Antibacterial agents

Abstract

The present invention relates to antibacterial protein complexes derived from nuclease bacteriocins and their production and use, and to related products having antibacterial surfaces. The complexes comprise a nuclease bacteriocin polypeptide and an immune polypeptide linked to a bacterial binding moiety.

Description

Technical Field

[0001] The present invention relates to a modified complex of a bacterial nuclease and an immune polypeptide and its use as an antibacterial agent. The present invention also relates to a pharmaceutical composition and medical use of the antibacterial complex and a method for producing the same. The present invention also relates to a product having an antibacterial surface containing a bacterial nuclease-immune protein complex and a method for producing the same. Background Art

[0002] The widespread emergence of antibiotic-resistant bacteria and the slowdown in the discovery of new classes of antibiotics are considered serious public health issues. Overuse of antibiotics over a long period of time has made infectious organisms resistant to antibiotics. As many antibiotics have lost their effectiveness, there is increasing pressure to identify new types of antibacterial molecules. Infections caused by Gram-negative bacteria are particularly worrying. Gram-negative pathogens have an impermeable outer membrane that prevents many classes of antibiotics from entering. However, some antibacterial agents can pass through the outer membrane through porins (e.g., OmpF and OmpC) present on the surface of bacteria.

[0003] Protein bacteriocin (PB) is a class of narrow-spectrum antimicrobial peptides produced by Gram-negative bacteria that target closely related bacteria. One class of PBs is the nuclease bacteriocin. These typically contain three domains - a central receptor binding domain, an N-terminal domain that interacts with the outer membrane protein translocator in the target bacterium, and a C-terminal cytotoxic domain / nuclease domain that must be translocated into the target bacterium. Bacteriocins are produced as heterodimeric complexes with specific immunity proteins (Im), which inactivate the cytotoxic domain. The nuclease PB binds to the receptor of the nuclease PB as a heterodimeric complex with the inhibitory Im, which dissociates from the PB when it is translocated into the target cell (Vankemmelbeke et al., 2009)(Farrance et al., 2013)( Figure 1 ). Many bacteriocins form an initial high affinity interaction with their receptors before crossing the outer membrane via the translocator protein. Bacteriocin translocation is mediated by mechanical unfolding of the toxin, driven by the proton motive force across the inner membrane, resulting in bacteriocin import and release of the Im at the cell surface. As the PB dissociates from the Im as it crosses the outer membrane, the nuclease is reactivated and becomes cytotoxic to the cell.

[0004] PB receptors are usually outer membrane proteins that are usually used for active uptake of nutrients (TonB-dependent receptors), passive diffusion of nutrients and metabolites (trimeric porins), or active efflux of antibiotics and other toxic compounds (TolC) (Cascales et al., 2007). However, the use of bacteriocins as antibacterial agents is limited by their high specificity for target receptors (which can be strain-specific) and high rates of acquired resistance (e.g. due to receptor mutations).

[0005] Therefore, there is still a need for new antibacterial agents having a wider range of uses and a lower resistance rate. Summary of the invention

[0006] The present inventors unexpectedly found that the receptor binding function of the nuclease bacteriocin heterodimer complex can be transferred from the nuclease bacteriocin polypeptide to the immune polypeptide component of the PB-Im complex without losing the function of the nuclease bacteriocin as an antibacterial agent. In other words, although the receptor domain is rearranged from PB to Im, the immune polypeptide is still combined with the nuclease domain, the translocation domain still interacts with the outer membrane protein translocation protein, and during the translocation of the nuclease domain, the immune protein is still discarded / dissociated together with the receptor binding domain. Therefore, PB-Im can still target bacterial cells, and the nuclease domain still passes through the membrane, and becomes cytotoxic when releasing the immune protein. Importantly, in this way, the cell killing function is separated from the surface binding function, so that the receptor specificity of a given bacteriocin and therefore the strain susceptibility are reprogrammed and / or expanded. By replacing or supplementing the receptor binding domain with different receptor binding domains connected to the immune polypeptide instead of the nuclease polypeptide, specificity and / or effectiveness can be changed or expanded. The inventors further recognized that moving the receptor binding function from the nuclease to the immune protein allows the use of different receptor binding parts instead of the receptor binding domains of existing bacteriocins. In particular, the receptor binding domain of the natural bacteriocin polypeptide must be easy to unfold to allow translocation into the target bacterial cell together with the translocation domain and the nuclease domain. However, the receptor binding part that is connected to the immune polypeptide and is discarded at the surface when the bacteriocin polypeptide is translocated does not require this unfolding property. Therefore, different types of receptor binding parts can be used, further expanding the ability of the modified protein bacteriocin to target different ligands / receptors and different bacterial strains.

[0007] Thus, in a first aspect, the present invention provides an antibacterial protein complex comprising (i) a nuclease bacteriocin polypeptide, and (ii) an immunity polypeptide linked to a bacteria binding moiety, wherein the nuclease bacteriocin polypeptide comprises a translocation domain and a nuclease domain, or comprises a translocation domain, a receptor binding domain, and a nuclease domain; and wherein the bacteria binding moiety binds to a ligand on the surface of a Gram-negative bacterium.

[0008] In some embodiments, the bacteria binding moiety is a receptor binding domain of a bacteriocin. In some embodiments, the receptor binding domain of the nuclease bacteriocin is different from the receptor binding domain connected to the immunity polypeptide, and / or binds to different Gram-negative bacterial surface ligands and / or different Gram-negative bacterial strains. In some embodiments, the immunity polypeptide is connected to multiple bacteria binding moieties or to multiple bacteriocin receptor binding domains, optionally wherein each bacteria binding moiety binds to a different bacteria surface ligand and / or a different bacterial strain, and / or wherein each bacteria binding moiety binds to a different ligand and / or bacterial strain with the receptor binding domain of the nuclease bacteriocin polypeptide.

[0009] The present invention also provides an antibacterial composition comprising the antibacterial protein complex. In some embodiments, the composition is a pharmaceutical composition.

[0010] The present invention also provides an antibacterial protein complex or pharmaceutical composition of the present invention for use in a method of treating a human or animal body by therapy. The present invention also provides the use of an antibacterial protein complex of the present invention in the preparation of a medicament. The present invention also provides a method of treating a human or animal subject by therapy. The method comprises administering an antibacterial protein complex or pharmaceutical composition of the present invention to a subject. The treatment in each of these embodiments can be used to prevent or treat bacterial infection or complications associated therewith.

[0011] The present invention also provides a medical device comprising, coated and / or impregnated with the antibacterial protein complex of the present invention. The medical device can be used in vivo in a subject in need thereof. The present invention also relates to the in vivo use of a medical device comprising, coated and / or impregnated with the antibacterial protein complex of the present invention, i.e., in a subject in need thereof.

[0012] The present invention also provides one or more polynucleotides encoding the antibacterial protein complex of the present invention. The present invention also provides one or more vectors comprising the one or more polynucleotides. The present invention also provides a host cell comprising the one or more vectors. The present invention also provides a method for producing the antibacterial protein complex of the present invention, the method comprising culturing the host cell of the present invention, and isolating the antibacterial protein complex from the culture.

[0013] The present invention also provides a product with an antibacterial surface or coating, wherein the surface or coating comprises an antibacterial protein complex comprising an immune polypeptide and a protein bacteriocin nuclease. The present invention also provides a method for providing a product with an antibacterial surface, the method comprising (a) providing an antibacterial protein complex comprising an immune polypeptide and a protein bacteriocin nuclease; and (b) binding the antibacterial protein complex to a surface. The complex can be bound to the surface through an immune polypeptide. In another embodiment, the method comprises (a) binding an immune polypeptide to the surface of the product; and (b) binding a protein nuclease bacteriocin to a surface-bound immune protein. In these embodiments, the immune polypeptide can be connected to a bacterial binding portion that binds to a ligand on the bacterial surface. The complex can be any antibacterial protein complex of the present invention.

[0014] The present disclosure will now be described in more detail by way of example and not limitation, and by reference to the accompanying drawings. When the present disclosure is given, many equivalent modifications and variations will be apparent to those skilled in the art. Therefore, the exemplary embodiments of the present disclosure set forth are considered to be illustrative and not restrictive. Without departing from the scope of the present disclosure, various changes may be made to the embodiments. Whether above or below, all documents cited herein are expressly incorporated by reference as a whole.

[0015] The present disclosure includes the combination of the described aspects and preferred features, unless such a combination is clearly not allowed or is pointed out to be explicitly avoided. Unless the content clearly indicates otherwise, the nouns used in this specification and the appended claims without quantifiers are intended to mean one / kind or more / kind.

[0016] The section headings used herein are for convenience only and are not to be construed as limiting in any way.

[0017] The experimental methods without specific conditions in the following examples generally follow conventional conditions or conditions recommended by the manufacturer. The various common chemical reagents used in the examples are generally commercially available products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1- Left: The colicin E9:Im9 complex forms a translocon complex at the cell surface of E. coli, where it binds BtuB with high affinity (Housden et al., 2005), allowing its unstructured N-terminus to pass through OmpF, allowing it to bind to TolB in the periplasm (Francis et al., 2021; Housden et al., 2013). (Middle) TolB binding leads to energized unfolding of colicin E9 and release of Im9 on the outside of the cell as colicin passes through OmpF. Colicin is processed by FtsH in the inner membrane (Walker et al., 2007), resulting in translocation of the cytotoxic domain into the cytoplasm. Right: Cytotoxic activity of colicin E9:Im9 against E. coli NEB5α cells grown as a lawn on agar plates.

[0019] Figure 2 -Im9-ColB TR nucleotide and amino acid sequences.

[0020] Figure 3 - (Left) E.coli E9, (Middle) Im9-ColB TR and (right) colicin E9:Im9-ColB TR Cytotoxic activity against E. coli BL21 (DE3) cells grown as a lawn on agar plates.

[0021] Figure 4 - (Left) E9ΔR, (Middle) Im9-ColB TR and (right) colicin E9 ΔR :Im9-ColB TR Cytotoxic activity against E. coli NEB5α cells grown as a lawn on agar plates.

[0022] Figure 5 -Im9-ColE9 R The nucleotide and amino acid sequence of colicin E9 comprises residues 294 to 455.

[0023] Figure 6 -(Left) colicin E9 ΔR 、(middle)Im9-ColE9 R and (right) colicin E9 ΔR :Im9-ColE10 R Cytotoxic activity against E. coli NEB5α cells.

[0024] Figure 7-Im9-CloDF13 R The nucleotide and amino acid sequence of cloacin DF13 comprises residues 301 to 460.

[0025] Figure 8 -(Left) Enterobacter cloacae DF13 ΔR 、(Medium)Im9-CloDF13 R and (right) Enterococcin DF13 ΔR :Im9-CloDF13 R Cytotoxic activity against Klebsiella quasipneumoniae M1-977 cells.

[0026] Fig. 9 - (Left) KlebC-E9 and (Right) Im9-CloDF13 R The cytotoxic activity of the composite KlebC-E9 against SG96, SR3 and SR68 Klebsiella strains shows how pre-binding to the lutA receptor (via the enterocin DF13 receptor binding domain) enhances the killing efficiency of KlebC.

[0027] Fig.10 -ColE9 ΔR :Im9-ColE9 R -ColB TR Activity against soft agar lawns seeded with E. coli fepA- BW25113 (left) and BL21(DE3) (right) cells, where bacteriocin function results in clear zones. FepA- cells lack the colicin B receptor, while BL21(DE3) cells lack the colicin E9 receptor BtuB. ColE9 ΔR :Im9-ColE9 R -ColBT R It is active against both strains, so Im9-ColE9 R -ColB TR Both receptor binding domains are functional.

[0028] Fig.11 - A lawn of E. coli JM83 on which sepharose beads were placed. Left: Activated sepharose blocked with cysteine. Right: Activated sepharose derivatized with Im9 bound to colicin E9.

[0029] Figure 12-OMP-lipid-OMP complex is the functional unit of the supramolecular OMP assembly across the entire outer membrane (OM) of E. coli. A: MD simulation snapshots of the OmpF-LPS / PL-BtuB complex, showing how the mutual sharing of asymmetric lipids produces a tightly packed interface. The barrel (mid-barrel) residues L259 and I273 in OmpF are highlighted. UV-activated crosslinking at these sites is crosslinked with LPS or PL. B: MD simulation snapshots of heterologous lipid-mediated complexes formed between trimer OmpF and three different monomeric β-barrels (FepA, BtuB and FhuA). The three-fold symmetry of OmpF (and most likely OmpC) allows different OMPs to be recruited to porins, and its complex becomes sufficiently stable through BPA lipid crosslinking to enable purification. C: OM imaging of live E. coli MG1655 cells by AFM (tapping mode) labeled with FepA-bound ColB-mCherry (marked as gray balls). Phase images show a trimeric porin network, with holes marked with small gray balls. The peaks in the height image show the positions of the ColB-mCherry fluorescent markers. Overlapping the FepA position with the trimeric porin indicates that FepA is embedded in the porin network. In addition, some areas do not have OMPs, and may be previously determined regional patches rich in LPS. The scale bar is 50nm. The color (phase / height) scales are 1.2 degrees / 2nm and 1.2 degrees, respectively. D: OMP island model, in which OmpF accommodates heterologous OMPs in its hexagonal array. The size of the island is similar to the size observed by live cell imaging. In this work, it was determined that the monomeric β-barrels incorporated into the island are BtuB, FhuE, LeptDE, FepA and FhuA. The components of the OMP biogenesis machine BAM represented by BamA are also determined in the OMP island. E: Cartoon diagram of an E. coli cell, which shows the OMP island distributed throughout the cell. The high copy number of OmpF causes the porin to spread to areas much more extensive than the island itself.

[0030] Figure 13 - KvarM-cys dimer. [A] Schematic diagram of the domain organization of the KvarM-cys dimer. [B] Gel filtration of KvarM-cys 280 Absorbance spectrum. The dimer peak eluted at 200.47 ml and the monomer peak eluted at 230.56 ml. [C] Non-reducing SDS-PAGE gel of wild-type KvarM, monomeric KvarM-cys and dimeric KvarM-cys.

[0031] Figure 14-2Polymeric KvarM-cys retains cytotoxic activity. Panel showing the cytotoxic activity of wild-type KvarM (left), monomeric KvarM-cys (middle), and dimeric KvarM-cys (right) against K. pneumoniae-like SG96 cells.

[0032] Fig.15 - Nucleotide and amino acid sequences of KvarM-Im9.

[0033] Figure 16 - KvarM-Im9 retains cytotoxic activity. [A] Panel showing the cytotoxic activity of wild-type KvarM (left) and KvarM-Im9 (right) against K. pneumoniae-like SG96 cells. [B] Schematic representation of the KvarM-Im9 fusion binding to the outer membrane receptor FhuA.

[0034] Fig.17 - Nucleotide and amino acid sequences of KlebC-E9.

[0035] Figure 18 - KvarM-Im9 and KlebC-E9 form a stable complex. [A] Gel filtration of KvarM-cys 280 Absorbance spectra. Left = complex peak, right = monomer peak. [B] SDS-PAGE of fractions containing KvarM-Im9:KlebC-E9 complex (middle) and excess KvarM-Im9 (right).

[0036] Figure 19 - KvarM Im9:KlebC-E9 complex exhibits enhanced killing activity. [A] Cytotoxic activity of KlebC-E9, KvarM-Im9 and KvarM-Im9:KlebC-A9 complex against K. pneumoniae SR3 cells (left) and K. quasipneumoniae SG96 cells (right). [B] KlebC-E9 receptor binding and cytotoxic activity. [C] KvarM-Im9 receptor binding and cytotoxic activity. [D] KvarM-Im9:KlebC-E9 complex receptor binding and cytotoxic activity.

[0037] Fig. 20 - Alphafold 2 structure predictions and schematics of Im9 (above / right of the barrel structure) fused to the C-terminus of KvarM (barrel and structure below the barrel) via no linker (left), a flexible glycine-serine linker (middle), and a rigid helical linker (right).

[0038] Fig.21- Cytotoxic activity of wild-type KvarM, KvarM-Im9, KvarM fused to Im9 with a flexible glycine-serine linker (KvarM-GS5-Im9), and KvarM fused to Im9 via a rigid helix (KvarM-helix-Im9) against K. pneumoniae-like SG96 cells.

[0039] Fig. 22 - Nucleotide and amino acid sequences of KvarM-GS5-Im9.

[0040] Fig.23 - Nucleotide and amino acid sequences of KvarM-helix-Im9.

[0041] Description of the sequence

[0042] SEQ ID NO: 1 shows the amino acid sequence of colicin E9 (ColE9).

[0043] SEQ ID NO: 2 shows ColE9 with residues 317 to 448 (corresponding to the R domain) deleted (ColE9 ΔR )'s amino acid sequence.

[0044] SEQ ID NO: 3 shows the amino acid sequence of cloacin DF13 (CloDF13).

[0045] SEQ ID NO: 4 shows the amino acid sequence of the enterocloacin DF13-E9 chimera (CloDF13-E9) comprising the DNase domain of E9.

[0046] SEQ ID NO: 5 shows the R domain (residues 324 to 459) of Enterocloacin DF13-E9 (DF13-E9 ΔR )'s amino acid sequence.

[0047] SEQ ID NO: 6 shows the amino acid sequence of Klebicin C (KlebC) from Klebsiella pneumoniae.

[0048] SEQ ID NO: 7 shows the amino acid sequence of Klebsiella pneumoniae klebsiella pneumoniae C (KlebC)-E9 chimera (KlebC-E9) comprising the DNase domain of E9.

[0049] SEQ ID NO: 8 shows the amino acid sequence Im9.

[0050] SEQ ID NO: 9 shows the amino acid sequence of the R domain of CloDF13.

[0051] SEQ ID NO: 10 shows the amino acid sequence of the NTR domain of ColB.

[0052] SEQ ID NO: 11 shows the amino acid sequence of the R domain of ColE9.

[0053] SEQ ID NO: 12 shows ColE9 R -ColB TR The amino acid sequence of

[0054] SEQ ID NO: 13 shows Im9-ColE9 R The amino acid sequence of

[0055] SEQ ID NO: 14 shows Im9-ColB TR The amino acid sequence of

[0056] SEQ ID NO: 15 shows Im9-CloDF13 R The amino acid sequence of

[0057] SEQ ID NO: 16 shows Im9-ColE9 R -ColB TR The amino acid sequence of

[0058] SEQ ID NO: 17 shows the polynucleotide sequence encoding ColE9.

[0059] SEQ ID NO: 18 shows the polynucleotide sequence encoding ColB.

[0060] SEQ ID NO: 19 shows the polynucleotide sequence encoding CloDF13-E9.

[0061] SEQ ID NO: 20 shows the polynucleotide sequence encoding KlebC-E9.

[0062] SEQ ID NO: 21 shows the polynucleotide sequence encoding Im9.

[0063] SEQ ID NO: 22 shows the polynucleotide sequence encoding KvarM-Im9.

[0064] SEQ ID NO: 23 shows the amino acid sequence of KvarM-Im9.

[0065] SEQ ID NO: 24 shows the polynucleotide sequence encoding KvarM-GS5-Im9.

[0066] SEQ ID NO: 25 shows the amino acid sequence of KvarM-GS5-Im9.

[0067] SEQ ID NO: 26 shows the polynucleotide sequence encoding KvarM-Helix-Im9.

[0068] SEQ ID NO: 27 shows the amino acid sequence of KvarM-helix-Im9.

[0069] SEQ ID NO: 28 shows the amino acid sequence of KvarM. DETAILED DESCRIPTION

[0070] Antibacterial protein complex

[0071] The initial step in protein bacteriocin (PB)-mediated bacterial killing is the formation of a high-affinity complex between the receptor binding (R) domain of the PB and the outer membrane receptor of the target organism. The inventors have shown that the R domain can be excised from the PB and fused to the immune protein (Im) of the PB, resulting in an Im-R fusion. The inventors have also shown that such modified Im-R fusions can deliver PB tangentially to bacteria (so-called frankincins). This allows new antimicrobial agents to be engineered to target one or more species-specific protein receptors on cell surfaces.

[0072] At present, the surface receptor library that can be targeted is limited by the PB that has been determined. This method is limited because the species coverage of PB (i.e., how many strains of a given species can be killed) is determined by the frequency of receptors present on the cell surface. If the receptor is rarely present in the outer membrane, or if its expression is regulated by growth conditions, this will limit the strain coverage of PB. However, according to the present invention, the surface binding of PB is separated from the ability to pass through the membrane and kill a given bacterial species, and it is not necessary to use only pre-existing receptor binding domains in hybrid PB. In addition, multiple outer membrane receptors can be targeted via multiple or tandem receptor binding parts through a single construct, thereby ensuring a wide range of species coverage without the need to prepare PB mixtures. The inventors also recognize that outer membrane proteins (outer membrane protein, OMP) gather on the surface of Gram-negative bacteria. The arrangement of OMP in these clusters brings together a large number of selected surface ligands (these surface ligands can serve as receptors of PB-Im complexes), and translocation proteins are required for input into cells and cytotoxicity. The specific interaction of the R domain with its receptor serves to concentrate the bacteriocin at the surface of the target bacteria, and thus, by varying the properties of this domain, hybrid bacteriocins with switched receptor specificity can be constructed.

[0073] Therefore, the inventors have found how to separate the function of surface binding from the function of cell killing, which currently limits the use of nuclease PB as an antibacterial agent. The key to this discovery is that the immune protein from nuclease PB can be fused with the R domain of PB. These Im-R fusions can be compounded with nuclease PB to produce bacteriocin: Im-R complexes. The R domain concentrates bacteriocins at the outer membrane. Then the translocation of PB occurs. Im-R fusions are usually left at the cell surface, except that the Im-R fusions themselves can be translocated to some embodiments (as described elsewhere herein) in the cell. This new method is applicable to any type of surface receptors, including the R domains of known PB or customized nano antibodies or other binding parts such as aptamers produced for the outer membrane protein of selection.

[0074] Nuclease bacteriocin peptide

[0075] The present invention provides an antibacterial protein complex comprising (i) a nuclease bacteriocin polypeptide, and (ii) an immunity polypeptide connected to a bacterial binding portion. Therefore, the nuclease bacteriocin polypeptide is complexed with the immunity polypeptide. The complex is generally a heterodimer complex. The nuclease bacteriocin polypeptide generally comprises a translocation domain and a nuclease domain, or comprises a translocation domain, a receptor binding domain and a nuclease domain. The nuclease bacteriocin can be any suitable naturally occurring nuclease bacteriocin or a variant thereof, or comprises the NT, R and / or C domains of any suitable naturally occurring nuclease bacteriocin. Some examples can be found in, for example, Sharp et al. (2017) PLoS Comp Biol. 13, e1005652. Variants can be as described herein, and are generally used to improve functional aspects while maintaining other activities of the polypeptide or domain, as described herein. For example, a variant can be derived from another bacterial species, thereby avoiding the inherent immunity to a given bacterial population.

[0076] Nuclease bacteriocin polypeptides generally contain a central receptor binding domain (R domain), an N-terminal region that interacts with bacterial surface translocation proteins (NT domain), and a (C-terminal) cytotoxic nuclease domain (C) (see Figure 1). However, in the complex according to the present invention, the R domain may or may not be present, since the receptor binding function is connected to (provided by) the immune polypeptide component of the complex. The NT region generally comprises a largely unstructured N-terminal region (N) that initially interacts with a bacterial surface translocation protein and a translocation domain (T) that facilitates translocation of the nuclease bacteriocin polypeptide by the translocation protein. The translocation protein may be, for example, an OmpF translocation protein, or any one of OmpC, PhoE, OmpK35 (OmpF homolog), OmpK36 (OmpC homolog), or TolC.

[0077] The C domain usually has DNA enzyme or RNA enzyme (rRNA enzyme or tRNA enzyme) activity. The C domain is cytotoxic to Gram-negative bacteria when in bacterial cells and does not bind to the immune polypeptide. When bound to the immune polypeptide, its cytotoxic activity is neutralized. The complex of the nuclease bacteriocin and the immune polypeptide is formed by the binding between the C domain of the nuclease bacteriocin polypeptide and the immune polypeptide.

[0078] The R domain is combined with bacterial surface ligand / outer membrane protein (OMP). Some examples of outer membrane proteins combined by naturally occurring bacteriocin include BtuB, Tsx, TolC, OmpA, FepA, Cir, FhuA, IutA, FpvAI, FpvAII, FptA, FiuA, Hur and porins (e.g., OmpF, OmpC, OmpK35 and OmpK36). The R domain can be a natural R domain related to the NT domain and / or C domain of the same bacteriocin in the natural protein. Alternatively, the nuclease bacteriocin can be a chimeric protein comprising NT, C and / or (optionally) R domains from different naturally occurring nuclease bacteriocins, or variants thereof.

[0079] Exemplary nuclease bacteriocin polypeptides useful in the present invention include ColE2, ColE7, ColE8 and ColE9 (DNA enzymes), ColE3, ColE4, ColE6, Klebsiella pneumoniae C and Enterococcus DF13 (16S RNase), ColE5 and ColD (tRNA RNase). The nuclease bacteriocin polypeptides used in the present invention may comprise the N, T, NT, R and / or C domains of any of these bacteriocins or any other suitable bacteriocin known to those skilled in the art, or variants thereof. Exemplary sequences are provided. In some embodiments, the nuclease bacteriocin polypeptide is colicin E9 (ColE9) (SEQ ID NO: 1), or ColE9 lacking its native R domain (e.g., having the amino acid sequence of SEQ ID NO: 2), or ColE9 having an R domain of a different nuclease bacteriocin polypeptide (e.g., any of the nuclease bacteriocins mentioned herein), such as the R domain of any of ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColE8. In other embodiments, the nuclease bacteriocin polypeptide is enterocin DF13 (CloDF13) (SEQ ID NO: 3), or CloDF13 lacking its native R domain, or CloDF13 having an R domain of a different nuclease bacteriocin polypeptide (e.g., any of those mentioned herein). In other embodiments, the nuclease bacteriocin polypeptide is Klebsiella pneumoniae (SEQ ID NO: 6), or Klebsiella pneumoniae having an R domain of a different nuclease bacteriocin polypeptide (e.g., any of those mentioned herein). The functional domain (e.g., R domain) of the nuclease bacteriocin polypeptide can be determined based on the primary sequence of the polypeptide, the solved structure when available, comparing the primary sequence to the sequences of other bacteriocins with solved structures, or using prediction software (e.g., AlphaFold and AlphaFold 2 programs).

[0080] Variants or their functional domains of known nuclease bacteriocins can be used as long as the variant still acts as an antibacterial agent. Therefore, the variant is usually still translocated through the outer membrane and maintains cytotoxic nuclease activity. The variant can also maintain the ability to bind to the surface of Gram-negative bacteria cells through the R domain, but in some embodiments, this function can be provided entirely by the part connected to the immune protein, as described herein. Otherwise, in some cases, the variant has, for example, at least 70%, or more usually at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity with known naturally occurring nuclease bacteriocins, or the nuclease bacteriocin polypeptide may contain NT, R and / or C domains having at least 70%, or more typically at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity to the corresponding domains of known naturally occurring nuclease bacteriocins, e.g., as described herein. The same applies to variants of the R domains of known bacteriocins (including pore-forming bacteriocins and nuclease bacteriocins) linked to the immunity polypeptides of the complex according to some embodiments of the present invention, as further discussed below.

[0081] For the purposes of the present invention, in order to determine the percent identity or similarity of two sequences (e.g., two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence to optimally align with the second sequence). The amino acids at each position are then compared. When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, the amino acid at that position is identical. The percent identity or similarity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in a reference sequence (e.g., SEQ ID NO: 1) × 100, or % identity = number of identical positions / total number of positions in either sequence × 100).

[0082] Sequence comparison is usually performed on the length of a reference sequence, such as SEQ ID NO:1 herein. If the sequence is shorter than the reference sequence, then the gap or missing position should be considered as a non-identical position. However, in some cases, sequence comparison can alternatively be performed on the length of the sequence compared with the reference sequence. If the reference sequence is shorter than the comparator sequence, then the gap or missing position should be considered as a non-identical position.

[0083] The skilled person is aware of different computer programs that can be used to determine the homology or identity between two sequences using a mathematical algorithm. In one embodiment, the Needleman and Wunsch (1970) algorithm, which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.con / products / gcg / ), is used to determine the percent identity between two amino acid or nucleic acid sequences using a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Other examples of suitable programs are the BESTFIT program provided by the UWGCG package (e.g., used under its default settings) (Devereux et al (1984) Nucleic Acids Research 12, 387-395) and PILEUP and BLAST algorithm c (e.g., used under its default settings), e.g., as described in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is available from the National Center for Biotechnology Information ( http: / / www.ncbi.nlm,nih.gov / ) are publicly available.

[0084] Variants as described herein may comprise one or more modifications of an amino acid sequence from a reference sequence by substitution, deletion and / or addition. For example, where appropriate, the modification may comprise substitution, addition and / or deletion of up to 50 or up to 40, 30, 20, 15, 10, 8, 6, 5, 4, 3, 2 or 1 amino acid from an amino acid sequence of a reference sequence. For example, the modification may comprise an amino acid replaced by an alternative amino acid having similar properties. This may be referred to as a "conservative amino acid substitution". For example, an amino acid with an aliphatic side chain is replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is replaced by another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain is replaced by another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is replaced by another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is replaced by another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced by another hydrophobic or hydrophilic amino acid, respectively. For a variety of reasons, making conservative substitutions can be useful, such as improving stability, improving the ability to prepare polypeptides synthetically, or introducing groups that allow additional functionalization, such as conveniently cross-linking polypeptides into trimers.

[0085] The percentage similarity between two sequences (e.g., amino acid sequences) is calculated in the same manner as the percentage identity, as described above, except that replacement of an amino acid with a different but similar amino acid at the same position in the aligned sequences is counted in the same manner as the same amino acid. Some properties of the 20 main amino acids that can be used to select suitable replacements are as follows:

[0086]

[0087] As used herein, sequences with a possible range of "identity or similarity" percentages include sequences with both a minimum identity percentage and a different, higher minimum similarity percentage, each within a disclosed range of percentages. For example, the sequence may have at least 70% sequence identity and at least 80%, 85%, or 90% sequence similarity, or the sequence may have at least 80% sequence similarity and at least 85%, 90%, 95%, or 100% sequence similarity.

[0088] Immune peptide

[0089] "Immunity proteins" / "immunity polypeptides" / "bacterial immunity polypeptides" are inhibitors of nuclease bacteriocins that bind to cytotoxic nuclease domains and neutralize their activity. In the context of the present invention, immunity polypeptides are "nuclease-specific immunity polypeptides". Any suitable nuclease-specific immunity polypeptides or variants thereof known in the art, such as those described above, can be used in the present invention. Typically, the immunity polypeptide and the C domain of the nuclease bacteriocin will be a naturally occurring pair or variants thereof, i.e., variants that retain their complex-forming activity. For example, in some embodiments, the immunity polypeptide is Im9 (e.g., a sequence having SEQ ID NO:8) or a variant thereof. Im9 binds to the C domain of ColE9. Therefore, in some embodiments, the immunity polypeptide comprises the amino acid sequence of Im9 or a suitable variant thereof, and the nuclease bacteriocin comprises the amino acid sequence of the C domain of ColE9 or a suitable variant thereof. Other examples of known immunity proteins include Im2, Im7, ImD, Im9, Im3, Im4, Im5, and Im6. Typically, immunity proteins bind with very high affinity to the C domain of the nuclease bacteriocin polypeptide in the complex, e.g., for the RNase-Im complex, K d For at least 10 -10 M or K d For at least 10 -10 M, 10 -11 M or 10 -12 M (e.g. measured at pH 7 and 25°C, e.g. by stopped-flow fluorescence, e.g. as described in Walker et al. (2003) Biochemistry 42, 4161), or for the DNase-Im complex, K d For at least 10 -10 M or K d For at least 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or 10 -14 M (e.g. as measured for the RNase-Im complex, or as described in Wallis et al. (1995) Biochemistry 34, 13743-13750). However, when the nuclease bacteriocin polypeptide is translocated across the outer membrane, the immunity polypeptide dissociates from the nuclease bacteriocin polypeptide. The immunity polypeptide is typically a small polypeptide of about 8 kDa to about 20 kDa.

[0090] Bacteria binding part

[0091] The antibacterial protein complex comprises an immunity polypeptide linked to a bacteria binding moiety (also referred to as a "bacterial targeting moiety"). The bacteria binding moiety binds to a ligand (e.g., an OMP) on the surface (expressed) of a Gram-negative bacterium. Thus, the bacteria binding moiety binds the complex to the surface of a bacterial cell expressing the ligand on its surface. The bacteria binding moiety thus also brings the complex to the vicinity of a translocation protein, through which the nuclease bacteriocin can enter the cell. In some embodiments, the affinity constant (K) of the bacteria binding moiety for the ligand is D ) value may be ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.5 nM, ≤0.4 nM, ≤0.3 nM, ≤0.2 nM, ≤0.1 nM or ≤0.05 nM. K D The values ​​may be measured by any suitable means known in the art, such as by ELISA, surface plasmon resonance (Biacore), or stopped-flow fluorescence at 25°C.

[0092] The bacteria binding moiety can be selected to target the complex to one or more specific strains of Gram-negative bacteria. For example, in some embodiments, the targeted strain is one or more Enterobacteriaceaespp, Pseudomonaceae spp, and / or Acinetobacter spp bacteria; or one or more strains of Escherichia, Salmonella, Serratia, Shigella, and / or Enterobacter, such as Escherichia coli, Salmonella enterica, Serratia marcescens, Shigella sonnei, Acinetobacter baumanii, and Enterobacter cloacae.

[0093] Some examples of ligands that can be bound by bacterial binding moieties include common ligands that are bound by the R domains of known nuclease bacteriocins as described above (e.g., BtuB, Tsx, TolC, OmpA, FepA, Cir, FhuA, IutA, FpvAI, FpvAII, FptA, FiuA, Hur, and porins (e.g., OmpF, OmpC, OmpK35, and OmpK36)). However, a key benefit of the present invention is that it opens up the possibility of targeting a more diverse set of surface ligands / OMPs. Thus, in some embodiments, the ligand is a ligand that is bound by the R domain of any bacteriocin, and includes, for example, Tsx, OmpF, OmpA, Cir, FhuA, and Tip-pilus F / N. Other ligands that can be bound include OmpC, LptD, OmpF, BtuB, FhuE, FhuA, FepA, and BamA, as described in Example 7 herein. There are also other surface-expressed TonB dependent transporters (TonB dependenttransporter, TBDT). It is also considered that the bacterial binding moieties combined with other ligands (such as LPS). Targeting essential proteins (such as BamA and LptD) are particularly attractive options because this will minimize the chance of resistance. However, the ligand that can be combined is basically only limited by the ability to produce a suitable binding moiety, and in general, for basically any ligand / OMP, this is within the skills of those skilled in the art. Therefore, the technician can select suitable target ligand and suitable binding moiety to suit its needs.

[0094] In some embodiments, the bacterial binding portion is a receptor binding domain of a bacteriocin, or a suitable variant thereof, such as described above. In some embodiments, the bacterial binding portion is a receptor binding domain of a nuclease bacteriocin, such as the R domain of any one of colicin E2 to E9 (ColE2 to ColE9), enterocin DF13 (CloDF13) or klebsiella pneumoniae C (KlebC), or a suitable variant thereof. Another example is colicin G. Another example is pyocin / pyocin S2. In some embodiments, a translocation domain (T) and / or an N domain, or part or all of the region from the N-terminus of a bacteriocin to the R domain may also be included. For example, ColB binds to FepA as its receptor and is subsequently translocated by the same FepA molecule. Therefore, ColB-TR is a single domain. In some embodiments, the bacteria binding moiety is or comprises a receptor binding domain of a non-nuclease bacteriocin (e.g., an R domain of a pore-forming bacteriocin) or a receptor binding domain of colicin M or KvarM, such as any of colicin A, B, E1, Ia, Ib, N, K, U, 5, and 10. Another example is pyocin / pyocin S5. Other suitable examples are provided in Sharp et al. (2017) PLoS Comp Biol. 13, e1005652.

[0095] In some embodiments, the immunity polypeptide connected to the bacteria binding part (Im-R) can provide a second cytotoxic domain for the complex. In some embodiments, Im-R can also be translocated into the target cell. Therefore, in some embodiments, Im-R can be a second toxin, as well as a targeting device for the composite PB. In some embodiments, the bacteria binding part can be or include the entire PB or a variant thereof. Specifically, the bacteria binding part can be or include an M-type bacteriocin or a colicin M homologue, such as colicin M itself or KvarM, or a functional variant thereof, such as described herein, such as those retaining its receptor binding, translocation and / or cytotoxic activity. Example 8 herein shows that even in the case of a large amount of fusion with the C-terminus of the M-type bacteriocin, the M-type bacteriocin can also maintain receptor binding, translocation and important cytotoxic activity. Therefore, Im-R can include an M-type bacteriocin, such as KvarM, which has an immunity polypeptide fused to the C-terminus of the M-type bacteriocin sequence. A specific example is the KvarM sequence in SEQ ID NO.23, 25 and 27 or SEQ ID NO:28. Other sequence elements may also be included, such as linkers (eg, those described in Examples 8 and 9), or one or more additional bacteria binding moieties, eg, as described herein.

[0096] In other cases, the bacteriocin-binding moiety is not the R domain of a bacteriocin, but is selected or generated to bind to a specific target ligand (eg, an antibody or antigen-binding fragment thereof) or an aptamer.

[0097] Nanobodies / single-domain antibodies (single-domainantibody, sbAb) / antibody fragments composed of a single monomer variable antibody domain are particularly suitable for use as bacterial binding moieties. In addition, since Im is not translocated into target cells, there is no need for the immune polypeptide-bacteria binding moiety to have mechanical instability, thereby allowing the use of nanobodies (containing internal disulfide bonds). For example, nanobodies are described in Holt et al. (2003), Trends in Biotechnology 21 (11): 484-490). In some embodiments, nanobodies are human or humanized nanobodies. Full human antibodies are those in which the variable regions and constant regions (if present) of both heavy and light chains are of human origin, or are substantially identical to sequences of human origin, but not necessarily from the same antibody.

[0098] Suitable aptamers can be generated using SELEX (Stoltenburg, R. et al., (2007), Biomolecular Engineering 24, p381-403; Tuerk, C. et al., Science 249, p505-510; Bock, L. C. et al., (1992), Nature 355, p564-566) or NON-SELEX (Berezovski, M. et al. (2006), Journal of the American Chemical Society 128, p1410-1411). Typically, the length of the aptamer can be at least 15 nucleotides, such as about 15 to about 50, about 20 to about 40, or about 25 to about 30 nucleotides in length.

[0099] In some cases, the antibacterial protein complex of the present invention comprises more than one bacterial binding portion. In some embodiments, the antibacterial protein complex comprises 2 to 10, or 2 to 5 or 2 to 4 bacterial binding portions. In particular, the antibacterial protein complex may comprise two or more bacterial binding portions connected to an immunity polypeptide, as further described below. As an alternative or in addition, the nuclease bacteriocin polypeptide of the complex may comprise a receptor binding domain, which is also a bacterial binding portion. Therefore, in a typical example, a complex with two bacterial binding portions may have a bacterial binding portion (i.e., the R domain of the nuclease bacteriocin) and a bacterial binding portion connected to an immunity protein; or there may be no R domain in the nuclease bacteriocin, but there are two bacterial binding portions connected to the immunity polypeptide. In another typical example, a complex with three bacterial binding portions may have a bacterial binding portion (i.e., the R domain of the nuclease bacteriocin) and two bacterial binding portions connected to the immunity protein; or there may be no R domain in the nuclease bacteriocin, and there are three bacterial binding portions connected to the immunity polypeptide.

[0100] Typically, multiple bacteria binding moieties have different properties. Typically, multiple bacteria binding moieties are combined with different surface ligands. Different surface ligands can be expressed by the same target Gram-negative bacteria. Such a complex is particularly useful for avoiding resistance in target cells, because if the target cell mutates or otherwise stops expressing the ligand combined by one of the bacteria binding moieties, one or more other bacteria binding moieties can still be used to target bacteria. As an alternative or supplement, the complex can include multiple bacteria binding moieties, which are combined with different surface ligands on different target bacterial cells or strain surfaces. Such a complex can target a wider range of target cells than a complex that does not include multiple bacteria binding moieties, which are combined with different surface ligands on different target bacterial cells or strain surfaces.

[0101] In some embodiments, the immunity polypeptide is linked to multiple bacterial targeting moieties. The inclusion of multiple bacterial targeting moieties linked to the immunity polypeptide is particularly helpful because the immunity polypeptide does not translocate into the target cell and therefore does not require the bacterial targeting moiety to be able to translocate, such as the case of the receptor binding domain of the naturally occurring nuclease bacteriocin. On the other hand, the inclusion of multiple bacterial binding moieties can increase the range of targetable bacterial strains, increase targeting efficiency and / or reduce resistance, for example due to mutations generated in one or more ligands that prevent binding by one or more bacterial binding moieties.

[0102] In some cases, one or more or each of the multiple bacteria targeting moieties can be a bacteriocin receptor binding domain, or more specifically, a bacteriocin receptor binding domain. In some cases, both a nuclease bacteriocin receptor binding domain and a non-nuclease bacteriocin receptor binding domain (e.g., a receptor binding domain of a pore-forming bacteriocin) can be included. In some embodiments, one or more or each of the multiple bacteria targeting moieties can be a bacteria targeting moiety other than a bacteriocin receptor binding domain, as described elsewhere herein. In some cases, multiple bacteria targeting moieties can be multiple copies of the same moiety. In other cases, multiple bacteria targeting moieties will be different and / or combined with different bacterial surface ligands and / or different bacterial strains, as described above.

[0103] In some embodiments, the immune polypeptide and the bacteria binding portion are fused together, i.e., as a chimeric polypeptide, which comprises the amino acid sequences of both the immune polypeptide and one or more bacteria targeting portions / domains. In a typical example, the immune polypeptide (sequence) is located at the N-terminal end (of the chimeric polypeptide sequence), and the bacteria binding portion (sequence) is at the C-terminal end (e.g., of the chimeric polypeptide). However, other arrangements are also contemplated, including having one or more bacteria binding portions (sequences) at the N-terminal end of the immune polypeptide (sequence), or having a central immune polypeptide (sequence) with one or more bacteria binding portions (sequences) on both sides (i.e., the flanks or N-terminal and C-terminal ends of the immune polypeptide sequence). When there are multiple bacteria targeting portions, these portions can be arranged in series. Therefore, in some cases, the immune polypeptides described herein that are connected to multiple bacteria binding portions can be connected to one or more bacteria binding portions through one or more other bacteria binding portions. In addition, where appropriate, the term "bacteria binding portion" may refer to a portion or domain having a bacterial surface ligand binding activity, or a portion or domain of a larger polypeptide / single amino acid chain. Likewise, the term "immunity polypeptide" may refer to a portion or domain having the properties described herein for an "immunity polypeptide", or a portion or domain of a larger polypeptide / single amino acid chain, where appropriate.

[0104] The bacteria-binding portion does not interfere with the binding of the immunity polypeptide to the C domain of the nuclease bacteriocin polypeptide; or the interaction of the nuclease bacteriocin polypeptide with the translocation protein; or the dissociation of the immunity polypeptide from the cytotoxic domain of the nuclease bacteriocin when the immunity polypeptide is translocated across the outer membrane.

[0105] In some specific exemplary embodiments, the bacterial binding moiety or one of the bacterial binding moieties linked to the immunity polypeptide is the receptor binding domain of bacteriocin CloDF13 (having the amino acid sequence of SEQ ID NO: 9) or a variant thereof, or the receptor binding domain (or T / R domain of ColB (having the amino acid sequence of SEQ ID NO: 10) or a variant thereof, or a variant thereof, or the receptor binding domain of ColE9 (having the amino acid sequence of SEQ ID NO: 11) or a variant thereof. In one embodiment, the immunity polypeptide is linked to both the R domain of ColE9 (SEQ ID NO: 11) and the T / R domain of ColB (SEQ ID NO: 10). In one embodiment, the immunity protein linked to the bacterial binding moiety comprises the sequence of SEQ ID NO: 12 (ColE9 R -ColB TR ).

[0106] In some specific exemplary embodiments, the immunity polypeptide is Im9 (SEQ ID NO: 8) or a variant thereof, and the bacteria-binding moiety is the R domain of ColE9 (SEQ ID NO: 11) or a variant thereof, the T / R domain of ColB (SEQ ID NO: 10) or a variant thereof, the R domain of CloDF13 (SEQ ID NO: 9) or a variant thereof, or the immunity polypeptide is linked to two bacteria-binding moieties comprising the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 10, or variants thereof, or to a sequence comprising SEQ ID NO: 12 (ColE9 R -ColB TR ) or an immunity polypeptide linked to a bacterial binding moiety is a fusion / chimeric polypeptide comprising SEQ ID NO: 13 (Im9-ColE9 R )、14(Im9-ColB TR )、15(m9-CloDF13 R ) or 16(Im9-ColE9 R -ColB TR ), for example, as described in Examples 1 to 3 and 5. In some specific embodiments, the complex further comprises ColE9 (SEQ ID NO: 1) or a variant thereof, or ColE9 with a deletion of the R domain (SEQ ID ID: 2) or a variant thereof, or the complex further comprises CloDF13-E9 (SEQ ID NO: 4) or a variant thereof, such as a variant in which the R domain is deleted (SEQ ID NO: 5), for example, as described in Examples 1 to 3 and 5 herein.

[0107] In another specific embodiment, the immunity polypeptide is Im9 (SEQ ID NO: 8) or a variant thereof, and the bacteria-binding moiety is the R domain of CloDF13 (SEQ ID NO: 9) or a variant thereof, or the immunity polypeptide linked to the bacteria-binding moiety is a fusion / chimeric polypeptide comprising the sequence of SEQ ID NO: 15 (Im9-CloDF13 R ) and the nuclease bacteriocin KlebC-E9 (SEQ ID NO: 7), or a variant thereof, e.g., as described in Example 4 herein.

[0108] In another specific embodiment, the immunity polypeptide is Im9 (SEQ ID NO: 8) or a variant thereof, and the bacteria binding moiety is KvarM or a variant thereof, or the immunity polypeptide linked to the bacteria binding moiety is a fusion / chimeric polypeptide comprising the sequence of SEQ ID NO: 23 (KvarM-Im9), SEQ ID NO: 25 (KvarM-MS5-Im9), or SEQ ID NO: 27 (KvarM-helix-Im9), or a variant thereof, as described herein in Example 9. In some specific exemplary embodiments, the immunity polypeptide, one or more bacteria binding moieties, the immunity polypeptide linked to the bacteria binding moiety, the nuclease bacteriocin, the nuclease bacteriocin R domain, and / or the antibacterial protein complex can be any of those described in the Examples herein or provided in the sequence listing.

[0109] Polynucleotides, vectors and host cells

[0110] The polypeptide of the present invention can be produced by any suitable means. For example, the polypeptide of the present invention comprises the product of chemical synthesis procedure and the product produced by recombinant technology from prokaryotic or eukaryotic host, and the prokaryotic or eukaryotic host includes, for example, bacteria, yeast, higher plants, insects and mammalian cells. Depending on the host used in the recombinant production process, the polypeptide of the present invention can be glycosylated or can be non-glycosylated. In addition, the polypeptide of the present invention can also include an initial methionine residue. This methionine residue can be derived from the initiation codon for initiating translation in the encoding nucleic acid.

[0111] The present invention provides one or more (isolated) polynucleotides (e.g., DNA or RNA) encoding an antibacterial protein complex of the present invention as described herein. Exemplary polynucleotide sequences that can be used in combination with the disclosure provided herein to design suitable exemplary polynucleotides of the present invention are provided herein as SEQ ID NOs: 17 to 21. The skilled person is aware that the DNA codon is degenerate and can easily envision alternative sequences or corresponding RNA sequences encoding the same polypeptides or variants thereof as described herein.

[0112] The polynucleotide of the present invention can be provided in the form of an expression cassette, which comprises a control sequence operably connected to an insertion sequence, thereby allowing the polypeptide to be expressed in vivo. Therefore, the present invention also provides one or more expression cassettes encoding one or more polynucleotides of the present invention. Then, these expression cassettes are usually provided in a vector (e.g., a plasmid or a recombinant viral vector). Therefore, in one embodiment, the present invention provides one or more vectors comprising the polynucleotide of the present invention. The vector can be a cloning vector or an expression vector. A suitable vector can be any vector that can carry a sufficient amount of genetic information and allow the expression of the encoded polypeptide.

[0113] Polynucleotides of the present invention, expression cassettes or vectors can be introduced into host cells, for example, by transfection. Therefore, the present invention also provides host cells, which comprise one or more polynucleotides of the present invention, expression cassettes or vectors. Polynucleotides, expression cassettes or vectors can be introduced into host cells instantaneously or permanently, allowing polypeptide expression. Such host cells include transient or more typically stable cells, such as higher eukaryotic cell lines (such as mammalian cells or insect cells), lower eukaryotic cells (such as yeast) or more typically prokaryotic cells (such as bacterial cells). Suitable host cells can be easily determined by technicians.

[0114] The present invention also provides a method for producing the antibacterial protein complex of the present invention, comprising culturing a host cell comprising one or more vectors, expression cassettes or polynucleotides of the present invention under conditions suitable for polypeptide expression. The polypeptide or complex can then be isolated from the culture.

[0115] General methods for constructing vectors or expression cassettes, transfection methods and culture methods are well known to those skilled in the art. See "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed), Wiley Interscience, New York and Maniatis Manual published by Cold Spring Harbor Publishing.

[0116] Products, compositions and uses

[0117] The polypeptides of the present invention have both medical and non-medical uses and can be incorporated into medical and non-medical products and compositions, as long as the antibacterial properties of the complexes of the present invention are useful. Some examples of compositions comprising the antibacterial protein complexes of the present invention include antibacterial surface sprays, wound cleansers, and medical lubricants. Some examples of other products that can effectively encompass the antibacterial protein complexes of the present invention include food preservatives and animal feeds.

[0118] Pharmaceutical compositions and modes of administration

[0119] In some aspects, the present invention relates to pharmaceutical compositions. The compositions comprise the antibacterial protein complexes of the present invention. The compositions typically also comprise at least one pharmaceutically acceptable excipient, carrier, diluent, buffer, stabilizer, preservative, adjuvant or other substance known to those skilled in the art. Such substances are preferably non-toxic and preferably do not interfere with the pharmaceutical activity of the active ingredient. The pharmaceutical carrier or diluent may be, for example, an aqueous solution. The exact nature of the carrier or other substance may depend on the route of administration, such as oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intradermal and intraperitoneal routes.

[0120] "Pharmaceutically acceptable carriers" are typically large, slowly metabolized macromolecules, such as proteins, sugars, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al., 2001, Vaccine, 19: 21188), trehalose (WO 00 / 56365), lactose, and lipid aggregates (e.g., oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The pharmaceutical composition may also contain a diluent, such as water, saline, glycerol, etc. In addition, auxiliary substances may be present, such as wetting agents or emulsifiers, pH buffer substances, etc. Sterile pyrogen-free phosphate-buffered saline is a typical carrier (Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472).

[0121] The pharmaceutical composition of the present disclosure can be lyophilized or in aqueous form, i.e., solution or suspension. This type of liquid formulation allows the composition to be used directly from its packaged form without the need for reconstruction in an aqueous medium, and is therefore ideal for injection. The pharmaceutical composition may be present in a vial, or it may be present in a pre-filled syringe (readyfilled syring). A syringe will contain a single dose, and a vial may contain a single dose or multiple doses.

[0122] The liquid formulations of the present disclosure are also suitable for reconstitution of other drugs from lyophilized forms. When the pharmaceutical composition is used for such extemporaneous reconstitution, the present invention provides a kit that may contain two vials, or may contain a prefilled syringe and a vial, wherein the contents of the syringe are used to reconstitute the contents of the vial prior to injection.

[0123] The pharmaceutical compositions of the present disclosure may contain antibacterial agents as preservatives, particularly when packaged in multi-dose form. Some examples include 2-phenoxyethanol or parabens (methyl, ethyl, propyl paraben). Any preservative is preferably present at low levels.

[0124] The pharmaceutical compositions of the present disclosure may include detergents, such as Tween (polysorbate), DMSO (dimethyl sulfoxide), DMF (dimethylformamide). Detergents are typically present at low levels, such as <0.01%, but may also be used at higher levels, such as 0.01% to 50%.

[0125] Pharmaceutical compositions of the present disclosure may comprise a sodium salt (eg, sodium chloride) and free phosphate ions in solution (eg, by using a phosphate buffer).

[0126] In certain embodiments, the pharmaceutical composition can be encapsulated in a suitable carrier, for example to improve stability. As will be appreciated by those skilled in the art, a variety of carriers are suitable for delivering the pharmaceutical composition of the present disclosure. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendritic polymers and other phospholipid-containing systems. Methods for incorporating pharmaceutical compositions into delivery vehicles are known in the art.

[0127] Some examples of suitable compositions and administration methods are provided in Esseku and Adeyeye (2011) and Van den Mooter G. (2006). In addition, some examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th edition 2000 published by Lippincott, Williams & Wilkins.

[0128] Administration routes include, but are not limited to, intranasal, oral, subcutaneous, intradermal, and intramuscular. Subcutaneous administration can be, for example, by injection into the abdomen, the outer and anterior sides of the upper arms or thighs, the back scapular region, or the upper buttocks ventral dorsal region.

[0129] The compositions of the present disclosure can be administered in one or more dosages and / or by a variety of administration routes. For example, such other routes include percutaneous, intravenous, intravascular, intraarterial, intraperitoneal, intrathecal, intratracheal, intracardiac, intralobally, intramedullary, intrapulmonary, and intravaginal. Depending on the desired treatment duration, compositions according to the present disclosure can be administered once or several times, or intermittently.

[0130] Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, pellets and granules. In such solid dosage forms, the active ingredient is usually combined with one or more pharmaceutically acceptable excipients (examples of which are described in detail above). Oral formulations can also be administered as aqueous suspensions, elixirs or syrups. For these, the active ingredient can be combined with a variety of sweeteners or flavoring agents, coloring agents, and, if desired, emulsifiers and / or suspending agents, and diluents such as water, ethanol, glycerol, and combinations thereof.

[0131] One or more compositions of the disclosure may be administered alone or in combination with other pharmaceutical compositions or treatments (eg, in combination with antibiotics), or methods and uses for treatment according to the disclosure may be performed.

[0132] Kits comprising the antibacterial protein complex of the invention and instructions for use, such as in any of the methods of the invention, are also within the scope of the invention. The kits may also include one or more additional agents, such as additional therapeutic or prophylactic agents.

[0133] Treatment

[0134] The antibacterial protein complexes of the present invention can be used in methods for treating the human or animal body by treatment. The term "treatment" as used herein includes therapeutic treatment and prophylactic treatment (although prevention can be considered as treatment (therapy) / treatment). It is usually administered in a "prophylactically effective amount" or "therapeutically effective amount", which is sufficient to cause a clinical response or show clinical benefit to the individual. For example, treatment can be to prevent, delay or shorten the onset of infection, or disease or condition, improve one or more symptoms, induce or prolong remission, delay relapse or reappearance, or reduce the bacterial load of infection. The polypeptide can be used in a method for treating or preventing bacterial infection or a disease or complication associated therewith in a subject, such as sepsis, pneumonia, wound infection, medical device infection (e.g., on a catheter) or biofilm. The polypeptide can also be used to reduce or improve any condition, symptom or side effect associated with the use of antibiotics, such as as described herein, for example, when the polypeptide is used in combination with a reduced dose, concentration or frequency of administration of an antibiotic.

[0135] The methods generally comprise administering (a therapeutically effective amount of) an antibacterial protein complex to a subject in need thereof. In some cases, the methods and uses of the invention may result in a reduction in bacterial load, e.g., a reduction of ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90% or 100% compared to before treatment. Methods for determining bacterial load are well known in the art, e.g., infection assays.

[0136] In some cases, the antibacterial protein complex can be administered in combination with administration of an antibiotic. In some cases, the polypeptide is used in a method of treating a subject, wherein the method comprises administering the antibacterial protein complex to the subject and administering the antibiotic to the subject. The polypeptide and the antibiotic can be co-administered, for example from a pharmaceutical composition comprising both agents, or can be administered consecutively in either order over an effective time frame, for example, both administrations are within a period of one week or 5, 4, 3, 2, or one day, or within 20, 15, 12, 10, 8, 6, 5, 4, 3, 2, 1 hour, or within 30, 20, 10, or 5 minutes.

[0137] Bacterial infection is a Gram-negative infection, such as infection with the following bacteria: from the class of gammaproteobacteria, or from the order of Enterobacteriaceae, or from the family of Enterobacteriaceae, Pseudomonas, Acinetobacter or Yersiniaceae. Some examples include pathogenic Salmonella, Escherichia, Shigella, Yersinia and Klebsiella bacteria. As described herein, one or more receptor binding domains and / or bacterial targeting moieties of the complex are capable of binding to a ligand or a portion thereof (e.g., at least 10%, 20%, 30%, 40%, %0%, 60%, 70%, 80% or 90%) of the bacterial surface causing infection. Most commonly, at least 50% of the target bacteria express ligands on their surface and / or are sensitive to the antibacterial activity of the complex.

[0138] When the complex is administered in combination with an antibiotic, the antibiotic is preferably an antibiotic active against Gram-negative bacteria. The antibiotic can be selected based on the type of subject (e.g., human subject) or for a particular type of application (e.g., for treating wound infections) or for a particular mode of administration as described herein.

[0139] "Subject" refers to an animal, a plant, a unicellular organism, or a cell culture. For example, the term "subject" is intended to include organisms, such as prokaryotes and eukaryotes, that are susceptible to bacterial infections (e.g., gram-negative infections) or suffer from bacterial infections (e.g., gram-negative infections). Some examples of objects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. Most commonly, the subject is a human, for example, suffering from a gram-negative infection, being at risk of suffering from a gram-negative infection, or being susceptible to a gram-negative infection. The bacterial infection to be treated or prevented can be systemic or localized, or otherwise concentrated or confined to a specific organ or tissue.

[0140] The present invention also relates to a method of formulating a pharmaceutical composition, for example for treating bacterial infection or a disease or complication associated therewith, for example as described herein. The method comprises mixing the antibacterial protein complex of the present invention with an acceptable carrier to prepare a composition.

[0141] The present invention also relates to the use of a polypeptide of the present invention for the preparation of a medicament for the treatment of a bacterial infection or a disease or complication associated therewith, for example as described herein.

[0142] Dosage and Administration

[0143] The dosage of the antibacterial protein complex administered may depend on many factors, such as the activity of the infection being treated; the activity of the specific complex of the invention; the nature and activity of the antibiotic (if any) paired with the polypeptide according to the invention; and the combined effects of such pairings. The dosage may also vary according to parameters related to the subject to be treated, such as age, weight and physical condition; the route of administration; and the desired regimen. The optimal dosage may be determined by conducting preliminary in vitro and in vivo efficacy experiments. The physician will be able to determine the route of administration and dosage required for a particular individual.

[0144] For the polypeptides disclosed herein, the therapeutically effective dose can be initially estimated in cell culture assays or in animal models (usually mice, rabbits, dogs or pigs). Animal models can also be used to achieve the desired concentration range and route of administration. The information obtained can then be used to determine the effective dose in other subjects (e.g., humans) and the route of administration. Dosage and administration can also be adjusted to provide sufficient levels of active ingredients or to maintain the desired effect. Additional factors that may be considered include the severity of the disease state; the age, weight and sex of the patient; diet; the desired duration of treatment; method of administration; time and frequency of administration; drug combinations; reaction sensitivity; tolerance / response to treatment; and the judgment of the treating physician.

[0145] Proteins and protein complexes are typically administered at 1 pg to 1 mg, more typically 1 pg to 10 μg for particle-mediated delivery, and 1 μg to 1 mg, more typically 1 to 100 μg, more typically 5 to 50 μg for other routes. Typically, it is expected that each dose will contain 0.01 to 3 mg. The optimal amount for a particular treatment can be determined by studies involving observation of clinical responses in subjects.

[0146] Administration of the antibacterial protein complex or pharmaceutical composition may be local, ie, applied directly to the site where its effect is desired (eg, directly to a wound), or systemic.

[0147] Prevent, destroy or eradicate biofilms

[0148] The antibacterial protein complexes of the present invention can also be used in compositions and methods for preventing, destroying or eradicating bacterial biofilms (e.g., Gram-negative biofilms, such as those caused by Klebsiella pneumoniae). Bacterial biofilms are clusters of bacteria attached to surfaces and / or to each other and embedded in a self-generated matrix. The biofilm matrix contains substances such as proteins (e.g., fibrin), polysaccharides (e.g., alginate), and extracellular DNA. For example, Klebsiella pneumoniae has the ability to aggregate into biofilms and is one of the main pathogens in nosocomial infections, showing a high resistance rate to antibiotics. Therefore, the polypeptides of the present invention can be used in methods for preventing, destroying or eradicating bacterial biofilms, and such methods are provided by the present invention.

[0149] The method may comprise contacting a surface (e.g., a biological or non-biological surface) with a composition comprising an antibacterial protein complex of the invention. The surface may be contacted so that biofilm is prevented, destroyed, reduced, or eradicated. The antibacterial protein complex of the invention may be used in combination with an antibiotic, as disclosed herein.

[0150] The surface may be a biological surface, such as a solid biological surface, such as skin. Alternatively, the surface may be a non-biological surface, such as the surface of a medical device. Some of such medical devices include contact lenses; drug pumps; implants, including dental implants, cardiac implants such as pacemakers, prosthetic heart valves, ventricular assist devices, artificial vascular grafts and stents; catheters, including peritoneal dialysis catheters, indwelling catheters for hemodialysis and for long-term administration of chemotherapeutic agents (Hickman catheters), urinary catheters and prosthetic devices, including urinary tract prostheses, prosthetic / artificial joints (e.g., hips); orthopedic materials; and endotracheal and ventilator catheters.

[0151] In some embodiments of the methods of treatment described herein, which are related subjects (e.g., pharmaceutical compositions, medicaments, and their production and use), a subject may suffer from a Gram-negative bacterial infection associated with a biofilm. Such bacterial infections include tonsillitis, osteomyelitis, bacterial endocarditis, sinusitis, corneal infection, urinary tract infection, biliary tract infection, infected kidney stones, urethritis, prostatitis, middle ear infection, dental plaque formation, gingivitis, periodontitis, cystic fibrosis, wound infection, particularly wounds associated with diabetes, and medical device infection, such as catheter infection and joint prosthesis and heart valve infection.

[0152] Products with antibacterial surfaces

[0153] The inventors have shown that nuclease bacteriocin: immune protein complexes can be bound to solid surfaces (e.g., agarose gel beads) via immune polypeptides and still retain their antibacterial properties (see Example 6 herein). Before the bacteriocin translocation, the immune polypeptide can remain bound to the C domain of the nuclease bacteriocin, and the bacteriocin can still translocate across the bacterial outer membrane and be cytotoxic to the bacterial cells. Therefore, the antibacterial protein complexes of the present invention can be attached or coated on products to provide them with an antibacterial surface. For example, the C-terminal cysteine ​​on the immune polypeptide can be connected to the surface of a suitable product, such as through a maleimide bond. The present invention provides methods for providing products with antibacterial surfaces, and products with such antibacterial surfaces. In some embodiments, the method comprises providing an antibacterial protein complex comprising an immune polypeptide and a protein bacteriocin nuclease; and binding the antibacterial protein complex to the product surface. In other embodiments, the method for providing a product with an antibacterial surface comprises binding the immune polypeptide to the product surface; and binding the protein nuclease bacteriocin to the surface-bound immune protein.

[0154] Another advantage of these embodiments of the invention is that once the complex is attached via the immunity protein, if the antibacterial properties of the surface become depleted, i.e. due to loss of nuclease bacteriocin polypeptides (due to translocation into contacting bacteria or other reasons), the surface can be replenished by contacting the surface with fresh nuclease bacteriocin polypeptides, which will bind to the available immunity polypeptides on the surface and provide renewed antibacterial activity. Thus, the invention also provides a method of replenishing the antibacterial activity of an antibacterial surface of a product, the method comprising providing a surface bound to an immunity protein, and contacting the surface with a nuclease bacteriocin (composition comprising a nuclease bacteriocin), wherein the nuclease bacteriocin (C domain of the nuclease bacteriocin) is bound to the immunity protein.

[0155] The present invention also provides a product having an antibacterial surface, wherein the surface comprises an antibacterial protein complex comprising an immune polypeptide and a protein bacteriocin nuclease. In some embodiments, the product is any one described herein.

[0156] In some embodiments, the immune polypeptide, nuclease bacteriocin, or antibacterial protein complex can be any of those described herein.

[0157] Medical devices and related products

[0158] The present invention also provides medical devices incorporating the antibacterial protein complexes of the present invention. For example, the polypeptide can be coated or (covalently or non-covalently) attached to the surface of the device (e.g., as described herein), or the device or its surface can be impregnated with the antibacterial protein complex. The method of incorporation will depend on the type of device and its use, and can be determined by a skilled person. Incorporation of the antibacterial protein complex can prevent or reduce the risk of bacterial infection (e.g., biofilm formation) with or without antibiotics.

[0159] Exemplary medical devices that can incorporate a polypeptide of the invention include, for example, catheters, tracheostomy tubes, wound drainage devices / catheters, stents, implants, introducers, stylets, sutures, shunts, gastrostomy tubes, cardiovascular stents, prostheses, pacemakers and ICD pulse generators, grafts, valves and implants, surgical guidewires, medical tubing, intravenous catheters, urinary catheters, Foley catheters, vascular access and dialysis catheters, peritoneal dialysis catheters, pacing leads, urinary catheters, wound dressings, medical sheets, endotracheal tubes, tracheostomy tubes and surgical repair constructs and meshes, wound dressings, sutures, sterile packaging, or any of the medical devices mentioned above.

[0160] Additional Definitions

[0161] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art in the technical field of this application. The terms used in the description of this application are only for the purpose of describing specific embodiments and are not used to limit this application. The term "and / or" used in this application includes any and all combinations of one or more related listed items.

[0162] Numerical ranges include the numbers that define the range. Therefore, each numerical range disclosed herein is intended to encompass each narrower numerical range that falls within such a wider numerical range, as if such narrower numerical ranges were explicitly written herein. Each maximum (or minimum) numerical limitation disclosed herein is also intended to include each lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were explicitly written herein.

[0163] As used herein, the term "about" means plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20% of the quantitative term.

[0164] "Antibiotics" and variants thereof refer to intermediates or metabolites of metabolic pathways that can kill or inhibit the growth of at least one microbial cell. Some antibiotics can be produced by microbial cells (e.g., bacteria). Some antibiotics can be chemically synthesized. It should be understood that bacteriocins are different from antibiotics, at least in that bacteriocins refer to gene products (in some embodiments, they undergo additional post-translational processing) or synthetic analogs thereof, while antibiotics refer to intermediates or products of metabolic pathways or synthetic analogs thereof.

[0165] "In combination with" means that two or more agents are administered to a subject together as a mixture, administered to a subject simultaneously as a single agent, or administered to a subject sequentially in any order as single agents.

[0166] Example

[0167] Example 1-

[0168] Im9 fused to the R domains of three different PBs, two of which target E. coli (ColE9 and ColB) and one targets Klebsiella pneumoniae (CloDF13), is used to illustrate the present invention. In the following, these constructs are denoted Im9-ColE9. R 、Im9-ColB TR and Im9-CloDF13 R .

[0169] Colicin E9 binds to the vitamin B12 transporter BtuB (receptor) before passing through the trimeric porin OmpF (translocator) at its N-terminus, allowing colicin E9 to interact with TolB in the periplasm. TolB is a component of the stimulated transperiplasmic Tol-Pal system. By binding to TolB, colicin E9 translocates across the E. coli cell envelope in an energy-dependent manner, allowing the delivery of the cytotoxic DNA enzyme domain to the cytoplasm.

[0170] Colicin B binds to the TonB-dependent receptor FepA (receptor and translocation protein) of ferric colicin and directly enters the pore through FepA in a TonB-dependent manner (Cohen-Khait et al., 2021; Hilsenbeck et al., 2004). Colicin B inserts the depolarized pore from the periplasm into the inner membrane.

[0171] Due to a frameshift within the btuB gene, BL21(DE3) cells do not express BtuB and are therefore resistant to colicin E9. Figure 2 As shown, Im9-ColB was designed TRFusion protein in which ColB (lacking its C-terminal cytotoxic domain) is linked to the C-terminus of Im9. Despite the N-terminal His-tag and approximately 37 kDa fused to its C-terminus, Im9-ColB TR Still bound to colicin E9.

[0172] Preparation of colicin E9, Im9-ColB in a concentration range of 1 μM to 5.7 pM TR and Im9-ColB TR Composite colicin E9 (colicin E9:Im9-ColB TR ) were prepared by serial dilutions of the bacteriocin. 5 μl of each dilution was spotted on a soft agar lawn seeded with BL21(DE3) cells. The plates were grown overnight at 37°C, where bacteriocin activity resulted in Figure 3 Transparent areas are shown. Colicin E9 and Im9-ColB TR Neither showed activity against BL21(DE3), but when combined as a complex, killing was observed at concentrations as low as 50 pM.

[0173] To verify that the R domain within colicin E9 had no effect on killing, residues 317 to 488 corresponding to the R domain (colicin E9 ΔR ) is missing. Figure 4 As shown, colicin E9 was tested in a concentration range of 1 μM to 5.7 pM. ΔR 、Im9-ColB TR and colicin E9 ΔR :Im9-ColB TR The activity of the complex against soft agar lawn inoculated with NEB5α cells. ΔR :Im9-ColB TR ) are active as toxins.

[0174] Example 2

[0175] By constructing Im9 linked to the R domain of colicin E9 (Im9-ColE9 R ), further exploring the use of Im9-R fusions to target bacteriocins ( Figure 5 ).

[0176] like Figure 6 As shown, colicin E9 was tested in a concentration range of 1 μM to 5.7 pM. ΔR 、Im9-ColE9 R and colicin E9 ΔR :Im9-ColE9 RActivity of the complex against soft agar lawns seeded with NEB5α cells. E9 only ΔR :Im9-ColE9 R The complex is active as a toxin.

[0177] Example 3

[0178] The R domain of enterocloacin DF13 (residues 301 to 460) was fused to Im9 to produce Figure 7 Im9-CloDF13 R .

[0179] Colicin E9:Im9-CloDF13 R The complex showed no activity against Klebsiella pneumoniae M1-977, however, the complex was active against E. coli expressing the CloDF13 receptor IutA (cloned from Klebsiella pneumoniae). Therefore, the lack of killing of Klebsiella pneumoniae may be due to the inability of colicin E9 to interact with Klebsiella porins or Klebsiella TolB. To verify this, CloDF13-E9 was prepared by deleting the receptor binding domain (residues 324 to 459) from enterocin DF13-E9 (a chimera containing the DNA enzyme domain of E9). ΔR .like Figure 8 As shown, in the presence and absence of Im9-CloDF13 R In the case of ΔR Ability to kill Klebsiella pneumoniae.

[0180] Deletion of the R domain of Enterocloacin DF13 rendered it inactive against Klebsiella pneumoniae M1-977, which is sensitive to intact Enterocloacin DF13. Activity could be restored by providing the R domain as a fusion to Im9.

[0181] Example 4

[0182] The cytotoxic activity of klebsiella pneumoniae C-E9 is limited by its low binding rate to its receptor TolC (Housden et al., 2021). Here we show that the killing efficiency of Im-R fusions, especially Im9-CloDF13 binding to IutA, can be enhanced by first binding to another outer membrane receptor ( Fig. 9 This was accomplished by preparing KlebC-E9:Im9-CloDF13 at 10 μM to 41 nM. R Serial dilutions of 50 μg / mL were prepared and spotted on soft agar lawns of nutrient broth (Merck) inoculated with SR3, SR68, or SG96 Klebsiella pneumoniae. Fig. 9 Comparison of these data with previous equivalent experiments with KlebC-E9 showed that KlebC-E9:Im9-CloDF13 R Enhanced killing of constructs.

[0183] Example 5

[0184] The above examples demonstrate for the first time that the PB R domain does not have to be within the bacteriocin molecule, but can be fused to the Im of the nuclease PB. A logical extension of this observation is that multiple different bacteriocin R domains can be arranged in tandem, allowing a single PB to target multiple outer membrane receptors, significantly broadening the strain selectivity of the PB. A past criticism of PBs as protein antibacterial agents has been their sometimes limited strain coverage. This development negates this problem.

[0185] Im9 and ColE9 R and ColB TR Connect in series as Im9-ColE9 R -ColB TR and with ColE9 ΔR complex.

[0186] ColE9 ΔR :Im9-ColE9 R -ColB TR The complex showed activity against E. coli fepA-BW25113 lacking the colicin B receptor and BL21(DE3) lacking the colicin E9 receptor BtuB ( Fig.10 ). Thus, both receptor binding domains linked in tandem to the immunity protein are functional. Thus, the specificity of a bacterial strain can be expanded by including multiple receptor binding domains linked to an immunity protein.

[0187] Example 6-Im-Surface Functionalization

[0188] The ColE9:Im9 complex is bound to the Sepharose 4B beads by covalent attachment of the Im9 polypeptide of the complex, which has a C-terminal cysteine ​​residue. The C-terminal cysteine ​​of Im is covalently attached to the Sepharose beads via a maleimide bond. The beads were added to a lawn of E. coli JM83 cells. ColE9:Im9 is active against E. coli. Activated Sepharose 4B blocked with cysteine ​​was used as a negative control. See Fig.11 .

[0189] Example 7 - Promiscuous associations between heterologous outer membrane proteins (OMPs)

[0190] This study explains how to target one OMP / bacterial surface ligand with a bacteriocin R domain to bring it into close proximity with other OMPs that can also be targeted / bound by additional R domains and have the necessary translocation proteins. Therefore, it is feasible to use the receptor binding domain from one bacteriocin to deliver a bacteriocin that has a different receptor binding domain in its native form. This also makes it feasible to use different bacterial binding moieties that target different surface ligands to replace the native bacteriocin receptor binding domain attached to the immunity protein.

[0191] β-barrel outer membrane proteins (OMPs) are thought to aggregate into heterogeneous supramolecular assemblies that provide functionality to the impermeable outer membrane (OMP) of Gram-negative bacteria (Rassam et al (2015) Nature 523, 333-336). However, the molecular basis of OM organization was previously unknown. As shown herein, by photoactivatable crosslinking into E. coli OM, coupled with simulations, biochemistry and biophysical analysis for studying OMP organization, the basis of such organization has now been established. A photoactivatable crosslinking strategy was designed, thereby capturing OMP neighbor contacts in living E. coli cells, limiting crosslinking species, reconstructing higher-order OMP complexes in vitro, and incorporating the association principles derived from these data into molecular dynamics and coarse-grained simulations. This approach shows that the asymmetric lipids that make OM an effective impermeability barrier also mediate promiscuous OMP interactions, acting as adhesives to stabilize the OMP network across the bacterial surface.

[0192] A molecular simulation model of supramolecular OMP islands constrained by cross-linking, native MS, fluorescence microscopy, and AFM data was developed to understand how OMP-lipid-OMP complexes lead to higher-order OMP assemblies (Figure 12). The model is based on six principles / assumptions. First, each OMP is surrounded by an asymmetric outer membrane lipid layer. Second, OMPs primarily associate with other OMPs through interfacial lipids rather than being present in the bulk LPS ( Fig. 12A ). Third, networks formed by a large number of porins OmpF / C dominate the OM landscape. Therefore, low-abundance OMPs such as TBDT and LptD are present in these networks. ( Fig. 12C ). Fourth, it is proposed that the “guest” OMPs not only reside in porin-rich regions but also associate with these porins through shared annular lipids, perhaps exploiting the threefold symmetry of the porins ( Fig. 12B Fifth, previous AFM data have shown that the imperfect hexagonal arrays formed by OmpFs in OM are often interspersed with small triangular arrangements of OMPs. The supramolecular model of the present invention involves two aspects of these geometries, the distance between the OmpF-OmpF centers of mass (about 80 to ) and the internal angle of the triangle (about 57 to 63° in our model). Sixth, OMP clusters may be highly diverse in their composition, containing both monomeric OMPs and trimeric OMPs.

[0193] The diameter of the E. coli OMP island is estimated to be about 300 to 500 nm. For the purpose of reducing the complexity of the simulated system, the diameter of the model of the present invention is 200 nm, but this is the size of the BamA cluster observed by super-resolution fluorescence microscopy. This results in an island containing 208 OMPs in a double layer consisting of 16,631 LPS and 56,304 PL molecules. Typically, the trimeric OmpF is surrounded by up to 20 LPS molecules, while the monomer β-barrel is surrounded by 12 to 15 LPS molecules. Assuming that these ratios are reasonable estimates of the E. coli OM structure, they mean that LPS is almost two orders of magnitude more than OMP.

[0194] The simulated OMP island (SOI) was used to explore two aspects of this supramolecular assembly, its stacking and internal mobility. Long-term simulations showed that the interfacial PL was mobile within the island, which is consistent with experimental single-particle tracking data showing that lipoylated mCherry in the OM is diffusive. In contrast, OMP and related LPS molecules are largely immobile, which is also consistent with previous experimental data. As a result, no new LPS-LPS or OMP-LPS interactions were formed or interrupted during the simulation. The original OMP island hypothesis assumes that OMP-OMP associations dominate in the OM to drive OMP aggregation, but in this work, only 1 / 36 BPA mutants determined direct OMP-OMP contacts. SOI is used to explore why such direct contacts are rare. Artificially remove the interfacial LPS and PL from the system, leaving "holes" in the OM. After 2 microseconds of simulation, some OMPs have moved to interact directly with each other, but the holes between them retain the size of the antibiotic vancomycin to some extent. Thus, in the absence of LPS-mediated interactions, OMP-OMP stacking is poor, potentially compromising the barrier function of the membrane.

[0195] The cross-linking data show that although both lipids of OM mediate the promiscuous association between OMPs, LPS is more effective. To understand why, the long-term simulation outcomes of 48-OMP clusters in which OMPs are present in symmetric PL / PL or asymmetric PL / LPS membranes were compared. OMPs and PLs show increased mobility in symmetric membranes, but, as in previous simulations, OMPs and LPSs are stationary in asymmetric membranes. As a result, in symmetric PL bilayers, the triangular and hexagonal lattice arrangements of OMPs disappear rapidly. A closer examination of the OMP-lipid lifetime in the two simulations shows that LPS interacts with OMPs for 97.3% of the duration of the simulation, with contributions from three parts of the molecule (6 lipid tails, head groups, and glycans) being substantially equal. In contrast, the PL interaction lasted for 5.6% of the simulation, and the transient nature of the interaction was the result of its smaller size and reduced tendency to interact with OMPs hydrophobic, polar, and electrostatic interactions. Therefore, SOI explains why BPA-mediated cross-linking with LPS helps stabilize the interconnections between OMPs more effectively than cross-linking with PL.

[0196] The accepted dogma is that Gram-negative bacteria have evolved LPS in the outer leaflet of the OM because its dual hydrophobic / hydrophilic nature serves as an effective permeation barrier for molecules of either polarity. This study suggests that an additional evolutionary driving force may be the preference of LPS to stabilize interactions between neighboring OMPs, which, by reducing fluidity in the membrane, supports the lattice-like supramolecular structure of the OM and thus its integrity.

[0197] This embodiment shows that the basic organizational unit of Gram-negative OM is a non-covalent OMP-lipid-OMP complex. These units are the building blocks of larger OMP islands, in which low-abundance monomeric β-barrel OMPs are accommodated in a broad network formed by trimeric porins. The resulting heterologous structure contains functionally diverse OMPs, which include LptD and BamA. As a result, the molecules deposited by these biogenesis machines (LPS and OMP) do not have to diffuse far to be incorporated into the extended OM, which avoids the problem of restricted diffusion. Individually, lipid-mediated OMP complexes are not very stable and are easily dissociated by detergents. However, in general, when spanning the entire bacterial surface, they constitute a powerful mode of cell envelope stability, which may contribute to the carrying (29,30) of OM. For example, there are 1,418 OMP-LPS-OMP contacts in the 200nm×200nm OMP island model. The island constitutes about 0.06% OM surface area of ​​a typical Escherichia coli cell. Thus, in addition to the stabilization provided by divalent metal ions bridging adjacent LPS molecules, there are a total of >2 million LPS-mediated cross-bridges interconnecting OMPs of the OM.

[0198] Thus, this latest cross-linking and simulation data suggests that OMPs cluster on the bacterial surface and that these clusters are heterogeneous with respect to the OMPs within them. Using the antibacterial complex of the present invention as a means of delivering PBs to kill bacteria achieves two important outcomes. First, it minimizes the risk that the OMP specificity of any one R domain may be lost from the surface. Second, it exploits the avidity of different OMPs that have multiple binding events when clustered together.

[0199] Example 8 - KvarM-Im9 fusion

[0200] M-type bacteriocins kill target cells by degrading peptidoglycan precursors in the periplasm, leading to cell lysis (Schaller et al., 1982). They are found in a range of different species, including Escherichia coli, Pseudomonas, Pectobacterium, Klebsiella, and Burkholderia (Chérier et al., 2021). Colicin M from Escherichia coli is the best-studied example of this type of bacteriocin. KvarM is a new bacteriocin identified from Klebsiella varicola by Dekovskiené et al. (2019) through homology with colicin M. It is a 30.8 kDa protein that binds to and translocates into the periplasm of the cell through the outer membrane ferric pigment receptor FhuA in a process driven by the Ton system. It is of particular interest because it has been shown to be active against a broad spectrum of Klebsiella strains, including those that are multidrug resistant in plate, liquid and biofilm killing assays.

[0201] KvarM and colicin M are organized into three characteristic regions of bacteriocins: an N-terminal unstructured translocation region (Pilsl et al., 1993), a central globular portion that interacts with its outer membrane receptor FhuA, and a C-terminal catalytic region that hydrolyzes the lipid II precursor (Sham et al., 2014). However, the relatively small size and compact folding of the molecules mean that these regions do not form independent folded domains, and attempts to truncate the molecules result in misfolded proteins (Barreteau et al., 2010).

[0202] result

[0203] KvarM retains cytotoxic activity in the presence of C-terminal fusions

[0204] To facilitate fluorophore conjugation for cell labeling assays, a cysteine ​​residue was added to the C-terminus of KvarM (KvarM-cys), followed by a His6 tag. When expressed in E. coli BL21 (DE3) cells, large amounts of protein were produced. At these high concentrations, the added cysteine ​​residue was able to form intermolecular disulfide bonds, resulting in the formation of dimerized KvarM, in which the C-termini of the two molecules were covalently fused ( FIG. 13[A] ). Gel filtration was used to separate monomeric and dimeric KvarM-cys species ( FIG. 13[B] ), and the presence of disulfides was determined by running the protein on a non-reducing SDS-PAGE gel (keeping the disulfide bonds intact) ( FIG. 13[C] ).

[0205] Serial dilutions of wild-type KvarM, monomeric and dimeric KvarM-cys (spanning concentrations from 10 μM to 169 pM) were spotted on a soft agar lawn of K. pneumoniae SG96. Once dry, the plates were incubated overnight at 37°C, with clear areas indicating bacteriocin-mediated killing.

[0206] Unexpectedly, dimeric KvarM-cys, monomeric KvarM-cys, and wild-type KvarM all showed comparable killing efficiency, and killing was observed at concentrations ranging from 10 μM to 14 nM ( Fig.14 ). This suggests the possibility that KvarM, and by extension other homologous M-type bacteriocins, could retain receptor binding, translocation, and significant cytotoxic activity even when bulky fusions are attached to their C-termini.

[0207] KvarM-Im9 fusion retains killing activity

[0208] The observation that KvarM can remain active with a C-terminal fusion inspired the design of a KvarM-immunity protein fusion. Im9 (which provides immunity to the E9 DNase domain) was fused to the C-terminus of KvarM, with two residues (leucine and glutamic acid from the restriction clone scar) separating the two proteins ( Fig.15 SEQ ID NO: 23). The His6 tag used for nickel affinity purification is located at the C-terminus of Im9.

[0209] Serial dilutions of wild-type KvarM and KvarM-Im9 (spanning 10 μM to 169 pM) were spotted on soft agar lawns seeded with Klebsiella pneumoniae SG96. KvarM-Im9 showed clear zones at concentrations as low as 41 nM. This is a concentration range comparable to wild-type KvarM, which showed clear killing zones as low as 14 nM and fuzzy zones as low as 0.5 nM under the same conditions ( FIG. 16 ). This suggests that KvarM-Im9 retains the ability to bind to its receptor FhuA. However, the clear zones produced by KvarM-Im9 are fuzzy, indicating that its cytotoxic activity is impaired when compared to wild-type KvarM. This example demonstrates that Im9, which normally dissociates from colicin E9 upon binding to outer membrane receptors and prior to cellular import (Vankemmelbeke et al., 2009), can translocate to the periplasm of target cells.

[0210] KvarM-Im9 forms a complex with KlebC-E9

[0211] Klebsiella C is a bacteriocin targeting Klebsiella, which binds to cells through the outer membrane protein TolC and translocates into cells during activation by the Ton-system (Housden et al., 2021). KlebC-E9 hybrid ( Fig.17 The sequence shown in Figure 2; SEQ ID NO: 23) kills target cells via the DNase activity of the E9 cytotoxic domain of colicin. The Im9 and E9 DNase domains form a high affinity complex that dissociates when the bacteriocin binds to its receptor on the surface of the target cell (Vankemmelbeke et al., 2009). Using the Im9:E9 DNase interaction, a complex can be formed between the KvarM-Im9 fusion and the KlebC-E9 hybrid. The KvarM-Im9:KlebC-E9 complex should be able to bind to two different outer membrane receptors: FhuA (via the receptor binding domain of KvarM) and TolC (via the receptor binding domain of KlebC). The complex should also have dual cytotoxic activity, using both the peptidoglycan precursor degradation activity of the C-terminal cytotoxic domain of KvarM and the DNase activity of the KlebC-E9 hybrid to kill target cells.

[0212] KlebC-E9 was mixed with 1.5× molar excess of KvarM-Im9 and then run under a gel filtration column ( FIG. 18 [A] ). Fractions from the two peaks in the SEC profile were run on an SDS-PAGE gel ( FIG. 18 [B] ), where both the KvarM-Im9 band and the KlebC-E9 band were observed in the fractions from the first peak, indicating the formation of a complex.

[0213] The KvarM Im9:KlebC-E9 complex displays enhanced killing against Klebsiella strains SG96 and SR3

[0214] Serial dilutions of KlebC-E9, KvarM-Im9 and KvarM-Im9:KlebC-E9 complex were prepared from 6 μM to 0.3 nM and 5 μl were spotted on soft agar lawns seeded with K. pneumoniae SR3 or K. pneumoniae-like SG96.

[0215] The KvarM-Im9:KlebC-E9 complex showed enhanced killing against both K. pneumoniae SR3 and K. pneumoniae-like SG96. In K. pneumoniae SR3 (Figure 19[A]), the complex showed a clear zone at concentrations as low as 0.9 nM - 3 orders of magnitude lower than the lowest concentration at which KlebC-E9 killing activity was observed. Since the rate-limiting step in the cytotoxic activity of Klebsiella C is its slow binding to the outer membrane receptor Tol C (Housden et al., 2021), the ability of the complex to also bind to the outer membrane receptor FhuA concentrates KlebC-E9 on the target cell surface, enhancing killing at lower concentrations. Similarly, in K. pneumoniae-like SG96, an enhancement of killing activity was observed. KlebC-E9 showed a clear zone of inhibition at concentrations as low as 2 nM (a fuzzy zone was observed at concentrations as high as 0.3 nM), while the complex produced a clear zone of inhibition at all concentrations tested. The KvarM-Im9 spot also produced a visible clearing zone at as low as 74 nM, indicating that it was able to direct complex binding by FhuA and maintain both its translocation and cytotoxic activity.

[0216] in conclusion

[0217] The results show that when the nuclease bacteriocin immunity protein is fused to its C-terminus, the M-type bacteriocin can maintain both receptor binding and translocation activity. Through the strong interaction of the fused immunity protein and the nuclease bacteriocin, the M-type bacteriocin-immunity fusion can form a complex with the nuclease bacteriocin. This complex combines the receptor binding and cytotoxic activity of its component bacteriocins, showing improved killing efficiency and expanded strain coverage.

[0218] Example 9 - Different KvarM-Im9 joints

[0219] Three KvarM-Im9 fusions were generated ( Fig. 20 ): KvarM-Im9, wherein KvarM and Im9 are linked by two amino acids (EL) ( Fig.15; SEQ ID NO: 23); KvarM-GS5-Im9, wherein KvarM and Im9 are connected by a flexible linker of five alternating glycine and serine residues and E and L residues ( Fig. 22 ; SEQ ID NO:25); and KvarM-helix-Im9, wherein KvarM and Im9 are connected by a rigid helical linker comprising 5 residues (EAAAK)3 repeated 3 times, followed by E and L residues ( Fig.23 ; SEQ ID NO:27). Although all three KvarM-Im9 fusions retained cytotoxic activity, they showed impaired killing efficiency compared to wild-type KvarM ( Fig.21 ).

[0220] The observed reduction in killing efficiency of the KvarM-Im9 fusions may be caused by steric clashes, in which the presence of the immunity protein interferes with the peptidoglycan precursor hydrolysis activity of the C-terminal cytotoxic region of KvarM. AlphaFold 2 projections of these fusion structures were generated to compare the proximity of the fused immunity protein to the C-terminal active site of KvarM ( Fig. 20 ). In the KvarM-Im9 fusion, the two proteins are linked by 2 amino acids (EL), with the Im9 protein in close proximity to the active site of KvarM. Addition of a flexible glycine-serine linker may exacerbate steric clashes, as Im9 was shown to be pulled further toward the cytotoxic region of KvarM. Conversely, a rigid helical linker ((EAAAK)3EL) was shown to pull Im9 away from the C-terminal cytotoxic region of KvarM, suggesting that this fusion is unlikely to interfere with catalysis.

[0221] These structural predictions are consistent with experimental results ( Fig.21 ), the experimental results showed that KvarM-helix-Im9 maintained the greatest killing activity (characterized by how the killing region was defined), followed by KvarM-Im9, while KvarM-GS5-Im9 showed the lowest activity.

[0222] sequence

[0223] SEQ ID NO: 1—Amino acid sequence of colicin E9 (ColE9).

[0224]

[0225] SEQ ID NO:2 - ColE9 with residues 317 to 448 (corresponding to the R domain) deleted (ColE9 ΔR )'s amino acid sequence.

[0226]

[0227] SEQ ID NO:3—Amino acid sequence of Enterocloacin DF13 (CloDF13).

[0228]

[0229] SEQ ID NO:4 - Amino acid sequence of the enterocloacin DF13-E9 chimera (CloDF13-E9) in which the C domain of cloDF13 is replaced by the C domain of E9.

[0230]

[0231] SEQ ID NO:5—Enterocloacin DF13-E9 (CloDF13-E9) with residues 324 to 459 (corresponding to the R domain) deleted ΔR )'s amino acid sequence.

[0232]

[0233] SEQ ID NO:6—Amino acid sequence of Klebsiella pneumoniae KlebC.

[0234]

[0235] SEQ ID NO:7 - Amino acid sequence of a Klebsiella pneumoniae KlebC-E9 chimera (KlebC-E9) in which the C domain of KlebC is replaced with the C region of E9.

[0236]

[0237] SEQ ID NO:8-Amino acid sequence of Im9

[0238] SEQ ID NO:9—Amino acid sequence of the R domain of CloDF13.

[0239]

[0240] SEQ ID NO: 10—Amino acid sequence of the TR domain of ColB.

[0241]

[0242] SEQ ID NO: 11 - Amino acid sequence of the R domain of ColE9.

[0243]

[0244] SEQ ID NO:12-ColE9 R -ColB TR The amino acid sequence of

[0245]

[0246] SEQ ID NO:13-Im9-ColE9 R The amino acid sequence of

[0247]

[0248] SEQ ID NO:14-Im9-ColB TR The amino acid sequence of

[0249]

[0250] SEQ ID NO:15-Im9-CloDF13 R The amino acid sequence of

[0251]

[0252] SEQ ID NO:16-Im9-ColE9 R -ColB TR The amino acid sequence of

[0253]

[0254] SEQ ID NO: 17—Polynucleotide sequence encoding ColE9.

[0255]

[0256] SEQ ID NO: 18—Polynucleotide sequence encoding ColB.

[0257]

[0258] SEQ ID NO: 19—Polynucleotide sequence encoding CloDF13-E9.

[0259]

[0260]

[0261] SEQ ID NO:20—Polynucleotide sequence encoding KlebC-E9.

[0262]

[0263] SEQ ID NO:21—Polynucleotide sequence encoding Im9.

[0264]

[0265] SEQ ID NO:22-KvarM-Im9 DNA sequence

[0266] SEQ ID NO:23—KvarM-Im9 amino acid sequence

[0267] SEQ ID NO:24-KvarM-GS5-Im9 DNA sequence

[0268] SEQ ID NO:25-KvarM-GS5-Im9 amino acid sequence

[0269] SEQ ID NO:26—KvarM-Helix-Im9 DNA sequence

[0270] SEQ ID NO:27—KvarM-Helix-Im9 amino acid sequence

[0271] SEQ ID NO:28—KvarM amino acid sequence

Claims

1. An antibacterial protein complex comprising (i) a nuclease bacteriocin polypeptide, and (ii) an immunity polypeptide linked to a bacteria binding moiety, wherein the nuclease bacteriocin polypeptide comprises a translocation domain and a nuclease domain, or comprises a translocation domain, a receptor binding domain, and a nuclease domain; and wherein the bacteria binding moiety binds to a ligand on the surface of a Gram-negative bacterium.

2. The antibacterial protein complex according to claim 1, wherein the bacteria-binding portion is a receptor binding domain of a bacteriocin.

3. The antibacterial protein complex of claim 1 or claim 2, wherein the receptor binding domain of the nuclease bacteriocin and the receptor binding domain linked to the immunity polypeptide are different, and / or wherein the receptor binding domain of the nuclease bacteriocin and the bacteria binding portion bind to different Gram-negative bacterial surface ligands and / or different Gram-negative bacterial strains.

4. The antibacterial protein complex according to any one of claims 1 to 3, wherein the immunity polypeptide is linked to multiple bacterial targeting moieties or to multiple bacteriocin receptor binding domains, optionally wherein each bacterial binding moiety binds to a different bacterial surface ligand and / or a different bacterial strain, and / or wherein each bacterial binding moiety binds to a different ligand and / or bacterial strain with the receptor binding domain of the nuclease bacteriocin polypeptide.

5. The antibacterial protein complex according to any one of claims 1 to 4, wherein the bacteria binding moiety linked to the immunity polypeptide is a nanobody.

6. The antibacterial protein complex according to any one of claims 1 to 5, wherein the immunity polypeptide is linked to an M-type bacteriocin.

7. The antibacterial protein complex according to claim 6, wherein the M-type bacteriocin is KvarM.

8. An antibacterial composition comprising the antibacterial protein complex according to any one of claims 1 to 7.

9. The composition of claim 8, which is a pharmaceutical composition.

10. The antibacterial protein complex according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 9 for use in a method of treatment of the human or animal body by therapy.

11. The antibacterial protein complex according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 9, for use in a method for treating or preventing bacterial infection.

12. A method of treating a subject, the method comprising administering to the subject the antibacterial protein complex of any one of claims 1 to 7 or the pharmaceutical composition of claim 9.

13. The method of claim 12, wherein the method is used to treat or prevent a bacterial infection.

14. A medical device comprising the antibacterial protein complex according to any one of claims 1 to 7.

15. One or more polynucleotides encoding the antibacterial protein complex according to any one of claims 1 to 7.

16. One or more vectors comprising one or more polynucleotides of claim 15.

17. A host cell comprising one or more vectors of claim 16.

18. A method for producing the antibacterial protein complex according to any one of claims 1 to 7, the method comprising culturing the host cell according to claim 17, and isolating the antibacterial protein complex from the culture.

19. A product having an antibacterial surface, wherein the surface comprises an antibacterial protein complex comprising an immune polypeptide and a protein bacteriocin nuclease.

20. A method of providing a product having an antibacterial surface, the method comprising: (I) (a) providing an antibacterial protein complex comprising an immunity polypeptide and a protein bacteriocin nuclease; as well as (b) binding the antibacterial protein complex to the surface of the product; or (II) (a) binding the immune polypeptide to the surface of the product; as well as (b) allowing the protein nuclease bacteriocin to bind to the surface-bound immune protein.

21. The product of claim 19 or the method of claim 20, wherein the immunity polypeptide is linked to a bacteria binding moiety which binds to a ligand on the surface of a bacterium.

22. The product according to claim 21, wherein the antibacterial protein complex is the antibacterial protein complex of any one of claims 1 to 7.

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