers (recipient residues of the corresponding A-ENA partner subunit are shown next to each highlighted residue, with the arrows running from the nucleophilic to the electrophilic residue); (b) Frontal view of Bti A-ENA (SEQ ID NO: 1; 90° rotation with respect to panel a) with residues involved in intermolecular isopeptide bonds highlighted and shown in stick representation: inter-protofibrillar contacts of monomer designated i are made with monomer i' (i.e. within the same plane in protofibril f') and with monomer i'+l (shifted one unit upwards in protofibril f'); intra-protofibrillar contacts are made with the A-ENA monomers directly above (i+1) or below (i-1) subunit i, as well as with monomer i- 5 via residue 2 in the N-terminal connector. As such, each subunit i is implicated in as much as 10 IPBs, crosslinking 7 different protomers within and across the protofibrils (c) Top (C-pole) and bottom (N-pole) view of a single A-ENA monomer in surface representation: both surfaces represent the terminal poles of the A-ENA fibril and form the contact sites for subunits above and below, respectively. These contact surfaces are primarily hydrophobic, with the exception of residues involved in IPB formation, highlighted and annotated by arrows; (d) Ultrastructure of the A-ENA fibril with both protofibrils (f' and f) shown in light grey and grey, with a single A-ENA monomer highlighted in cartoon representation; (e) Close-up of boxed area in panel d and with cartoon representation of the subunits in protofibril f (i+1 to i-5), and illustrated as sticks are the residues involved in the intra-protofibrillar IPBs between i / i+1, i / i-1 and i / i-5; (f) Cartoon representation of the inter-protofibrillar IPBs between i / i' and i / i'+l.

[0081] Figure 5. Helix cross-linking by autocatalytic units for isopeptide bond formation. A-ENA forms ten isopeptide bonds interactions with neighbouring A-ENA subunits. These isopeptide bonds are formed by autocatalytic units that consist of an acid-base catalyst (preferably Glu), a nucleophilic residue (preferably Lys or the N-terminal amine) and an electrophilic residue (preferably Glu, Gin, Asn or Asp). Autocatalytic isopeptide bond formation relies on close proximity and defined relative positioning of the acid-base catalysts, which activates the nucleophilic subunits for attack and nucleophilic substitution (i.e. dehydration) of the electrophilic residues. The defined positioning of nucleophilic and electrophilic residues on adjacent helices results in spontaneous, autocatalytic cross-linking of A-ENA subunits under physiological conditions, (a) overview of a subunit 'i' and its neighbouring subunits containing the isopeptide bond units that mediate intra- and interf ibrillar crosslinking of helix hairpins, (b) close-up view of the five IPB units (IPB-U1 - IPB-U5) implicated in subunit cross-linking.

[0082] Figure 6. Sequence conservation in A-ENA (like) proteins. A-ENA consensus weblogo derived from a multiple sequence alignment of the top 250 blast hits using Q8KNV8 (SEQ ID NO: 1) as a query sequence, filtered down to a maximum 95 % sequence identity redundancy; Residues involved in IPB formation (derived from the cryoEM structure of recombinant Q8KNV8 fibrils) have been highlighted with an asterisk. Catalytic acid / base residues (primarily Glu) are highlighted with a plus symbol. The residue numbering presented here does not correspond to the residue numbering frame used for Q8KNV8 (shifted by +4). This discrepancy stems from the multiple sequence alignment that served as a source to create the consensus logo.

[0083] Figure 7. Helical wheel representation of the A-ENA interaction network. Helical secondary structure results in defined positioning of sidechains and defined knobs-in-hole interaction of helix-helix contacts. When viewed along the long axis of the helix, the position of side chains can be portrayed in a helical wheel. The uniform helical rise and twist in an alpha-helix of 1.5 A and 100°, respectively, results in the positioning of residues such that every 8thresidue is located on the equivalent position on the helical wheel, adopting a translation along the helix by 10.5 A. As such, helices can be defined as consecutive heptameric units or 'heptads' (H), with side chains named 'a' to 'g'. Side chain projections on the alphahelix results in the formation of ridges and valleys referred to as 'knobs' and 'holes' located in systematically defined positions, and resulting in defined knobs-in-holes interactions when two adjacent alpha-helices interact. Within the A-ENA subunit alpha-helices al and a2 are each made of five heptads (Hl-1 to Hl-5, and H2-1 to H2-5, resp.), and form a helix hairpin by joining into an antiparallel packing contact by hydrophobic knobs-in-hole interactions (i.e. position 'd' on the helical wheels as defined above) along the length of the helices. During the stacking of A-ENA subunits (i.e. i / i+1 and i / i-1 interactions), alpha-helices al and a2 in consecutive subunits go into pairwise parallel packing by the primarily hydrophobic knobs-in-hole interactions along the length of the helices, involving heptad positions 'a' and 'e' on the C-pole facing side and 'g' and 'c' on the N-pole facing side of the A-ENA hairpins. Exceptions in the hydrophobic contact are formed by residues going into isopeptide bond formations (highlighted by solid arrows, pointing from the nucleophile towards the electrophile) and / or forming the acid-base catalyst (highlighted in red circle and labelled) in the IPB units. The packing of the f and f' protofibrils in A-ENA is mediated by knobs-in-holes and IPB interactions of positions 'b', 'e' and 'f' of Heptads H2-3 and H2-4 of a2 in subunits i with i' and i'+l. Bottom: alignment of A-ENA orthologues with sequence identities to A-ENA (UniprotKB Q8KNV8; SEQ ID NO: 1) as low as 24% (see Figure 7). A- ENA subunits adopt a generic structure formed of a variable N-terminal lock (dubbed 'NTL'), followed by a-helix 'al' consisting of 5 Heptad repeats (Hl-1 to Hl-5), followed by a variable length linker sequence (dubbed 'L'), followed by a second a-helix 'a2' , and ending in a variable length C-terminal tail (dubbed CT). Alignment of A-ENA orthologues according to the Heptad repeats demonstrates a strong conservation of the position of hydrophobic residues implicated in the intra- and inter-helical knob-in- holes interactions (labelled (|) and \| / for hydrophobic and short chain residues, respectively) and forming the interhelical isopeptide bond units (labelled y, 8 and s for respectively the acid / base catalyst, the nucleophilic residue and the electrophilic residue).

[0084] Figure 8. Recombinant production and fibril formation A-ENA orthologues. (a) Uniprot accession codes of putative A-ENA orthologues (SEQ ID NOs: 3-5) and the corresponding sequence identity to Bti A-ENA (Uniprot: Q8KNV8, SEQ ID NO: 1); (b) nsTEM micrographs of the self-assembled fibrils formed by recombinant expression of the corresponding A-ENA orthologues in the cytoplasm of E. coli. Fibrils were purified from the cytoplasm of E. coli C43 (DE3) after enzymatic digestion for 18h at 37°C in 1 mg.mL1lysozyme, 5mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris pH 6.8, 50mM NaCI, followed by detergent extraction for 30 minutes in 1% (w / v) sodium dodecyl sulfate (SDS) at 100°C (see Example 2 for full description). Fibrils obtained for the 3 tested orthologues have a diameter that is in accordance to the diameter observed for recombinant Q8KNV8 and recombinant Q8KNV7. The nsTEM images were obtained from samples that were derived from the insoluble fraction after SDS / heat extraction, demonstrating extreme physical-chemical robustness of the resulting fibrils. The latter is indicative of the autocatalytic formation of one or more I PBs.; (c) AlphaFold2 multimer vl.3 models of the respective A-ENA orthologue hexameric sequences as indicated in (a); (d) Multiple sequence alignment of the tested A-ENA orthologue sequences, and Q8KNV8 (A-ENA; SEQ ID NO: 1) and Q8KNV7 (A-ENA-1; SEQ ID NO: 2): residues involved in IPB formation and catalytic acidic glutamate residues are highlighted with black asterisks and red plus signs, respectively.

[0085] Figure 9. Conserved architecture and isopeptide bond pattern in A-ENA like protein nanofibrils, (a, b) A-ENA like fibril assembly units adopt a generic structure comprising, in consecutive order: an N-terminal lock (NTL) of variable length and sequence, followed by an a-helix (al) comprising preferentially five helix Heptads (Hl-1 to Hl-5), a variable length linker region (L; frequently, but not limited to a 4-5 residue turn), an a-helix (a2) comprising preferentially five helix Heptads (H2-1 to H2-5), and a variable length C-terminal tail (CT). In said fibril assembly units, the al and a2 Heptads hold a consensus sequence motif of hydrophobic and short side chain residues as shown in Figure 10, thereby facilitating the adoption of an antiparallel helix hairpin within the A-ENA like units, and facilitating the pairwise stacking of the fibril assembly units (i / i+1 and i / i-1) into helical protofibrils (labelled f) with approximate twist and rise of 12° and 10.8 A. In addition, a conserved sequence motif (see Figure 10) of hydrophilic residues in helix heptads Hl-2, Hl-3, Hl-4 and Hl-5 in al, and H2-2, H2-3, H2-4 and H2-5 in a2 form isopeptide bond formation units consisting of acid-base catalists (preferably Glu or Asp), nucleophilic residues (preferably Lys or the N-terminal amine) and an electrophilic residue (preferably Glu, Gin, Asp or Asn). Isopeptide bond pairs are highlighted in boxes, indicating the conserved Heptad positions (e.g. Hl-3a in subunit 'i', etc.) in the different subunits of protofibril f (i, i+1, i-1, i-5) and protofibril f' (i' and i'+l), with arrows pointing from the nucleophile residue towards the electrophilic residue. Schematic drawing shows the position amenable for maximum isopeptide bond pairing capacity. Individual IPB pairs can be considered facultative and substituted for hydrophobic knobs-in-holes interaction. Global removal of all IPB pairs results in poor A-ENA like self-assembly (see Figure 12) and poor fibril stability. The N-terminal amine in the NTL can be implicated in IPB formation with residue Hl-5b of non-adjacent subunits, i.e. subunit i-5 in the case of Bti A-ENA (SEQ. ID NO: 1). The exact subunit 'i-x' implicated in the IPB can be varied by extending or shortening the NTL; (c, d) Side and top (C-pole) view of a cartoon representation of a hexadecameric fragment of an A-ENA-like nanofibril showing the helical stacking of A-ENA like units (i, i+1, i-1 etc.) and the association of two A-ENA-like protofibrils (f and f'). Fibrils extend by addition of new A-ENA like monomeric units at the C- or N-pole of the fibril. Shown in sphere representation are those residues implicated in intra- and inter-fibrillar isopeptide bonds cross-linking A-ENA like subunits according the schematic drawing in panels a and b.

[0086] Figure 10. Design principles and sequence motifs of self-assembling and self cross-linking protein nanofibrils of helical hairpins, (a, b) A-ENA like fibril assembly units adopt a generic structure comprising, in consecutive order: an N-terminal lock (NTL) of variable length and sequence, followed by an a-helix (al) comprising preferentially five helix Heptads (Hl-1 to Hl-5), a variable length linker region (L), an a-helix (a2) comprising preferentially five helix Heptads (H2-1 to H2-5), and a variable length C- terminal tail (CT)). In said A-ENA like fibril assembly units, the al and a2 Heptads hold a consesus sequence motif of hydrophobic and short side chain residue, thereby facilitating the adoption of an antiparallel helix hairpin within the A-ENA like units, and facilitating the pairwise stacking of the fibril assembly units (i / i+1 and i / i-1) into helical protofibrils (see Figure 7). In addition, a conserved sequence motif of hydrophilic resideus in helix heptads Hl-2, Hl-3, Hl-4 and Hl-5 in al, and H2-2, H2-3, H2-4 and H2-5 in a2 form isopeptide bond formation units consisting of acid-base catalists (y; preferably Glu or Asp), nucleophilic residues (8; preferably Lys or the N-terminal amine) and an electrophilic residue (e; preferably Glu, Gin, Asp or Asn). Said isopeptide bond formation units (shown in Figures 5, 7 and 9) can be individually replaced by hydrophobic residues for hydrophobic knobs-in-holes interactions. The NTL, linker (L) and C-tail elements are variable in length and sequence. A prefered consensus sequence for the NTL, in— non-limiting manner, is shown in panel c.

[0087] Figure 11. HMIVI profiles of Helix 1 and Helix 2. Sequence logo of the 35 residue long hmm profiles of Helix 1 and Helix 2 build on a set of 593 A-ENA (like) sequences - with the accession numbers listed in Table 3. Although pairwise sequence identity in this list of proteins is as low as 20-30 %, there is a strong conservation in key positions of Helix 1 and Helix 2, as reflected in the formulas provided herein for the heptad elements of each helix.

[0088] Figure 12. Isopeptide bond formation is required for self-assembly of stable A-ENA like nanofibrils, (a) SDS-PAGE of a purified A-ENA mutant (i.e. A-ENA E29A Q44A N64A E78A Q82A; SEQ ID NO: 15) wherein residues E29, Q44, N64, E78 and Q82 were mutated to an alanine residue; (b) and (c) nsTEM micrographs of lmg.mL1A-ENA in lxPBS. Scalebars represent 1pm and 0.1pm, respectively.

[0089] Figure 13. Overview of A-ENA engineering sites, (a) Sites for insertion of single amino acids, peptides or full domains at the wild-type A-ENA N-terminus, C-terminus or into the loop connecting helices al and a2; (b) Sites for insertion of single amino acids, peptides or full domains at the N-terminus of A-ENA ANTL (e.g. SEQ ID NO: 12), C-terminus or into the loop connecting helices al and a2; (c) and (e) Sites for site-directed mutagenesis of surface exposed residues on the wild-type A-ENA fibril: indicated in stick on the cartoon representation of A-ENA (c) and indicated in bold on the five heptads of helices al and a2, respectively; (d) and (f) Sites for site-directed mutagenesis of surface exposed residues on the ANTL A- ENA fibril: indicated in stick on the cartoon representation of A-ENA (c) and indicated in bold on the five heptads of helices al and a2, respectively.

[0090] Figure 14. Post-polymerization functionalization of A-ENA fibrils using the SpyTag / SpyCatcher system.

[0091] (a) Required genetic building blocks: A-ENA with C-terminal SpyTag and SpyCatcher with C-terminal fusion protein (here: Superfolder green fluorescent protein (sfGFP); Pedelacq et al. 2006. Nat Biotechnol. 24(l):79-88) and molecular model of the SpyTagged A-ENA fibril with a bound SpyCatcher-sfGFP unit. For schematic representation, only a single bound SpyCatcher-sfGFP molecule is shown here; (b) nsTEM micrograph of purified, homopolymeric A-ENA-SpyTag fibrils; (c) SDS-PAGE analysis of sfGFP with and without prior incubation with A-ENA-(l)-SpyTag fibrils, showing that A-ENA fibril bound Spycatch-sfGFP fluorescent signal is present in the stacking gel (high MW-no separation) of lane (2-3); (d) Fluorescence image of a centrifuged suspension of A-ENA-SpyTag fibrils incubated with SpyCatcher-sfGFP: the fluorescent insoluble phase consists of A-ENA fibrils.

[0092] Figure 15. Optimized linker design strategy for surface display of heterologous domains on the A-ENA scaffold, (a) Molecular model of a homopolymeric A-ENA fibril displaying fusion domains on the fibril surface: for this strategy a domain of interest is fused to A-ENA (either at the N- or C-terminus or within the al-a2 loop linker region). When recombinantly expressed, a homopolymeric A-ENA fibril is produced where the A-ENA to fusion domain ratio is 1:1. Crucially, to avoid steric hindrance between the displayed domains, a flexible linker is required. Using this approach, fusion domains with a hydrodynamic radius that are smaller than or equal to 150 % of the A-ENA fibril pitch (i.e. 10.8 nm) can be efficiently displayed;

[0093] (b) Examples of surface display of different heterologous domains coupled to the A-ENA C-terminus: nsTEM micrographs of A-EN A fibrils displaying p66a (SEQ ID NO: 8), MBD2 (SEQ ID NO: 9) and rubredoxin (SEQ ID NO: 10), respectively; (c) AlphaFold2 model and molecular weight of the corresponding domains discussed in (b); (d) Eppendorf inversion test was performed to probe for hydrogelation (e) Melting curves for wild-type A-ENA and A-ENA-Rubredoxin (RR) fusion protein.

[0094] Figure 16. Block copolymer design strategy for surface display of heterologous domains on the A-ENA scaffold, (a) Molecular model of a heteropolymeric A-ENA fibril displaying fusion domains on the fibril surface: for this strategy, a domain of interest is genetically fused to A-ENA (either at the N- or C-terminus within the al-a2 loop linker region). When recombinantly expressed in a background strain that also expressed wild-type A-ENA, a heteropolymeric A-ENA fibril is produced where the A-ENA to fusion domain ratio is larger than one. Using this approach, fusion domains with a hydrodynamic radius that are larger than or equal to 150 % of the A-ENA fibril pitch (i.e. 10.8nm) can be efficiently displayed; (b) nsTEM micrograph of block copolymer A-ENA fibrils consisting of wild-type A-ENA (block A) and Nanobody-TEV-A-ENA (SEQ ID NO: 11) units; (c) Alphafold2 model of the displayed nanobody.

[0095] Figure 17. Functional Display of camelid antibodies. Schematic representation of the A-ENA like nanofibril, with alternatively the SpyCatcher (left) or SpyTag (right) sequences in fusion to the NTL or CT of the A-ENA monomer (only a single fusion is shown for clarity). SpyCatcher and SpyTag A-ENA fusions form fibrils that can be used to capture, respectively, SpyTagged and SpyCatcher constructs. Shown here is SpyTagged (left) or SpyCatcher fused (right) Nb bound to the modified A-ENA nanofibrils.

[0096] Figure 18. A-ENA sporesilk networks associate Bacillus thuringienis Sv. israelensis spores with parasporal bodies, (a) Stain-free TEM images of A-ENA biofilm in Bti, grown on LB agar, scraped from the plate and resuspended in water. The sample was then deposited onto a Formvar / Carbon grid. The observed fibrils emerging from the spores correspond to A-ENA based on their diameter (=10 nm); (b) Stain-free TEM images of BTI AA-ENA strain grown on LB agar, scraped from the plate, resuspended in water and finally deposited onto a Formvar / Carbon grid. This strain shows a complete lack of A-ENA fibrils; (c, d) Stain-free TEM images of isolated spores from Bti WT and Bti AA-ENA strain. To isolate spores, 200 pl of spore suspension were added on top of 1ml of 50 % histodenz and the sample was centrifuged for 40 minutes at 16000 xg. The supernatant was removed and the pellet was washed 3 times with water (5 min at 16000 xg) before deposition onto a Formvar / Carbon grid; (e) Bti AA-ENA strain was complemented with a plasmid carrying the ORF of A-ENA gene under its native promoter. A- ENA biofilm is observed encompassing spores and PSBs; (f) Based on TEM images, spores and PSB were manually counted and the ratio PSB / spore is shown in the plot comparing spore suspension and spore isolation. The experiment was repeated 4 times, each time counting more than 100 entities (spore or PSB) per group. Panels (a) to (e) are representative images of each group. Figure 19. Exogenous A-ENA forms sporesilk networks that associate spores and parasporal toxin crystals in Bacillus thuringiensis Sv. Kurstaki (Btk). (a) Stain-free TEM images of WT Btk strain grown on LB agar, scraped from the plate and resuspended in water. PSB crystals are free in the media and lack direct attachment to the spores, (b) Stain-free TEM images of Btk strain expressing A-ENA gene from Bti under its native promoter. The images clearly show that spores are linked to PSB by A-ENA filaments and that A-ENA forms a biofilm as observed in Bti. (c, d) Isolated spores of WT Btk and +A-ENA Btk. Spores were isolated by sucrose cushion. Three different sucrose concentrations were added in a 1.5 ml tube (80 %, 70 % and 60 %) and 200 pl of spore suspension were added on top. The sample was centrifuged at 16000 x g for 20 minutes. Free PSB were found at the interface between 70 % and 80 % sucrose whereas spores were found at the pellet. The supernatant was completely removed and the pellet was washed 3 times with water (5 min at 16000 x g) before deposition onto a Formvar / Carbon grid, (e) Detailed PSB from Btk connected by A-ENA bundles, (f) Based on TEM images, spores and PSB were manually counted and the ratio PSB / spore is shown in the plot comparing spore suspension and spore isolation. The experiment was repeated 4 times, each time counting more than 100 entities (spore or PSB) per group. Panels (a) to (d) are representative images of each group.

[0097] Figure 20. A-ENA-mediated association of spores and parasporal toxins enhances the entomopathogenic activity of Bacillus thuringiensis. (a) The survival curve of Chironomus aprilinus in the presence of isolated spores, as illustrated in Figure 18, was determined; (b) Similarly, the survival curve of Chironomus aprilinus in the presence of spore suspension was assessed. For both experiments a) and b), each group consisted of a minimum of 8 larvae. Prior to commencing the experiment, the viability of all larvae was confirmed. Larvae of varying sizes representing different maturation stages were included. Isolated spores and spore suspension were administered at a final ODgoo of 0.02. As a negative control, phosphate saline buffer (PBS) was used. Larvae survival was monitored daily over a period of one week. The experiments were conducted in triplicate under room temperature conditions. In both plots, the three curves are statistically significative (p<0.0001) according to the log-rank (mantelcox) test; (c) An image displaying an example of both living and deceased larvae from the Chironomus aprilinus cohort used in the experimental setup.

[0098] Figure 21. In vivo functionalization of A-ENA sporesilk networks in Bacillus thuringiensis. Bacillus thuringiensis Israelensis (Bti) and Bacillus thuringiensis kurstaki (Btk) strains were subjected to electroporation using the pA180 plasmid, which carries A-ENA fused to the C-terminus of SpyTag under the control of its native promoter and terminator (SEQ. ID NO:7). Following spore isolation as described above, the spores were incubated with 100 pM of purified sfGFP N-terminally fused to SpyCatcher for 10 minutes at room temperature, while being continuously agitated. Subsequently, the spores were centrifuged at 10000 x g for 5 minutes and washed with PBS to eliminate any unbound sfGFP:SpyCatcher. This washing step was repeated three more times before imaging the spores. Fluorescence microscopy revealed that spores transformed with A-ENA:SpyTag displayed a robust fluorescence signal from the spore cluster and PSB in both Bti and Btk strains. To account for potential autofluorescence originating from the spores, wild-type (WT) Bti and Btk strains were included as negative controls. The scale bar in the images represents a length of 2.5 pm.

[0099] Figure 22. Stability analysis of recombinant A-ENA. Figure panels show A-ENA fibrils subjected to following treatments: (a) incubation for lh at 99°C in 2 % (w / v) SDS, (b) incubation for lh at room temperature in 8M urea, (c) high temperature desiccation; i.e. A-ENA fibers suspended in miliQ water were desiccated at 200°C for 15min, and rehydrated in miliQ water for imaging, (d) incubation for lh at room temperature in 2M NaOH, (e) autoclaving for 20min at 121 °C, (f) incubation for 30min at room temperature in 100 % (v / v) formic acid.

[0100] Figure 23. Enhanced entomopathogenic activity of Bacillus thuringiensis on Trichoderma ni (cabbage looper) larvae with purified recombinant A-ENA fibrils, a. Representative ns-EM images of wildtype Btk spore suspensions incubated with purified recombinant A-ENA fibrils (4.18 mg / mL, produced in E. coli) show that the exogenous addition of fibrils to spore preparations results in the clustering of spores and Cry toxin crystals in a network of A-ENA fibrils, b. representative picture of dead and alive Trichoderma ni (cabbage looper) larvae, following feeding on medium supplemented with Btk spore suspensions, c. Survival curve of Trichoderma ni larvae feeding on media supplemented with different formulations: spore suspensions of wild-type B. thuringiensis Sv. Kurstaki ('BTK WT'), recombinant B. thuringiensis Sv. Kurstaki expressing A-ENA ('BTK + A-ENA'), or wild-type B. thuringiensis Sv. Israelensis ('BTI'), and phosphate buffered saline as a negative control ('PBS'), d. Survival curves of T. ni larvae feeding on media supplemented with spore suspensions of wild-type B. thuringiensis Sv. Kurstaki ('BTK WT'); spore suspensions of wild-type B. thuringiensis Sv. Kurstaki ('BTK WT + A-ENA fibrils') incubated with purified recombinant A-ENA (4.18 mg / ml, produced in E. coli); and as a negative control, purified recombinant A- ENA fibrils (4.18 mg / ml), to rule out any toxic effects.

[0101] Figure 24. Functionalized A-ENA fibrils engineered by insertions in the linker region, a.-c. Schematic representation of the A-ENA linker insertion mutants (A-ENA_LI). d. negative stain EM image of cell suspension of E. coli DH5a expressing A-ENA_LI-R4.5-2RFD (SEQ ID NO:67); scale bar = 100 nm. The image shows the presence of assembled A-ENA_LI-R4.5-2RFD fibrils in the cell suspension e. nsEM images of purified A-ENA_LI-R4.5-2RFD, showing the presence of abundant, micrometer-long A-ENA-like nanofibrils of approximately 10 nm thickness, confirming that linker insertion mutants retain the capacity to self-assembly into SDS-stable protein nanofibrils; scale bars = 200 nm (left) and 100 nm (right). Figure 25. Mutant A_ENA_E28Q_E39Q_E41Q with a single IPB for A-ENA fibril self-assembly. A-ENA was mutated in residues E28, E39, and E41 to a Gin (Q), thereby removing the acid base catalyst of I PB1, 1, 4 and 5 which normally form in wild type A-ENA fibrils, hence only allowing IPB3 as interconnection between the A-ENA monomeric subunit, which showed to be sufficient for fibril assembly, (a, b). Ribbon (a) and schematic (b) diagram of A-ENA fibril with residues forming the IPB3 isopeptide bond (K76 -> Q82; SEQ ID NO: 68) shown as spheres (a) or indicated as arrows (b). IPB3 cross-links the f and f' protofibrils in an i -> i'-l fashion. Accordingly, a single isopeptide bond across the f - f' protofibril interface results in a nanofibril wherein all protomers are cross-linked to form a single covalent unit. To generate an A-ENA variant with a single IPB3, the acid base catalysts in A-ENA (SEQ ID: NO: 1) that are required for the formation of IPB1 (E41), IPB2 (E39) and IPB4 and 5 (E28) (Figure 5) were mutated to the similar, but non-catalytic residue Gin, resulting 'A_ENA_E28Q_E39Q_E41Q' (SEQ ID NO 68). (c). Alphafold 3 prediction of a protofibril of A_ENA_E28Q_E39Q_E41Q and associated PAE plot. (d). negative stain EM image of purified, SDS-resistant mutant-A-ENA fibrils formed by A_ENA_E28Q_E39Q_E41Q, demonstrating that a single, cross-fibrilar IPB (i.e. IPB3) is sufficient for the formation of robust protein nanofibrils. Scale bar = 100 nm.

[0102] Figure 26. General overview of A-ENA production and processing pipeline.

[0103] DESCRIPTION

[0104] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases 'in one embodiment' or 'in an embodiment' in various places throughout this specification are not necessarily all referring to the same embodiment but may. Definitions

[0105] Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where the term "essentially consisting of" or "consisting essentially of" or "comprising substantially" is used herein for chemical matter or compounds such as proteins, this means that specific further components or matter can be present, namely those not materially affecting the essential characteristics of the chemical matter. Where specifically mentioned herein, "comprising substantially" or "essentially consisting of" or "consisting essentially of" refer to the majority or bulk of the chemical matter , such as a polypeptide, wherein the functional outcome is defined thereby, but may contain further matter, such as further amino acids. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, structural biology, and / or computational biology).

[0106] The term "nucleic acid sequence", "DNA sequence" or "nucleic acid molecule(s)" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and singlestranded DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analog. By "nucleic acid construct" it is meant a nucleic acid molecule that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like. "Coding sequence" is a nucleotide sequence, which is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances. "Promoter region of a gene" or "regulatory element" as used here refers to a functional DNA sequence unit that, when operably linked to a coding sequence and possibly placed in the appropriate inducing conditions, is sufficient to promote transcription of said coding sequence. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A promoter sequence "operably linked" to a nucleic acid molecule that is a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the promoter sequence. "Gene" as used here includes both the promoter region of the gene as well as the coding sequence. It refers both to the genomic sequence (including possible introns) as well as to the cDNA derived from the spliced messenger, operably linked to a promoter sequence. The term "terminator" or "transcription termination signal" encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the natural gene, or from a variety of other (bacterial) genes. With a "chimeric gene" or "chimeric construct" or "chimeric gene construct" is meant a recombinant nucleic acid sequence molecule in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA, such that the promoter or regulatory nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid coding sequence. The regulatory nucleic acid sequence of the chimeric gene is not operatively linked to the associated nucleic acid sequence as found in nature, and may be heterologous to the encoding nucleic acid sequence molecule, meaning that its sequence is not present in nature in the same constellation as presented in the chimeric construct. More general, the term "heterologous" is defined herein as a sequence or molecule that is different in its origin.

[0107] The terms "protein", "polypeptide", and "protein domain" are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. A monomeric or protomer is defined as a single polypeptide chain from amino-terminal end (also referred to herein as N-term or N-terminus or N-terminal end) to carboxy-terminal end (also referred to herein as C-term or C-terminus or C-terminal end). A "protein subunit" as used herein refers to a monomer or protomer, which may form part of a multimeric protein complex or assembly. A "protein domain" as used herein refers to a folded protein, or folded part of a protein as a distinct functional and / or structural unit in a protein. Usually a protein domain is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains may exist in a variety of biological contexts, where similar domains can be found in proteins with different functions. Protein secondary structure elements typically spontaneously form as an intermediate before the protein folds into its three dimensional tertiary structure. The two most common secondary structural elements of proteins are alpha helices and beta (P) sheets, though p-turns and omega loops occur as well. Beta sheets consist of beta strands (also p-strand) connected laterally by at least two or three back-bone hydrogen bonds, forming a generally twisted, pleated sheet. A p-strand is a stretch of poly-peptide chain typically 3 to 10 amino acids long with backbone in an extended conformation. A p-turn is a type of non-regular secondary structure in proteins that causes a change in direction of the polypeptide chain. Beta turns (P turns, p- turns, p-bends, tight turns, reverse turns) are very common motifs in proteins and polypeptides, which mainly serve to connect p-strands.

[0108] "Coiled coils" are tertiary protein structures in which alpha helices are wound around each other to form superhelical bundles, usually consisting of two or three helices in parallel or antiparallel orientation. The coiled coil is usually formed by multimeric proteins either of the same (homo) or of different chains (hetero). Consecutive helices from the same polypeptide chain may as well fold into a coiled coil structure, typically the helices of the same monomer are then in antiparallel orientation. The helices constituting the coiled coil interact via a knobs-into-holes geometry of amino-acid sidechains at their interface (Crick FHC (1952) Nature 170:882-883), wherein a residue from one helix (knob) packs into a space surrounded by four sidechains of the facing helix (hole). This regular packing also provides for the necessity of the heptad repeat, defined as precisely recurrent positions of the side-chains every seven residues along the helix interface. The heptad regularity of side-chain positions of coiled coils is thus indicated by seven-residue sequence repeats whose positions are labelled a-g; wherein typically the core-forming positions (a and d) are usually occupied by hydrophobic residues, whereas the remaining, solvent-exposed positions (b, c, e, f, and g) are dominated by hydrophilic residues. Through this strict patterning, coiled coils are predictable based on their amino acid sequence to a level of detail that permits the assignment of individual residues to the positions of the heptad repeat. Prediction tools are known to the skilled person, as for instance described in Lupas et al. (2017; The Structure and Topology of a-Helical Coiled Coils. Subcell Biochem; 82: 95-129).

[0109] In the context of the present invention, 'self-assembly' refers to the spontaneous organization of molecules in ordered supramolecular structures thanks to their mutual non-covalent interactions without external control or template. The chemical and conformational structures of individual molecules carry the instructions of how these are assembled. The same or different molecules may constitute the building blocks of a molecular self-assembling system. Generally, interactions are established in a less ordered state, such as a solution, random coil, or disordered aggregate leading to an ordered final state, which can be a crystal or folded macromolecule, or a further assembly of macromolecules. The association of small molecules or proteins into well-ordered structures is driven by thermodynamic principles, thus, based on energy minimization. The interactions involved in the molecular assembly process are electrostatic, hydrophobic, hydrogen bonding, van der Waals interactions, aromatic stacking, and / or metal coordination. Although non-covalent and individually weak, these forces can generate highly stable assemblies and govern the shape and function of the final assembly (Lombardi et al., 2019; Pharmaceutics, 11, 166). Said self-assembling protein subunits described herein, and called A-ENA monomeric proteins herein, are capable of self-assembling into monomers, further multimerize and fold and engage into protein fibrils as described herein. The fibrous assemblies can be obtained from the pre-existing components termed building blocks, or subunits, more specifically the isolated self-assembling A-ENA monomeric proteins as described herein.

[0110] The terms "chimeric polypeptide", "chimeric protein", "chimer", "fusion polypeptide", "fusion protein", or "heterologous fusion", are used interchangeably herein and refer to a protein that comprises at least two separate and distinct polypeptide components that may or preferably may not originate from the same protein. The term also refers to a non-naturally occurring molecule which means that it is manmade. The term "fused to", and other grammatical equivalents, such as "covalently linked", "connected", "attached", "ligated", "conjugated", and as specifically used herein 'inserted in' when referring to a chimeric or fusion polypeptide (as defined herein) refers to any chemical or recombinant mechanism for linking two or more polypeptide components. The fusion of the two or more polypeptide components may be a direct fusion of the sequences or it may be an indirect fusion, e.g. with intervening amino acid sequences or linker sequences, or chemical linkers. The fusion of amino acid residues or (poly)peptides to an Ena protein or insertion into an Ena protein sequence, or to another protein of interest as described herein, may be a covalent peptide bond, or also refer to a fusion obtained by chemical linking. The term "fused to", as used herein, and interchangeably used herein as "connected to", "conjugated to", "ligated to" refers, in particular, to "genetic fusion", e.g., by recombinant DNA technology, as well as to "chemical and / or enzymatic conjugation" resulting in a stable covalent link.

[0111] As used herein, the term "protein complex" or "protein assembly" or "multimer" refers to a group of two or more associated macromolecules, whereby at least one of the macromolecules is a protein. A protein complex or assembly, as used herein, typically refers to binding or associations of macromolecules that can be formed under physiological conditions. Individual members of a protein complex, such as protein subunits or protomers, are linked by non-covalent or covalent interactions. "Binding" means any interaction, be it direct or indirect. A direct interaction implies a contact between the binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more molecules. The binding or association maybe non-covalent - wherein the juxtaposition is energetically favoured by for instance hydrogen bonding or van der Waals or electrostatic interactions - or it may be covalent, for instance by peptide bonds, disulphide bonds or isopeptide bonds.

[0112] It will be understood that a protein complex can be multimeric. Protein complex assembly can result in the formation of homo-multimeric or hetero-multimeric complexes. Moreover, interactions can be stable or transient. The term "multimer(s)", "multimeric complex", or "multimeric protein(s) or assemblies" comprises a plurality of identical or heterologous polypeptide monomers. Polypeptides can be capable of self-assembling into multimeric assemblies (i.e.: dimers, trimers, pentamers, hexamers, heptamers, octamers, etc.) formed from self-assembly of a plurality of a single polypeptide monomers (i.e., "homo-multimeric assemblies") or from self-assembly of a plurality of different polypeptide monomers (i.e. "hetero-multimeric assemblies"). As used herein, a "plurality" means 2 or more. The multimeric assembly comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or preferably more polypeptide monomers. The multimeric assemblies can be used for any purpose and provide a way to develop a wide array of protein "nanomaterials." In addition to the finite, cage-like or shell-like protein assemblies, they may be designed by choosing an appropriate target symmetric architecture. The monomers or protomers and / or multimeric assemblies of the invention can be used in the design of higher order assemblies, such as protofibrils, fibrillar assemblies or fibrils and further fibers, with the attendant advantages of hierarchical assembly. The resulting multimeric or fibrous assemblies are highly ordered materials with superior rigidity and monodispersity, and can be functional as a multimer or fibrous structure itself, or form the basis of advanced functional materials, such as modified surfaces containing multimeric assemblies or fibrillar structures, and custom-designed molecular machines with wide-ranging applications. More specifically, a multimer as used herein refers to homo- or heteromultimeric, or homo- or heteropolymeric protein complexes which are associated with each other to form an organized structure, though non-covalent and / or covalent interactions; and / or further modified to grow or develop into self-assembling or triggered formation of nanofibrils. Said multimeric assemblies may contain monomeric ENA fusion proteins as defined herein, or ENA protein variants, mutant and / or engineered ENA proteins, such as those exemplified and described herein, as well as other proteins that may associate to said ENA protein-based, or in particular A-ENA-based multimers, called engineered multimers, thereby expanding said multimer towards further modifications required for certain applications.

[0113] By "recombinant polypeptide" is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide, which may be obtained in vitro and / or in a cellular context. When the chimeric polypeptide or fusion polypeptide or biologically active (i.e. functional) portion thereof is recombinantly produced, it is also preferably enriched, purified or made substantially free of culture medium, i.e., the impurities represent less than about 20 %, more preferably less than about 10 %, and most preferably less than about 5 % of the volume of the protein preparation. By "isolated" or "purified" is meant material that is substantially or essentially free from components that normally accompany it in its native state.

[0114] "Homologue", "Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and / or insertions relative to the unmodified or wild-type protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met, also indicated in the conventionally known one-letter code herein) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. A "substitution", or "mutation" as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity. The percentage of amino acid identity as provided herein is preferably in view of a window of comparison corresponding to the total length of the native or natural wild-type protein, or of the specific amino acid sequence referred to.

[0115] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source, or included in a cell, cell line or organism. A wild-type gene or gene product is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene or gene product a observed in nature. In contrast, the term "modified", "engineered", "mutant" or "variant" refers to a gene or gene product that displays modifications in sequence, post-translational modifications and / or functional properties (i.e. altered characteristics) when compared to the wild-type or naturally-occurring gene or gene product. A knock-out refers to a modified or mutant or deleted gene as to provide for non-functional gene product and / or function. It is noted that naturally occurring mutants or variants may be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product, and a different sequence as T1 compared to the reference gene or protein. With the term 'functional homologue' or 'functional variant' is herein referred to a modified, engineered or mutant gene product with retained functionality as compared to the A-ENA wild type or original sequence on which the functional homologue or functional variant is based for introduction of modifications or mutations. With 'functionality' is herein referred to the self-assembling characteristic of the monomeric protein subunits, and the further spontaneous assembly of said monomers into nanofibril lar structures, as described herein.

[0116] The term "vector", "vector construct", or "recombinant vector", as used herein, can be double-stranded or single- stranded and may be DNA, RNA, or DNA / RNA hybrid molecules, in any conformation including but not limited to linear, circular, coiled, supercoiled, torsional, nicked and the like. These vectors of the invention include but are not limited to plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenoviral, AAV or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC), all of which are well-known and can be purchased from commercial sources. Any vector may be used to construct and express the fusion molecules used in the invention. General classes of vectors of particular interest include prokaryotic and / or eukaryotic cloning vectors, expression vectors, fusion vectors, phage and yeast display vectors, shuttle vectors for use in different hosts, mutagenesis vectors, transcription vectors, vectors for receiving large inserts and the like. Most of the requisite methodology can be found in Ausubel et al. 2007. Vector constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system recognized by the host, including the intended DNA fragment encoding the nucleic acid molecule of the present invention, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the molecule-encoding segment. Expression systems may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Signal peptides may also be included, where appropriate, from secreted polypeptides of the same or related species, which allow the protein to cross and / or lodge in cell membranes, or be secreted from the cell. An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host. Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Examples of workable combinations of cell lines and expression vectors are described in for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). Many useful vectors for expression in bacteria, yeast, fungal, mammalian, insect, plant or other cells are well known in the art.

[0117] "Host cells" can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. For all standard techniques see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). Recombinant host cells, in the present context, are those which have been genetically modified to contain an isolated DNA molecule, nucleic acid molecule or expression construct or vector of the invention. The DNA can be introduced by any means known to the art which are appropriate for the particular type of cell, including without limitation, transformation, lipofection, electroporation or viral mediated transduction. A DNA construct capable of enabling the expression of the chimeric protein of the invention can be easily prepared by the art-known techniques such as cloning, hybridization screening and Polymerase Chain Reaction (PCR). Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (2012), Wu (ed.) (1993) and Ausubel et al. (2016). Representative host cells that may be used with the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells. Bacterial host cells suitable for use with the invention include Escherichia spp. cells, Bacillus spp. cells, Pasteuria spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, Lactococcus spp. cells, Lactobacillus spp. cells, and Salmonella spp. cells. Animal host cells suitable for use with the invention include insect cells and mammalian cells (most particularly derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa. Yeast host cells suitable for use with the invention include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts. The host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively, the host cells may also be transgenic animals, or plants or parts or materials derived thereof. The term "medicament", or "medicine", as used herein, refers to a substance / composition used in therapy, i.e., in the prevention or treatment of subject potentially suffering from a disease or disorder. The terms "disease" or "disorder" refer to any pathological state, in particular to the diseases or disorders as defined herein.

[0118] The term "subject", "individual" or "patient", used interchangeably herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, for whom diagnosis, therapy or prophylaxis is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In one embodiment, the subject is a human, a rat or a non-human primate. Preferably, the subject is a human. In one embodiment, a subject is a subject with or suspected of having a disease or disorder, or is expected to be at high risk of developing a disease or disorder, in particular a disease or disorder as disclosed herein, also designated "patient" herein.

[0119] The terms "treatment" or "treating" or "treat", or "a method to treat", can be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. Therapeutic treatment is thus designed to treat an illness or to improve a person's or animal's health, rather than to prevent an illness. Treatment may also refer to a prophylactic treatment which relates to a medication or a treatment designed and used to prevent a disease from occurring, herein referred to as "prevention".

[0120] A "composition" relates to a combination of one or more active molecules, preferably including one or more protein nanofibrils of the present invention, and may further include buffered solutions and / or solutes such as pH buffering substances, water, saline, physiological salt solutions, glycerol, preservatives, etc. for which a person skilled in the art is aware of the suitability to obtain optimal performance. Suitable conditions as used herein could also refer to conditions for using, applying or administering a protein nanofibril as described herein, or may include bacterial culture or endospore materials or remains from bacterial cultures when protein nanofibrils are produced in and / or isolated from sporulating bacteria.

[0121] A "pharmaceutical composition" is a therapeutically active composition comprising the therapeutically active agents or therapeutically active compositions provided by the present invention and optionally comprising a carrier, diluent or excipient. A "carrier", or "adjuvant", in particular a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable adjuvant" is any suitable excipient, diluent, carrier and / or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. Preferably, a pharmaceutically acceptable carrier or adjuvant enhances the immune response elicited by an antigen. Suitable carriers or adjuvantia typically comprise one or more of the compounds included in the following non-exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. The term "excipient", as used herein, is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, stabilizing agents, flavouring agents or colorants. A "diluent" includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, or preservatives may be included in such vehicles. A pharmaceutically effective amount of polypeptides, or conjugates of the invention and a pharmaceutically acceptable carrier is preferably that amount which produces a result or exerts an influence on the particular condition being treated. For therapy, the pharmaceutical composition of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including orally, parenterally, topically, nasally, ophthalmically, intrathecally, intracerebroventricularly, sublingually, rectally, vaginally, and the like. Still other techniques of formulation as nanotechnology and aerosol and inhalant are also within the scope of this invention. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter-indications and other parameters to be taken into account by the clinician. The pharmaceutical composition of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. When prepared as lyophilization or liquid, physiologically acceptable carrier, excipient, stabilizer need to be added into the pharmaceutical composition of the invention (Remington's Pharmaceutical Sciences 22nd edition, Ed. Allen, Loyd V, Jr. (2012). The dosage and concentration of the carrier, excipient and stabilizer should be safe to the subject (human, mice and other mammals), including buffers such as phosphate, citrate, and other organic acid; antioxidant such as vitamin C, small polypeptide, protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as PVP, amino acid such as amino acetate, glutamate, asparagine, arginine, lysine; glycose, disaccharide, and other carbohydrate such as glucose, mannose or dextrin, chelate agent such as EDTA, sugar alcohols such as mannitol, sorbitol; counterions such as Na+, and / or surfactant such as TWEEN™, PLURONICS™ or PEG and the like.

[0122] Detailed description

[0123] The present invention is based on a new type of Endospore appendage (ENA) proteins, in particular Alpha-helical Endospore Appendage proteins, or A-ENA, which were found to function as self-assembling proteins forming multimeric fibrous structures associated with Bacillus thuringiensis (Bt) endospores and parasporal bodies, and contributing to the pathogenicity of these bacterial spores, for instance as insecticidal agents. These fibrous structures appearing in Bt endospore cultures were resolved herein for the first time, to reveal a proteinaceous content constituting A-ENA proteins, appearing in a highly- organized, compelling protein fibril structure spontaneously folded to provide for an extremely high stability and rigidity to strongly associate the spore with the parasporal body. Investigating the biological function of these fibrous assemblies through the application of Bt A-ENA knock-out strains and complementation of speculating bacteria lacking endogenous A-ENA proteins indeed indicated that the association of the parasporal bodies, which comprehend the bipyramidal crystals containing toxic protei ns to kill lepidopterous insects, are associated to the Bt spores through the A-E N A-based fibrils.

[0124] The rigidity, thermal and mechanical stability and tensile strength of those A-ENA-based protein fibrils appeared to derive from the presence of Several isopeptidic covalent interactions present within the fibrils, resulting in a biological structured material with huge potential in different application areas. However, ex v / vo isolation of A-ENA fibrils separated from parasporal bodies was principally impossible since these naturally occurring A-ENA-based fibrils, as appearing in Bt spore cultures, always strongly associated with parasporal bodies among potentially further components of said sporulated cultures, thereby playing a key role in the toxicity of Bt spores to insects, though complicating their isolation and structural analysis. Finally, through more advahced technical improvements in the Cryo-EM field, the structural ana lysis of Bacillus thuringiensis israelensis (Bti) spore cultures allowed to identify these Bti fibrils as A-ENA proteih-based fibrils, represented in the genome of this Bacillus species by two A-ENA genes encoding two proteins (depicted A-ENA and A-ENA-1 herein, SEQ ID NO:1 and 2, resp.). Further comparative analysis of homologous bacterial protein sequences, and based on the novel structural insights, allowed to define the structural features of this newly annotated 'A-ENA' protein family.

[0125] Alphahelical Endospore Appendage (A-ENA) proteins

[0126] The A-ENA proteins are defined herein as protein monomers which appear as self-assembling alphahelical (a-helical) antiparallel coiled-coil monomers providing for a helical pattern in its structure which is determined by its amino acid sequence, as known for coiled-coil structured proteins, and as further defined herein, consisting of a consensus sequence determined by the fragments NTL-helix 1, L, helix 2, CT, wherein each helix is defined by the sequence at least 5 heptad elements (H 1-5), wherein each heptad element contains 7 amino acids, of which the side chains are defined as 'abcdefg', and as further defined herein. Furthermore, in addition to their sequence-based alpha-helical cbiled-cOil structural features, A-ENA proteins are characterized by the presence of a number of amino acid residues that, within said self-assembled antiparallel coiled-coil hydrophobic environment, position geometrically as such that autocatalytic formation of one or more isopeptide covalent bonds within or with other A-ENA monomers is facilitated, and resulting in a 'functional' A-ENA protein, which is capable of spontaneously forming a fibrillar assembly, as described herein.

[0127] So the present invention provides for a newly annotated bacterial protein family which is defined as 'A- ENA proteins' functional in spontaneous formation of protein fibrils, which intermolecularly connect through isopeptide bonds shown herein to be responsible for the irreversible nature of the strongly assembled fibrous structures. Further resolution of the recombinantly produced fibrils using exogenous introduction of Bti A-ENA into F.co / / , led to the matriculate structural design of the fibril build-up required to obtain the protein nanofibril as described herein, based on a coiled-coil monomeric Structure defined by its heptad repeat elements and consensus formula as described herein, thereby providing the required rigidity through the autocatalytic association of at least one or more isopeptide (IPB) bonds upon its self-assembly. Although the wild type A-ENA identified herein as SEQ ID NO: 1, assembling these Bti protein fibrillar structures connecting the spore and parasporal body contains multiple I PBs, interestingly, mutation of all but one of the conserved acid / base autocatalytic residues revealed that a nanofibril still spontaneously assembles when at least one IPB can be formed between the A-ENA protein monomer subunits of the two protofibrils. This need for at least one (cross-fibrillar) IPB supports the further finding that A-ENA structural homologues with low sequence identity still allow to self-assemble into nanofibrils if they comply to the sequence motif provided for the heptad elements and contain at least one of the IPB acid / base catalyst (y), donor (6), and acceptor (s) positions in said heptads complying to the E / D (for y), K as donor, and E / Q / D / N as acceptor amino acid identity, resp., as to produce the at least one covalent IPB connection for establishing the necessary stability of the assembled protein nanofibril.

[0128] So, a first aspect of the invention relates to a protein fibril built from or consisting of two protofibrils, wherein each protofibril contains two or more monomer protein subunits, based on or derived from or consisting of A-ENA protein subunits, which spontaneously fold as alpha-helical antiparallel coiled-coil structured monomers and have an amino acid sequence which consists of protein fragments according to the formula: NTL-helixl(hl)-L-helix2(h2)-CT, wherein each fragment NTL, L, and CT, contains at least 1, 1 and 4 amino acids, respectively, and wherein fragments hl and h2 (also called helix al and helix a2 herein) contain at least 5 heptad elements to allow formation of the helix hairpin, and wherein the amino acids are defined as further detailed below, and wherein said monomeric protein subunits are interconnected through at least one or more isopeptidic bonds (IPBs). Said self-assembling monomer protein subunits are A-ENA proteins, as defined herein, or are A-ENA-based proteins or proteins derived from A-ENA protein subunits, as also further defined herein, wherein said protein fibril thus constitutes the basic A-ENA protein structure scaffold as defined herein, and when A-ENA-based or derived protein monomer sequences are applied for forming the fibril, these are also functional in assembling into the fibril similar as for wild type A-ENA proteins, but may contain modifications, such as insertions, mutations, fusions, or conjugations to the A-ENA monomer (therefore called an 'A-ENA-based' monomer or 'A-ENA derived' monomer sequence, i.e. functional homologues, or functional variants, or engineered A-ENA proteins), which are provided by engineering the A-ENA monomer sequences or proteins, as described further below herein.

[0129] Specific embodiments may also relate to said protein fibril built from two protofibrils, wherein each protofibril contains two or more monomer protein subunits, based on or derived from or consisting of A-ENA protein subunits, wherein said protein nanofibril is an 'A-ENA-based protein nanofibril' in that beyond the A-ENA protein subunits, and / or derived or variants thereof, the fibril may as well contain further components derived from the production host, from attached sporulation culture, or which are purposely attached to or brought in contact with the protein nanofibril. Said components may be synthetic materials such as chemicals, plastics, surfaces, etc, or natural materials, such as other proteins, carbohydrates, lipids, LPS, metabolites, bacterial components, etc.

[0130] As to clearly define the basic structural features of said A-ENA protein monomeric subunits, the features considered necessary to allow for a folded protein functional in spontaneous fibrous assembly were deducted based on the structural information disclosed herein for Bti A-ENA nanofibrils and validated by structural modelling of potential A-ENA orthologues followed by their recombinant production and self-assembly into fibrils. Indeed, alignment of A-ENA orthologues based on the heptad repeat elements demonstrated a strong conservation of the position of hydrophobic residues implicated in the intra- and inter-helical knob-in-holes interactions (labelled (|) and \| / for hydrophobic and short chain residues, respectively), required for obtained a coiled-coil structure, and residues involving the interhelical isopeptide bond units (labelled y, 8 and s for respectively the acid / base catalyst, the nucleophilic residue and the electrophilic residue).

[0131] The protein nanofibril of the present invention comprises or (at least) consists of two protofibrils, each composed of at least two monomeric A-ENA or A-ENA-based protein subunits comprising covalently connected amino acid sequence fragments according to the formula NTL-helixl(hl)-L-helix2(h2)-CT, wherein said monomeric protein subunit spontaneously folds in aqueous solution into two alpha helices, named helix 1 (hl) and helix 2 (h2), as an alpha-helical antiparallel coiled-coil structure, as defined herein. The helices hl and h2, or alternatively called alpha-helices al and a2, are defined herein as consecutive heptameric units or 'heptads' or 'heptad repeats' or 'heptad elements' (H), comprising amino acid residues with side chains designated a to g, thus 'abcdefg'. Each of said two helices contains a continuous sequence of at least 5 heptad (H) elements, which are defined according to the formula Hl-1 - Hl-2 - Hl-3 - Hl-4 - Hl-5 for helix 1, and H2-1 - H2-2 - H2-3 - H2-4 - H2-5 for helix 2, wherein said heptad elements each comprise seven amino acid residues designated 'abcdefg' with each heptad element having a consensus sequence as defined here below, as to form a helix hairpin by joining into an antiparallel packing contact by hydrophobic knobs-in-hole interactions which is a required pattern to provide for an antiparallel coiled-coil structure, and to create a hydrophobic environment with the provision of the residues positioned to autocatalytically form intermolecular IPBs in the presence of further monomers.

[0132] So within the A-ENA subunits, the primary amino acid sequence of said hl and h2 helices of said NTL-hl- L-h2-CT formula determine the 3D structure to obtain said helical wheels along the length of the helices, to assemble into a coiled-coil structure, wherein IPB formation is triggered in the presence of further A- ENA or A-ENA-based monomers.

[0133] Coiled-coils are conventionally known to fold from heptad elements present in the amino acid sequence to form helices that pack as knobs-in-holes structures, due to the side chain systematic appearance of said a to g heptad element residue pattern forming ridges and valleys. Thus the spontaneous folding of said coiled-coil structure of the monomer is predictable by the skilled person in the possession of the amino acid sequence, for instance by applying a prediction tool as for instance described in Lupas et al. (2017; The Structure and Topology of a-Helical Coiled Coils. Subcell Biochem; 82: 95-129). So dictated by the conserved motif in each of the subsequently defined heptad elements of the monomeric amino acid sequence, and using the available tools in the art, a person skilled in the art can deduce the coiled-coil structural feature for a polypeptide. In the present invention, a helix hairpin is thus formed by the monomers as a result of the presence of the intramolecular antiparallel coiled-coil structure induced by the sequence of hl and h2 in the NTL-hl-L-h2-CT consensus sequence of the A-ENA proteins, as defined herein.

[0134] The A-ENA monomeric protein subunits comprised in the protofibril and nanofibrils of the present invention are based on the initial characterization of the Bti fibrous assemblies structurally analyzed from the spore cultures, thereby revealing the main constituent being the subunit provided by the A-ENA protein sequences (SEQ. ID NOs: 1 and 2), as disclosed herein, forming the fibrils on the endospore connection that tightly associates the spore with the parasporal bodies. Based on the structural resolution and the protein sequence, the monomeric protein sequence consensus protein for providing the coiled-coil basis of the self-assembling protein nanofibril structure of the present invention is presented to constitute the NTL-hl-L-h2-CT fragments, wherein said fragments NTL, hl, L, h2 and CT are connected to each other to form one polypeptide chain, so through peptide bonds, and wherein helix 1 and helix 2 are composed of at least 5 heptad elements (H), wherein the consensus sequence for each of said heptad elements is provided herein to result in a monomeric protein subunit of the A-EN A protein family, as defined herein, that is capable of forming an antiparallel coiled-coil which further assembles into a protofibril which in its turn intertwines to result in the protein nanofibril of the present invention. Furthermore, said heptad elements as defined herein include the necessary provisions to allow the spontaneous intermolecular isopeptide interactions irreversible connecting said monomers within said protein nanofibril.

[0135] The monomeric protein subunit of the protein nanofibril provided herein is called Alpha-helical- Endospore appendage (A-ENA) polypeptide, a protein family originating from bacteria, particularly from sporulating bacteria, most particularly from Bacillus, and is defined herein as a spontaneously folding monomeric protein subunit forming 2 alpha-helices which assemble in an antiparallel coiled-coil structure, packed as knobs into holes, with an amino acid sequence comprising fragments according to the following formula NTL-hl-L-h2-CT, which are covalently connected via peptide bonds to form one polypeptide chain, and wherein:

[0136] NTL refers to the N-terminal lock, or N-terminal amino acid sequence, which contains at least 1 amino acid preceding the sequence constituting helix 1, and which provides for a variable fragment; L refers to the linker fragment which forms a loop or turn in the monomer 3D structure, and comprises at least four amino acid residues, located between the helix 1 and helix 2, providing the covalent connection (through peptidic bonds) to allow an alpha-helical intramolecular coiled-coil, and typically said linker is variable in its sequence;

[0137] CT refers the C-terminal tail or C-terminal end of the polypeptide of the A-ENA monomer and contains at least one amino acid residue following the sequence constituting helix 2, and which provides for a variable fragment; and hl and h2 referring to helix 1 and helix 2, resp., also referred to as al helix or a2 helix, of said coiled- coil, which are further defined by the present of (at least) 5 heptad elements (depicted Hl-5) in each of said helix 1 and helix 2, and are defined for the A-ENA protein family as follows: Helix 1 comprises or consists of Hl-1 -Hl-2 -Hl-3 -Hl-4 -Hl-5 wherein said heptad elements are defined by residues 'abcdefg' and connected in this particular order from N-terminal end to C- terminal end of said helix, and wherein the consensus amino acid sequence 'a-b-c-d-e-f-g' of each heptad element (Hl-1 to Hl-5) is, or respectively corresponds to,:

[0138] -

[0139] Helix 2 comprises or consists of H2-1 -H2-2 -H2-3 -H2-4 -H2-5 wherein said heptad elements are defined by residues 'abcdefg' and connected in this particular order from N-terminal end to C- terminal end of said helix, and wherein the consensus amino acid sequence 'a-b-c-d-e-f-g' of each heptad element is, or respectively corresponds to,:

[0140] - and wherein said consensus amino acid sequence residues are defined as follows:

[0141] 'O' represents a hydrophobic amino acid residue selected form the list of M, V, I, L, A, G, H, W, Y, F for at least 70 % of said O> positions indicated in said heptad elements; preferably from the list of L, M, I, V, F and W;

[0142] 'tp' represents a short side chain amino acid residue selected from the list of V, C, G, A, P, S, T, N, D; preferably from the list of A, G, S, and T;

[0143] 'y' is an amino acid residue for isopeptide interactions functioning as acid / base catalyst, selected from E , or D, for at least one or more of said y positions indicated in said heptad elements; preferably the amino acid representing y is E;

[0144] '6' is an amino acid residue for isopeptide interactions functioning as 'donor' or nucleophile, which is preferably a Lysine residue (K), for at least one or more of said 6 positions indicated in said heptad elements; 's' is an amino acid residue for isopeptide interactions functioning as 'acceptor' or electrophile, selected from the list of E, Q, D, and N, for at least one or more of said s positions indicated in said heptad elements; preferably from the list of E, Q. and N; and wherein X represents an amino acid that can be any type of amino acid residue known in the art, and of which the identity is not restrictive for the functionality of the A-ENA protein.

[0145] Amino acids 'X' or 'any type of amino acid residue' as used herein may thus be defined as any one of the 20 naturally occurring amino acid residues conventionally known and listed herein (below), as well as different isomeric forms or enantiomers thereof and synthetic analogues or variants thereof. The amino acids are presented herein by their 3- or 1-lettercode nomenclature as defined and provided also in the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides. Eur. J. Biochem. 138: 9-37 (1984)); as follows: Alanine (A or Ala), Cysteine (C or Cys), Aspartic acid (D or Asp), Glutamic acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or He), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q. or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Vai), Tryptophan (W or Trp), and Tyrosine (Y or Tyr).

[0146] In a further specific embodiment, 'O' represents a hydrophobic amino acid residue selected form the list of M, V, I, L, A, G, H, W, Y, F for at least 75 %, 80%, 85 %, 90 %, 95%, or 99 %, or 100% of said <D positions indicated in said heptad elements.

[0147] In a specific embodiment wherein said monomeric protein subunit is interconnected in the protein nanofibril described herein through a single IPB per monomer, the positions in the monomer defined as Y, '6', and 's' will provide the respective acid / base catalyst, donor and acceptor residues at least at H2- 5 'y' being an E or D; at least the H2-3 '6' provides a Lysine, and at least the H2-4 's' provides for an E, Q, D or N acceptor, thereby allowing cross-protofibrillar covalent IPB formation (see figure 25). The positions of residues defined as Y, '6', and 's' elsewhere in the heptad motifs may in this case be any amino acid (herein defined as 'X' above), preferably an amino acid not sterically hindering the folding or fibril formation, such as a Q instead of E or D for the acid / base catalyst.

[0148] In more specific embodiments, the monomeric protein subunit of said protein nanofibril described herein is covalently interconnected with further monomers through multiple IPBs per monomer, wherein the monomer sequences are defined in their Y, '6', and 's' residues in the heptad elements to provide for the respective acid / base catalyst, donor and acceptor residues, resp., required to form said IPBs (as also indicated in figure 7) between the specific side chain residues connecting one or more of : NTL of (i) and Hl-5b of (i-5), (i-4), (i-3), or (i-2);

[0149] Hl-4g of (i) and H2-2a of (i-1);

[0150] H2-3g of (i) and H2-4a of (i-1);

[0151] H2-3f of (i) and H2-4e of (i');

[0152] H2-4g of (i) and Hl-3a of (i-1);

[0153] H2-4g of (i+l)and Hl-3a of (i);

[0154] NTL of (i+5), (i+4), (i+3), or (i+2) and Hl-5b of (i);

[0155] Hl-4g of (i+1) and H2-2a of (i);

[0156] H2-3g of (i+1) and H2-4a of (i); and / or

[0157] H2-3f of (i'+l) and H2-4e of (i).

[0158] As shown in Figure 7 , the 'y', '6' , and 's' residues of these positions required to form said IPB between some or all of these IPB connections are thus present in these monomers as an E or D as catalyst, a Lysine as donor, and an E, Q, D or N as acceptor. For monomers wherein not all of the 'y', '6', and 's' residues in the heptad elements are involved in IPB connections, said positions may hence also contain alternative amino acids which are not functional as catalyst (e.g. Gin), as donor, or as acceptor, so any type of amino acid preferably an amino acid wherein the side chain is similar in structure to the limitations of amino acid side chains that are functional as catalyst, donor or acceptor.

[0159] In a further embodiment, the protein nanofibril as described comprises or consists of two protofibrils, each comprising at least two A-ENA monomers, as defined herein above, and wherein said helix 1 and helix 2 of said monomeric protein subunits comprises, or consists of, or is defined by the consensus sequence of the heptad elements according to the formula: and wherein said consensus amino acid sequence residues are defined as follows:

[0160] O is a hydrophobic amino acid residue selected form the list of M, V, I, L, A, G, H, W, Y, F; preferably L, M, I, V, F or W;

[0161] O is a short side chain amino acid residue selected from the list of V, C, G, A, P, S, T, N, D; preferably A, G, S, or T; at least one of said y positions is an amino acid residue for isopeptide interactions functioning as acid / base catalyst, selected from E, or D; preferably E; the remaining y residues can be any type of amino acid residue; at least one of said 6 positions is an amino acid residue for isopeptide interactions functioning as 'donor' or nucleophile, selected from K; the remaining 6 residues can be any type of amino acid residue; at least one or more of said s positions is an amino acid residue for isopeptide interactions functioning as 'acceptor' or electrophile, selected from E, Q, D, or N; preferably E, Q. or N; the remaining s residues can be any type of amino acid residue;

[0162] X can be any type of amino acid residue.

[0163] In a specific embodiment, the initially characterized A-ENA protein monomers as described herein are applied for forming an isolated protein nanofibril of the present invention. Said isolated protein nanofibril may thus be obtained from a host wherein said A-ENA proteins are produced recombinantly by expression of a nucleic acid molecule encoding said A-ENA protein, and / or wherein said A-ENA protein monomers are provided as monomeric subunits selected from the list of A-ENA proteins according to the protein sequences represented by any one of the accession numbers listed in Table 3.

[0164] The list of this group of 593 accession numbers depicting 593 different proteins classified herein as A- ENA(-like) proteins or A-ENA protein orthologues was composed by analysis of the structurally identified Bti A-ENA, in alignment with the finding on the potential orthologues as tested in Example 7, also exemplified herein as SEQ. ID NOs:3-5; and based on the proposed consensus sequence motifs for the NTL-hl-L-h2-CT fragments and heptad elements as described herein, to provide for a self-assembling protein unit that spontaneously forms fibrils irreversibly crosslinked through the presence of one ore more autocatalytically formed IPBs, preferably formed cross-protofibrils.

[0165] Moreover, alignment of the A-ENA protein family, including the members for which the accession numbers are provided in Table 3, resulted in a pairwise sequence identity as low as 20-30 % for the overall sequence (NTL-hl-L-h2-CT). Despite this low sequence conservation, the consensus motifs required for structurally allowing self-assembly and IPB formation are conserved, as discussed herein, and in particular, the residues present as heptad elements in Helix 1 and Helix 2 allowed to establish a methodology for determining whether a protein sequence may be classified as an A-ENA or A-ENA-like protein. An A-ENA-like protein is defined herein as an A-ENA protein complying to contain the scaffold motifs of the NTL-hl-L-h2-CT formula wherein the heptad elements are defined as described herein, though with potential variation in NTL, L and CT length of nature, as well as potential modifications or additions to the protein which do not impact the A-ENA scaffold or self-assembling structural feature. Indeed, the data in Table 1 and Table 2 contain the Hidden Markov model (HMM) profile that was established for helix 1 and helix2 of the A-ENA monomers composed of the formula NTL-hl-L-h2-CT, as defined herein. The HMM calculation was based on the 593 A-Ena(-like) sequence and resulted in the sequence logos of the 35 residue long HMM profiles of Helix 1 and Helix 1, resp. and should be interpreted as in Wheeler et al. (2014): 'hidden Markov models are shown by drawing a stack of letters for each position, where the height of the stack corresponds to the conservation at that position, and the height of each letter within a stack depends on the frequency of that letter at that position.'

[0166] In a particular embodiment, said A-ENA protein monomer subunit is defined by an amino acid sequence according to the formula NTL-hl-L-h2-CT, wherein NTL , CT , and L comprise at least 1 , 1, or 4 amino acids, resp. and hl and h2 provide for helix 1 and helix 2 each comprising or essentially consisting of 5 heptad elements Hl, H2, H3, H4, H5, according to the consensus motif provided below. The heptad elements composed of residues a-b-c-d-e-f-g may more specifically be defined as follows: wherein said consensus amino acid sequence residues of said heptad elements are defined as follows:

[0167] O is a hydrophobic amino acid residue selected form the list of M, V, I, L, A, G, H, W, Y, F; preferably L, M, I, V, F or W;

[0168] Ψ is a short side chain amino acid residue selected from the list of V, C, G, A, P, S, T, N, D; preferably A, G, S, or T; y is an amino acid residue for isopeptide interactions functioning as acid / base catalyst, selected from E , or D; preferably E; s is an amino acid residue for isopeptide interactions functioning as 'acceptor' or electrophile, selected from E, Q, D, or N; preferably E, Q or N; X can be any type of amino acid residue, the amino acid residue one-letter code as indicated, and the amino acids between brackets provide for alternatives (indicated by 'or') of said 'abcdegf' residues at the said respective position.

[0169] In a further specific embodiment, said A-ENA protein monomeric subunits are defined by the NTL-hl-L- h2-CT consensus formula as defined herein, and are further specified by the following hl and h2 polypeptide sequences as those comprising or essentially consisting of heptad elements Hl-l-Hl-5 and H2-1 -H2-5 corresponding to the following sequences:

[0170] Hl-1 is DQAINII, Hl-2 is LASIGLE, Hl-3 is ELGLAHV, Hl-4 is INAEGEK, Hl-5 is VQAVVAG, H2-1 is

[0171] FDQLLAT, H2-2 is NESVTQT, H2-3 is LKTVIKK, H2-4 is EMLLQFK, and H2-5 is LEEAKSL (corresponding to SEQ ID NO: 24-33, resp.) or

[0172] -Hl-1 is EQAINII, Hl-2 is LASIGLE, Hl-3 is ELGLAHV, Hl-4 is INAEGEK, Hl-5 is VQAVVTE, H2-1 is LDQLLAT, H2-2 is NESTVDT, H2-3 is LKTVIKK, H2-4 is EMLLQLK, and H2-5 is LEETKSI (corresponding to SEQ ID NO: 34-43, resp.) which are the heptad element sequences 'abcdefg' corresponding to those heptad elements as present in the A-ENA proteins of Bacillus thuringiensis Sv Israelensis strain ATCC 35646 (defined by UniprotKB Q8KNV8- SEQ ID NO: 1 and UniprotKB Q8KNV7- SEQ ID NO:2, respectively); and wherein said monomers spontaneously fold into an alphahelical antiparallel coiled coil structure, and allow for autocatalytically-induced isopeptide bond formation upon further assembly into fibrous structures.

[0173] The fragments referred to herein as NTL, CT, and L, are used to define the polypeptide sequence of the monomer in the N-terminal end NTL preceding helix 1, the C-terminal end CT following helix 2, and the internal linker sequence L connecting helix 1 and helix 2, respectively. The minimal number of amino acid residue constituting said fragments has been defined herein as one amino acid for the NTL and CT fragments, and minimally 4 amino acids for the Linker. The maximal number of amino acids is not limiting herein, with as a sole requirement that the A-ENA monomeric protein should spontaneously fold into an alpha-helical antiparallel coiled-coil structure, to allow further self-assembly in a protofibril, and ultimately a nanofibril.

[0174] The NTL has been observed to play a role in covalent linking of the protein nanofibril, thus acting as a 'lock' for the fibrillar structure, and depending on its sequence this fragment may thus be applied to tune the structure of the fibril. In a specific embodiment, the length of the NTL sequence is at least one and up to (or maximally) 3 amino acids, or is at least one and up to 4 amino acids, or is at least one and up to 5 amino acids, or is at least one and up to 6 amino acids, or is at least one and up to 7 amino acids, or is at least one and up to 8 amino acids, or is at least one and up to 9 amino acids, or is at least one and up to 10 amino acids, or is at least one and up to 11 amino acids, or is at least one and up to 12 amino acids, or is at least one and up to 13 amino acids, or is at least one and up to 14 amino acids, or is at least one and up to 15 amino acids, or is at least one and up to 16 amino acids, or is at least one and up to 17 amino acids, or is at least one and up to 18 amino acids, or is at least one and up to 19 amino acids, or is at least one and up to 20 amino acids, or is at least one and up to 21 amino acids, or is at least one and up to 22 amino acids, or is at least one and up to 23 amino acids, or is at least one and up to 24 or more amino acids, or is at least 2, 3, 4, 5, 6, or 7 amino acids and up to 3, 4, 5, 6, 7, or 8 amino acids or more. The total length or number of amino acids of the NTL may, or may not, include the amino acid sequence of an additional tag, protein domain, or peptide, attached to the A-ENA NTL fragment to provide for a modified A-ENA or A-ENA-based monomeric protein.

[0175] The C-terminal tail (CT) provides for a fragment structurally exposed at the surface of the monomer, or fibril, therefore also suitable for attaching, connecting or fusing to a further moiety such as a protein domain, tag or other molecule. So in another specific embodiment, the length of the C-terminal tail sequence is at least one and up to (or maximally) 2 amino acids, or is at least one and up to 3 or 4 amino acids, or is at least one and up to 5 amino acids, or is at least one and up to 6 amino acids, or is at least one and up to 7 amino acids, or is at least one and up to 8 amino acids, or is at least one and up to 9 amino acids, or is at least one and up to 10 amino acids, or is at least one and up to 11 amino acids, or is at least one and up to 12 amino acids, or is at least one and up to 13 amino acids, or is at least one and up to 14 amino acids, or is at least one and up to 15 amino acids, or is at least one and up to 16 amino acids, or is at least one and up to 17 amino acids, or is at least one and up to 18 amino acids, or is at least one and up to 19 amino acids, or is at least one and up to 20 amino acids, or is at least one and up to 21 amino acids, or is at least one and up to 22 amino acids, or is at least one and up to 23 amino acids, or is at least one and up to 24 or more amino acids, or is at least 2, 3, 4, 5, 6, or 7 amino acids and up to 3, 4, 5, 6, 7, or 8 amino acids or more. The total length or number of amino acids of the CT may, or may not, include the amino acid sequence of an additional tag, protein domain, or peptide, attached to the A-ENA CT fragment to provide for a modified A-ENA or A-ENA-based monomeric protein.

[0176] The linker (L) sequence will appear in the structure as a loop or turn, providing for a covalent linking between the 2 helices of the hairpin, and in parallel allowing to insert or conjugated further proteins or molecules or tag on said A-ENA protein. So in a specific embodiment, the length of the Linker sequence, positioned between the of helixl and helix2 sequences is at least 4 and up to (or maximally) the number of amino acids resulting in a 3D structure that would prevent the helixl and helix2 from forming a coiled coil. So preferably, the Linker fragment is at least 4 and up to 30 amino acids, or is at least 4 and up to 25 amino acids, or is at least 4 and up to 20 amino acids, or is at least 4 and up to 15 amino acids, or is at least 4 and up to 10 amino acids, or is at least 5 and up to 9 amino acids, or is at least 6 and up to 8 amino acids, or is at least 7 amino acids. The total length or number of amino acids of the linker may, or may not, include the amino acid sequence of an additional tag, protein domain, or peptide, inserted into the A-ENA Linker fragment to provide for a modified A-ENA or A-ENA-based monomeric protein.

[0177] A-ENA-based monomeric protein subunits forming said protein nanofibril of the present invention as described herein, provide for A-ENA proteins with a sequence that is based on or derived from original bacterial A-ENA proteins, as annotated herein, though wherein parts of the consensus sequences of fragments may be adapted, modified, or altered by mutating, substituting, adding or deleting further amino acids or peptides or proteins, to provide for functionalized A-ENA proteins or fibrous structures of the same. In certain embodiments, a heterologous amino acid sequence or protein or tag can be fused, added, or inserted in any one of said NTL, CT, or L fragments, which results in an A-ENA-based protein monomer or modified or engineered A-ENA, ultimately providing for a functionalized protein that selfassembles into a functionalized protein fibril, and / or a fibril with a modified surface.

[0178] Specifically, the N-terminal lock or N-terminal end of the A-ENA or A-ENA-based monomeric protein subunit may further be defined as an amino acid sequence of at least 1 amino acid, preferably at least seven amino acids, wherein said NTL fragment comprises or consists of a consensus sequence defined as M-Ψ -Φ-Z-X-Φ-P, wherein: tp is a short side chain amino acid residue, selected from the list of V, C, G, A, P, S, T, N, D; preferably A, G, S or T

[0179] O is a hydrophobic amino acid residue, selected from the list of M, V, I, L, A, G, H, W, Y, F; preferably from the list of L, M, I, V, F, or W;

[0180] X = any amino acid residue;

[0181] Z is Ala or Proline, and M and P are one letter code for the respective amino acids Methionine and Proline.

[0182] In a further specific embodiment said NTL fragment comprises or consists of the consensus sequence defined as M- Ψ -Φ-Z-X-Φ-P, wherein tp is selected from S, G, or T;

[0183] O is a hydrophobic amino acid residue, selected from the list of M, V, I, L, A, G, H, W, Y, F;

[0184] X = any amino acid residue;

[0185] Z is Ala or Proline, and M and P are one letter code for the respective amino acids Methionine and Proline. A-ENA proteins with an NTL defined by said consensus motif are providing the option to form an IPB wherein the donor or nucleophilic residue is provided by the N-terminally free amino (NH2-)group present at the peptide backbone. The NTL consensus motif forms a structural element that helps pack the NTL on the surface of the fibrils, and thereby helps position the N-terminal amine into optimal orientation and distance for isopeptide bond formation.

[0186] In a specific embodiment said NTL comprises or consist of M-G-(M or I )-P-(T or N)-l-P, wherein the one- letter codes are applied for each amino acid, and the amino acids between brackets provide for alternatives (indicated by 'or') at said positions, and wherein said NTL may comprise further amino acids, such as those NTL fragments of SEQ ID NO:1 and SEQ ID NO:2 for example, which further contain EGLDIT (SEQ ID NO:44) as part of the NTL.

[0187] In certain embodiments, the A-ENA monomeric protein subunit comprising or composed of the amino acid sequence corresponding to the formula NTL-hl-L-h2-CT as described herein, comprises further amino acid residues in the hl and / or h2 helix sequence located N-terminally and / or C-terminally from the heptad elements Hl to H5, though not part of NTL , CT or Linker region, wherein the helix 1 and helix 2 thus correspond to a formula for helix 1: [X]ni-Hl-1 -Hl-2 -Hl-3 -Hl-4 -Hl-5 - [X]n2, and / or for helix 2: [X]m-H2-1 -H2-2 -H2-3 -H2-4 -H2-5 - [X]n2, wherein the heptad elements are defined by residues 'abcdefg', as described herein, are covalently linked in said order, and wherein [X]nindicates such additionally present amino acids as part of the helix structure hl and / or h2, wherein X can be any amino acid, and nl and n2 refer to the number of different amino acids of said helix preceding or following the heptad elements, respectively, wherein each X of said n amino acids may be identical or different, as to result in the helix 1 or helix 2 polypeptide chain forming said alpha (a)-helix, resp.

[0188] In a specific embodiment, said [X]nifor hl is a single Arginine (R), Leucine (L), Asparagine (N), or Lysine (K) residue, and / or said [X]n2 of helix 1 comprises or consists of 3 amino acids, such as FEK, or FKK, and / or said [X]n2 of helix 2 comprises or consists of 3 amino acids , such as IQS or LKL.

[0189] A-ENA protein fibrils

[0190] The present invention relates to a protein fibril, preferably a nanofibril, wherein a 'nanofibril' is defined herein as a fibrous assembly with a diameter in the nanoscale and significantly of a length up to several micrometers. The term 'fibril', 'filament', 'fibrous assembly', or 'fibrous structure', as used interchangeably herein, refers to structured biochemical compounds, such as protein assemblies or protein-based assemblies, preferably composed of protein material, forming long-shaped ordered structures with diameters up to 100 nanometers, and potentially part of larger hierarchical structures.

[0191] In view of the terminology used herein for defining (nano)fibrils, we refer herein to 'fibers' as a potential plurality of nanofibrils, wherein fibers are generally considered to represent rather larger diameter (in the micro- to m ill i-scale) structures as compared to fibrils, and wherein 'fibers' thus preferably provide for a higher-ordered hierarchical structure of said plurality of fibrils. In a specific embodiment, such a fiber comprises a plurality of A-ENA nanofibrils, wherein each fibril makes further lateral associations to another fibril, to provide for a structured fiber. A further example of a fiber is a woven assembly of protein nanofibrils.

[0192] N a nof i bri 11 a r structures provide for unique and interesting characteristics to form subject of investigation in several areas of research and applications, such as microbiology, biomechanics, and material science. The present invention provides for protein nanofibrils which are obtained upon the spontaneous intertwining of two protofibrillar structures, wherein the term 'protofibril' refers to a fibrous selfassembly of multimers or polymers, which is fundamentally composed of the monomeric protein subunits as described herein, called A-ENA proteins or A-ENA-based proteins, comprising protein fragments according to the formula defined herein as NTL-helixl(hl)-L-helix2(h2)-CT, as further detailed herein, interconnected through at least one or more isopeptidic bonds. In particular said protofibrils are composed of functional A-ENA proteins, or orthologues thereof, with functional referring to their capability to self-assemble as coiled-coil structures into protofibrillar structures that intertwine forming the nanofibril as described herein.

[0193] Previously, self-assembling S- and L-ENA proteins have been reported based on the resolution of Bacillus cereus endospore appendages (Pradhan et al., 2021; Remaut et al. WO2022 / 029325), which also provide for self-assembling proteins that multimerize into protein fibrils. These S- and L-ENA protein-based fibrous structures actually also provide for nanofibrillar structures in the meaning of the present invention, wherein the S- and L-ENA fibrils are composed of a number of polypeptide chains appearing as helical multimers or discs rather which stack into a helical (S-ENA) or ladder-type (L-ENA) of multimeric assemblies or fibrils, wherein the multimers of said fibrils are interconnected by disulfide bridges as covalent intermolecular rigidifying connections. Although Pradhan et al. (2021) and Remaut et al. (WO2022 / 029325) refer to S- or L-ENA 'fibers', we chose to herein redefine these structures as nanofibrillar or 'fibrils' to be more in line with the conventional use of the term fiber, referring in several fields to a plurality of fibrils.

[0194] In the present invention, the Alphahelical-Endospore appendage proteins, as firstly annotated in Bacillus thuringiensis herein, called A-ENA proteins, are very different in their protein structure as compared to the previously found S- and L-ENA proteins. Also for A-ENA proteins, the structural appearance of the resulting assembled fibrils is dictated or driven by the amino acid sequence of the monomeric proteins, which self-assemble not only into monomers forming a protofibril, but wherein said monomers also intermolecularly connected through one or more isopeptide bonds. The resulting A-ENA multimerous assemblies have a (proto)fibrous structure, whereas S-ENA and L-ENA multimeric assemblies form helical arcs or discs, respectively, which can visibly be distinguished from fibrous structures (under a microscope). As a consequence of said structural differences, the nanofibrils that are composed of S- or L-ENA proteins are considered as a plurality of stacked helical arcs or discs, resp., which further elongate into a fibrous assembly that is interconnected covalently by disulfide bridges between the different multimeric assemblies; whereas the nanofibrils composed of A-ENA proteins contain at least two intertwined alpha-helical protofibrils, covalently connected by isopeptide bonds.

[0195] One advantage for at least some biotechnological applications wherein said fibrous materials may be used is that the isopeptide bonds form irreversible, and thus provide for an extremely high tensile strength of said assembled fibrils, and incredible mechanical and thermal stability of the protein nanofibril. Even though also the S- and L-ENA-based fibrils are considered as rigid stable bionanomaterials, the nature of the isopeptide covalent connection is stronger as compared to disulfide connections.

[0196] From a phenotypic point of view, the A-ENA fibrils are thus easily distinguished from any other fibrils in bacterial or more specifically bacillus endospore samples by simply microscopically analyzing them. Indeed, when inspecting a 2D image of a microscopic analysis as shown herein, e.g. in Figure 2, the A- ENA nanofibrils reveal a highly ordered unique fibril structure as compared for instance to the fibrous structures described for S- and L-ENA proteins (see Pradhan et al., 2021 or Remaut et al. WO2022 / 029325).

[0197] In one embodiment described herein, the protein nanofibril comprises or consists of two protofibrils wherein each protofibril comprises or consists of at least two monomeric protein subunits, wherein each monomeric protein subunit comprises covalently interconnected fragments according to the formula NTL-hl-L-h2-CT, spontaneously folding in aqueous solution into two helices, hl and h2, as an alphahelical antiparallel coiled-coil structure, and each helix comprising 5 or more heptad elements according to the formula Hl- to H5 as previously described herein, according to the consensus sequences provided for their 'abcdefg' side chain format, and wherein the L, NTL, and CT are defined as described herein, and wherein the monomeric protein subunits interact through at least one or more isopeptidic covalent bond. In a specific embodiment, said nanofibril is thus composed of A-ENA monomers, wherein said A- ENA monomers are defined as described herein above, and / or wherein said A-ENA monomers may also be A-ENA-based monomers, which are defined as A-ENA proteins containing one or more modifications, required to functionalize the (nano)fibril, though wherein the functionality to self-assemble into protein nanofibrils is thus retained. The fibril of the present invention is thus preferably formed from 2 protofibrils of each at least two or more A-ENA or A-ENA-based monomers, preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or more A-ENA or A-ENA-based monomers. Thus the basic structure of a protein nanofibril as described herein contain two protofibril of each at least 2 monomers, and is thus a tetrameric structure. In the embodiment wherein said protein nanofibril contains two protofibrils or each at least 3 monomers, a hexameric structure is provided, and so on. Said A-ENA monomer or A-ENA-based monomers of said protofibril may be identical or may be different, as further described herein.

[0198] In view of visualizing the 3D structure of said protein nanofibril, the A-ENA monomers of said fibril are called i and i' in the f and f' protofibril respectively, which form the intertwined helical fibril, and incremental (i.e. i+1; i+2, i+3, etc., or i'+l; i'+2; i'+3, etc.) or decremental (i.e. i-1, i-2, i-3, etc. or i'-l; i'-2; i'-3, etc.) numbering is used for further stacked protomers towards the C- and N-pole of said fibril, respectively. Here and throughout the application, A-ENA fibrils are oriented as such in the drawings with their subunits' C-termini pointing upwards (referred to as C-terminal or pointed fibril end, i.e. C- pole), and their subunits' N-terminal extensions or ending pointing down (referred to as N-terminal or barbed fibril end, i.e. N-pole).

[0199] In a further specific embodiment, said protein nanofibril is an isolated protein nanofibril, wherein 'isolated' refers to the nanofibril being devoid of other compounds such as materials from bacterial spore culture or from bacterial constituents like parasporal bodies, spores, or other bacterially derived material. In reality, ex vivo extraction or isolation of an A-ENA fibril from naturally occurring bacterial endospores was not possible. In hindsight, this is considered as caused by their particular structure and the presence of the isopeptidic bonds. Since the A-ENA nanofibrils cannot be obtained from the bacterial spores 'as such', so the fibrils isolated from any further bacterial compounds (e.g. without parasporal body or spores attached), the protein nanofibrils of the present invention are isolated or obtained from other means, such as from recombinantly produced fibrils, allowing isolation without sporulation and presence of the associating bacterial compounds. So in a specific embodiment, the protein nanofibrils of the present invention, also called the A-ENA protein nanofibril or A-ENA-based protein nanofibril, is a recombinantly produced A-ENA nanofibril or A-ENA-based nanofibril.

[0200] Isopeptide bonds (IPBs)

[0201] The present invention relates to protein nanofibrils, as described herein, with an extremely high strength and stability, which is a consequence of the presence of spontaneously formed covalent linkages of each of the monomeric protein subunits present in the protofibrils to one or more other monomeric proteins subunits of said protein fibril as isopeptidic bonds. An isopeptide bond is an amide bond that forms between carboxyl / carboxamide and amino groups, where at least one of the carboxyl or amino groups is outside of the protein main-chain (the backbone of the protein). In contrast to the common peptide (eupeptide) bonds, isopeptide bonds can form covalent links between (different) polypeptide backbones, which is a quite scarce covalent interaction, though very commonly known for instance for post-translational events like ubiquitination, wherein ubiquitin at its C-terminal glycine residue interacts with the lysine side chain of the substrate protein forming intermolecular isopeptide bonds; or glutathione, including an isopeptide bond between the side chain of a glutamate residue and the amino group of a cysteine residue, providing an autocatalytically formed intramolecular isopeptide bond. An isopeptide bond is chemically irreversible under biological conditions and resistant to most proteases. It may be formed through enzyme catalysis; however isopeptide bonds can also be formed spontaneously or autocatalytically, as used interchangeably herein. Spontaneous isopeptide bond formation was first observed in the bacteriophage HK97, wherein the HK97 capsid structure revealed to be assembled by the polymerization of a subunit that was covalently cross-linked, as a result of autocatalyzed isopeptide bond formation between Lys and Asn side chains in neighbouring subunits, which formed covalently assembled 5 and 6 member rings that catenated during capsid assembly to form a structure referred to as protein chainmail (Wikoff et al. 2000; Science 289, 2129- 2133).

[0202] Spontaneous isopeptide bond formation typically occurs after protein folding, through nucleophilic attack of the e-amino group from a lysine on the Cy group of an asparagine, promoted by a nearby glutamate.

[0203] The term "spontaneous" as used herein refers to a bond e.g. an isopeptide or covalent bond which can form in or between protein(s) without any other agent or enzyme being present and / or without chemical modification of the protein e.g. without native chemical ligation or chemical coupling. So, a 'spontaneous isopeptide bond' or 'autocatalytically formed isopeptide bond' is formed when one or more protein is isolated on its own (intramolecularly) or between two proteins when in isolation (without further agents or without chemical modification). A spontaneous isopeptide or covalent bond may therefore form of its own accord in the absence of enzymes or other exogenous substances or without chemical modification. Particularly however, a spontaneous isopeptide or covalent bond may require the presence of an 'autocatalytic residue' such as a glutamic acid or an aspartic acid residue, which is part of the polypeptide chain of the isopeptide bond-forming protein to allow formation of the bond in a proximity induced manner.

[0204] For the autocatalytic formation of isopeptide cross-links or bonds or interactions, as used interchangeably herein, a conservation of three amino acids is thus responsible: one acting as a nucleophilic donor, one as an electrophilic acceptor, and as a catalytically important acidic Glu or Asp residue, mediating the transfer of protons. The requirement of the latter and the particular positioning of residues within a hydrophobic environment near the active site was observed in numerous Immunoglobulin-like pilin subunits of Gram-positive bacterial pili, wherein these internal crosslinks provide stabilisation against chemical, thermal and mechanical stress. The hydrophobic environment is important as this changes the pKa values of the amino acid side chains resulting in an inverse protonated state, where the Lysine side chain or donor residue side chain is unprotonated and the Glu / Asp side chain is protonated (Kang and Baker, 2011- Trends Biochem. Sci. 36, 229-237; Hu et al., 2011- J Am Chem Soc. 133(3):478-85). Furthermore, isopeptide bond formation is proximity-induced and hence the three residues involved in the reaction must have the proper geometry. The protein nanofibril as described herein is composed of said monomeric protein subunits appearing as coiled coils wherein, upon selfassembly into the protofibrils and further intertwined nanofibril, the positions of said three residues provides for the geometrical needs to form autocatalytic intermolecular isopeptide bonds, as described herein. Indeed, the amino acid sequences of A-ENA or A-ENA-based protein monomers, as defined above, comprise the necessary limitations within their heptad elements to first assemble into an alphahelical antiparallel coiled-coil structure, and thereby provide the required hydrophobic environment and geometry to allow proximity-induced isopeptide connections when further monomers are present as protofibril subunits. So besides the heptad element based limitations required for coiled-coil assembly, A-ENA protein sequences are also extremely conserved in the positions containing the donor, acceptor, and catalytic residues for isopeptide bond formation, as observed in the protein nanofibril.

[0205] So, after that a monomeric coiled coil structure is assembled, the protofibril multimeric fibrous structures assemble, and isopeptide bonds are formed between monomers of said protofibril, as well as between monomers of a second protofibril, which has intertwined with the first protofibril, resulting in a tight, strong irreversible protein nanofibril. Specifically, the mechanism of autocatalytic isopeptide bond formation involves, first the Cy carbonyl group of the acceptor or electrophile (preferably Asn / Asp) being polarized via hydrogen bonding to the protonated catalytic Glu / Asp residue, next, the unprotonated Lys e-amino group of the donor residue nucleophilically attacks said polarized Cy carbonyl group of the acceptor residue, to result in the formation of a tetrahedral intermediate, from which a proton is then transferred from the Lys e-amino group to the catalytic Glu / Asp residue, as well as a proton is transferred from the catalytic Glu / Asp residue to the leaving group of the acceptor residue. Subsequently, depending on the nature of the side chain of the acceptor residue, the formation of a Lys- Asn isopeptide bond results in the release of a NH3 molecule, while a Lys-Asp linkage leads to the release of a H2O molecule (Kang and Baker, 2011). So, the protein nanofibril of the present invention provides for protein structures wherein the isopeptide bonds are formed intermolecularly, which means between two protein molecules. Typically, an isopeptide bond herein forms between a lysine residue and glutamine, glutamic acid, an asparagine, or aspartic acid residue or the terminal carboxyl group of the protein; or forms between the alpha-amino terminus of a protein and an asparagine, aspartic acid, glutamine or glutamic acid. So, an isopeptide bond as provided in the protein nanofibril described herein may form between a lysine residue and an asparagine residue or between a lysine residue and an aspartic acid residue. Particularly, isopeptide bonds can occur between the side chain amine of lysine and carboxamide group of asparagine. Each residue of the pair involved in the isopeptide bond is referred to herein as a reactive residue.

[0206] The presence of covalent, in particular isopeptide bond, interactions within or between protein(s) may be determined by bioinformatic sequence analysis, or preferably by mass spectrometry or structure determination, such as by X-ray crystallography, Cryo-EM, or NMR.

[0207] Spontaneously forming isopeptide bonds as a method of covalently linking amino acids is often observed intramolecularly, but when appearing intermolecularly, as is also the case for the protein nanofibril described herein, this is an advantageous approach for cross-linking molecules. In contrast to the other methods of covalently linking polypeptides, spontaneously forming isopeptide bonds do not require enzymatic catalysis or cofactors; are expected to form specifically, due to the requirement of three reactive residues positioned in the correct conformation surrounded by a hydrophobic environment; are chemically extremely stable thanks to the amide type of bond; are typically resistant to proteolytic degradation, since these are outside of the protein main chain; and finally their formation is not redoxdependent, and is an irreversible interaction.

[0208] These advantages have evolutionary probably played a role in bacteria which lack disulfide bond forming 'machinery' to strengthen bacterial proteins that get for instance exported (Dutton et al., 2008- Proc. Natl. Acad. Sci. U. S. A 105, 11933-11938), since many Gram-positive bacteria were shown to incorporate intramolecular isopeptide bonds as a means to stabilize protein structures, for instance also of physiological importance for outer-membrane proteins in these bacteria (Dutton et al., 2008; Hu et al., 2011- J Am Chem Soc. 133(3):478-85).

[0209] In a specific embodiment, the protein nanofibril as described herein, wherein the monomeric protein subunits, preferably A-ENA monomeric protein subunits, are numbered as i, i + n and i-n for protomers towards the C- or N-pole of said fibril f, respectively, (and subsequently i', i' +n and i-n for the protofibril f' monomeric units), each monomer i, i+ / -n, i', and i'+ / -n, is covalently linked to at least one further monomer i, i+ / -n, i', and / or i'+ / -n, by the presence of an isopeptidic bond, more preferably, wherein each monomer is covalently linked through one or more IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked by the presence of one IPB to one further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked through two IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked via two IPBs to one or more further monomers of said nanofibril, which may be inter- or intra-protofibrillar connections, wherein intra-protofibrillar connections may also be called cross-fibrillar connections. In a further embodiment, each monomer is covalently linked by the presence of three IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked via four IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked via five IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked via six IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked via seven IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked via eight IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked via nine IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked via ten IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked via eleven IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked via twelve IPBs to one or more further monomers of said protein nanofibril, more preferably, wherein each monomer is covalently linked via thirteen or more IPBs to one or more further monomers of said protein nanofibril. The monomers having more than 1 IPB in connections or cross-links to one or more monomers herein may mean that the one monomer is linked to one other further monomer of the fibril via more than 1 IPB, or may as well meant that the one monomer is linked to several other further monomer of the fibril, each connected via 1 or more IPBs to the one monomer.

[0210] In a particular embodiment, each monomer of said fibril is thus connected to more than one further monomers of the fibril, via the presence of IPBs, wherein the crosslink between two monomers can involve 1, 2, 3, or more IPBs. In further particular embodiments, the protein nanofibril comprises A-ENA monomers wherein each monomer is involved in at least one IPB that covalently connects both protofibrils (f) and (f') of said nanofibril. In another embodiment, said protein nanofibril comprises A- ENA monomers wherein each monomer is involved in more than one IPB, of which at least one IPB covalently connects both protofibrils (f) and (f') of said nanofibril, and at least one covalently connects the monomer subunit with further monomer subunits of the same protofibril of said nanofibril. In a further embodiment said protein nanofibril comprises A-ENA monomers wherein each monomer is involved in 9 or 10 isopeptidic bonds with other monomers of said fibril, more specifically, connecting said monomer to 3, 4, 5, or 6 other A-ENA monomers of said nanofibril.

[0211] In a further specific embodiment, said protein nanofibril described herein, comprises monomeric protein subunits wherein each monomeric protein subunit, preferably the A-ENA or A-ENA-based monomeric protein subunits, has covalent connections to further monomers of the fibril wherein the IPBs are formed between the side chain of the amino acid acting as a nucleophile and the side chain of the amino acid acting as electrophile, respectively, at positions:

[0212] • the N-terminal residue of the NTL (as nucleophilic donor via the free amino-terminal group of the polypeptide backbone) of (i) and Hl-5b of (i-5), (i-4), (i-3), or (i-2);

[0213] • Hl-4g of (i) and H2-2a of (i-1);

[0214] • H2-3g of (i) and H2-4a of (i-1);

[0215] • H2-3f of (i) and H2-4e of (i');

[0216] • H2-4g of (i) and Hl-3a of (i-1);

[0217] • H2-4g of (i+l)and Hl-3a of (i);

[0218] • NTL of (i+5), (i+4); (i+3), or (i+2) and Hl-5b of (i);

[0219] • Hl-4g of (i+1) and H2-2a of (i);

[0220] • H2-3g of (i+1) and H2-4a of (i); and / or

[0221] • H2-3f of (i'+l) and H2-4e of (i), which is read as the position of the nucleophilic donor its side chain position of the first mentioned monomer, in connection with the electrophilic acceptor its side chain position of the second mentioned monomer (e.g. the donor side chain is at position g of heptad element Hl-4 of monomer (i) in connection with the acceptor side chain at position a of heptad element H2-2 of monomer (i-1)).

[0222] In another alternative embodiment, said protein nanofibril described herein, comprises monomeric protein subunits wherein each monomeric protein subunit, preferably the A-ENA or A-ENA-based monomeric protein subunits, has covalent connections to further monomers of the same protofibril wherein the IPBs are formed between the side chain of the amino acid acting as a nucleophile and the side chain of the amino acid acting as electrophile, respectively, at the heptad element positions Hl-4g of (i) and H2-2a of (i-1), and / or H2-3g of (i) and H2-4a of (i-1), and / or H2-4g of (i) and Hl-3a of (i-1), and / or H2-4g of (i+l)and Hl-3a of (i), and / or Hl-4g of (i+1) and H2-2a of (i), and / or H2-3g of (i+1) and H2-4a of (i).

[0223] In another specific embodiment, said protein nanofibril described herein, comprises monomeric protein subunits wherein each monomeric protein subunit, preferably the A-ENA or A-ENA-based monomeric protein subunits, has covalent connections to further monomers of the other protofibril of the nanofibril wherein the IPBs are formed between the side chain of the amino acid acting as a nucleophile and the side chain of the amino acid acting as electrophile, respectively, at the heptad element positions H2-3f of (i) and H2-4e of (i'), and / or H2-3f of (i'+l) and H2-4e of (i).

[0224] In another alternative embodiment, said protein nanofibril described herein, comprises monomeric protein subunits wherein each monomeric protein subunit, preferably the A-ENA or A-ENA-based monomeric protein subunits, has covalent connections to further monomers, wherein the IPBs are formed between the side chain of the amino acid acting as a nucleophile and the side chain of the amino acid acting as electrophile, respectively, at the N-terminal residue of the NTL (as nucleophilic donor via the free amino-terminal group of the polypeptide backbone) of (i) and Hl-5b of (i-5) via its N-terminal lock amino-terminal group as nucleophilic donor from monomer (i) and Hl-5b of (i-5), and / or at the N- terminal residue of the NTL (as nucleophilic donor via the free amino-terminal group of the polypeptide backbone) of monomer (i+5) and Hl-5b of (i).

[0225] In a specific embodiment, wherein the A-ENA monomeric protein is described by the formula NTL-hl-L- h2-CT, and wherein the heptad elements are provided by consensus sequences of residues a-b-c-d-e-f-g defined as follows: wherein said consensus amino acid sequence residues of said heptad elements are defined as follows:

[0226] O is a hydrophobic amino acid residue selected form the list of M, V, I, L, A, G, H, W, Y, F; preferably L, M, I, V, F or W;

[0227] Ψ is a short side chain amino acid residue selected from the list of V, C, G, A, P, S, T, N, D; preferably A, G, S, or T; y is an amino acid residue for isopeptide interactions functioning as acid / base catalyst, selected from E , or D; preferably E; s is an amino acid residue for isopeptide interactions functioning as 'acceptor' or electrophile, selected from E, Q, D, or N; preferably E, Q or N;

[0228] X can be any type of amino acid residue, the amino acid residue one-letter code as indicated, and the amino acids between brackets provide for alternatives (indicated by 'or') of said 'abcdegf' residues at the said respective position,

[0229] The isopeptide bond forming 'donor' or nucleophile is a Lysine (K) in each heptad, though if in H2-3 this donor K would be substituted by any other type of amino acid (X), So if H2-3 heptad residue f and / or g are not a Lysine (K), these residues will not form an IPB, and likely not a protein nanofibril, as this IPB provides for the inter-protof ibrillar connection, providing stability to the nanofibril assembly.

[0230] So in a preferred embodiment, the H2-3 f and / or H2-3 g residues are K, and in alternative embodiments, A-ENA(-like) proteins are provided wherein each of the donor residues of the heptad elements, more specifically H l-4g and / or H2-4g, may be replaced with any other amino acid (X), resulting in fewer IPBs when self-assembled into fibrils. However, it is essential that at least one K is still present, to form at least one IPB, preferably the H2-3 f or g donor position is a K.

[0231] Functionalized A-ENA protein assemblies

[0232] As defined and introduced above, the A-ENA proteins may be provided as A-ENA proteins according to the formula NTL-hl-L-h2-CT, as defined and described herein above, wherein the monomer is modified or engineered as to obtain a 'modified A-ENA protein', or 'A-ENA-based monomeric protein', which thus refer to A-ENA proteins which are based on naturally-occurring or native A-ENA protein sequences, or at least are defined by the A-ENA definitions as provided herein for their sequence and functionality, but which are modified or engineered to result in an A-ENA protein that functionalized the spontaneously assembled A-ENA protein nanofibrils upon production in a host.

[0233] Said modifications or engineering strategies are further exemplified and discussed herein, in a nonlimiting manner, and serve as support to enable the skilled person in designing such modified A-ENA proteins.

[0234] With 'modified' or 'engineered' A-ENA protein, or 'A-ENA-based protein' (as used interchangeably herein) is thus meant herein that the reference A-ENA protein to which the 'modified' A-ENA protein is compared to is a protein subunit with a sequence comprising the interlinked fragments according to the formula NTL-hl-L-h2-CT as previously described herein, wherein the at least 5 heptad elements of the hl and h2 helices are defined as described herein and thus allow for a spontaneous folding of the monomer in a helix hairpin, with the Linker forming a loop connecting the two antiparallel helices, and the NTL and CT present at the surface, and which further self-assembles into fibrous structures as described herein, wherein each monomer is involved in at least one intermolecularly formed IPB, to optimally 10 I PBs, with further monomers (which may be identical or different in sequence from each other). In said comparison, the 'modified' may then be further specified by the presence of any one of the following engineering formats:

[0235] (1) the NTL is truncated as compared to the native A-ENA protein of said modified A-ENA monomer; preferably wherein the truncation results in an NTL of less than 4 amino acids, preferably 3 amino acids, 2 amino acids, or 1 amino acid.

[0236] One advantage of providing a modified A-ENA wherein said modification is a truncation of the NTL as compared to the naturally occurring NTLs is the said A-ENA monomer is more accessible in its folded state for post-fibril-formed modifications of the nanofibril.

[0237] (2) the A-ENA heptad element sequence conservation is altered -and provides for an A-ENA mutant variant or surface mutant variant- to allow production of an A-ENA protein nanofibril with a modified surface structure or properties as compared to the native A-ENA sequence of said modified A-ENA; more specifically, wherein:

[0238] (2a) the modified A-ENA sequence contains an NTL of at least 4 amino acids and the heptad elements of the modified A-ENA monomer are substituted with a (natural or synthetic) amino acid not falling under the consensus sequence of the herein defined 'conventional' A- ENA heptad at said position (i.e. a mutant variant) in at least one or more of the following positions: Hl-la, Hl-lb, Hl-lc, Hl-3f, Hl-4f, Hl-5b, Hl-5f, H2-lb, H2-lc, and / or H2-5b; or

[0239] (2b) the modified A-ENA sequence contains an NTL of less than 4 amino acids and the heptad elements of the modified A-ENA monomeric protein are mutated to a (natural or synthetic) amino acid not falling under the consensus sequence of the herein defined 'conventional' A- ENA heptad at said position in at least one or more of the following positions: Hl-la, Hl-lb, Hl-lc, Hl-lf, Hl-2f, Hl-3b, Hl-3f, Hl-4b, Hl-4f, Hl-5b, Hl-5f, H2-lb, H2-lc, H2-2b, H2-2f, H2-3f, H2-4f, H2-5b, H2-5c and / or H2-5f;

[0240] (3) the A-ENA protein contains a modification in that it is further fused to a protein tag or protein domain at its N-terminus, C-terminus or inserted within the Linker region, wherein said fusions are typically obtained through expression of a genetic fusion wherein a heterologous (poly)peptide sequence, which in itself may form a tag or folded protein domain, is fused to or inserted within the native A-ENA sequence (or A-ENA mutant variant); (4) the A-ENA protein is conjugated to another protein or chemical moiety, such as a tag, protein domain, small molecule, or a label, among others, wherein said conjugation may be performed post-fibril formation, and / or wherein a conjugation is defined as a covalent connection formed with one or more side chain residues or atoms (in an orthogonal manner).

[0241] It is envisaged herein that modifications which involve an A-ENA fusion or conjugation with or to heterologous or further peptides, proteins, protein domains, tags, labels or other functional moieties, said modifications may be obtained by connecting said heterologous moiety directly or through the use of a linker to the A-ENA subunit(s).

[0242] In a specific embodiment disclosed herein, the A-ENA subunit is fused or conjugated to a tag or protein binding partner, for post-fibril formation of a specific tag-protein binding interaction on the fibril surface. In further specific embodiments, said tag / protein binding partner pair comprises a protein and tag which are capable to covalently interact, such as interact via the formation of an isopeptide bond, more specifically, as known for the herein exemplified tag / protein binding partner pair SpyTag / SpyCatcher, wherein the tag or the catcher may thus be fused to one or more A-ENA proteins, resulting in one counterpart of said pair being displayed on the surface of the protein nanofibril, and the other counterpart forming a covalent interaction, specifically an isopeptide bond, with said fibril-associated part of the pair (preferably post-fibril formation).

[0243] Further embodiments thus relate to protein nanofibrils comprising at least one modified A-ENA monomer, wherein the modified format is provided according to (1), (2), (3) or (4) as provided above. More specific embodiment relate to the protein nanofibril as described herein, wherein at least two different sequences of monomeric proteins form a heteropolymeric fibril upon self-assembly; or alternatively, wherein all monomers are identical to form a homopolymeric fibril.

[0244] In a further specific embodiment, said protein nanofibril comprises or consists of two protofibrils wherein each monomeric protein further comprises a flexible linker and / or a protein domain with a hydrodynamic radius below 11 nm for surface display on homopolymeric fibrils, as to prevent protein domains clashing when nanofibril assembly occurs.

[0245] Flexible linkers as used herein are provided by amino acid sequences typically with a length in the range of 1-30 amino acids and generally rich in small or polar amino acids such as Ser or Thr, or non-polar amino acids, such as Gly, to provide good flexibility and solubility. The small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaction between the linker and the protein moieties. The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues ("GS" linker). An example of the most widely used flexible linker has the sequence of (Gly-Gly-Gly-Gly-Ser)n. By adjusting the copy number "n", the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions (Chen et al. Adv Drug Deliv Rev. 2013, 65(10):1357-69). Besides the GS linkers, many other flexible linkers have been designed for recombinant fusion proteins, also rich in small or polar amino acids, but possibly also containing amino acids amino acids such as Lys and Glu to improve solubility. Alternatively, the linker peptide is built using computational design by adding single amino acids or short peptides extending from the scaffold (see also example 8).

[0246] In specific embodiments, said protein nanofibril comprises a modified A-ENA monomer containing a heterologous protein domain (according to the modified format of (3) as provided above, wherein said heterologous protein domain is fused to said A-ENA monomer via its N- or C-terminal end or inserted in the linker region of A-ENA, resulting in a 3D protein structure wherein the heterologous protein domain is displayed on the surface of the nanofibril.

[0247] In a further specific embodiment, the protein nanofibril comprises or consists of two protofibrils, each comprising or consisting or at least two A-ENA monomers, wherein at least one A-ENA monomer is a modified A-ENA, and wherein said modified A-ENA is provided in the format of (3) or (4) as provided above, or specifically is a fusion of A-ENA with a heterologous protein domain or tag, as described herein (and according to (3)). Said protein nanofibrils may be obtained through combining or mixing nonmodified A-ENA proteins (i.e. native A-ENA proteins) or mutant variant A-ENA proteins (according to modified format (2) provided above) and modified A-ENA proteins which are A-ENA heterologous fusion proteins (as format (3) provided above) or conjugates (as format (4) provided above), to result in the self-assembly of heteropolymeric nanofibrils which can be considered as co-polymers appearing as alternating blocks of surface-displaying parts and non-displaying parts (e.g. as visualized in Figure 16a).

[0248] The mixture of different A-ENA and modified A-ENA monomers of said heteropolymeric protein nanofibril may be obtained through recombinant expression of a modified A-ENA protein sequence (e.g. a genetic fusion of an A-ENA to a heterologous peptide or protein tag or domain at the A-ENA N-term, C-term or inserted in the linker region), and a non-modified or conventional A-ENA in a host.

[0249] Alternatively said mixture of different A-ENA and modified A-ENA monomers of said heteropolymeric protein nanofibril may be obtained through recombinant expression of a modified A-ENA protein sequence (e.g. a genetic fusion of an A-ENA to a heterologous peptide or protein tag or domain at the A-ENA N-term, C-term or inserted in the linker region), in a host which endogenously expressed a native A-ENA, such as an endospore-forming bacterial strain, preferably a Bt strain. Alternatively, said mixture of different A-ENA and modified A-ENA monomers of said heteropolymeric protein nanofibril may be obtained through recombinant expression of a modified A-ENA protein sequence (e.g. a genetic fusion of an A-ENA to a heterologous peptide or protein tag or domain at the A-ENA N-term, C-term or inserted in the linker region) providing for the heterologous moiety (displayed) on the surface of the fibril, and a modified A-ENA which is modified in the meaning of modification format (1) (truncated NTL) or (2) (mutant variant with modified surface), as both provided above, in a host.

[0250] Further aspects related to the A-ENA protein nanofibril and the production thereof

[0251] A further aspect of the invention relates to nucleic acid molecules or nucleic acid sequences which code for the A-ENA-based monomer or the modified A-ENA protein monomer as described herein, for forming the recombinantly produced protein nanofibril as described herein.

[0252] Furthermore, a vector comprising a nucleic acid molecule coding for a naturally occurring A-ENA protein, or for an A-ENA-based monomer, or a modified A-ENA protein monomer as described herein is disclosed, which may be for cloning or for expression of the recombinant A-ENA proteins modified A-ENA proteins as described herein. Said vector may be introduced in a host through techniques for transforming said host transiently or stably, as known by the skilled person, as to produce the exogenously introduced A- ENA or modified A-ENA proteins in said host, specifically in the cytoplasm of the host cells, or alternatively as secretory proteins for obtaining the A-ENA or modified A-ENA proteins in the culture medium. Upon self-assembly of the recombinantly expressed A-ENA or modified A-ENA or A-ENA-based monomers, fibrous structures form in said host or host culture, allowing extraction of A-ENA multimeric assemblies and optimally A-ENA or A-ENA-based protein nanofibrils from said host.

[0253] So in a specific embodiment the disclosure relates to a host cell comprising the nucleic acid molecule as described herein, coding for an A-ENA protein, or a modified A-ENA protein, or for an A-ENA-based protein. Another specific embodiment relates to said host cell comprising the protein nanofibril as described herein, wherein said protein nanofibril comprises the A-ENA protein as a result from recombinant production of an exogenously introduced A-ENA protein or A-ENA protein-encoding nucleic acid molecule, or modified A-ENA protein-encoding nucleic acid molecule, expression cassette or vector (comprising a chimeric gene construct to encode said A-ENA protein assembly).

[0254] In a preferred embodiment said host cell is a bacterial cell, more preferably, said host cell is E.coli.

[0255] In another preferred embodiment, said host cell is a bacterial cell capable of sporulating, or is a Bacillus cell, more preferably a Bacillus thuringiensis cell. In another preferred embodiment, the bacterial host cell has no endogenous A-ENA protein-encoding gene. Said host cell may as well be provided by other organisms such as plants, fungi or animals. A further aspect relates to a production method for recombinantly producing the A-ENA or A-ENA-based protein nanofibril as described herein, comprising the steps of: a) Introducing the nucleic acid molecule encoding the A-ENA protein, or A-ENA-based or modified A-ENA protein as described herein for recombinant expression of the A-ENA monomeric protein in a host cell, and / or b) Incubating the host cell expressing said A-ENA protein in suitable conditions for cultivating the production of the A-ENA proteins or A-ENA-based or modified A-ENA proteins self-assembling, and preferentially spontaneously forming protein nanofibrils, in said host cell, or in said host cell culture, c) Releasing said A-ENA protein assemblies from said host cell, preferably through cell lysis, and / or d) Isolating the self-assembled protein nanofibrils, preferably through resuspension from the insoluble fraction and / or further purification from the cell lysate.

[0256] In a preferred embodiment said introducing in step a) is obtained by transforming said host cell with a chimeric gene comprising said A-ENA (or modified A-ENA) protein-encoding sequence. In another embodiment, said host cell is a bacterial or a fungal cell, in a more preferred embodiment a microbial production host such as E.coli for cytosolic expression of the A-ENA protein nanofibril, or such as Lactococcus lactis for secretion of the A-ENA protein nanofibril in the cultivation medium.

[0257] In another preferred embodiment, said host is a spore-forming strain, more preferably a Bacillus strain, more preferably a Bt strain. In another preferred embodiment, the A-ENA protein produced in said bacterial strain, preferably in said Bacillus strain, is a heterologous A-ENA protein. Another embodiment provides for a host wherein said Bacillus strain expresses an endogenous A-ENA protein, which may be used to introduce a heterologous A-ENA protein, as to produce the (heteropolymeric) protein nanofibril described herein.

[0258] The invention further provides for recombinantly produced protein nanofibrils, preferably A-ENA or A- ENA-based or modified-A-ENA protein nanofibrils, obtainable by or from said method to produce the nanofibril as described herein.

[0259] The invention further provides for isolated protein nanofibrils, preferably A-ENA or A-ENA-based or modified-A-ENA protein nanofibrils, obtainable by or isolated from said host as provided by the method to produce the nanofibril as described herein.

[0260] Bacterial endospores with recombinant A-ENA protein nanofibrils

[0261] As disclosed in the present application, Bti spore cultures have been found to contain protein nanofibrils composed of A-ENA or A-ENA-1 proteins (as depicted in SEQ. ID NOs:l and 2, resp.), which function in associating the spores and parasporal bodies in contact to guarantee their pathogenic activity towards for instance lepidopteran insects.

[0262] Since the presence of these protein nanofibrils may be optimized or altered or modified for specific purpose of potency as to provide modified endospores, another aspect of the invention relates to modified endospores or modified bacillus endospores, or modified Bt endospores, wherein the term 'modified' refers to the presence of an non-endogenous A-ENA protein or modified A-ENA protein or A- ENA-based protein in said Bacillus strain, and / or Bacillus endospore. In a further specific embodiment, said modified Bacillus endospore or endospore-forming Bacillus strain or cell comprises an non-naturally occurring or modified or engineered A-ENA protein or a nucleic acid molecule encoding the same.

[0263] As disclosed herein, a number of Bacillus strains lacking an endogenous or native gene for producing an A-ENA protein or protein nanofibril have been identified. Hence, in a specific embodiment, recombinantly (or exogenously) introducing or expressing an A-ENA protein or A-ENA-based protein in said bacterial strain or cell lacking the gene or capacity to produce a native A-ENA is provided herein, for the production of the A-ENA protein nanofibril as described herein.

[0264] A further specific embodiment relates to a modified Bacillus endospore, which displays a portion of its modified A-ENA monomeric protein on the surface of the assembled protein nanofibril as described herein. Said modified bacillus endospore nanofibrils displaying a heterologous protein part of the A-ENA protein (as described herein , modified by insertions or fusion of a protein tag or protein domain to an A-ENA protein monomer) are thus provided herein.

[0265] Finally, as also demonstrated herein said modified bacterial endospore, preferably Bacillus endospore, may be used for aiding, facilitating, improving, increasing, or gaining bacterial spore activity, preferably pathogenic activity, more preferably pesticidal activity, most preferably insecticidal activity. In alternative embodiments, said modified bacterial spores, preferably Bacillus spores, as described herein, may be used in gaining, facilitating, increasing, improving a functional effect that is obtained by the protein nanofibril recombinantly produced in said modified spore culture.

[0266] In certain embodiments, the protein nanofibrils produced in said modified bacillus endospores may be present in a modified form or environment which allows for ex vivo extraction or isolation of the protein nanofibrils as such from the culture or bacillus spores, which is also envisaged from the described modified bacillus endospores disclosed herein.

[0267] A further alternative use of the protein nanofibril has been disclosed herein wherein said protein nanofibril in its recombinantly produced purified form has been applied tighter or in combination with, or sequentially to applying a bacterial endospore culture, preferably a Bt endospore culture or suspension, as to enhance or improve the pathogenicity of said endospores on their targets as compared to applying the endospore culture without addition of the purified protein nanofibril.

[0268] Thus, in a specific embodiment, said protein nanofibril as described herein, and preferably as recombinantly produced by the method described herein, provides for an additional or synergistical virulence when applying pesticidal spore substances to their respective pests.

[0269] The protein nanofibril of the present invention thus further provides for an alternative use in agricultural crop treatments or crop improvements and may contribute to future improved pest management strategies.

[0270] Pharmaceutical compositions and medical use

[0271] A further aspect of the invention relates to a pharmaceutical composition comprising the protein nanofibril described herein, or the nucleic acid molecule encoding the monomer of the nanofibril as described herein, and optionally comprising a carrier, diluent or excipient. In a further specific embodiment said pharmaceutical composition comprises a therapeutically active composition comprising the protein nanofibril as described herein, and / or a pharmaceutical composition wherein the therapeutically active composition is presented or provided as an agent coupled, inserted, or attached to the protein fibril of the present invention.

[0272] In a further aspect, the protein nanofibril or the nucleic acid molecule, or the pharmaceutical composition as described herein, is applied as (part of) a medicine or medicament. Specific embodiments relate to the protein nanofibril or the nucleic acid molecule, or the pharmaceutical composition as described herein, for use in therapeutic treatment of a subject, in preventive or prophylactic treatment of a subject. So in specific embodiments, a method of treatment is disclosed comprising the step of administering the protein nanofibril, the nucleic acid molecule or the pharmaceutical composition, as described herein, to a subject suffering or possibly suffering from a disease or disorder, for curing or preventing said disease or disorder.

[0273] In a particular embodiment, the protein nanofibrils are modified by the covalent or non-covalent addition of bioactive or therapeutic proteins, preferably Nanobodies. In said embodiment, the modified protein nanofibrils and their associations, conglomerates or hydrogels thereof provide for a high density, highly multivalent (several hundreds to several thousands) presentation of the bioactive protein. When modified through a reversible bond, by non-covalent association or proteolytic release, nanofibril aggregates and hydrogels can be used for slow release of the bio-active protein. A preferred methodology of a reversible, non-covalent modification of said protein nanofibril and bioactive protein is through the genetic fusion of a receptor - peptide tag pair on the protein nanofibril monomers and bioactive protein, wherein the receptor or peptide tag are attached to the NTL or CT of the nanofibril monomers by a peptide bond, and wherein the second component of the receptor - peptide tag pair is attached to the N- or C-terminus of the bioactive protein. In a preferred embodiment, the receptor - peptide tag pair consists of a Strep tag - (Strept)avidin pair, a SUMO tag - SUMO pair, a coiled - coiled helix pare, or a SpyTag - SpyDock (a mutant SpyTag - SpyCatcher pair unable to form a isopeptide bond) pair.

[0274] Aspects of the disclosure

[0275] The disclosure relates in a first aspect to an isolated protein nanofibril comprising two protofibrils each comprising at least two monomer protein subunits, wherein each monomer protein subunit comprises covalently connected amino acid sequence fragments according to the formula: NTL - helix 1- L - helix 2 - CT, wherein said monomer protein subunit spontaneously folds in aqueous solution into two helices, helix 1 and helix 2, as an alpha-helical antiparallel coiled coil structure, and wherein helix 1 and helix 2 each comprise a continuous sequence of at least 5 heptad (H) elements according to the formula Hl-1 - Hl-2 - Hl-3 - Hl-4 - Hl-5 for helix 1, and H2-1 - H2-2 - H2-3 - H2-4 - H2-5 for helix 2, wherein said heptad elements each comprise seven amino acid residues designated 'abcdefg' with the following consensus sequences: wherein: Φ is a hydrophobic amino acid selected form the list of M, V, I, L, A, G, H, W, Y, F; tp is a short side chain residue, selected from the list of V, C, G, A, P, S, T, N, D; y is a residue that serves as acid / base catalyst, selected from E, D, Q; 6 is a residue capable to serve as isopeptide bond 'donor' or nucleophile, selected from K; s is a residue capable to serve as isopeptide bond 'acceptor' or electrophile, selected from E, Q, D, N; X can be any amino acid, and wherein the linker (L) fragment comprises at least 4 amino acids, and N-terminal lock (NTL) and C-terminal tail (CT) fragments comprise at least 1 amino acid, and wherein said monomeric protein subunits are interconnected through at least one or more isopeptidic bonds (IPBs). In a further embodiment, said protein nanofibril is disclosed wherein the monomeric protein subunits (i and i + / - n) of the first protofibril (f) and the monomer subunits (i' and i' + / - n) of the second protofibril (f') are covalently connected through at least one or more I PBs, wherein the IPBs are formed between the side chain of the amino acid acting as a nucleophile and the side chain of the amino acid acting as electrophile, respectively, at positions:

[0276] NTL of (i) and Hl-5b of (i-5), (i-4), (i-3), or (i-2);

[0277] Hl-4g of (i) and H2-2a of (i-1);

[0278] H2-3g of (i) and H2-4a of (i-1);

[0279] H2-3f of (i) and H2-4e of (i');

[0280] H2-4g of (i) and Hl-3a of (i-1);

[0281] H2-4g of (i+l)and Hl-3a of (i);

[0282] NTL of (i+5), (i+4), (i+3), or (i+2) and Hl-5b of (i);

[0283] Hl-4g of (i+1) and H2-2a of (i);

[0284] H2-3g of (i+1) and H2-4a of (i); and / or

[0285] H2-3f of (i'+l) and H2-4e of (i).

[0286] Further embodiments disclose any of said protein nanofibrils, wherein said Heptad elements have the following consensus sequences: ,

[0287] A more specific embodiment further discloses any of said protein nanofibrils, wherein the NTL comprises a consensus sequence: M-Ψ -Φ-Z-X-Φ-P, wherein: tp is a short side chain residue, selected from the list of V, C, G, A, P, S, T, N, D;

[0288] Φ is a hydrophobic amino acid, selected from the list of M, V, I, L, A, G, H, W, Y, F X is any amino acid,

[0289] Z is Ala or Proline, and

[0290] M and P are one letter code for the respective amino acids Methionine and Proline.

[0291] Anotherembodimentdiscloses any of said protein nanofibrils, wherein the monomer is an A-EN A protein selected from the list of proteins depicted in Table 3, and / or consists of an amino acid sequence selected from the list of SEQ. D NO: 1-6.

[0292] In specific embodiments, said protein nanofibril has an NTLthat is less than 4 amino acids. Alternatively, said protein nanofibril is disclosed, wherein the monomeric protein subunit comprises: an NTL of at least 4 amino acids and the monomeric protein is a mutant variant in at least one or more of the following positions: Hl-la, Hl-lb, Hl-lc, Hl-3f, Hl-4f, Hl-5b, Hl-5f, H2-lb, H2- lc, and / or H2-5b; or the NTL is less than 4 amino acids and the monomeric protein is a mutant variant in at least one or more of the following positions: Hl-la, Hl-lb, Hl-lc, Hl-lf, Hl-2f, Hl-3b, Hl-3f, Hl-4b, Hl-4f, Hl-5b, Hl-5f, H2-lb, H2-lc, H2-2b, H2-2f, H2-3f, H2-4f, H2-5b, H2-5c and / or H2-5f, wherein the protein nanofibril has a modified surface .

[0293] A further embodiment discloses any of said protein nanofibrils, wherein the monomeric protein further comprises a protein tag or domain which is fused or conjugated to the N-terminus, C-terminus or within the Linker region for forming a functionalized fibril.

[0294] An alternative embodiment discloses said protein nanofibril, comprising identical monomer proteins forming a homopolymeric fibril upon self-assembly, or comprising at least two different monomeric proteins forming a heteropolymeric fibril upon self-assembly.

[0295] In a further embodiment any of said protein nanofibrils is disclosed, wherein said fibril is a recombinantly produced protein nanofibril.

[0296] Another aspect discloses a modified Bacillus endospore, which comprises and / or displays any of said monomeric protein subunits or (modified) protein nanofibrils.

[0297] An alternative embodiment discloses a modified Bacillus endospore, wherein the Bacillus strain lacks an endogenous monomeric protein of said protein nanofibril, and comprises an exogenously introduced monomeric protein subunit, or comprises said protein nanofibril discloses herein.

[0298] Further embodiments disclose the use of said modified Bacillus endospores, for increasing bacterial spore activity, preferably pathogenic activity. A further aspect discloses a nucleic acid molecule encoding the monomer protein subunit for forming said (modified) protein nanofibril.

[0299] A final aspect relates to a method for producing any of said disclosed protein nanofibrils, comprising the steps of: recombinant expression of the of said disclosed nucleic acid molecule encoding said protein, or expression of said monomeric protein for forming said nanofibril in a host cell; and releasing the selfassembled protein nanofibrils from the host cell, preferably through cell lysis; and isolation of the selfassembled protein nanofibrils, preferably through resuspension from the insoluble fraction and / or further purification from the cell lysate.

[0300] It is to be understood that although particular embodiments, specific configurations as well as materials and / or molecules, have been discussed herein for methods and products according to the disclosure, various changes or modifications in form and detail may be made without departing from the scope of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

[0301] EXAMPLES

[0302] Example 1. Characterization of A-ENA nanofibrils within a Bacillus thuringiensis serovar israelensis spore biofilm.

[0303] Here, we summarize the characterization of nanofibrils found in the spore biofilm of Bacillus thuringiensis serovar israelensis (Bti). A bacterial lawn of Bti spores was produced on LB agar as follows: 10 mL liquid LB was inoculated starting from a single Bti colony pre-grown on LB agar, and grown to stationary phase overnight at 37°C. Next, 2 mL of the liquid culture was spread on a 250 mL square LB agar plate and incubated at 30°C for 1 week. The resulting spore biofilm was harvested with a cell scraper and resuspended in 10 mL deionized water (miliQ). The spore suspension was used for brightfield microscopy and negative stain Transmission Electron microscopy (nsTEM). Phase contrast microscopy imaging using a lOOx oil objective revealed the presence of two types of refractile bodies (quasi-spherical and elliptical) with a typical diameter of 0.5 pm and 1.5 pm, respectively. The latter species are engulfed with a sac-like structure and correspond to Bti spores. Spores were associated with the former class of refractile bodies, which we identified as parasporal bodies (PSBs; see below). PSBs from Bti are condensed, agglomerated structures that consist of different crystalline subdomains composed of multiple types of proteins that infer larvicidal activity against the larvae of insects such as mosquitoes and black flies. To investigate the spore-PSB association in further detail, we proceeded with a nsTEM analysis of the resuspended spore biofilm samples. For this, we collected nsTEM micrographs on Bti spore samples that were deposited on Formvar / Carbon grids (400 Mesh, Cu; Electron Microscopy Sciences) and stained using 2% (w / v) uranyl acetate. Low magnification TEM images (2500x) reveal the presence of a fibrillar network that permeates the entire biofilm engulfing both spores and PSBs (Figure 1). Based on high-magnification cryoEM imaging (60k), we calculated 3D reconstruction maps that allowed a partial, de novo residue assignment of the fibril monomer. This subsequently enabled a sequence-based search of the Bti genome, and an unambiguous identification of the component subunits as UniProt: Q8KNV8 (SEQ ID NO:1), here dubbed as "A-ENA". Genome analysis reveals the presence of a second A-ENA like sequence adjacent to A-ENA, consisting of Q8KNV7 (SEQ ID NO:2), here dubbed "A-ENA1". Based on the nsTEM analysis, we conclude that the abundant fibril network is composed of individual A-ENA protein filaments consisting of A-ENA and / or A-ENA-1. A-ENA nanofibrils are characterized by an apparent diameter of ± 8 nm, and a distinct projection map in nsTEM or cryoEM. A-ENA as illustrated in the models in Figure 3, A-ENA fibrils are composed of two protofibrils (f and f' ) that intertwine into a two-start helical superstructure with a twist and rise of approximately 12° and 10.8 A, respectively. A-ENA monomeric subunits (labeled i and i') self-assemble into an alpha-helical hairpin with an N-terminal extension projecting downwards along the fibril axis. The A-ENA monomer interact laterally between protofibrils to form a dimeric entity, which then stacks axially, thereby extending each protofibril, resulting in a tetrameric complex, as shown in Figure 3c, providing for the minimal essential fibril unit formed by self-assembling A-ENAs.

[0304] We identify clear coupling of A-ENA filaments to the outermost layers of the endospores, but also to the surface of the PSBs, e.g. clusters of A-ENA fibrils emanate from both the PSB and / or the spore exosporium. Moreover, PSBs are often found coupled to the mature spores via the A-ENA filaments. Such a fibril-mediated PSB-spore or spore-spore connection can span multiple micrometers and effectively functions as a reinforced tether between the various structures.

[0305] Example 2. Recombinant production and purification of in cellulo assembled A-ENA and A-ENA-1 fibrils.

[0306] Native wild-type sequences of A-ENA (gene: ATN07_33990; protein: Q8KNV8; SEQ ID NO:1) and A-ENA- 1 (gene: ATN07_33980 ; protein: Q8KNV7; SEQ ID NO:2) were PCR amplified from the pBtoxis plasmid (CP013279; pAM65-52-4-128K) using primer setl (SEQ ID NO:16-17) and primer set2 (SEQ ID NO:18-19), respectively. Amplified fragments were cloned into the pASK vector using Gibson Assembly (https: / / doi.org / 10.1038 / nmeth.1318) and introduced into E. coli ToplO. Resulting transformants were screened by colony PCR and Sanger sequencing (pASK sequencing primers; SEQ ID NO:2Q-21). The obtained plasmids (pASK_A-ENA; pASK_A-ENA-l) were used to transform competent cells of E. coli C43(DE3). Single colonies were used to start overnight (ON) LB cultures. 10 mL ON culture was used to inoculate 1 L LB, 50 pg / ml ampicillin at 37°C. Recombinant expression was induced at an ODeoo of 0.8 by the addition of 50 pM anhydrotetracycline and cultures were left to incubate ON at 18°C. Cells were resuspended in 1 mg.mL1lysozyme, 5mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris pH 6.8, 50 mM NaCI and incubated for 18h under stirring conditions at room temperature. Next, sodium dodecyl sulfate (SDS) was added to the lysate to a final concentration of 1% (w / v) and the lysate / SDS mixture was heated to 100°C. Next, the mixture was left to cool at room temperature for 15 min and centrifuged in a Beckman JA 14.50 50 mL falcon rotor at 20.000 ref for 30 minutes. The resulting supernatant was discarded and the pellet (i.e. insoluble fraction consisting of A-ENA(-l) fibrils) was brought back up into suspension (5 mL miliQ per gram of wet fibril pellet) using an IKA overhead stirring system. The resulting suspension was subjected to three additional rounds of centrifugation (20.000 ref, 30 min) and resuspension of the respective pellets into miliQ to remove residual SDS. The suspension obtained after the last wash step was diluted ten-fold in miliQ, deposited on Formvar / Carbon grids (400 Mesh, Cu; Electron Microscopy Sciences) and stained using 2% (w / v) uranyl acetate and blotted dry. Negative staining transmission electron microscopy (nsTEM) analysis revealed the presence of micrometer long fibrils with a diameter of approximately 8 nm (Figure 2). 2D classification of boxed fibril segments (Figure 2a) was in agreement with 2D class average images that had been previously obtained on ex vivo fibrilspore suspensions extracted from a Bacillus thuringiensis serovar israelensis spore biofilm (Figure lh-i).

[0307] CryoEM structural analysis of these recombinant A-ENA fibrils confirmed the A-ENA monomer subunit alpha-helical antiparallel coiled-coil structure as observed for the wild-type A-ENA fibril monomers, as well as their inter-protofibrillar contacts and isopeptide bonds (Figure 4, 5). Also intra-protofibrillar contacts and isopeptide bonds were visible from A-ENA (i) with the A-ENA monomers directly above (i+1) or below (i-1) subunit i, as well as with monomer i-5 via residue 2 in the N-terminal connector. These observations demonstrate that the formation of A-ENA nanofibrils does not rely on accessory factors in the native Bti cells, and occurs spontaneously and efficiently in an unrelated recombinant host cell. Thus, A-ENA subunits contain intrinsic self-assembling properties, as well as the capacity for autocatalytic formation of intermolecular and interfibrillar isopeptide bonds. The contact surfaces of the A-ENA monomers are primarily hydrophobic, however except for a number of residues involved in covalent interactions. Moreover, the observation of the highly stable nature of the wild type A-ENA fibrils could be explained by the nature of these covalent interactions as being formed as an irreversible isopeptide bond (IPB), leading to a stable fibrillar structure wherein each subunit i is implicated in as much as 10 IPBs, crosslinking 7 different subunits within and across the protofibrils (Figure 4, 9).

[0308] Example 3. Isopeptide bond formation is required for self-assembly of stable A-ENA like nanofibrils. A-ENA monomeric subunits self-assembling into nanofibrils contain ten isopeptide bonds with neighbouring A-ENA subunits. These isopeptide bonds are formed by autocatalytic units that consist of an acid-base catalyst (preferably Glu), a nucleophilic residue (preferably Lys or the N-terminal amine) and an electrophilic residue (preferably Glu, Gin, Asn or Asp), and is proximity-induced as defined by the relative positions of each acid-base catalyst to activate the nucleophilic residue for attack of the electrophilic residue. Besides the Lysines in the helices, also the N-terminal amine in the NTL can be implicated as a nucleophilic donor in IPB formation, wherein the exact subunit 'i-x' implicated in the IPB can be varied by extending or shortening the NTL (Figure 4a, 9). The exact residue positions of each type of residue were annotated for A-ENA (SEQ ID NO:1) as shown in Figure 4a, 5 and 7.

[0309] To gauge for the role of the isopeptide bonds in the self-assembly of A-ENA monomers into stable A-ENA nanofibrils, we mutated the electrophilic residues of the isopeptide-bond pair. For this, E29, Q44, N64, E78 and Q82 were mutated to an alanine residue (SEQ ID NO: 15). To facilitate purification from an E.coli lysate, a 7x Histag was also added onto the C-terminus. The resulting DNA sequence was ordered as a double stranded synthetic DNA fragment (gblock, IDT), and cloned into the pASK vector and recombinantly expressed as outlined in Example 2. Harvested cells were resuspended in 100 mM K- phosphate pH 7.2 buffer supplemented with 8 M urea and 1 M NaCI at a concentration of 10 ml buffer per gram of wet cell pellet. The resulting cell suspension was sonicated on ice for 5 min using a 500W Qsonica probe sonicator using a 30 s / 15 s on / off pulse set at 50 % amplitude. Next, the cell lysate was centrifuged for 45min at 20.000rcf to pellet insoluble cell debris. The cleared lysate was loaded onto a pre-equilibrated prepacked 5mL Histrap column and washed with 20 column volumes of 100 mM K- phosphate pH 7.2 , 8 M urea and 1 M NaCI. The column was eluted with 100 mM K-phosphate pH 7.2, 8 M urea and 400mM imidazole and the resulting fractions were analyzed using SDS-PAGE analysis. Fractions containing A-ENA molecules were pooled and dialyzed against lOOmM Hepes pH 8.0 and 8 M urea. The dialyzed protein was loaded onto a prepacked 1ml Q anion exchange column that was preequilibrated with 100 mM Hepes pH 8.0, 8 M urea and the flow-through was collected. Next, the column was eluted with 100 mM Hepes pH 8.0, 8 M urea and 1 M NaCI. SDS-PAGE analysis of the flow-through and eluate fractions showed that A-ENA was found as a pure monomeric species in the flow-through (Figure 12 a). Next, the A-ENA monomers were buffer exchanged from 100 mM Hepes pH 8.0, 8 M urea to lxPBS using a 40K MWCO Zeba spin desalting column and left to incubate at room temperature for 24h. The desalted A-ENA sample was applied onto a Formvar / Carbon grid, stained with 2% uranyl acetate and imaged using a 120kV JEOL electron microscope (Figure 12 b and c). The resulting micrographs were devoid of any fibrillar structures. Rather, the A-ENA E29A Q44A N64A E78A Q82A mutant abundantly forms fractal-like aggregates. From this we conclude that the isopeptide bonds are crucial for the formation of stable A-ENA fibrils.

[0310] Example 4. A-ENA sporesilk networks induce Bacillus thuringiensis spore biofilms and associate spore with parasporal bodies.

[0311] To generate the A-ENA KO (AA-ENA) in Bacillus thuringiensis serovar israelensis (Bti), we followed the procedure described in Wang et al. (2019 Front Microbiol.10:1932) based on the pJOE8999 plasmid carrying the CRISPR / Cas9 system (Altenbuchner, 2016. Appl Environ Microbiol. 82(17):5421-7) purchased from the Bacillus Genetic Stock Center. First, the plasmid pJOE8999 was linearized with pair of primers p551 and p552. Around lkb upstream and lkb downstream of the A-ENA gene were amplified with the pair of primers p588 & p633 and p634 & p635 respectively using Bti spore stock as a gDNA donor template. Both PCR fragments were cloned into the linearized pJOE8999 vector by Gibson assembly following the manufacturer's instructions. Oligos p525 and p526 were used to sequence verify the plasmid. Next, we cloned the sgRNA as designed using the web server: http: / / www.rgenome.net / cas-designer / . The chosen sgRNA (SEQ. ID NO:22) was cloned into the abovedesigned plasmid by PCR using the pair of primers p594 & p595. sgRNA was sequenced verified using oligo p531. Plasmid was transformed into the E. coli strain dam- / dcm- and grown in LB supplemented with 50 pg / ml of kanamycin to produce an unmethylated plasmid. The plasmid was then electroporated in Bti WT and transformants were selected at 30 °C on BHI plates supplemented with 0.5 % glycerol and 25 pg / ml of kanamycin. The next day, a single colony was inoculated into 10 ml of liquid BHI supplemented with and 25 pg / ml of kanamycin and incubated for 3h at 37 °C. In order to induce the expression of Cas9, mannose was added at a final concentration of 0.4% w / v and incubated for an additional 3h at 37 °C. Serial dilutions of the culture were plated on LB agar containing 25 pg / ml of kanamycin and 0.4 % w / v mannose at 37 °C. The next day, mutants were selected by colony PCR using pair of oligos p602 and p650. A single colony was selected and the A-ENA locus was sequence verified using oligos p602 and p650. Finally, the KO A-ENA strain was imaged via TEM to ensure the absence of A-ENA fibrils (Figure 18b).

[0312] Complementation of the A-ENA KO was done by replacing the cas9 gene in pJOE8999 plasmid with the promoter, CDS and terminator of the A-ENA gene from Bti. First, plasmid pJOE8999 was linearized using pair of oligos p622 & p623. The A-ENA locus including promoter, CDS and terminator was amplified using pair of oligos p683 & p684 and cloned into the above-linearized plasmid by Gibson assembly following the manufacturer's instructions. The mixture was transformed into E. coli dam / dcm- strain and selected in LB plates supplemented with 50 pg / ml of Kanamycin and incubated at 37 °C. Next day, positive colonies were selected by colony PCR using pair of primers p617 & p631 and grown in 10 ml LB with of 50 pg / ml of Kanamycin, plasmid was extracted using NucleoSpin Plasmid EasyPure kit leading to plasmid A176. Plasmid was sequenced verified (Eurofins) and transformed into electrocompetent Bti cells. A single positive colony was inoculated in BHI supplemented with 25 pg / ml of Kanamycin and grown overnight at 30 °C. The next day, 2 mL of the liquid culture was spread on a 250 mL square LB agar plate supplemented with 25 pg / ml of Kanamycin and incubated at 30°C for 1 week. Spores we resuspended in water and the presence of A-ENA fibrils was assessed via TEM. Spore samples were deposited on Formvar / Carbon grids (400 Mesh, Cu; Electron Microscopy Sciences) and stained using 2% (w / v) uranyl acetate. TEM images revealed the presence of a fibrillar network that holds the entire biofilm encompassing both spores and PSBs as observed for the WT (Figure 18e) and confirmed the correct complementation of the A-ENA KO.

[0313] Example 5. Exogenous A-ENA forms sporesilk networks that associate spores and Cry toxin crystals in Bacillus thuringiensis Sv. kurstaki.

[0314] The A176 plasmid, described in Example 4, that was used to complement the A-ENA knockout (KO), was electroporated into Bacillus thuringiensis Sv. kurstaki cells, which naturally lack A-ENA. Following electroporation, the transformed cells were selected on brain heart infusion (BHI) plates supplemented with 25 pg / ml of kanamycin and incubated overnight at 30 °C. The next day, a single colony was inoculated into 10 ml of BHI medium containing 25 pg / ml of kanamycin and grown overnight at 30 °C. Subsequently, 200 pl of the overnight culture was plated on a square plate measuring 245 x 245 x 25mm, containing Luria-Bertani (LB) medium supplemented with 25 pg / ml of Kanamycin, and allowed to sporulate for a week at 30 °C. The resulting spore biofilm was harvested using a cell scraper, resuspended in 10 mL of deionized water (miliQ) and used for TEM imaging and light microscopy. The Btk strain expressing A-ENABti exhibited a biofilm similar to Bti in addition to bipyramidal PSB crystals connected to A-ENA bundles, as depicted in Figure 19.

[0315] Example 6. A-ENA mediated association of spores and parasporal toxins enhances entomopathogenesis in Bacillus thuringiensis.

[0316] To investigate the role of A-ENA in the association between PSB and spores, we assessed the virulence of Bacillus thuringiensis Sv. israelensis strains expressing or lacking A-ENA (wild-type vs A-ENA knockout) using the Chironomus aprilinus insect model. Fresh Chironomus aprilinus larvae were procured from Zooschatz (Berlin, Germany) with batch number 1223153. Bti wild-type (WT) and A-ENA knockout (KO) strains were cultured on LB plates for one week. The resulting spore biofilm was scraped and resuspended in 10 ml of deionized water, creating a spore suspension. In parallel, spores were isolated from 1 ml of the spore suspension by centrifugation at 16,000 x g for 40 minutes using a 50 % histodenz cushion. The isolated spores were subsequently washed three times with deionized water by centrifugation at 5000 x g for 10 minutes. To determine the mortality rate, a minimum of 8 Chironomus aprilinus larvae were included in each group, with a final volume of 3 ml containing sea salt and a vitamin premix provided with the larvae. Spore suspension and spore isolation samples were added to a final optical density (ODgoo) of 0.02. As a negative control, phosphate saline buffer (PBS) was used. The survival of the larvae was monitored daily over a period of 7 days. The study was performed in biological triplicates under room temperature conditions. As illustrated in Figure 20 the loss of A-ENA fibrils results in a significant reduction of the killing activity of Bti spores. The point of death of larvae exposed to Bti AA-ENA is extended by at least one day compared to larvae exposed to WT Bti, and insect larvae have a higher probability to survive at least a weak exposure to Bti endospores. The effect was most pronounced in isolated spore preps of Bti AA-ENA, where the precipitation and isolation of the endospores from the growth medium results in a drastic reduction of PSBs due to the lack of A-ENA fibrils (Figure 18 f). A mild delay in killing activity is still seen in spore suspensions of Bti AA-ENA, where the PSB remain present in suspension, though are no longer associated with the endospores (Figure 18d, f). Thus, these experiments demonstrate that an inability of Bt spores to retain their secreted toxins in close proximity to the endospores, results in marked reduction in virulence and entomotoxic activity.

[0317] Example 7. A-ENA protein sequence conservation.

[0318] A multiple sequence alignment was performed based on the top 593 blast hits using Q8KNV8 (SEQ ID NO: 1) as a query sequence, to create an A-ENA consensus web logo as shown in Figure 6. Looking at the alignment of A-ENA orthologues with only 24 % sequence identity to A-ENA (SEQ ID NO: 1) (see Figure 7 lower panel), it was found that A-ENA subunits adopt a generic structure formed of a variable N-terminal lock ('NTL'), followed by a-helix 'al' consisting of 5 Heptad repeats (Hl-1 to Hl-5), followed by a variable length linker sequence (dubbed 'L'), followed by a second a-helix 'al', and ending in a variable length C- terminal tail ('CT'). Alignment of A-ENA orthologues according to the Heptad repeats demonstrates a strong conservation of the position of hydrophobic residues implicated in the intra- and inter-helical knob-in-holes interactions and forming the isopeptide bond units.

[0319] These residues involved in IPB formation (derived from the cryoEM structure of recombinant Q8KNV8 fibrils) were found to be largely conserved in the conserved sequence logo, and the conservation of the hydrophobic residues in the helices indicated that these A-ENA orthologues are likely also functional in the formation of self-assembled nanofibrils. The A-ENA orthologues (SEQ ID NOs: 3-5, as shown in Figure 7 and Figure 8) and Bti A-ENA (Uniprot: Q8KNV8, SEQ ID NO: 1) and A-ENA- 1 (Uniprot: Q8KNV7; SEQ ID NO: 2), were recombinantly produced to verify this hypothesis. As shown in the images of Figure 8, selfassembled fibrils were indeed formed in the cytoplasm of E. coli, which likely contain IPBs, since the sequence conservation allows for that and the fibrils shown represent the insoluble fraction after SDS / heat extraction, and therefor indicative of the autocatalytic formation of one or more IPBs.

[0320] Furthermore, based on the alphahelical coiled-coil structure of the A-ENA monomers, we could outline the link between the amino acid sequence of the A-ENA protein and its structure by representing the monomers as helical wheels wherein the intermolecular interactions are delineated (Figure 7). Indeed, knobs-in-hole interactions of helix-helix contacts are precisely definable by the sidechains at each position of the sequence. Based on the insights from the cryo-EM structure showing a fibril with a uniform helical rise and twist in an alpha-helix of 1.5 A and 100°, respectively, the residues of the monomers were positioned such that every 8thresidue is located on the equivalent position on the helical wheel, adopting a translation along the helix by 10.5 A. So herein we defined the helices hl and h2, or al and a2, each by a series of 5 heptameric units or 'heptads' (H), with side chains named 'a' to 'g', providing for a conserved sequence motif that allows self-assembly into the helix hairpin or alphahelical coiled-coil, as used interchangeably herein (see Figure 7 and 10). For further fibril formation, A-ENA subunits further engage in stacking interaction (i.e. i / i+1 and i / i-1 interactions) by alpha-helices al and a2 in consecutive subunits going into pairwise parallel packing by the primarily hydrophobic knobs-in-hole interactions along the length of the helices, involving heptad positions 'a' and 'e' of the Heptads on the C-pole facing side and 'g' and 'c' on the Heptads of the N-pole facing side of the A-ENA hairpins. Through the presence of said consensus sequence motif of hydrophobic and short side chain residues in the al and a2 Heptads as shown in Figure 10, the adoption of an antiparallel helix hairpin within the A-ENA like units tis facilitated, as well as the pairwise stacking of the fibril assembly units (i / i+1 and i / i-1) into helical protofibrils (labelled f) with approximate twist and rise of 12° and 10.8 A.

[0321] Exceptions in the hydrophobic contact are formed by residues going into isopeptide bond formations and / or forming the acid-base catalyst in the IPB units (Figure 9). Also the packing of the f and f' protofibrils in A-ENA is mediated by knobs-in-holes and IPB interactions of positions 'b', 'e' and 'f' of Heptads H2-3 and H2-4 of a2 in subunits i with i' and i'+l.

[0322] Example 8. Engineering of A-ENA fibrils.

[0323] Based on the structural insights we gained from analyzing the A-ENA nanofibrils, different positions within the A-ENA protein sequence were tested for the addition / insertion of single amino acids, peptides or folded domains, as well as different sites were tested for site-directed mutagenesis in view functionalization of the A-ENA nanofibrils. Given that both the N-and C-terminus are surface exposed in the final double protofibril assembly, both termini are labeled as potential sites for the addition of single amino acids, peptides or folded domains. In addition to the termini, we also identified the loop (L, linker) that connects helices al and a2 as a potential site for insertion of single amino acids, peptides and folded domains (Figure 13a). In addition to these sites, we also proposed solvent exposed residues found on the surface of the A-ENA fibril as potential mutation possibilities to alter the surface properties of the resulting A-ENA fibrils (hydrophobicity, electrostatics etc.) and form sites for targeted modification by change to natural or non-natural amino acids that are amenable to chemical conjugation (i.e. residues containing in a non-exhaustive way, primary amines, thiols, azide, alkyn groups, ...) known to the person skilled in the art. As shown in Figure 13c, these possible mutation sites are presented herein in stick representation annotated with the residue number according to the A-ENA sequence (SEQ. ID NO: 1). In Figure 13e, we mapped these residues onto the five heptads of helices al and a2 as defined previously and shown in Figures 9 and 10. For the wild-type A-ENA fibril, heptads Hl-2 (and to a lesser extent Hl-1 and Hl-3) are buried from the solvent by the N-terminal lock that docks onto the fibril surface. However, deletion of the N-terminal lock via truncation of the residues 1 to 12 exposed these residues to the solvent, and renders them more amenable to alteration. In Figure 13b, we defined A-ENA-delta NTL (ANTL; SEQ ID NO: 12), and its corresponding sites for the addition of single amino acids, peptides or folded domains. This in turn allowed us to determine additional residues for site-directed mutagenesis (Figure 13d) and the corresponding positions in the respective heptads (Figure 13f).

[0324] -Post-polymerization functionalization ofA-ENA fibrils:

[0325] As an example of A-ENA engineering, we cloned the SpyTag sequence (SEQ ID NO:23) onto the C -terminus of A-ENA and A-ENA-1, and probed for the ability of the resulting constructs to selfpolymerize and to form a covalent complex with a two domain construct, consisting of SpyCatcher003 and superfolder green fluorescent protein (sfGFP) separated by an SG linker (Figure 14a). Here, A-ENA- SpyTag and A-ENA-l-SpyTag were recombinantly expressed in the cytoplasm of E.coli and the resulting fibrils were purified using the protocol outlined in Example 2 (Figure 14b). Next, we tested whether the fibrils could selectively capture SpyCatcher-sfGFP. For this, we co-incubated a suspension of A-ENA- SpyTag and A-ENA-l-SpyTag fibrils with purified SpyCatcher-sfGFP for 30 min at room temperature. 10 pl aliquots of the reaction mixture were analyzed on SDS-PAGE and the resulting gel was analyzed using a Li-COR M Odyssey imaging system (green fluorescence channel at 488nm). The SpyCatcher-sfGFP sample (lane 1 in Figure 14c) runs at an apparent molecular weight of 40 kDa which is in agreement with the theoretical molecular mass of 40.19kDa. We also point out that the SpyCatcher-sfGFP band still fluoresces at the expected wavelength, demonstrating that the sfGFP moiety is still folded when coupled to the fibrils. Upon co-incubation with A-ENA-SpyTag (lane 2) and A-ENA-l-SpyTag (lane 3) a second fluorescent band became visible, specifically in the region of the gel that corresponds to the stacking gel. This section of the SDS-PAGE is well beyond the high molecular weight range that can calibrated with the a molecular marker, but it is clear that these are high molecular weight species. The fact that these species (i) remain trapped within the pore network of the stacking gel after boiling in NuPage LDS sample buffer and (ii) are fluorescent leads us to conclude that they correspond to A-ENA and A-ENA-1 fibrils that have been covalently coupled to SpyCatcher-sfGFP. This is further exemplified by a larger scale coincubation experiment of A-ENA-SpyTag fibrils with soluble SpyCatcher-sfGFP. After an incubation of 30 min at room temperature, the fibril suspension was centrifuged for 30min at 20.000rcf. The resulting pellet (insoluble) and supernatant (soluble) were illuminated using a Clare chemical DR46B blue LED transilluminator which excites the samples using pure visible blue light and filters the signals through an amber screen, allowing for visualization of the fluorescence (Figure 14d). We record a clear fluorescent signal in the fibril pellet after centrifugation which demonstrates that the SpyCatcher-sfGFP molecules were efficiently sequestered by the A-ENA fibrils. -Pre-polymerization functionalization ofA-ENA fibrils - optimized linker strategy:

[0326] The SpyTag-approach outlined above can be used for post-polymerization functionalization of A-ENA fibrils. Using SpyCatcher-fusion constructs, virtually any passenger domain -without any restrictions on size and molecular weight- can be efficiently coupled onto the A-ENA scaffold. Because the display spacing or 'rise' of SpyTag peptides is approximately 10.8A per protofibril, we do anticipate that this approach will not yield full stochiometric labelling of all the available binding sites for display domains with a hydrodynamic radius that greatly exceeds 10.8A. For those cases where a one to one ratio is required between A-ENA and the display domain of interest (e.g. to minimize the distance between the domains along the length of the fibril), or for those case where pre-polymerization functionalization is preferred, we developed a second method of A-ENA based functional display (Figure 15a). For this, we constructed genetic fusions between A-ENA and various display domains separated by a flexible linker. As this approach yields homopolymers with a display density of 0.108 A1, optimized linkers are required to avoid steric clashes between the display domains of the neighboring units. By inclusion of a flexible linker, inserted display domains obtain the conformational freedom to pack in a zigzag or staggered stacking rather than a purely translational stacking along the length of the fibril. In this way, domains with a height exceeding the 10.8 A rise of A-ENA subunits in the protofibrils can be accommodated by a staggered stacking. Preferred linkers are between 1 and 20 amino acid residues in length, but can be longer if higher degrees of freedom and / or a longer distance from the A-ENA fibril backbone are desired. Preferred linkers will have primarily small and hydrophilic amino acids (G, A, S, T, N, D) in no particular preferred sequence, but can include larger hydrophilic residues (K, E, R, Q, H, Y), and tend to be low or negative in Proline. Linker design can be done at random, or guided through state- of-the-art protein modelling and design tools such as protein MPNN (Dauparas et al. Science 378, 49- 56(2022)), Rosetta (https: / / github.com / RosettaCommons), among others, and as known by the skilled person. In one approach, insertion domains are placed in silico along the A-ENA backbone in the preferred orientation and position, and the spacing between the N-terminal, C-terminal or Linker residue connecting to the C- or N-terminal residue of the insertion domain can be built in silico by protein design tools. Validation of linker design can be done in first instance through de novo structure prediction tools such as Alphafold (Jumper, J., Evans, R., Pritzel, A. et al. Nature 596, 583-589 (2021)) or RosettaFold (Baek et al. Science 373,871-876(2021)) among others, as known by the skilled person, and / or by experimental validation through negative stain EM inspection of the culture supernatants. Here we report the successful recombinant production and polymerization of three different A-ENA fusion constructs (p66a, MBD2 and rubredoxin; SEQ. ID NOs: 8, 9, 10). In Figure 15b, nsTEM micrographs are shown of self-assembled A-ENA-TEV-p66a, A-ENA-TEV-pMBD2 and A-ENA-TEV-Rubredoxin fibrils purified from the cytoplasm of E.coli. Next, we performed functional assays to demonstrate the folded nature of the display domains. p66a and MBD2 are two alpha-helical proteins from Homo sapiens that form an antiparallel coiled coil complex with nanomolar affinity. Here, we anticipated that a co-mixture of A-ENA-TEV-p66a and A-ENA- TEV-pMBD2 would result in a non-covalently cross-linked fibril network driven by the p66a and MBD2 i nterf i bri 11 a r complex formation. To demonstrate such in terf ibri 11 a r coupling, both A-ENA coiled coil fibrils were purified as described Example 2, after which the samples were subjected to an additional freeze thaw cycle using 12% (w / v) urea and 8% (w / v) NaOH to achieve a higher purity. After three wash steps, through consecutive centrifugation (30 min at 20.000 ref) and resuspension in lxPBS buffer, the insoluble pellet of the fibrils was harvested, and 100 mg of protein pellet of A-ENA-p66a and A-ENA-MBD2 was resuspended separately in 180 pl of 50 mM Tris buffer pH 7.0. From each solution, 60 pl was combined and mixed thoroughly. After 30 minutes an eppendorf inversion test was performed to probe for hydrogelation (Figure 15d). The suspensions of A-ENA-p66a and A-ENA-MBD2 did not remain fixed at the base of the Eppendorf during the inversion test, whereas the mixed suspension formed a turbid, stable hydrogel that did stably remain at the Eppendorf base. This shows that the respective display domains are (i) folded and (ii) accessible for complexation, leading to interf ibrillar coupling, kinetic arrest of the polymer network and the establishment of a mixed A-ENA hydrogel.

[0327] To establish the folded nature of the rubredoxin passenger domain (from Desulfovibrio vulgaris), thermal denaturation assays based on SYPRO orange fluorescence were performed. For this, 25 pl samples containing the fibrils of interest in combination with IX SYPRO™ Protein Gel Stains (Thermo fisher scientific; Waltham, Massachusetts, United states) were loaded onto a Hard-Shell 96 well microplate (Bio-rad; Hercules, California, United states) plate. Using a CFX opus 96 qPCR plate reader system (Bio-rad; Hercules, California, United states), the temperature was increased from 25°C to 100°C by 0.5°C every 10 seconds and fluorescent emission spectra were measured at every temperature interval. Melt curves are calculated as the first derivative of the RFU in order of temperature (Figure 15e). The reported melting temperatures correspond to local maxima of the melt curves, when values are >1000 RFU / dT. For A-ENA wild-type fibrils, no clear maxima larger than 1000 RFU / dT were measured, indicating no significant unfolding events within the measured temperature range (Figure 15e). For the A-ENA-TEV_rubredoxin sample, however, a clear maximum (1435 dRFU / dT) was detected at 94.5°C, indicating the unfolding of the fused rubredoxin domain. From this we conclude that the rubredoxin domain grafted onto the A-ENA scaffold is natively folded, and shows an unfolding Tm of 94.5 °C. This Tm is similar to thermal unfolding temperatures reported previously for bacterial Rubredoxin. While no exact unfolding data is available on the Desulfovibrio vulgaris Rubredoxin protein used here, it is known to rapidly unfold at 100°C (Lazaridis, I. Lee and Karplus 1997, Protein Sci. 6(12):2589-605). By comparison, a homologous Rubredoxin from Clostridium pasteurianum (70 % amino acid sequence identity) has an unfolding temperature of 69°C for the reduced state and 83°C for the oxidized state (Bonomi et al. 2000, Protein Sci. 9(12):2413-26).

[0328] -Pre-polymerization functionalization of A-ENA fibrils- with loop insertions

[0329] To evaluate if A-ENA fibers can be modified by insertion of recombinant sequences into the linker loop connecting Helix 1 and Helix 2 of A-ENA-like fiber subunits (Figure 24), a genetic fusion construct was made where a foreign sequence was inserted between residues valine 55 and threonine 56 of B. thuringiensis Sv. Israelensis A-ENA (UniprotKB Q8KNV8; SEQ ID NO: 1). As a non-limiting example, we inserted a sequence corresponding to a beta solenoid domain comprising two curlin-like repeats, with the amino acid sequence as shown in SEQ ID NO: 66 (referred to as 'R4.5-2RFD'). The inserted curlin-like repeat domain and the A-ENA monomer are connected with a two amino acid linker (GG) connecting the C-terminus of Helix 1 with the N-terminus of the insertion sequence, and a two amino acid linker (SG) connecting the C-terminus of the insertion sequence with the N-terminus of Helix 2, resulting in a fusion protein here referred to as 'A-ENA_LI-R4.5-2RFD' (SEQ ID NO: 67). In these A-ENA loop insertion (LI) designs, the length and sequence of the linking residues connecting Helix 1 and 2 with the insertion sequence are variable and can be shortened or increased in function of the desired distance between the A-ENA fibril scaffold and the inserted sequence displayed on the ridge of the A-ENA fibrilrs (Figure 24).

[0330] The construct with coding sequence for A-ENA_LI-R4.5-2RFD (SEQ ID NO:67) was made through PCR linearization of a plasmid vector holding the coding sequence for A-ENA (pASK-A-ENA) with outward facing primers flanking residues 55 and 56 (primer Ll_l and primer Ll_2 -SEQ ID NOs: 69-70, resp.). This was followed by Gibson assembly of the vector and a DNA fragment corresponding to the coding sequence for the desired insertion sequence and linkers, flanked by the equivalent complementary sequences to Ll_l and Ll_2.

[0331] To evaluate whether A-ENA_LI insertion mutants (also called herein A-ENA sequences wherein a heterologous or foreign sequence is inserted in the linker connecting Helix 1 and Helix 2) retain their self-assembling properties, E. coli DH5a cells were transformed with the plasmid encoding the sequence for A-ENA_LI-R4.5-2RFD (SEQ ID NO: 67). A-ENA_LI-R4.5-2RFD expression was induced at an OD60o of 0.8 by the addition of 50 pM anhydrotetracycline and cultures were left to incubate overnight at 18°C. Cells were resuspended in 1 mg.mL-1 lysozyme, 5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris pH 6.8, 50 mM NaCI and incubated for 18h under stirring conditions at room temperature. Next, sodium dodecyl sulfate (SDS) was added to the lysate to a final concentration of 1 % (w / v) and the lysate / SDS mixture was heated to 100°C. The mixture then was left to cool at room temperature for 15 min and centrifuged in a Beckman JA 14.50 50 mL falcon rotor at 20.000 ref for 30 minutes. The resulting supernatant was discarded and the pellet consisting of A-ENA fibrils was brought back into suspension at a concentration of 5 mL miliQ water per gram of wet fibril pellet. The resulting suspension was subjected to three additional rounds of centrifugation (20.000 ref, 30 min) and resuspension of the respective pellets into miliQ water to remove residual SDS. The suspension obtained after the last wash step was diluted ten-fold in miliQ water, deposited on Formvar / Carbon grids (400 Mesh, Cu; Electron Microscopy Sciences) and stained using 2 % (w / v) uranyl acetate and blotted dry. Negative staining transmission electron microscopy (nsTEM) analysis revealed the presence of micrometer long fibrils with a diameter of approximately 10 nm (Figure 24d,e), demonstrating the A-ENA linker insertion variants retain their capacity to self-assemble into SDS-stable A-ENA like nanofibrils.

[0332] -Pre-polymerization functionalization of A-ENA fibrils - block copolymer strategy:

[0333] The optimized linker design strategy outlined above is ideally suited for the surface display of heterologous domains with a molecular weight that is comparable to or smaller than A-ENA. For the display of proteins that are considerably larger than A-ENA, we developed a block copolymer design strategy (Figure 16a). At its core, we performed co-expression in E. coll of wild-type A-ENA and genetic fusion constructs of A-ENA. As a proof of principle, we worked with a camelid VHH antibody (nanobody) that was coupled to the A-ENA N-terminus using the TEV-protease cleavage site as a linker (designated hereafter as Nanobody-TEV-A-ENA; SEQ ID NO: 11). Nanobody-TEV-A-ENA was cloned into the pBAD vector, and transformed into a ToplO E.coli strain that was harboring the pASK-A-ENA wild-type construct. The resulting co-expression leads to the formation of heteropolymeric A-ENA fibrils composed of A-ENA and Nanobody-TEV-A-ENA subunits (Figure 16b). We anticipated that the distribution of both subunit types within the A-ENA fibril is random and resembles the topology of a block copolymer. The average distance between two display domains depends on the block sizes within the copolymer. Via careful control of expression levels of both proteins (through the choice of the respective promoter strength, plasmid copy number, inducer concentration, induction time point), the ratio between A-ENA and A-ENA fusion construct is controlled. This strategy has the benefit that it does not impose an upper limit on the size of the passenger domain because of the random distribution of A-ENA and A-ENA fusion constructs within the fibril assembly.

[0334] Example 9. In vivo functionalization of A-ENA sporesilk networks in Bacillus thuringiensis.

[0335] In the experiments shown in Figure 14, 15, 16 and 17 we have shown how A-ENA subunits can be functionalized by direct fusion of a polypeptide, including, in a non-limiting way, affinity reagents such as nanobodies, metal-binding proteins such as rubredoxin, affinity tags such as SpyTag or SpyCatcher, coiled-coil pairs. We demonstrated that said modified A-ENA retain the properties to self-assemble into homopolymeric and / or heteropolymeric (i.e. block copolymers) nanofibrils, and that the fused polypeptides retain their activity. In this way, A-ENA nanofibrils can be formed that are functionalized by genetic fusion or by covalent or non-covalent addition of functional polypeptides through means like, in a non-limiting way, the SpyTag - SpyCatcher technology, StrepTag - Streptavidin technology, coiled- coil pairing. These principles are shown in Figure 14-17 for the in vitro assembly of A-ENA nanofibrils.

[0336] In the experiments shown in Figure 21, we aimed to determine whether A-ENA fusion proteins could form in vivo during the sporulation process, yielding Bt endospores decorated with engineered A-ENA fibrils as described above. To assess this, Bti and Btk were genetically modified with a plasmid harboring an expression construct for A-ENA:SpyTag, under the regulation of the native A-ENA promoter and terminator (SEQ. ID NOs: 45 and 46, resp.). Transformed cells show the expression of A-ENA fibrils with morphological features indistinguishable from wild-type (WT) A-ENA fibrils, similar to what was observed for in vitro grown A-ENA-SpyTag nanofibrils (Figure 14). Subsequently, isolated endospores derived from these transformed cells were exposed to purified SpyCatcher-sfGFP (approximately 240 pM, ~5 mg / mL) for 30 minutes, followed by thorough PBS washing (four times). In parallel, we included WT Bti and Btk spores as a negative control. Upon completion of the wash steps, we observed fluorescent labeling exclusively on the surface of endospores expressing A-ENA:SpyTag, whereas endospores from WT Btk or Bti displayed no fluorescence.

[0337] These experiments show that similar to what was seen for in vitro A-ENA:SpyTag nanofibrils, A- ENA:SpyTag subunits get incorporated into the WT A-ENA nanofibrils in vivo, and are accessible for modification by SpyCatcher and SpyCatcher fusions. In this way, and by extension using the alternative labelling methodologies described above, A-ENA nanofibrils on Bt endospores can be functionalized in vivo. Using such approaches, Bt endospores can be functionalized with affinity reagents that contain specific binding properties for biological and non-biological surfaces, such as, in a non-limiting way, the gastrointestinal epithelium of specific insects and insect larvae, the surface of specific plants and crops. In such ways, functionalized Bt spores can be derived that have an increased tropism for specific insect groups, and / or have an increased affinity and retention time on desired crops or surfaces.

[0338] Example 10. Physicochemical stability of A-ENA fibrils.

[0339] To test the chemical stability of recombinant A-ENA fibrils, fibrils were harvested from a concentrated stock solution via centrifugation (20.000 ref, lh, supernatant discarded). The resulting pellets after centrifugation of 100 pl aliquots were resuspended and incubated in various conditions: 100 pl 2 % (w / v) SDS, 8 M urea, 2 M NaOH or 100 % (v / v) formic acid, prior to negative stain TEM sample preparation, as indicated in Figure 22. To test the physical robustness of recombinant A-ENA fibrils, a 100 pl aliquot of the fibril stock solution was desiccated at 200° C for 15 min in an open glass vial in an oven. The resulting dried material was rehydrated in 100 pl miliQ. water prior to nsTEM imaging. Additionally, a 100 pl aliquot was autoclaved for 20 min at 121°C. Next, 3 pl aliquots of the treated samples were deposited onto Cu- mesh formvar grids, washed twice with 20 pl miliQ. water and incubated for lmin with 2 % (w / v) uranyl acetate and blotted dry with Whatmann 2 filter paper. Micrographs were collected with a 120 kV JEOL 1400 microscope equipped with LaB6 filament and TVIPS F416 CCD camera operated at 60.000x magnification. As shown in Figure 22, A-ENA fibrils were intact and abundantly retained in each of these treatments wherein proteins typically denature or hydrolyse.

[0340] Example 11. A-ENA increases insecticidal activity of B. thuringiensis Sv. Kurstaki.

[0341] As shown in Example 6, A-ENA forms a virulence factor for the insecticidal activity of B. thuringiensis Sv. Israelensis, and as shown in Example 5 and 9 (Figures 19, 21), recombinant expression of A-ENA in Bt Sv. Kurstaki resulted in Bti-resembling sporesilk networks wherein bipyramidal PSB crystals are connected to A-ENA bundles. Next, recombinant expression of A-ENA was applied to test whether natural A-ENA presence in those Bt strains could result in a gain-of-function, potentially enhancing their virulence and thus their efficacy in pest control.

[0342] Trichoplusia ni, commonly known as the cabbage looper, a highly destructive leaf feeder with a devastating impact on cruciferous crops such as cabbage and broccoli was used as an insect model , as it is a natural target of Btk. The above-described Btk strain expressing A-ENA on a plasmid (gain of function approach) was tested and compared in their survival of the larvae feeding on medium supplemented with spores of Btk WT or the Btk WT strain recombinantly expressing A-ENA (Btk+A-ENA). Although Bti naturally contains A-ENA, its PSBs are not effective against T. ni, thus serving as a negative control (Figure 23c). We used 1-week-old larvae and followed their survival over a week after adding a suspension of spore on their solid feeding media. Results clearly showed that ~50 % (15 / 28) of larvae were still alive on day four in the Btk WT strain, whereas this number dropped to ~10 % (23 / 26) for the BTK strain expressing A-ENA (Figure 23c). Building on the observation that the clustering activity of A- ENA enhances virulence, we investigated whether adding purified A-ENA fibrils (recombinantly produced in E. coli) to WT Btk spores. ns-EM showed that the addition of purified A-ENA mimics the aggregation of spore and PSBs, similar to what is seen for Btk recombinantly expressing A-ENA (Figure 19 and 23a). The results demonstrate that the addition of recombinant A-ENA fibrils effectively clusters Btk spores and PSBs (Figure 23a). We then repeated the killing assay with WT Btk spores and PSB suspension, with and without the addition of recombinant A-ENA fibrils. Consistent with our earlier findings, approximately 50 % (8 / 16) of larvae remained alive on day four in the WT Btk strain, whereas survival dropped to approximately 18% (3 / 16) when recombinant A-ENA fibers were present (Figure 23d). As a negative control, we included recombinant A-ENA, confirming that in themselves, the A-ENA fibrils do not exert a direct toxic effect. Example 12. A single isopeptide bond is sufficient for self-assembly of SDS-resistant A-ENA-like nanofibrils.

[0343] To determine the minimal number of isopeptide bonds needed for the formation of A-ENA-type fibrils with a high physicochemical stability (see Example 10), we selectively mutated the acid / base catalysts in the IPB triads that are required for the formation of the five different IPBs present in A-ENA (UniprotKB Q8KNV8; SEQ ID NO: 1). Based on the structure of the A-ENA nanofibrils as disclosed herein, it emerges that IPB3 may suffice to form an IPB network that cross-links all protomers and the two A-ENA protofibrils f and f' into a single covalently linked A-ENA nanofibril molecule (Figure 25 a, b). Any of the other IPBs observed in the wild-type A-ENA nanofibril, as a single IPB or in combination with one or more IPBs other than IPB3 will result in the cross-linking of A-ENA subunits within, but not across the A-ENA protofibrils. Accordingly, we modified A-ENA (UniprotKB Q8KNV8; SEQ ID NO 1) to mutate residues E28, E39 and E41 to the similar, but catalytically inactive residue Gin, in essence resulting in the inactivation of, respectively IPB4 and 5, IPB2 and IPB1 (see Figure 5b). To do so, the coding sequence for A-ENA in the pASK_A-ENA plasmid was mutated by directed PCR-based mutagenesis, resulting in plasmid pASK_E- ENA E28Q_E39Q_E41Q. Alphafold 3 predicted the mutant protein to form protein fibrils near isomorphous to those of A-ENA wild type (Figure 25c). To evaluate the prediction, the A_ENA_E28Q_E39Q_E41Q mutant (SEQ ID NO:68) was produced by recombinant expression in E. coli C43 transformed with pASK_E-ENA_ E28Q_E39Q_E41Q and induced for at least 1 hour by the addition of 50 pM anhydrotetracycline at an ODgoo of 0.8. Cell pellets were processed as described above for A- ENA, and negative stain EM inspection of the resulting pellets demonstrated the presence of SDS- resistant nanofibrils with a diameter and morphology equivalent to that of wildtype A-ENA fibrils (Figure 25d).

[0344] Example 13. General flow and processes used in the production, processing and storage of ENA nanofibrils.

[0345] A-ENA nanofibrils are typically produced by biological fermentation through recombinant expression in a bacterial (preferred), yeast, plant, or animal host. To do so, A-ENA protein subunits are expressed from one or more plasmid-borne or genomically inserted copies of an A-ENA coding sequence, with expression guided to the host cytoplasm or export apparatus (i.e. general secretory (SEC) or twin arginine (TAT) pathways), where subunits self-assemble into robust nanofibrils of nano- to micrometer scale length (Figure 26 I.). A-ENA nanofibrils are harvested from the culture by mild lysis of the cells. Cells can be lysed in situ in the culture medium or in concentrated suspensions after sedimentation or centrifugation and resuspension in the desired buffer. Methods for mild cell lysis include, in a non-exhaustive way, spontaneous or induced autolysis (for example by co-expression of an endolysin, lysozyme, or other cell wall degrading proteins known in the art), suspension in hypotonic buffers, freeze-thaw cycles, mild sonication, detergent treatment, as well as EDTA - lysozyme treatment (Figure 26 IL). Cell lysates are then cleared from cell debris and other insoluble contaminants via methods known in the art, including, in a non-exhaustive manner, sedimentation, low speed centrifugation (i.e. 2.000 - 10.000 g), cross-flow filtration as well as enzymatic treatment (i.e. using proteases, glycosyl hydrolases, nucleases, cellulases, esterases etc.) (Figure 26 III.). Suspended fibrils are further purified from soluble and smaller suspended contaminants by means of detergent (i.e. SDS, LDS, DDM, tergitol, Tween™, Triton™, Elugent, among others) and / or chemical washes (i.e. NaOH, NH4OH, ureum, formic acid, alcohols and / or organic solvents) and / or enzymatic degradation (i.e. using proteases, glycosyl hydrolases, nucleases, cellulases, esterases, among others) (Figure 26 IV(a)). Fibrils are separated from soluble or small suspended contaminants sedimentation (including chemically induced precipitation) or higher speeds centrifugation (typically higher than 10.000 g), cross-flow or dead-end centrifugation (Figure 26 I V(b)). The desired A-ENA fibril purity is obtained by consecutive wash and separation steps as described above. Finally, purified A-ENA fibrils are collected and / or stored via desiccation and / or freeze drying (i.e. resulting in dry pellets, flakes, films or powders), or can be stored frozen, cooled or at ambient temperature, as suspension in pure water, aqueous buffers and / or acids, alkali, or salt solutions, as well as in organic solvents, or combinations thereof (Figure 26 V).

[0346] Table 1: HMM profile for Helix 1 (Hl)

[0347] HMMER3 / f [3.4 | Aug 2023]

[0348] NAME A_ENA_blast-580_currated_Hl LENG 35 ALPH amino RF no MM no CONS yes CS no MAP yes DATE Tue Aug 29 11 : 22 : 01 2023 NSEQ 594 EFFN 7.708694 CKSUM 134923421 -7.0325 0.71967 -7.4623 0.71967 -4.4263 0.71967 C D E F G H M N P Q R W Y m->i m->d i->m i->i d->m d- COMPO 2.00044 4.98713 3.54149 2.15203 4.08727 3.04489

[0349] 3.48939 2.21903 2.98377 1.99882 3.64094 3.12875 4.77844

[0350] 3.11407 3.91870 2.62243 3.27214 2.87075 6.02319 4.54348

[0351] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0352] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0353] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0354] 0.00485 5.72794 6.45028 0.61958 0.77255 0.00000

[0355] *

[0356] 1 3.05344 3.17800 2.14452 1.39477 4.74498 3.12482

[0357] 3.45791 4.34023 2.25019 4.06134 4.79603 2.75581 3.96873

[0358] 2.92236 3.01581 2.77339 3.27013 4.04636 6.18772 4.35641 1 e - - -

[0359] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0360] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0361] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0362] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0363] 0.95510

[0364] 2 3.06603 4.12392 1.85139 1.51570 5.10147 3.50821

[0365] 3.78857 4.58797 2.79878 4.06126 4.26842 2.97234 4.41013

[0366] 1.72153 3.03865 3.02828 3.41355 4.03103 6.18791 4.77649 2 e - - -

[0367] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0368] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0369] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0370] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0371] 0.95510

[0372] 3 1.27020 4.84209 5.06091 4.48430 4.00148 4.10089

[0373] 4.90247 2.64704 4.33831 3.08201 3.82029 4.58887 4.88374

[0374] 4.52963 4.46211 2.55547 2.06728 1.43361 5.52344 4.33929 3 a - - -

[0375] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0376] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0377] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0378] 0.95510 4 2.62500 4.55659 5.29022 4.67550 3.44110 4.01612 4.83675 1.04055 4.03609 1.82034 3.42960 4.41270 4.86734 4.59513 3.90074 3.74477 3.42879 1.92067 5.33303 3.82271 4 i - - -

[0379] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0380] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0381] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0382] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0383] 0.95510 5 2.84095 5.57273 3.26975 2.94396 4.67448 3.92240 3.15918 2.88829 2.85158 3.27483 4.12235 1.30381 3.47252 3.38139 3.44930 2.40373 2.32414 3.25767 6.05036 4.53425 5 n - - -

[0384] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0385] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0386] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0387] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0388] 0.95510 6 3.03101 5.09856 1.95526 3.18603 3.82897 4.23211 4.27820 3.13149 3.43872 1.13008 2.89554 3.35569 4.61409 3.18238 3.67053 2.97695 3.43919 3.09340 5.66793 4.19837 6 1 -

[0389] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0390] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0391] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0392] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0393] 0.95510 7 4.79637 5.95345 6.85863 6.31681 4.32209 6.35069 6.75983 1.57505 6.22590 0.45970 3.62101 6.53971 6.31677 6.06154 6.16510 5.78967 4.34983 2.59505 6.63913 5.71156 7 L -

[0394] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0395] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0396] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0397] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0398] 0.95510 8 4.50688 6.20733 7.08770 6.50107 3.56191 6.59414 6.82702 1.93560 6.39706 0.32427 3.35228 6.79658 6.40151 6.04322 6.23074 6.04560 5.26142 3.49677 6.54969 5.70932 8 L -

[0399] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0400] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0401] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0402] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0403] 0.95510 9 1.40031 5.63796 3.45730 1.97206 4.93571 3.95328 4.08493 3.76425 2.91699 3.50102 4.27684 3.47974 4.43473 2.69510 3.47513 2.02173 2.41536 3.54845 6.10006 3.87478 9 a - - -

[0404] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0405] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0406] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0407] 0.95510 10 3.11154 4.22374 6.05365 6.03515 6.33062 3.82717 6.46755 5.84141 5.88188 5.54137 6.26254 5.16914 5.10019 5.92737 5.81661 0.17974 3.29432 4.86142 7.67290 6.69127 10 S -

[0408] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0409] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0410] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0411] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0412] 0.95510 11 3.56802 5.53956 6.53252 6.09030 5.03805 6.06536 6.92108 0.36229 6.06367 3.30933 4.04763 6.24215 6.26900 6.34958 6.24607 5.52179 4.02721 1.77636 7.20449 5.95632 11 I -

[0413] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0414] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0415] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0416] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0417] 0.95510 12 0.20346 5.43786 5.77833 5.81690 6.38029 2.50560 6.45108 5.90860 5.86447 5.60164 6.32120 5.13648 5.11411 5.89751 5.82784 3.35670 4.12004 4.90494 7.71266 6.72269 A -

[0418] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0419] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0420] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0421] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0422] 0.95510 13 3.16691 4.72119 5.21856 4.60847 2.76107 4.01118 4.59520 2.79196 3.98920 0.92183 1.74969 4.63058 4.85897 3.57582 4.17308 3.67164 3.56618 2.92382 5.34158 4.04032 13

[0423] 1 -

[0424] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0425] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0426] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0427] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0428] 0.95510 14 3.50095 6.12883 3.23955 0.49556 5.45675 4.19060 4.49810 4.95080 3.15284 4.42441 4.55703 3.38672 4.66869 2.60376 3.81050 3.38341 3.83862 4.25096 6.55835 5.12222 E -

[0429] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0430] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0431] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0432] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0433] 0.95510 15 3.33404 6.80987 3.11186 0.33119 6.10577 3.66794 4.91764 5.65213 3.17635 5.09529 5.93362 3.82657 5.00689 3.44095 4.51619 3.99563 4.41897 5.18679 7.23079 5.69261 15 E -

[0434] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0435] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0436] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0437] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0438] 0.95510 16 2.54955 4.71642 5.28636 4.67256 3.58090 4.22241 4.84123 1.53629 4.46151 1.28344 2.35694 4.32640 4.87142 4.59608 4.47597 3.56973 2.34456 2.76702 5.33977 4.16344 16 1 -

[0439] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0440] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0441] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0442] 0.95510 17 0.89999 5.44070 5.37954 5.16461 6.14896 1.13017 6.04289 5.64690 5.14424 5.30522 6.05115 4.92611 5.05819 5.36438 3.72137 1.80583 4.06618 4.79026 7.45416 6.35826 17 a - - -

[0443] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0444] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0445] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0446] 0.95510 18 3.73035 5.60241 6.27919 5.67423 3.81284 5.59207 4.85298 2.52336 5.49568 0.31666 3.28503 5.75922 5.76762 5.45381 5.45207 4.93964 4.53289 3.17675 6.09079 5.05936 18 L -

[0447] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0448] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0449] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0450] 0.95510 19 0.74027 5.40864 5.90511 5.82467 6.26370 3.87773 6.37606 5.75823 5.72555 5.45924 6.19341 5.12335 5.09188 5.80735 5.72625 0.88068 3.75553 4.03311 7.60750 6.59630 19 a - - -

[0451] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0452] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0453] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0454] 0.95510 20 3.56346 6.91948 2.95287 2.92807 6.19556 4.41445 0.37951 5.74922 4.07012 5.20366 6.07177 2.92291 5.06838 3.84668 4.70112 3.55283 4.53657 5.29137 7.35382 5.79351 20 H -

[0455] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0456] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0457] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0458] 0.95510 21 4.46689 5.66371 6.55732 6.03712 3.99411 6.00306 6.52104 0.49501 5.94140 1.73234 3.48208 6.17836 6.13588 5.97842 5.97190 5.41407 4.15431 2.25794 6.65116 5.58911 21 I -

[0459] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0460] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0461] 0.95510

[0462] 22 4.17137 5.43853 6.19981 4.76516 4.28282 5.52895 5.93354 1.02756 5.47319 1.07576 2.33002 5.69848 5.75970 5.53565 5.48219 4.88763 4.11606 2.27827 6.22478 5.12539 i - - -

[0463] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0464] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0465] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0466] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0467] 0.95510

[0468] 23 3.86762 6.20204 3.76516 3.32640 5.53420 4.44414 3.95859 4.63643 2.72849 4.46141 5.28592 0.41601 4.89726 3.76589 3.42336 3.48281 4.08562 4.61529 6.55825 4.62194 23 N -

[0469] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0470] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0471] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0472] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0473] 0.95510

[0474] 24 0.21449 5.39780 5.93606 5.85691 6.17234 3.49717 6.36327 5.63550 5.74131 5.35656 6.11198 5.13059 5.09220 5.81553 5.72557 3.00967 3.37977 3.45669 7.53647 6.51551 A -

[0475] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0476] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0477] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0478] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0479] 0.95510

[0480] 25 4.39822 7.20334 2.95445 0.28616 6.45325 3.88075 5.10091 6.05106 3.84186 5.46632 6.37553 3.88628 5.14458 2.78964 4.94328 4.20783 4.71297 5.56040 7.60534 5.97615 25 E -

[0481] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0482] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0483] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0484] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0485] 0.95510

[0486] 26 0.98390 5.43495 5.77243 5.80558 6.38317 0.66669 6.44618 5.91346 5.85886 5.60301 6.31994 5.13106 5.11039 5.88911 5.82665 3.10318 4.11586 4.90457 7.71500 6.72509 G -

[0487] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0488] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0489] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0490] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0491] 0.95510

[0492] 27 4.35103 7.03947 3.13050 0.25409 6.34792 4.48020 5.02637 5.88707 3.58459 5.29324 6.18261 3.91896 5.13247 3.72725 3.60742 4.18454 4.63819 5.42906 7.39500 5.87596 27 E -

[0493] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0494] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0495] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0496] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0497] 0.95510 28 3.51585 6.34628 4.26383 3.44061 5.85363 4.66285 4.64700 5.20715 0.35301 4.59072 5.42590 3.91698 5.04639 3.17694 3.12883 3.80684 3.50309 4.83950 6.60414 5.40083 28 K -

[0498] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0499] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0500] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0501] 0.95510 29 3.67440 5.31841 6.06401 5.49213 3.84643 5.37006 5.77721 0.73622 5.32714 1.46179 2.90248 5.53883 5.63748 5.42269 5.34513 4.72037 3.51770 2.30738 6.12973 4.99968 29 i - - -

[0502] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0503] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0504] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0505] 0.95510 30 3.85492 6.36294 3.11792 2.71878 5.73220 4.36543 3.53762 5.20048 2.99404 4.63409 5.43089 3.82096 4.85651 0.45908 3.31068 3.77615 4.08827 4.77399 6.72357 5.33785 30 Q -

[0506] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0507] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0508] 0.01081 5.72794 4.89346 0.61958 0.77255 0.48576

[0509] 0.95510 31 1.70045 3.99413 3.73010 2.95151 2.64869 3.79149 3.15641 3.77849 2.08857 3.07808 4.03336 3.71989 4.52603 3.25840 2.54639 3.02191 3.24696 3.50043 4.50404 2.34725 31 a - - -

[0510] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0511] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0512] 0.00487 5.72200 6.44435 0.61958 0.77255 0.46510

[0513] 0.98906 32 1.76258 4.71250 5.30195 4.68684 2.14557 3.75355 4.84205 1.97041 4.47190 2.60656 3.21974 4.67049 4.87158 4.60325 4.48024 3.55134 3.32445 1.35572 5.33568 3.62393 32 v - - -

[0514] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0515] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0516] 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0517] 0.95510 33 2.72280 4.72418 5.30686 4.69318 3.72689 4.20848 4.85703 1.93030 4.26358 1.21461 3.48331 4.68162 4.88474 4.61431 4.32496 3.67542 2.90478 1.43659 5.35254 4.17615 33 1 -

[0518] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0519] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.00485 5.72794 6.45028 0.61958 0.77255 0.48576

[0520] 0.95510

[0521] 34 2.36259 4.61144 2.75487 2.47312 4.77259 1.43857

[0522] 3.88885 4.13036 2.00610 3.64947 4.79171 3.02482 4.41051

[0523] 2.81735 3.15967 2.78587 3.26293 3.75158 6.18413 4.77388 34 g -

[0524] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0525] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0526] 0.04016 5.72794 3.32124 0.61958 0.77255 0.48576

[0527] 0.95510

[0528] 35 2.61301 4.49784 2.80928 2.33002 4.34197 3.39358

[0529] 3.56375 4.18974 2.27222 3.12708 3.00716 3.33474 3.92297

[0530] 2.94481 3.00924 2.72821 1.57260 3.76750 4.82275 4.58273 35 t -

[0531] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0532] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0533] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0534] 0.00338 5.69116 * 0.61958 0.77255 0.00000

[0535] *

[0536] Table 2: HMM profile for Helix 2 (H2)

[0537] HMMER3 / f [3.4 | Aug 2023]

[0538] NAME A_ENA_blast-580_currated_H2 LENG 35 ALPH amino RF no MM no CONS yes CS no MAP yes DATE Tue Aug 29 11 : 38 : 15 2023 NSEQ 593 EFFN 593.000000 CKSUM 1270651096 -7.1671 0.72025 -7.4471 0.72025 -4.2809 0.72025 C D E F G H M N P Q R W Y m->i m->d i->m i->i d->m d- COMPO 3.14619 4.74729 2.88211 2.39766 3.45708 4.46199

[0539] 4.90632 2.47825 2.38261 1.73313 3.09926 2.72192 5.34805

[0540] 2.93929 3.37226 2.89058 3.06940 2.45561 5.72114 5.50002

[0541] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0542] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0543] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0544] 0.02336 9.96932 3.77021 0.61958 0.77255 0.00000

[0545] *

[0546] 1 3.16097 4.94151 9.14150 5.55778 2.70538 5.50944

[0547] 5.03908 1.60265 5.08501 1.36975 3.29123 2.39331 2.49634

[0548] 4.59539 8.31012 4.01766 2.84357 2.28054 6.05767 5.22740 1

[0549] L -

[0550] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0551] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0552] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0553] 0.00090 9.94603 7.06450 0.61958 0.77255 0.84487

[0554] 0.56144

[0555] 2 3.23772 3.19518 1.77982 1.48103 5.30818 3.68207

[0556] 3.51930 8.30661 2.22313 4.93790 5.86750 2.86042 4.21679

[0557] 3.13807 2.78044 2.13355 3.84849 4.35119 9.90502 4.17212 2 e - - -

[0558] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0559] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0560] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0561] 0.00007 9.95481 10.67716 0.61958 0.77255 0.05785 2.87866

[0562] 3 4.40136 9.48484 0.97921 1.36060 4.64513 4.17368

[0563] 4.88061 4.61397 4.20457 4.24536 5.45340 2.72244 8.14115

[0564] 2.02412 7.16220 3.01369 4.00018 4.34772 9.91746 8.50644 3 d -

[0565] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0566] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0567] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0. 00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0568] 0.95510 4 2.72818 5.43895 9.24500 8.63047 3.60001 8.44157 8.77849 1.58253 8.41410 0.68718 3.09435 8.60939 8.79977 8.53951 5.47237 7.75525 5.10314 2.21821 9.25983 3.56707 4 L -

[0569] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0570] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0571] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0572] 0.95510 5 4.79423 4.75486 9.16671 3.28788 3.19868 5.14895 8.69256 1.81327 8.33148 0.57446 2.85246 3.47674 8.72177 3.79141 8.33402 4.78865 4.63706 3.36687 5.91102 5.16393 5 L -

[0573] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0574] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0575] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0576] 0.95510 6 1.92076 4.48935 2.65318 1.75789 5.85244 4.82519 5.70302 5.49719 1.54925 2.92867 4.13281 3.24892 8.14066 2.45719 2.64824 3.32505 3.08281 4.04399 9.91865 5.45840 6 k -

[0577] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0578] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0579] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0580] 0.95510 7 3.36930 8.57814 9.18851 8.57310 2.14188 8.38190 8.71819 1.24496 8.35535 2.22171 3.30538 4.64980 8.74454 4.40490 8.35860 5.00605 2.82087 1.12353 9.20658 8.03076 7 v - - -

[0581] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0582] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0583] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0584] 0.95510 8 8.70735 4.29939 3.40530 4.56727 10.85268 4.90271 5.34186 10.51347 8.69286 3.38461 10.89650 0.25151 9.36218 2.29824 9.54834 4.76809 4.65271 9.98551 12.05791 10.32119 8 N -

[0585] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0586] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0587] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0588] 0.95510 9 3.16918 3.27146 1.95675 1.63546 8.83411 3.51192 3.98255 8.32108 1.48764 7.79351 8.52792 2.50269 8.14041 3.12705 2.39828 3.07472 3.28332 7.87902 9.91946 4.90140 9 k -

[0589] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0590] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0591] 0.95510

[0592] 10 2.97727 3.67770 4.35064 3.69391 8.83440 4.05452 4.76108 5.08954 4.85699 4.27762 8.52907 2.66140 8.14254 3.51898 7.16385 0.35791 3.45294 7.87962 9.92076 8.50921 10 S -

[0593] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0594] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0595] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0596] 0.95510

[0597] 11 2.98612 9.68291 10.78313 10.39961 9.32198 10.44931 11.68725 2.98321 10.45994 3.81613 4.16978 10.62219 10.58051 10.84894 10.74743 9.98084 2.92153 0.21428 11.78884 10.46524 11 V - -

[0598] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0599] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0600] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0601] 0.95510

[0602] 12 2.70786 5.16067 3.45148 1.49371 4.38880 3.76736 3.35895 3.19306 3.44455 4.10900 4.08527 2.08895 8.14063 2.31311 1.94226 2.62433 3.05974 4.73778 9.91872 8.50719 12 e - - -

[0603] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0604] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0605] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0606] 0.95510

[0607] 13 3.44987 4.43419 2.08330 2.72850 8.83403 5.27532 3.80509 5.67621 1.49846 3.87730 8.52784 2.53823 5.15235 2.60444 1.69230 2.21758 3.38350 5.23204 9.91939 8.50759 13 k -

[0608] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0609] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0610] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0611] 0.95510

[0612] 14 4.29985 8.63993 9.25941 8.64675 3.67149 8.45836 8.79929 2.53873 3.38452 2.25333 1.08411 8.62631 8.81701 8.56030 8.43632 4.04701 1.19078 2.50741 9.28264 8.10798 14 m - - -

[0613] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0614] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0615] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0616] 0.95510

[0617] 15 5.17113 9.80419 10.87392 3.38636 8.95509 10.51589 11.46971 1.97532 10.48112 0.51719 2.56512 10.69450 10.56868 10.64014 10.64424 10.03432 4.76817 1.96813 11.38747 10.27841 15 L - -

[0618] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0619] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0. 00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0620] 0.95510 16 3.69282 9.48755 2.11740 1.81266 8.83404 3.66649 4.47106 5.69381 2.14298 7.79344 3.31839 2.56894 8.14036 2.03501 1.47595 4.13019 3.74611 4.79706 9.91939 4.35237 16

[0621] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0622] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0623] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0624] 0.95510 17 1.78340 9.48576 2.49906 2.87318 8.83138 2.99656 7.93439 3.03677 2.98171 4.37183 3.84000 2.44102 8.14088 4.16307 3.82607 2.73549 1.29296 3.08767 9.91811 8.50683 17 t -

[0625] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0626] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0627] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0628] 0.95510 18 2.61154 8.85175 9.52428 8.93023 8.01418 8.75579 9.13288 1.22061 8.73262 2.15977 4.56261 8.92444 4.01807 8.87598 8.75165 5.39725 3.41486 0.82625 9.60828 4.50522 18 v - - -

[0629] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0630] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0631] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0632] 0.95510 19 2.47912 8.56072 9.15741 8.54156 4.04917 4.18595 8.69080 1.01371 4.25899 2.80864 3.75178 4.30772 8.72055 3.38512 5.15106 2.63639 1.94239 1.87472 9.18258 8.00678 19 i - - -

[0633] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0634] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0635] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0636] 0.95510 20 4.39509 5.82961 3.58784 3.53623 3.22433 4.66561 5.09033 4.49057 0.88052 3.23858 2.32019 2.81103 8.14205 2.63930 2.33197 3.17880 3.74508 5.17818 9.91523 4.66396 20 k -

[0637] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0638] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0639] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0640] 0.95510 21 7.00010 9.30252 7.34299 3.90681 3.78965 4.47570 4.91632 4.03070 0.79099 1.68034 4.04781 2.54188 8.20140 4.04434 3.90951 2.67534 2.76537 4.31784 9.78400 8.42482 21 k -

[0641] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0642] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0643] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0644] 0.95510

[0645] 22 8.76488 11.69520 4.70001 0.44427 10.90367 8.65054

[0646] 9.37248 10.54946 4.50469 3.38458 10.93152 8.07848 9.39121 1.21679 9.32804 8.52422 9.10580 4.93777 12.05314 10.34837 22

[0647] E -

[0648] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0649] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0650] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0651] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576 0.95510

[0652] 23 4.25138 5.12166 9.14345 3.07481 3.88819 4.31547

[0653] 5.32943 1.95046 3.94592 1.98644 1.01102 3.47468 8.71868

[0654] 4.72410 8.32531 3.69668 3.56514 3.47505 2.22957 8.00770 23 m - - -

[0655] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0656] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0657] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0658] 0.01075 9.96932 4.54267 0.61958 0.77255 0.48576

[0659] 0.95510

[0660] 24 3.45250 8.54922 9.15626 8.53977 3.87347 8.34770

[0661] 5.35806 1.79751 8.32101 0.59609 2.99303 3.20659 8.71124

[0662] 5.34930 8.32352 4.38842 3.34983 2.55612 9.17122 5.47757 24

[0663] L -

[0664] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0665] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0666] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0667] 0.00007 9.95864 10.68099 0.61958 0.77255 0.07521 2.62488

[0668] 25 4.71879 10.36066 11.22647 10.62830 4.82175 10.70343

[0669] 10.88598 3.07287 10.51461 0.09811 5.27374 10.91911 10.49623

[0670] 10.11867 10.31950 4.30984 5.12257 7.99505 10.59757 5.28859

[0671] 25 L -

[0672] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0673] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0674] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0675] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576 0.95510

[0676] 26 4.62291 9.47854 3.22741 2.82696 4.62303 4.79172

[0677] 2.97610 3.99878 2.26618 1.72110 5.32742 4.16890 8.14297

[0678] 0.84218 4.17202 4.11311 4.51784 3.83478 9.91295 5.20528 26 q -

[0679] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0680] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0681] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0682] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576 0.95510

[0683] 27 2.78903 4.09592 3.26544 2.61041 1.19029 3.68628

[0684] 4.90914 4.45068 3.08626 3.41478 2.64022 2.42167 8.14400

[0685] 3.73245 2.76211 2.25106 3.84277 5.34275 9.91045 5.44120 27 f -

[0686] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0687] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739

[0688] 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0689] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0690] 0.95510 28 9.04155 10.89006 4.54327 8.72215 10.72047 9.41261 8.95636 9.80696 0.10479 3.38298 9.96188 8.89771 9.66463 4.38793 3.71641 8.98943 4.07108 9.54966 10.88266 9.94223 28 K -

[0691] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0692] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0693] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0694] 0.95510 29 4.06336 8.65362 9.26559 3.38573 2.73082 8.46116 8.79439 3.24007 8.43247 0.31995 2.48292 8.62929 8.81608 5.07418 8.43237 4.89725 4.50103 4.78794 9.26869 8.10108 29 L -

[0695] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0696] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0697] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0698] 0.95510 30 3.02491 3.96033 1.98434 0.68912 8.83397 4.11756 5.52567 4.87985 2.85933 3.74170 8.52780 3.33068 8.14037 3.15289 3.81119 2.78459 4.37609 4.80709 9.91936 8.50757 30 E -

[0699] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0700] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0701] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0702] 0.95510 31 3.22134 3.96018 1.32472 2.90175 3.17489 5.23273 4.36651 3.91828 4.04418 2.24074 3.55303 1.81319 8.14813 3.28176 3.31999 2.84737 2.56476 4.42318 9.90041 5.55585 31 d -

[0703] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0704] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0705] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0706] 0.95510 32 2.05541 5.67944 8.91179 3.38480 2.65608 8.23656 8.52643 1.48454 4.91791 3.05990 5.02322 8.38345 8.55110 8.32586 8.21511 3.88914 3.15273 0.88719 8.92341 7.91908 32 v - - -

[0707] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0708] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0709] 0.00007 9.96932 10.69167 0.61958 0.77255 0.48576

[0710] 0.95510 33 3.12456 5.13806 3.84298 4.54699 3.50530 4.21952 8.33223 1.67060 2.72250 1.04878 2.89547 3.20062 5.45814 4.99721 3.80850 3.05947 3.70084 2.69176 9.40403 8.16941 33 1 -

[0711] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0712] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 0.00241 9.96932 6.04759 0.61958 0.77255 0.48576

[0713] 0.95510 34 3.06684 4.32309 1.53725 1.68199 3.07637 3.01392 5.54737 5.03930 2.55786 4.30005 6.08680 2.32579 3.28772 2.98598 2.79268 2.76080 3.78028 5.93369 9.43785 5.35837 d - - -

[0714] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0715] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0716] 0.00720 9.96698 4.94317 0.61958 0.77255 1.15360

[0717] 0.37907 35 2.50381 3.00062 3.41271 4.00930 2.31719 4.08475 4.87680 1.98673 5.25948 1.13004 2.99739 3.10059 8.59499 5.07520 3.77743 2.88847 4.65179 3.59363 9.25834 4.42657

[0718] 1 -

[0719] 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513

[0720] 3.72494 3.29354 2.67741 2.69355 4.24690 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503

[0721] 0.00005 9.95982 * 0.61958 0.77255 0.00000

[0722] *

[0723] Table 3: NCBI Reference Sequence accession numbers of A-ENA like proteins (n=593) >SFX69347.1; >WP_131849659.1; >WP_249075847.1; >WP_091176133.1; >WP_018885230.1;

[0724] >WP_238189196.1; >WP_238189194.1; >WP_041058496.1; >WP_133378365.1; >WP_147208463.1;

[0725] >NMA89768.1; >WP_036201651.1; >WP_121214240.1; >AIM 15264.1; >NEX80099.1;

[0726] >WP_126294795.1; >WP_071392824.1; >WP_151701241.1; >WP_210471316.1; >WP_040376149.1;

[0727] >WP_212924438.1; >WP_204668115.1; >WP_212976039.1; >WP_066186838.1; >WP_152657822.1;

[0728] >WP_040982409.1; >WP_188733585.1; >WP_066142189.1; >WP_115749335.1; >WP_020616168.1;

[0729] >WP_128658904.1; >WP_238650419.1; >WP_173140208.1; >WP_010268044.1; >WP_047980762.1;

[0730] >WP_263706280.1; >MBO2505129.1; >WP_016839178.1; >WP_141601888.1; >WP_208650703.1;

[0731] >WP_090774452.1; >WP_090776151.1; >PFB49380.1; >WP_257209516.1; >OQR53105.1;

[0732] >WP_068775901.1; >WP_142506947.1; >TQR39522.1; >WP_119150851.1; >WP_215175558.1;

[0733] >WP_215175557.1; >WP_211350104.1; >WP_175371047.1; >PGQ44283.1; >WP_181538938.1;

[0734] >WP_044896080.1; >MCL6586930.1; >WP_181538937.1; >WP_012957170.1; >KQU17358.1;

[0735] >KRF52451.1; >KRF52448.1; >KQU17360.1; >WP_066154797.1; >WP_155477750.1; >NLX02164.1;

[0736] >NLK52489.1; >NLV22477.1; >WP_134230475.1; >WP_134230476.1; >WP_224876227.1;

[0737] >WP_064094024.1; >WP_224876226.1; >WP_197315488.1; >WP_224809048.1; >WP_066265084.1;

[0738] >WP_270407384.1; >WP_134230544.1; >WP_069644379.1; >WP_069703205.1; >WP_069644377.1;

[0739] >WP_153725722.1; >NLF45454.1; >WP_221860360.1; >WP_019155143.1; >NLT40941.1;

[0740] >MBE3101226.1; >MBE3101227.1; >WP_235222005.1; >OQB14150.1; >MCL2703241.1;

[0741] >WP_242057465.1 / 6-7; >WP_019155144.1; >WP_080630428.1 / 4-4; >WP_183242236.1;

[0742] >WP_042536196.1 / 4-4; >KIP20212.1 / ll-ll; >HBR31256.1; >OON92172.1; >ONI43887.1; >HIS65799.1;

[0743] >HIU33750.1; >NLK86606.1; >WP_090446350.1; >QGU95879.1; >WP_072907480.1;

[0744] >MCL6617755.1 / 104-1; >WP_181520874.1; >WP_055441798.1; >AST05588.1; >WP_035048978.1;

[0745] >WP_184664076.1; >WP_184664075.1; >WP_110609538.1; >WP_093051849.1; >WP_106589654.1;

[0746] >WP_010239793.1; >MCK9536559.1; >WP_078665439.1; >NMB44602.1; >NU90181.1; >OQA15291.1;

[0747] >NLO10347.1; >WP_236914082.1; >WP_252225997.1; >WP_204489239.1; >HHU63352.1;

[0748] >WP_254495860.1; >HBI56858.1; >WP_008908033.1; >SEF39824.1; >WP_242971176.1;

[0749] >WP_122963797.1; >WP_122903756.1; >WP_007783061.1; >WP_122961155.1; >WP_122925796.1;

[0750] >WP_163859565.1; >WP_134757257.1; >WP_173140217.1; >WP_216857486.1; >WP_049741785.1;

[0751] >WP_088910016.1; >WP_106657270.1; >WP_172139574.1; >WP_137031666.1; >WP_016739500.1;

[0752] >WP_106784580.1; >WP_219661378.1; >WP_047070179.1; >TQR34900.1; >WP_087348525.1;

[0753] >WP_199929547.1; >WP_017246715.1; >WP_174226400.1; >WP_144618485.1; >WP_007720341.1;

[0754] >WP_056488977.1; >WP_197936189.1; >WP_010268048.1; >OAB30303.1; >WP_138224403.1;

[0755] >WP_091176138.1; >WP_130607412.1; >WP_087443534.1; >WP_127585513.1; >WP_161411873.1;

[0756] >WP_173215395.1; >WP_079412742.1; >WP_171414263.1; >WP_021254599.1; >WP_270170784.1;

[0757] >OBZ18017.1; >OBZ18016.1; >WP_028562891.1; >WP_209971550.1; >WP_028546586.1;

[0758] >WP_232275159.1; >WP_028595674.1; >WP_258279168.1; >WP_175373344.1; >WP_058303866.1;

[0759] >WP_048744837.1; >CDN41938.1 / 4-4; >WP_036651907.1; >WP_113029034.1; >MBD2860923.1;

[0760] >WP_223836348.1; >WP_144509072.1; >WP_098688323.1; >MBK5491851.1; >WP_252211459.1;

[0761] >PFI81502.1; >PGK33495.1; >WP_098650108.1; >WP_197722729.1; >WP_216636299.1 / 22-;

[0762] >WP_197478852.1 / 21-; >KXG08769.1; >WP_115749336.1; >WP_246020340.1; >WP_104059545.1;

[0763] >WP_151701235.1; >WP_212508844.1; >WP_174521724.1; >WP_191814960.1; >WP_244811852.1;

[0764] >WP_173660201.1; >WP_036198767.1; >WP_208650728.1; >WP_019155127.1; >WP_212924437.1;

[0765] >WP_040982410.1; >WP_229720117.1; >WP_235817440.1; >WP_244704997.1; >WP_244853042.1;

[0766] >WP_239587967.1; >SKB05896.1; >WP_244715222.1; >WP_185959608.1; >NMA90008.1;

[0767] >MBP1969341.1 / 5-5; >WP_209462530.1; >WP_257230192.1; >MBG0967840.1; >WP_236686903.1;

[0768] >WP_019244172.1; >ALP35341.1; >WP_238354028.1; >WP_044648569.1; >WP_212734477.1;

[0769] >WP_211556547.1; >SIS52057.1; >WP_234969477.1; >WP_245629781.1; >AEJ44700.1;

[0770] >WP_237700123.1; >ACV59513.1; >WP_245530800.1; >MCD8500378.1; >WP_071313875.1;

[0771] >WP_089023434.1; >WP_197315495.1; >WP_134230541.1; >WP_064094028.1; >WP_224876222.1;

[0772] >WP_217225283.1; >WP_246421570.1; >WP_242175596.1; >WP_274069240.1; >TQR39524.1;

[0773] >WP_255504946.1; >MCD8503411.1; >WP_166246166.1; >WP_131015779.1; >WP_270407379.1;

[0774] The unique accession code representative for an A-Ena-like protein sequence is provided herein between

[0775] Sequence Listing

[0776] >SEQ ID NO:1: Bacillus thuringiensis Sv. Israelensis strain ATCC35646 A-ENA amino acid sequence

[0777] (UniProt: Q8KNV8 )

[0778] >SEQ ID NO:2: Bacillus thuringiensis Sv. Israelensis strain ATCC35646 A-ENA-1 amino acid sequence (UniProt: Q8KNV7)

[0779] >SEQ ID NO: 3: A-ENA homologue UniprotKB A0A0R3K429

[0780] >SEQ ID NO: 4: A-ENA homologue UniprotKB A0A172TKT0

[0781] >SEQ ID NO: 5: A-ENA homologue UniprotKB A0A2X4WSQ5 > SEQ ID NO: 6: A-ENA homologue UniprotKB A0A0A3J768

[0782] > SEQ ID NO: 7: chimeric protein of Bacillus thuringiensis Sv Israelensis strain ATCC35646 A-ENA (SEQ ID NO:1) fused to SpyTag

[0783] >SEQ ID NO: 8: chimeric protein of Bti A-ENA (SEQ ID NO:1) fused to p66a

[0784] >SEQ ID NO: 9: chimeric protein of Bti A-ENA (SEQ ID NO:1) C-terminally fused to M BD2

[0785] >SEQ ID NO: 10: chimeric protein of Bti A-ENA (SEQ ID NO:1) fused to Rubredoxin Desulfovibrio vulgaris

[0786] >SEQ ID NO: 11: chimeric protein of anti-GFP Nanobody207 fused to truncated Bti A-ENA-ANTL

[0787] >SEQ ID NO: 12: A-ENA-ANTL

[0788] >SEQ ID NO:13: chimeric protein of Bacillus thuringiensis Sv Israelensis strain ATCC35646 A-ENA-1 (SEQ ID NO:2) fused to SpyTag

[0789] >SEQ ID NO: 14: chimeric protein of superfolder Green Fluorescent Protein fused to SpyCatcher003

[0790] >SEQ ID NO: 15: A-ENA mutant variant (E29A Q44A N64A E78A Q82A) with C-terminal 7x His-tag

[0791] >SEQ ID NO:16: FW primer setl

[0792] >SEQ ID NO:17: REV primer setl

[0793] >SEQ ID NO:18: FW primer set2

[0794] >SEQ ID NO:19: REV primer set2

[0795] >SEQ ID NO:20: pASK forward sequencing primer

[0796] >SEQ ID NO:21: pASK reverse sequencing primer

[0797] >SEQ ID NO:22: sgRNA

[0798] >SEQ ID NO:23: SpyTag amino acid sequence

[0799] >SEQ ID NO:24: Heptad Hl-1 Bti A-ENA

[0800] >SEQ ID NO:25: Heptad Hl-2 Bti A-ENA

[0801] >SEQ ID NO:26: Heptad Hl-3 Bti A-ENA

[0802] >SEQ ID NO:27: Heptad Hl-4 Bti A-ENA

[0803] >SEQ ID NO:28: Heptad Hl-5 Bti A-ENA

[0804] >SEQ ID NO:29: Heptad H2-1 Bti A-ENA

[0805] >SEQ ID NO:30: Heptad H2-2 Bti A-ENA >SEQ ID N0:31: Heptad H2-3 Bti A-ENA

[0806] >SEQ ID NO:32: Heptad H2-4 Bti A-ENA

[0807] >SEQ ID NO:33: Heptad H2-5 Bti A-ENA

[0808] >SEQ ID NO:34: Heptad Hl-1 Bti A-ENA-1

[0809] >SEQ ID NO:35: Heptad Hl-2 Bti A-ENA-1

[0810] >SEQ ID NO:36: Heptad Hl-3 Bti A-ENA-1

[0811] >SEQ ID NO:37: Heptad Hl-4 Bti A-ENA-1

[0812] >SEQ ID NO:38: Heptad Hl-5 Bti A-ENA-1

[0813] >SEQ ID NO:39: Heptad H2-1 Bti A-ENA-1

[0814] >SEQ ID NO:40: Heptad H2-2 Bti A-ENA-1

[0815] >SEQ ID NO:41: Heptad H2-3 Bti A-ENA-1

[0816] >SEQ ID NO:42: Heptad H2-4 Bti A-ENA-1

[0817] >SEQ ID NO:43: Heptad H2-5 Bti A-ENA-1

[0818] >SEQ ID NO:44: NTL of Bti A-ENA

[0819] >SEQ ID NO:45: Bacillus thuringiensis Sv. Israelensis strain ATCC35646 A-ENA promoter

[0820] >SEQ ID NO:46: Bacillus thuringiensis Sv. Israelensis strain ATCC35646 A-ENA terminator

[0821] >SEQ ID NO:47-65: oligo primer sequences used in Example 4.

[0822] >SEQ ID NO: 66: two curlin-like repeats (with calcium and calcium carbonate binding properties)

[0823] >SEQ ID NO: 67: A-ENAJJ-R4.5-2RFD (= SEQ ID NO:66 inserted between residues 55 and 56 of A-ENA (SEQ ID NO 1), flanked by GG and SG linkers at N- and C-terminal side of the insertion sequence)

[0824] >SEQ ID NO: 68: A_ENA_E28Q_E39Q_E41Q (=A-ENA SEQ ID NO:1 with Glu28, Glu39 and Glu41 substituted by Gin, as indicated in bold; K76 and Q82 for forming IPB3 with further subunits are underlined)

[0825] MGMPTIPEGLDITRDQAINIILASIGLQELGLAHVINAQGQKVQAVVAGFEKETVTFDQLLATNESVTQTLKTVIKKEM LLQFKLEEAKSLIQSSSPPSIS

[0826] >SEQ ID NOs:69-70: primers

Claims

CLAIMS1. An isolated protein nanofibril comprising two protofibrils each comprising at least two monomer protein subunits, wherein each monomer protein subunit comprises covalently connected amino acid sequence fragments according to the formula: NTL - helix 1- L - helix 2 - CT, wherein said monomer protein subunit spontaneously folds in aqueous solution into two helices, helix 1 and helix 2, as an alpha-helical antiparallel coiled coil structure, and wherein helix 1 and helix 2 each comprise a continuous sequence of at least 5 heptad (H) elements according to the formula Hl-1 - Hl-2 - Hl-3 - Hl-4 - Hl-5 for helix 1, and H2-1 - H2-2 - H2-3 - H2-4 - H2-5 for helix 2, wherein said heptad elements each comprise seven amino acid residues designated 'abcdefg' with the following consensus sequences:wherein:Φ is a hydrophobic amino acid selected form the list of M, V, I, L, A, G, H, W, Y, F, for at least 70 % of said <t> positions indicated in said heptad elements; tp is a short side chain residue, selected from the list of V, C, G, A, P, S, T, N, D; y is a residue that serves as acid / base catalyst selected from E, or D, , for at least one or more of said y positions indicated in said heptad elements;6 is a Lysine (K) residue capable to serve as isopeptide bond 'donor' or nucleophile, for at least one or more of said 6 positions indicated in said heptad elements; s is a residue capable to serve as isopeptide bond 'acceptor' or electrophile selected from E, Q, D, N, for at least one or more of said s positions indicated in said heptad elements;X can be any amino acid,and wherein the linker (L) fragment comprises at least 4 amino acids, and N-terminal lock (NTL) and C-terminal tail (CT) fragments comprise at least 1 amino acid, and wherein said monomeric protein subunits are interconnected through at least one or more isopeptidic bonds (IPBs).

2. The protein nanofibril of claim 1, wherein at least one IPB covalently interconnects the two protofibrils (f) and (f').

3. The protein nanofibril of claim 2, wherein said at least one IPB covalently interconnects the (f) and (f') protofibrils through the monomeric protein subunits (i1) of protofibril (f') and the monomer subunits (i and / or i + 1) of protofibril (f) between the side chain of the amino acid acting as a nucleophile and the side chain of the amino acid acting as electrophile, respectively, at positions:H2-3f of (i') and H2-4e of (i); and / orH2-3f of (i+1) and H2-4e of (i').

4. The protein nanofibril of claims 1 to 3, wherein the monomeric protein subunits (i and i + / - n) of protofibril (f) and the monomer subunits (i' and i' + / - n) of protofibril (f') are covalently connected through at least one or more IPBs, wherein the IPBs are formed between the side chain of the amino acid acting as a nucleophile and the side chain of the amino acid acting as electrophile, respectively, at positions:NTL of (i) and Hl-5b of (i-5), (i-4), (i-3), or (i-2);Hl-4g of (i) and H2-2a of (i-1);H2-3g of (i) and H2-4a of (i-1);H2-3f of (i) and H2-4e of (i'-l);H2-4g of (i) and Hl-3a of (i-1);H2-4g of (i+l)and Hl-3a of (i);NTL of (i+5), (i+4), (i+3), or (i+2) and Hl-5b of (i);Hl-4g of (i+1) and H2-2a of (i);H2-3g of (i+1) and H2-4a of (i); and / orH2-3f of (i') and H2-4e of (i).

5. The protein nanofibril of claims 1 to 4, wherein said Heptad elements have the following consensus sequences:

6. The protein nanofibril of any one of claims 1 to 5, wherein the NTL comprises a consensus sequence: M-Ψ -Φ-Z-X---P, wherein:Ψ is a short side chain residue, selected from the list of V, C, G, A, P, S, T, N, D;Φ is a hydrophobic amino acid, selected from the list of M, V, I, L, A, G, H, W, Y, FX is any amino acid,Z is Ala or Proline, andM and P are one letter code for the respective amino acids Methionine and Proline.

7. The protein nanofibril of any one of claims 1 to 6, wherein the monomer is an A-ENA protein selected from the list of proteins depicted in Table 3, and / or consists of an amino acid sequence selected from the list of SEQ. ID NO: 1-6 or a functional homologue or variant of any one thereof.

8. The protein nanofibril of any one of claims 1 to 7, wherein the NTL of the monomer protein subunit is less than 4 amino acids.

9. The protein nanofibril of any one of claims 1 to 7, wherein the monomeric protein subunit comprises: a. an NTL of at least 4 amino acids and the monomeric protein is a mutant variant in at least one or more of the following positions: Hl-la, Hl-lb, Hl-lc, Hl-3f, Hl-4f, Hl-5b, Hl-5f, H2-lb, H2-1C, and / or H2-5b; or b. the NTL is less than 4 amino acids and the monomeric protein is a mutant variant in at least one or more of the following positions: Hl-la, Hl-lb, Hl-lc, Hl-lf, Hl-2f, Hl-3b, Hl-3f, Hl-4b, Hl-4f, Hl-5b, Hl-5f, H2-lb, H2-lc, H2-2b, H2-2f, H2-3f, H2-4f, H2-5b, H2- 5c and / or H2-5f.

10. The protein nanofibril of any one of claims 1 to 9, wherein the monomeric protein further comprises a protein tag or domain which is fused or conjugated to the N-terminus, C-terminus or within the Linker region for forming a functionalized fibril.

11. The protein nanofibril of any one of claims 1 to 10, comprising identical monomer proteins forming a homopolymeric fibril upon self-assembly, or comprising at least two different monomeric proteins forming a heteropolymeric fibril upon self-assembly.

12. The protein nanofibril of any one of claims 1 to 11, wherein said fibril is a recombinantly produced protein nanofibril.

13. A modified bacterial endospore, preferably a Bacillus endospore, which comprises and / or displays the protein nanofibril of any one of claims 8 to 12.

14. A modified bacterial endospore, preferably a Bacillus endospore, wherein the bacterial strain lacks an endogenous monomeric protein of the protein nanofibril of claims 1 to 7, wherein a selfassembling monomeric protein forming the protein nanofibril of any one of claims 1 to 12 is exogenously introduced.

15. Use of the modified bacterial endospore, preferably the Bacillus endospore, of claims 13 or 14, for increasing bacterial spore activity, preferably pathogenic activity.

16. Use of the protein nanofibril of any one of claims 1 to 12, to enhance pesticidal activity of a bacterial endospore, preferably insecticidal activity of a B. thuringiensis endospore.

17. A host cell recombinantly expressing the self-assembling monomeric protein forming the protein nanofibril of any one of claim 1 to 12.

18. A method for producing the protein nanofibril of any one of claims 1 to 12, comprising the steps of: a. culturing the host cell of claim 17, b. releasing the self-assembled protein nanofibrils from the host cell, preferably through cell lysis, and c. isolation of the self-assembled protein nanofibrils, preferably through resuspension from the insoluble fraction and / or further purification from the cell lysate.

19. The method for producing the protein nanofibril of any one of claims 1 to 12, according to claim 18, wherein following the cell lysis of step b) and prior to the isolation in step c), the lysate is cleared from cell debris and other contaminants, preferably through sedimentation, centrifugation, crossflow filtration and / or enzyme treatment.

20. The method for producing the protein nanofibril of any one of claims 1 to 12, according to claim 18 or 19, wherein the releasing of the self-assembled protein nanofibrils in step b) is performed through cell lysis using a detergent, preferably SDS, DDM, Triton, Tergitol or Tween.

Images