antibacterial agents

Abstract

The present invention relates to nuclease bacteriocin-derived antimicrobial protein complexes, their preparation and use, and related products having antimicrobial surfaces. The complexes comprise a nuclease bacteriocin polypeptide and an immunity polypeptide linked to a bacteria-binding moiety.

Description

[Technical Field]

[0001] The present invention relates to modified conjugates of bacterial nucleases and immunity polypeptides and their use as antibacterial agents. The present invention also relates to pharmaceutical compositions and medical uses of the antibacterial conjugates, as well as methods for making the same. The present invention also relates to articles having antibacterial surfaces comprising bacterial nuclease-immunity protein conjugates, and methods for making the same. [Background technology]

[0002] The widespread emergence of antibiotic-resistant bacteria and the slowdown in the discovery of new classes of antibiotics are considered serious public health problems. Years of overuse of antibiotics have enabled infectious organisms to develop resistance to antibiotics. As many antibiotics have lost their effectiveness, there is increasing pressure to identify new types of antibacterial molecules. Of particular concern are infections caused by Gram-negative bacteria. Gram-negative pathogens have an impermeable outer membrane that prevents the entry of many classes of antibiotics. However, some antibacterial agents can cross the outer membrane through porins present on the bacterial surface, such as OmpF and OmpC.

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

[0004] PB receptors are typically outer membrane proteins that are usually involved in the active uptake of nutrients (TonB-dependent receptors), the passive diffusion of nutrients and metabolites (trimeric porins), or the 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 the target receptor, which can be strain-specific, and the high incidence of acquired resistance, for example, due to receptor mutations.

[0005] Therefore, there is still a need to develop new antibacterial agents with a broader spectrum of use and lower resistance rates. Summary of the Invention

[0006] Surprisingly, the inventors have found that the receptor-binding function of a nuclease bacteriocin heterodimeric complex can be transferred from the nuclease bacteriocin polypeptide to the immunity polypeptide component of the PB-Im complex without impairing the nuclease bacteriocin's function as an antibacterial agent. That is, despite the relocation of the receptor domain from PB to Im, the immunity polypeptide still binds to the nuclease domain, the translocation domain still interacts with the outer membrane protein translocator, and the immunity protein, along with the receptor-binding domain, still is released / dissociated during translocation of the nuclease domain. Thus, PB-Im still targets bacterial cells, and the nuclease domain still crosses the membrane, becoming cytotoxic upon release of the immunity protein. Importantly, this separation of cell-killing function from surface-binding function makes it possible to reprogram and / or broaden receptor specificity and, therefore, strain susceptibility to specific bacteriocins. Specificity and / or efficacy can be altered or expanded by replacing or supplementing the receptor-binding domain with another receptor-binding domain that binds to an immunity polypeptide rather than a nuclease polypeptide. Furthermore, the inventors recognized that transferring the receptor-binding function from a nuclease protein to an immunity protein allows the use of different receptor-binding moieties other than the receptor-binding domain of an existing bacteriocin. In particular, the receptor-binding domain of a naturally occurring bacteriocin polypeptide must easily unfold to enable translocation and translocation into the target bacterial cell along with the nuclease domain. However, the receptor-binding moiety that binds to an immunity polypeptide and is released to the surface when the bacteriocin polypeptide translocates does not require such unfolding properties. Thus, different types of receptor-binding moieties can be used, further expanding the ability to engineer protein bacteriocins to target different ligands / receptors and different bacterial strains.

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

[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 and the receptor-binding domain attached to the immunity polypeptide are different and / or bind to different Gram-negative bacterial surface ligands and / or different Gram-negative bacterial strains. In some embodiments, the immunity polypeptide is attached to multiple bacteria-binding moieties or multiple bacteriocin receptor-binding domains, optionally, each bacteria-binding moiety binds to a different bacterial surface ligand and / or different bacterial strain and / or each bacteria-binding moiety binds to a different ligand and / or bacterial strain than the receptor-binding domain of the nuclease bacteriocin polypeptide.

[0009] The present invention further provides antimicrobial compositions comprising the antimicrobial protein complex, hi some embodiments, the composition is a pharmaceutical composition. The present invention further provides an antimicrobial protein complex or pharmaceutical composition of the present invention for use in a method of therapeutically treating the human or animal body. The present invention further provides the use of an antimicrobial protein complex of the present invention in the manufacture of a medicament. The present invention further provides a method of therapeutically treating a human or animal subject, the method comprising administering an antimicrobial protein complex or pharmaceutical composition of the present invention to the subject. In each of these embodiments, the treatment may be for the purpose of preventing or treating a bacterial infection or a complication associated therewith.

[0010] The present invention further provides a medical device coated and / or impregnated with the antimicrobial protein complex of the present invention. The medical device may be for in vivo use in a subject in need thereof. The present invention also relates to the in vivo use of a medical device coated and / or impregnated with the antimicrobial protein complex of the present invention, i.e., use in a subject in need thereof.

[0011] The present invention further provides one or more polynucleotides encoding the antimicrobial protein complexes of the present invention. The present invention further provides one or more vectors comprising the polynucleotide(s). The present invention further provides host cells comprising the vector(s). The present invention further provides a method for producing the antimicrobial protein complexes of the present invention, comprising culturing a host cell of the present invention and isolating the antimicrobial protein complex from the culture.

[0012] The present invention further provides an article having an antimicrobial surface or coating, wherein the surface or coating comprises an antimicrobial protein complex comprising an immunity polypeptide and a protein bacteriocin nuclease. The present invention also provides a method of providing an article having an antimicrobial surface, comprising: (a) providing an antimicrobial protein complex comprising an immunity polypeptide and a protein bacteriocin nuclease, and (b) binding the antimicrobial protein complex to a surface. The complex may be bound to the surface via the immunity polypeptide. In another embodiment, the method comprises: (a) binding an immunity polypeptide to the surface of the article, and (b) binding a protein nuclease bacteriocin to the surface-bound immunity protein. In these embodiments, the immunity polypeptide may be bound to a bacteria-binding moiety that binds to a ligand on the bacterial surface. The complex may be any of the antimicrobial protein complexes of the present invention.

[0013] The present disclosure will now be described in more detail, by way of example and not limitation, with reference to the accompanying drawings. Many equivalent modifications and variations will be apparent to those skilled in the art upon reading the present disclosure. Accordingly, the exemplary embodiments shown in this disclosure are considered to be illustrative and not limiting. Various changes can be made to the embodiments described in this disclosure without departing from the scope of the present disclosure. All documents cited herein, whether supra or infra, are expressly incorporated by reference in their entirety.

[0014] The present disclosure includes combinations of the described aspects and preferred features unless such combinations are clearly impermissible or are expressly described as being avoided. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0015] Section headings are used herein for convenience only and should not be construed as limiting in any way. In the following examples, the experimental methods without specific conditions generally follow conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are generally all commercially available products. [Brief explanation of the drawings]

[0016] [Figure 1]Left: The colicin E9:Im9 complex forms a translocon complex on the E. coli cell surface and binds with high affinity to BtuB (Housden et al., 2005), allowing the unstructured N-terminus to pass through OmpF and bind to TolB in the periplasm (Francis et al., 2021; Housden et al., 2013). (Middle) TolB binding activates the unfolding of colicin E9, which then passes through OmpF, releasing Im9 extracellularly. The colicin is degraded 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 an agar plate. [Figure 2-1] Nucleotide and amino acid sequences of Im9-ColBTR. [Figure 2-2] Nucleotide and amino acid sequences of Im9-ColBTR. [Figure 3] Cytotoxic activity of (left) colicin E9, (center) Im9-ColBTR, and (right) colicin E9:Im9-ColBTR against E. coli BL21(DE3) cells grown as a lawn on agar plates. [Figure 4] Cytotoxic activity of (left) colicin E9ΔR, (center) Im9-ColBTR, and (right) colicin E9ΔR:Im9-ColBTR against E. coli NEB5α cells grown as a lawn on agar plates. [Figure 5] Nucleotide and amino acid sequences of Im9-ColE9R, which contains residues 294 to 455 of colicin E9. [Figure 6] Cytotoxic activity of (left) colicin E9ΔR, (center) Im9-ColE9R, and (right) colicin E9ΔR:Im9-ColE9R against E. coli NEB5α cells. [Figure 7] Nucleotide and amino acid sequences of Im9-CloDF13R, which contains residues 301 to 460 of cloacin DF13. [Figure 8]Cytotoxic activity of (left) cloacinDF13ΔR, (middle) Im9-CloDF13R, and (right) cloacinDF13ΔR:Im9-CloDF13R against Klebsiella quasipneumoniae M1-977 cells. [Figure 9] Cytotoxic activity of (left) KlebC-E9 and (right) KlebC-E9 complexed with Im9-CloDF13R against Klebsiella strains SG96, SR3, and SR68, showing how pre-binding to the IutA receptor (via the receptor-binding domain of cloacin DF13) enhances the killing efficiency of KlebC. [Figure 10] Activity of ColE9ΔR:Im9-ColE9R-ColBTR on soft agar lawns inoculated with E. coli fepA-BW25113 (left) and BL21(DE3) (right) cells, creating a zone of clearance due to bacteriocin function. FepA- cells lack the colicin B receptor, while BL21(DE3) cells lack the colicin E9 receptor BtuB. Both receptor-binding domains within Im9-ColE9R-ColBTR are functional, as ColE9ΔR:Im9-ColE9R-ColBTR is active against both strains. [Figure 11] A bacterial lawn of E. coli JM83 with Sepharose beads. Left: Activated Sepharose blocked with cysteine. Right: Activated Sepharose derivatized with Im9 to which colicin E9 is bound. [Figure 12-1]OMP-lipid-OMP complexes are the functional units of supramolecular OMP assemblies that span the outer membrane (OM) of Escherichia coli. A: Instantaneous images from MD simulations of the OmpF-LPS / PL-BtuB complex show how asymmetric lipids are covalently linked to each other, generating a tightly packed interface. OmpF middle barrel residues L259 and I273 are highlighted. UV-activated cross-linking at these sites results in cross-linking to either LPS or PL. B: Instantaneous images from MD simulations of heterogeneous lipid-mediated complexes formed between trimeric OmpF and three different monomeric β-barrels, FepA, BtuB, and FhuA. The threefold symmetry of OmpF (and possibly OmpC) allows various OMPs to be recruited to the porin, and BPA lipid cross-linking may sufficiently stabilize the complex, allowing purification. C: AFM (tapping mode) imaging of the OM of live E. coli MG1655 cells labeled with FepA-bound ColB-mCherry (shown in gray circles). The phase image shows the trimeric porin network, with pores indicated by small gray circles. Peaks in the height image indicate the location of ColB-mCherry fluorescent labeling. Superimposing the FepA position with the trimeric porin indicates that FepA is embedded within the porin network. Additionally, there are areas where no OMPs appear, likely representing patches of previously identified LPS-rich regions. The scale bar is 50 nm. The color (phase / height) scales are 1.2° / 2 nm and 1.2°, respectively. D: A model of an OMP island in which OmpF hosts heterologous OMPs within its hexagonal array. The island dimensions are approximately the same as those observed by live cell imaging. The monomeric β-barrels incorporated into the island are BtuB, FhuE, LeptDE, FepA, and FhuA, identified in this study. BAM, a component of the OMP biogenesis machinery, represented by BamA, has also been identified within the OMP island. E: A schematic diagram showing OMP islands distributed throughout the cell in E. coli cells. The high copy number of OmpF causes the porin to be spread over an area much larger than the island itself. [Figure 12-2]OMP-lipid-OMP complexes are the functional units of supramolecular OMP assemblies that span the outer membrane (OM) of Escherichia coli. A: Instantaneous images from MD simulations of the OmpF-LPS / PL-BtuB complex show how asymmetric lipids are covalently linked to each other, generating a tightly packed interface. OmpF middle barrel residues L259 and I273 are highlighted. UV-activated cross-linking at these sites results in cross-linking to either LPS or PL. B: Instantaneous images from MD simulations of heterogeneous lipid-mediated complexes formed between trimeric OmpF and three different monomeric β-barrels, FepA, BtuB, and FhuA. The threefold symmetry of OmpF (and possibly OmpC) allows various OMPs to be recruited to the porin, and BPA lipid cross-linking may sufficiently stabilize the complex, allowing purification. C: AFM (tapping mode) imaging of the OM of live E. coli MG1655 cells labeled with FepA-bound ColB-mCherry (shown in gray circles). The phase image shows the trimeric porin network, with pores indicated by small gray circles. Peaks in the height image indicate the location of ColB-mCherry fluorescent labeling. Superimposing the FepA position with the trimeric porin indicates that FepA is embedded within the porin network. Additionally, there are areas where no OMPs appear, likely representing patches of previously identified LPS-rich regions. The scale bar is 50 nm. The color (phase / height) scales are 1.2° / 2 nm and 1.2°, respectively. D: A model of an OMP island in which OmpF hosts heterologous OMPs within its hexagonal array. The island dimensions are approximately the same as those observed by live cell imaging. The monomeric β-barrels incorporated into the island are BtuB, FhuE, LeptDE, FepA, and FhuA, identified in this study. BAM, a component of the OMP biogenesis machinery, represented by BamA, has also been identified within the OMP island. E: A schematic diagram showing OMP islands distributed throughout the cell in E. coli cells. The high copy number of OmpF causes the porin to be spread over an area much larger than the island itself. [Figure 12-3]OMP-lipid-OMP complexes are the functional units of supramolecular OMP assemblies that span the outer membrane (OM) of Escherichia coli. A: Instantaneous images from MD simulations of the OmpF-LPS / PL-BtuB complex show how asymmetric lipids are covalently linked to each other, generating a tightly packed interface. OmpF middle barrel residues L259 and I273 are highlighted. UV-activated cross-linking at these sites results in cross-linking to either LPS or PL. B: Instantaneous images from MD simulations of heterogeneous lipid-mediated complexes formed between trimeric OmpF and three different monomeric β-barrels, FepA, BtuB, and FhuA. The threefold symmetry of OmpF (and possibly OmpC) allows various OMPs to be recruited to the porin, and BPA lipid cross-linking may sufficiently stabilize the complex, allowing purification. C: AFM (tapping mode) imaging of the OM of live E. coli MG1655 cells labeled with FepA-bound ColB-mCherry (shown in gray circles). The phase image shows the trimeric porin network, with pores indicated by small gray circles. Peaks in the height image indicate the location of ColB-mCherry fluorescent labeling. Superimposing the FepA position with the trimeric porin indicates that FepA is embedded within the porin network. Additionally, there are areas where no OMPs appear, likely representing patches of previously identified LPS-rich regions. The scale bar is 50 nm. The color (phase / height) scales are 1.2° / 2 nm and 1.2°, respectively. D: A model of an OMP island in which OmpF hosts heterologous OMPs within its hexagonal array. The island dimensions are approximately the same as those observed by live cell imaging. The monomeric β-barrels incorporated into the island are BtuB, FhuE, LeptDE, FepA, and FhuA, identified in this study. BAM, a component of the OMP biogenesis machinery, represented by BamA, has also been identified within the OMP island. E: A schematic diagram showing OMP islands distributed throughout the cell in E. coli cells. The high copy number of OmpF causes the porin to be spread over an area much larger than the island itself. [Figure 13-1]KvarM-cys dimerizes. [A] Diagram showing the structure of the dimer domain of KvarM-cys. [B] A280 absorbance profile of gel filtration of KvarM-cys. 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. [Figure 13-2] KvarM-cys dimerizes. [A] Diagram showing the structure of the dimer domain of KvarM-cys. [B] A280 absorbance profile of gel filtration of KvarM-cys. 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. [Figure 14] Dimeric KvarM-cys retains cytotoxic activity. Plate showing the cytotoxic activity of wild-type KvarM (left), monomeric KvarM-cys (middle), and dimeric KvarM-cys (right) against Klebsiella quasipneumoniae SG96 cells. [Figure 15-1] Nucleotide and amino acid sequences of KvarM-Im9. [Figure 15-2] Nucleotide and amino acid sequences of KvarM-Im9. [Figure 16] KvarM-Im9 retains cytotoxic activity. [A] Plate showing the cytotoxic activity of wild-type KvarM (left) and KvarM-Im9 (right) against Klebsiella quasipneumoniae SG96 cells. [B] Schematic diagram of the KvarM-Im9 fusion bound to the outer membrane receptor FhuA. [Figure 17-1] Nucleotide and amino acid sequences of KlebC-E9. [Figure 17-2] Nucleotide and amino acid sequences of KlebC-E9. [Figure 18-1]KvarM-Im9 and KlebC-E9 form a stable complex. [A] A280 absorbance profile of gel filtration of KvarM-cys. Left = complex peak, right = monomer peak. [B] SDS-PAGE of fractions containing the KvarM-Im9:KlebC-E9 complex (center) and excess KvarM-Im9 (right). [Figure 18-2] KvarM-Im9 and KlebC-E9 form a stable complex. [A] A280 absorbance profile of gel filtration of KvarM-cys. Left = complex peak, right = monomer peak. [B] SDS-PAGE of fractions containing the KvarM-Im9:KlebC-E9 complex (center) and excess KvarM-Im9 (right). [Figure 19-1] The KvarM Im9:KlebC-E9 complex has enhanced killing activity. [A] Cytotoxic activity of KlebC-E9, KvarM-Im9, and the KvarM-Im9:KlebC-E9 complex against K. pneumoniae SR3 cells (left) and K. quasipneumoniae SG96 cells (right). [B] Receptor binding and cytotoxic activity of KlebC-E9. [C] KvarM-Im9 receptor binding and cytotoxic activity. [D] KvarM-Im9:KlebC-E9 complex receptor binding and cytotoxic activity. [Figure 19-2] The KvarM Im9:KlebC-E9 complex has enhanced killing activity. [A] Cytotoxic activity of KlebC-E9, KvarM-Im9, and the KvarM-Im9:KlebC-E9 complex against K. pneumoniae SR3 cells (left) and K. quasipneumoniae SG96 cells (right). [B] Receptor binding and cytotoxic activity of KlebC-E9. [C] KvarM-Im9 receptor binding and cytotoxic activity. [D] KvarM-Im9:KlebC-E9 complex receptor binding and cytotoxic activity. [Figure 20] Alphafold2 structure prediction and schematic diagram of Im9 (above / right of barrel structure) fused to the C-terminus of KvarM (barrel and below-barrel structures) via no linker (left), a flexible glycine-serine linker (middle), or a rigid helical linker (right). [Figure 21] Cytotoxic activity of wild-type KvarM, KvarM-Im9, KvarM fused to Im9 by a flexible glycine-serine linker (KvarM-GS5-Im9), and KvarM fused to Im9 by a rigid helix (KvarM-helix-Im9) against K. quasipneumoniae SG96 cells. [Figure 22-1] Nucleotide and amino acid sequences of KvarM-GS5-Im9. [Figure 22-2] Nucleotide and amino acid sequences of KvarM-GS5-Im9. [Figure 23-1] Nucleotide and amino acid sequences of KvarM-helix-Im9. [Figure 23-2] Nucleotide and amino acid sequences of KvarM-helix-Im9. DETAILED DESCRIPTION OF THE INVENTION

[0017] Array Description SEQ ID NO: 1 shows the amino acid sequence of colicin E9 (ColE9). SEQ ID NO: 2 is ColE9 lacking residues 317 to 448, which correspond to the R domain (ColE9 ΔR ) amino acid sequence.

[0018] SEQ ID NO: 3 shows the amino acid sequence of cloacin DF13 (CloDF13). SEQ ID NO: 4 shows the amino acid sequence of the cloacin DF13-E9 chimera (CloDF13-E9), which contains the DNase domain of E9.

[0019] SEQ ID NO: 5 is cloacin DF13-E9 (DF13-E9) lacking the R domain (residues 324-459). ΔR ) amino acid sequence. SEQ ID NO: 6 shows the amino acid sequence of Klebsiella pneumoniae klebicin C (KlebC).

[0020] SEQ ID NO: 7 shows the amino acid sequence of Klebsiella pneumoniae klebicin C (KlebC)-E9 chimera (KlebC-E9), which contains the DNase domain of E9. SEQ ID NO: 8 shows the amino acid sequence of Im9.

[0021] SEQ ID NO: 9 shows the amino acid sequence of the R domain of CloDF13. SEQ ID NO: 10 shows the amino acid sequence of the NTR domain of ColB. SEQ ID NO: 11 shows the amino acid sequence of the R domain of ColE9.

[0022] SEQ ID NO: 12 is ColE9 R -ColB TR The amino acid sequence of SEQ ID NO: 13 is Im9-ColE9 R The amino acid sequence of SEQ ID NO: 14 is Im9-ColB TR The amino acid sequence of

[0023] SEQ ID NO: 15 is Im9-CloDF13 R The amino acid sequence of SEQ ID NO: 16 is Im9-ColE9 R -ColB TR The amino acid sequence of SEQ ID NO: 17 shows the polynucleotide sequence encoding ColE9.

[0024] SEQ ID NO: 18 shows the polynucleotide sequence encoding ColB. SEQ ID NO: 19 shows the polynucleotide sequence encoding CloDF13-E9. SEQ ID NO: 20 shows the polynucleotide sequence encoding KlebC-E9.

[0025] SEQ ID NO: 21 shows the polynucleotide sequence encoding Im9. SEQ ID NO: 22 shows the polynucleotide sequence encoding KvarM-Im9. SEQ ID NO: 23 shows the amino acid sequence of KvarM-Im9.

[0026] SEQ ID NO: 24 shows the polynucleotide sequence encoding KvarM-GS5-Im9. SEQ ID NO: 25 shows the amino acid sequence of KvarM-GS5-Im9.

[0027] SEQ ID NO: 26 shows the polynucleotide sequence encoding KvarM-helix-Im9. SEQ ID NO: 27 shows the amino acid sequence of KvarM-helix-Im9.

[0028] SEQ ID NO: 28 shows the amino acid sequence of KvarM. Antibacterial protein complexes The first step in bacterial protein bacteriocin (PB)-mediated killing is the formation of a high-affinity complex between the PB's receptor-binding (R) domain and the outer membrane receptor of the target organism. We demonstrated that the R-domain can be cleaved from the PB and fused to the PB's immunity protein (Im), resulting in an Im-R fusion. We also showed that such an engineered Im-R fusion enables tangential delivery of PB into bacteria (so-called frankincense). This enables new antibacterial agents that can be engineered to target one or more species-specific protein receptors on the cell surface.

[0029] The repertoire of surface receptors that can be targeted is currently limited by the PBs that have already been identified. This approach is limited because the species coverage of a PB (i.e., how many strains of a particular species it can kill) is determined by how frequently the receptor is found on the cell surface. If a receptor is rarely found on the outer membrane or its expression is regulated by growth conditions, this will limit the strain coverage of the PB. However, according to the present invention, surface binding of the PB is separated from its ability to cross the membrane and kill specific bacterial species, eliminating the need to use only existing receptor-binding domains in hybrid PBs. Furthermore, multiple outer membrane receptors can be targeted via multiple or tandem receptor-binding moieties through a single construct, thereby ensuring broad species coverage without the need to create a PB cocktail. Additionally, the inventors have recognized that outer membrane proteins (OMPs) are clustered on the surface of Gram-negative bacteria. The arrangement of OMPs in these clusters brings together many of the surface ligands that can function as receptors for the PB-Im complex, along with the translocators required for cellular uptake and cytotoxicity. Specific interactions between the R-domain and the receptor drive the concentration of the bacteriocin on the surface of target bacteria, and altering the identity of this domain allows the construction of hybrid bacteriocins with switchable receptor specificity.

[0030] Thus, the inventors have discovered a way to separate the surface-binding function from the cell-killing function, which currently limits the use of nuclease PB as an antibacterial agent. The key to this discovery is the discovery that an immunity protein derived from nuclease PB can be fused to the R domain of PB. This Im-R fusion can form a complex with nuclease PB, forming a bacteriocin:Im-R complex. The R domain concentrates the bacteriocin at the outer membrane. PB translocation occurs. The Im-R fusion typically remains on the cell surface, except in embodiments where the Im-R fusion itself can translocate into the cell (as described elsewhere herein). This novel approach can be adapted to any type of surface receptor, including known PB R domains, custom nanobodies, or other binding moieties, such as aptamers generated against any outer membrane protein of choice.

[0031] Nuclease Bacteriocin Polypeptides The present invention provides an antibacterial protein complex comprising (i) a nuclease bacteriocin polypeptide and (ii) an immunity polypeptide bound to a bacteria-binding moiety. Thus, the nuclease bacteriocin polypeptide is complexed with the immunity polypeptide. This complex is typically a heterodimeric complex. The nuclease bacteriocin polypeptide typically comprises a translocation domain and a nuclease domain, or a translocation domain, a receptor-binding domain, and a nuclease domain. The nuclease bacteriocin may be any suitable naturally occurring nuclease bacteriocin or variant thereof, or may comprise its NT, R, and / or C-domains. Examples are described, for example, in Sharp et al. (2017) PLoS Comp Biol. 13, e1005652. Variants are as described herein and are typically engineered to improve aspects of function while maintaining other activities of the polypeptide or domain as described herein. For example, variants may be derived from another bacterial species, thereby evading the inherent immunity of a particular bacterial population.

[0032] Nuclease bacteriocin polypeptides typically contain a central receptor-binding domain (R-domain), an N-terminal region (NT domain) that interacts with a bacterial surface translocator, and a (C-terminal) cytotoxic nuclease domain (C) (see Figure 1). However, in complexes of the present invention, the R-domain may or may not be present, as the receptor-binding function is associated with (or provided by) the immunity polypeptide component of the complex. The NT region typically contains a largely unstructured N-terminal region (N) that initially interacts with the bacterial surface translocator, and a translocation domain (T) that facilitates translocation of the nuclease bacteriocin polypeptide through the translocator. The translocator may be, for example, the OmpF translocator, or any one of OmpC, PhoE, OmpK35 (a homolog of OmpF), OmpK36 (a homolog of OmpC), or TolC.

[0033] The C domain typically has DNase or RNase (rRNase or tRNase) activity. The C domain is cytotoxic to Gram-negative bacteria when present within the bacterial cell and not bound to an immunity polypeptide. Its cytotoxicity is neutralized upon binding to an immunity polypeptide. A complex of a nuclease bacteriocin and an immunity polypeptide is formed by the binding of the C domain of the nuclease bacteriocin polypeptide to the immunity polypeptide.

[0034] The R domain binds to a ligand / outer membrane protein (OMP) on the bacterial surface. Examples of outer membrane proteins to which naturally occurring bacteriocins bind include BtuB, Tsx, TolC, OmpA, FepA, Cir, FhuA, IutA, FpvAI, FpvAII, FptA, FiuA, Hur, and porins such as OmpF, OmpC, OmpK35, and OmpK36. The R domain can be a natural R domain associated with the NT domain and / or C domain of the same bacteriocin in the natural protein. Alternatively, the nuclease bacteriocin can be a chimeric protein containing the NT domain, C domain, and / or (optionally) R domain of a different naturally occurring nuclease bacteriocin, or variants thereof.

[0035] Examples of nuclease bacteriocin polypeptides that can be used in the present invention include ColE2, ColE7, ColE8, and ColE9 (DNases), ColE3, ColE4, ColE6, klebicin C, and cloacin DF13 (16S RNase), ColE5, and ColD (tRNA RNase). Nuclease bacteriocin polypeptides used in the present invention can include the N, T, NT, R, and / or C domains of any of these bacteriocins, or other suitable bacteriocins known to those of skill 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 with an R domain of a different nuclease bacteriocin polypeptide, such as any of the nuclease bacteriocins mentioned herein, e.g., having the R domain of any of ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, or ColE8. In other embodiments, the nuclease bacteriocin polypeptide is cloacin DF13 (CloDF13) (SEQ ID NO: 3), or CloDF13 lacking its native R domain, or CloDF13 with an R domain of a different nuclease bacteriocin polypeptide, such as those mentioned herein. In other embodiments, the nuclease bacteriocin polypeptide is klevicin C (SEQ ID NO: 6) or klevicin C with the R domain of a different nuclease bacteriocin polypeptide, such as those referenced herein. The functional domain of a nuclease bacteriocin polypeptide, e.g., the R domain, can be determined based on the primary sequence of the polypeptide, available solved structures, by comparing the primary sequence to other bacteriocins with available solved structures, or using prediction software such as the AlphaFold and AlphaFold2 programs.

[0036] Variants of known nuclease bacteriocins, or functional domains thereof, can be used as long as the variants still have antibacterial activity. Thus, typically, the variants still translocate across the outer membrane and maintain cytotoxic nuclease activity. The variants may retain the ability to bind to the cell surface of Gram-negative bacteria via the R domain, although in some embodiments, this function may be entirely provided by the moiety attached to the immunity protein, as described herein. Otherwise, a variant may in some cases have, for example, at least 70%, 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 a known naturally occurring nuclease bacteriocin polypeptide, or a nuclease bacteriocin polypeptide. may comprise NT, R, and / or C domains having at least 70%, 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 domain(s) of a known naturally occurring nuclease bacteriocin, e.g., as described herein. As discussed further below, the same is true for variants of the R domain of known bacteriocins (including pore-forming and nuclease bacteriocins) that are attached to the immunity polypeptide of the complex, according to some embodiments of the invention.

[0037] For purposes of the present invention, 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 into the first sequence for optimal alignment with the second sequence). The amino acids at each position are then compared. If a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, the amino acids at that position are identical. The percent identity or similarity between two sequences depends on the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence (e.g., SEQ ID NO: 1) × 100, or % identity = number of identical positions / total number of positions in either sequence × 100).

[0038] Typically, sequence comparison is carried out over the entire length of the reference sequence, for example, SEQ ID NO: 1 herein.If a sequence is shorter than the reference sequence, gaps or missing positions should be considered as non-identical positions.However, in some cases, sequence comparison can alternatively be carried out over the entire length of the sequence compared with the reference sequence.If the reference sequence is shorter than the comparison sequence, gaps or missing positions should be considered as non-identical positions.

[0039] Those skilled in the art are aware of different computer programs available for determining the homology or identity between two sequences using mathematical algorithms. In one embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using a Blosum 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Other suitable programs include the BESTFIT program (for example, used with default settings) provided by the UWGCG package (Devereux et al. (1984) Nucleic Acids Research 12, 387-395), and the PILEUP and BLAST algorithms (for example, used with default settings), as described in, for example, 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 analysis is publicly available through the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ).

[0040] The variants described herein may contain one or more modifications from the amino acid sequence of the reference sequence by substitution, deletion, and / or addition. For example, the modifications may include, where appropriate, up to 50, up to 40, 30, 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions, and / or deletions from the amino acid sequence of the reference sequence. For example, the modifications may include replacing an amino acid with 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 with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is replaced with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain is replaced with 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 with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is replaced with 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 with another hydrophobic or hydrophilic amino acid, respectively. Making conservative substitutions can be useful for various reasons, such as improving stability, improving the ability to synthetically produce a polypeptide, or introducing a group that allows additional functionality, such as easily cross-linking a polypeptide into a trimer.

[0041] The percent similarity between two sequences, such as amino acid sequences, is calculated similarly to the percent identity described above, except that substitutions of an amino acid at the same position in the aligned sequences with a different but similar amino acid are counted as the same amino acid. Some properties of the 20 main amino acids that can be used to select appropriate substitutions are as follows:

[0042] [Table 1]

[0043] As used herein, sequences having a possible range of percentages of "identity or similarity" include sequences having both a minimum percentage identity and a different, higher minimum percentage similarity, each percentage within the disclosed percentage range. For example, the sequences may have at least 70% sequence identity and at least 80%, 85%, or 90% sequence similarity, or the sequences may have at least 80% sequence similarity and at least 85%, 90%, 95%, or 100% sequence similarity.

[0044] Immunopolypeptides An "immunity protein" / "immunity polypeptide" / "bacterial immunity polypeptide" is an inhibitor of a nuclease bacteriocin that binds to the cytotoxic nuclease domain and neutralizes its activity. In the context of the present invention, the immunity polypeptide is a "nuclease-specific immunity polypeptide." Any suitable nuclease-specific immunity polypeptide known in the art, or a variant thereof, such as those described above, can be used in the present invention. Typically, the immunity polypeptide and the C domain of the nuclease bacteriocin are a naturally occurring pair or a variant thereof, i.e., a variant that maintains their complex formation activity. For example, in some embodiments, the immunity polypeptide is Im9 (e.g., having the sequence of SEQ ID NO: 8) or a variant thereof. Im9 binds to the C domain of ColE9. Thus, 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. Examples of other known immunity proteins include Im2, Im7, ImD, Im9, Im3, Im4, Im5, and Im6. Typically, immunity proteins have very high affinity for the C-domain of the nuclease bacteriocin polypeptide of the complex, e.g., at least 10 for the RNase-Im complex. -10 K of M d , or at least 10-10 M, 10 -11 M, 10 -12 K of M d (e.g., at pH 7 and 25°C, using, e.g., stopped-flow fluorometry, as described, e.g., in Walker et al. (2003) Biochemistry 42, 4161), or at least 10 for DNase-Im complexes. -10 M or at least 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, or 10 -14 K of M d (e.g., as an RNase-Im complex or as described in Wallis et al. (1995) Biochemistry 34, 13743-13750). However, the immunity polypeptide dissociates from the nuclease bacteriocin polypeptide when the nuclease bacteriocin polypeptide translocates across the outer membrane. The immunity polypeptide is typically a small polypeptide of about 8 kDa to about 20 kDa.

[0045] Bacterial binding part The antimicrobial protein complex comprises an immunity polypeptide bound to a bacteria-binding moiety (which may also be referred to as a "bacterial targeting site"). The bacteria-binding moiety binds to a ligand, e.g., an OMP, present (expressed) on the surface of Gram-negative bacteria. The bacteria-binding moiety thus binds the complex to the surface of bacterial cells expressing the ligand on their surface. The bacteria-binding moiety also directs the complex to the vicinity of a translocator, which allows the nuclease bacteriocin to enter the cell. In some embodiments, the bacteria-binding moiety has an affinity constant (K) for the ligand. D ) value may be 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, or 0.05 nM or less. DValues can be measured by any suitable means known in the art, for example, ELISA, surface plasmon resonance (Biacore), or stopped-flow fluorescence at 25°C.

[0046] The bacteria-binding moiety can be selected to target the conjugate to a particular strain of one or more Gram-negative bacteria. For example, in some embodiments, the target strain(s) are one or more Enterobacteriaceae, Pseudomonaceae, and / or Acinetobacter bacteria; or one or more strains of Escherichia coli, Salmonella, Serratia, Shigella, and / or Enterobacter bacteria, such as E. coli, Salmonella enterica, Serratia marcescens, Shigella sonnei, Acinetobacter baumanii, and Enterobacter cloacae.

[0047] Examples of ligands that can bind to the bacterium-binding moiety include the common ligands that bind to the R domains of the known nuclease bacteriocins mentioned above (e.g., BtuB, Tsx, TolC, OmpA, FepA, Cir, FhuA, IutA, FpvAI, FpvAII, FptA, FiuA, Hur, and porins such as OmpF, OmpC, OmpK35, and OmpK36). However, one of the major advantages 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 one that is bound by the R domain of any bacteriocin, including, for example, Tsx, OmpF, OmpA, Cir, FhuA, and Tip-pilus F / N. Other ligands that can bind include OmpC, LptD, OmpF, BtuB, FhuE, FhuA, FepA, and BamA, as described in Example 7 herein. Also included are other surface-expressed TonB-dependent transporters (TBDTs). Bacterial binding moieties that bind other ligands, such as LPS, are also contemplated. Targeting essential proteins such as BamA and LptD is a particularly attractive option because it minimizes the potential for resistance. However, the ligands that can be bound are essentially limited only by the ability to generate appropriate binding moieties, which is generally within the skill of those skilled in the art for essentially any ligand / OMP. Thus, those skilled in the art can select appropriate target ligands and appropriate binding moieties to suit their needs.

[0048] In some embodiments, the bacterium-binding portion is a receptor-binding domain of a bacteriocin, or a suitable variant thereof, e.g., as described above. In some embodiments, the bacterium-binding portion is a receptor-binding domain of a nuclease bacteriocin, such as the R domain of any of colicins E2-E9 (ColE2-ColE9), cloacin DF13 (CloDF13), or klebicin C (KlebC), or a suitable variant thereof. Another example is colicin G. Another example is pyocin / pyosin S2. In some embodiments, the translocation domain (T) and / or the N domain, or part or all of the region N-terminal to the R domain of a bacteriocin, may also be included. For example, ColB binds to FepA as its receptor and is then translocated through the same FepA molecule. Thus, ColB-TR is a single domain. In some embodiments, the bacteria-binding moiety is or includes a receptor-binding domain of a non-nuclease bacteriocin, such as the R domain of any one of the pore-forming bacteriocins colicin A, B, E1, Ia, Ib, N, K, U, 5, and 10, or the receptor-binding domain of colicin M or KvarM. Another example is pyosin / pyosin S5. Other suitable examples are described in Sharp et al. (2017) PLoS Comp Biol. 13, e1005652.

[0049] In some embodiments, the immunity polypeptide (Im-R) bound to the bacterium-binding moiety may be complexed with a second cytotoxic domain. In some embodiments, Im-R may translocate into target cells. Thus, in some embodiments, Im-R may be both a second toxin and a targeting device for the complexed PB. In some embodiments, the bacterium-binding moiety may be or include the entire PB, or a variant thereof. Specifically, the bacterium-binding moiety may be or include an M-type bacteriocin or colicin M homologue, such as colicin M itself or KvarM, or a functional variant thereof, such as those described herein, that retain receptor binding, translocation, and / or cytotoxic activity. Example 8 herein demonstrates that an M-type bacteriocin can maintain receptor binding, translocation, and significant cytotoxic activity even with large fusions at its C-terminus. Thus, Im-R may include an M-type bacteriocin, such as KvarM, with an immunity polypeptide fused C-terminally to the M-type bacteriocin sequence. Specific examples are the KvarM sequences of SEQ ID NO: 23, 25, 27, or SEQ ID NO: 28. Other sequence elements may also be included, such as a linker as described herein (e.g., as described in Examples 8 and 9), or one or more additional bacteria-binding moieties.

[0050] In other cases, the bacteria-binding moiety is selected or generated to bind to a specific target ligand, such as an antibody or antigen-binding fragment thereof, or an aptamer, rather than the R domain of the bacteriocin.

[0051] Nanobodies / single-domain antibodies (sbAbs) / antibody fragments consisting of a single monomeric variable antibody domain are particularly suitable for use as bacteria-binding moieties. Furthermore, because Im is not translocated into target cells, the immune polypeptide-bacteria binding moiety does not need to be mechanically unstable; nanobodies (containing internal disulfide bonds) can be used. Nanobodies are described, for example, in Holt et al. (2003), Trends in Biotechnology 21(11):484-490. In some embodiments, the nanobody is a human or humanized nanobody. A fully human antibody is one in which the variable and constant regions (if present) of both the heavy and light chains are all human-derived or substantially identical to human-derived sequences, although not necessarily from the same antibody.

[0052] Suitable aptamers can be produced 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, aptamers are at least 15 nucleotides in length, e.g., about 15 to about 50, about 20 to about 40, or about 25 to about 30 nucleotides in length.

[0053] In some cases, the antimicrobial protein complex of the present invention comprises more than one bacterium-binding moiety. In some embodiments, the antimicrobial protein complex comprises 2 to 10, 2 to 5, or 2 to 4 bacterium-binding moieties. In particular, the antimicrobial protein complex may comprise two or more bacterium-binding moieties attached to an immunity polypeptide, as further described below. Alternatively or additionally, the nuclease bacteriocin polypeptide of the complex may comprise a receptor-binding domain, which is also a bacterium-binding moiety. Thus, in a typical example, a complex having two bacterium-binding moieties may have one bacterium-binding moiety that is the R-domain of the nuclease bacteriocin and one bacterium-binding moiety attached to an immunity protein; or the nuclease bacteriocin may lack an R-domain and have two bacterium-binding moieties attached to an immunity polypeptide. In another exemplary example, a complex having three bacteria-binding moieties may have one bacteria-binding moiety that is the R-domain of a nuclease bacteriocin and two bacteria-binding moieties attached to immunity proteins; or the nuclease bacteriocin may lack an R-domain and have three bacteria-binding moieties attached to immunity polypeptides.

[0054] Typically, the multiple bacterial binding moieties have different identities. Typically, the multiple bacterial binding moieties bind to different surface ligands. The different surface ligands may be expressed by the same target Gram-negative bacterium. Such complexes are particularly useful for avoiding the development of resistant bacteria in target cells, because even if the target cell mutates or otherwise stops expressing the ligand bound by one of the bacterial binding moieties, the bacterium can still be targeted using one or more other bacterial binding moieties. Alternatively or additionally, the complex may contain multiple bacterial binding moieties that bind to different surface ligands on the surface of different target bacterial cells or strains. Such complexes can target a broader range of target cells than complexes that do not contain multiple bacterial binding moieties that can bind to different surface ligands on the surface of different target bacterial cells or strains.

[0055] In some embodiments, the immunity polypeptide is bound to multiple bacterial targeting sites. Including multiple bacterial targeting sites bound to the immunity polypeptide is particularly useful because the immunity polypeptide is not translocated into the target cell, and therefore the bacterial targeting sites do not need to be translocated, as is the case with the receptor-binding domain of a naturally occurring bacteriocin nuclease. On the other hand, the inclusion of multiple bacterial binding moieties may broaden the range of bacterial strains that can be targeted, improve targeting efficiency, and / or reduce resistance, for example, by introducing mutations in one or more ligands that prevent binding by one or more bacterial binding moieties.

[0056] In some cases, one or more, or each, of the multiple bacterial target sites may 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) may be included. In some embodiments, one or more, or each, of the multiple bacterial target sites may be a bacterial target site other than a bacteriocin receptor binding domain, as described elsewhere herein. In some cases, the multiple bacterial target sites may be multiple copies of the same moiety. In other cases, the multiple bacterial target sites are different and / or bind to different bacterial surface ligands and / or different bacterial strains, as described above.

[0057] In some embodiments, the immunity polypeptide and the bacteria-binding moiety are fused, i.e., present as a chimeric polypeptide containing the amino acid sequences of both the immunity polypeptide and one or more bacterial target sites / domains. Typically, the immunity polypeptide (sequence) is N-terminal (of the chimeric polypeptide sequence) and the bacteria-binding moiety(s) is / are C-terminal (e.g., of the chimeric polypeptide). However, other arrangements are also contemplated, including those having one or more bacteria-binding moieties (sequences) at the N-terminus of the immunity polypeptide (sequence) or having one or more bacteria-binding moieties (sequences) on either side of a central immunity polypeptide (sequence) (i.e., adjacent to, or N- and C-terminal to, the immunity polypeptide sequence). When there are multiple bacterial target sites, these may be arranged in tandem. Thus, an immunity polypeptide referred to herein as being bound to multiple bacteria-binding moieties may, in some cases, be bound to one or more of the bacteria-binding moieties via one or more other bacteria-binding moieties. Furthermore, the term "bacteria-binding moiety" may refer, where appropriate, to a portion of a larger polypeptide / single amino acid chain, or to a domain, having bacterial surface ligand-binding activity. Similarly, the term "immunity polypeptide" may, where appropriate, refer to a portion or domain of a larger polypeptide / single amino acid chain having the properties described herein for "immunity polypeptide."

[0058] The bacteria-binding moiety(s) do not interfere with binding of the immunity polypeptide to the C-domain of the nuclease bacteriocin polypeptide; or interaction of the nuclease bacteriocin polypeptide with the translocator; or dissociation of the immunity polypeptide from the cytotoxic domain of the nuclease bacteriocin upon translocation across the outer membrane.

[0059] In certain exemplary embodiments, the bacteria-binding moiety that binds to the immunity polypeptide, or one of them, 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 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 binds 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 that binds to the bacteria-binding moiety is the receptor-binding domain of bacteriocin CloDF13 (having the amino acid sequence of SEQ ID NO:12) or a variant thereof, or the receptor-binding domain (or T / R domain) of ColB (having the amino acid sequence of SEQ ID NO:13) or a variant thereof. R -ColB TR ) array.

[0060] In certain 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 bound to two bacteria-binding moieties comprising the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 10, or a variant thereof, or SEQ ID NO: 12 (ColE9 R -ColB TR ), or the immunity polypeptide is linked to a bacteria-binding moiety, which may be SEQ ID NO: 13 (Im9-ColE9), as described, for example, in Examples 1-3 and 5. R ), 14(Im9-ColB TR ), 15(Im9-CloDF13 R ) or 16(Im9-ColE9R-ColB TR) is a fusion / chimeric polypeptide comprising the sequence of any one of the following: ColE9 (SEQ ID NO: 1) or a variant thereof, or R-domain deleted ColE9 (SEQ ID NO: 2) or a variant thereof, or CloDF13-E9 (SEQ ID NO: 4) or a variant thereof, such as an R-domain deleted (SEQ ID NO: 5), e.g., as described in Examples 1-3 and 5 herein.

[0061] 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 SEQ ID NO: 15 (Im9-CloDF13), e.g., as described herein in Example 4. R ) the nuclease bacteriocin KlebC-E9 (SEQ ID NO: 7) or a variant thereof.

[0062] 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-GS5-Im9), or SEQ ID NO:27 (KvarM-helix-Im9), or a variant thereof, as described herein in Example 9. In certain exemplary embodiments, the immunity polypeptide, one or more bacteria-binding moieties, immunity polypeptide linked to a bacteria-binding moiety, nuclease bacteriocin, nuclease bacteriocin R-domain, and / or antimicrobial protein complex may be those described in the Examples herein or provided in the Sequence Listing. Polynucleotides, vectors and host cells Polypeptides of the present invention can be produced by any suitable means. For example, polypeptides of the present invention include products of chemical synthesis procedures and products produced by recombinant technology from prokaryotic or eukaryotic hosts, including, for example, bacteria, yeast, higher plants, insects, and mammalian cells. Depending on the host used in a recombinant production procedure, polypeptides of the present invention may be glycosylated or non-glycosylated. Furthermore, polypeptides of the present invention may also include an initial methionine residue. This methionine residue may be derived from the start codon in the encoding nucleic acid used to initiate translation.

[0063] The present invention provides one or more (isolated) polynucleotides (e.g., DNA or RNA) encoding the antimicrobial protein complexes of the 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 invention are set forth herein in SEQ ID NOS: 17-21. Those skilled in the art will recognize that DNA codons are degenerate and can readily envision alternative sequences or corresponding RNA sequences that encode the same polypeptides or variants thereof as described herein.

[0064] The polynucleotides of the present invention may be provided in the form of an expression cassette comprising a control sequence operably linked to the inserted sequence, thereby enabling expression of the polypeptide in vivo. Thus, the present invention also provides one or more expression cassettes encoding one or more polynucleotides of the present invention. These expression cassettes are further typically provided within a vector (e.g., a plasmid or a recombinant viral vector). Thus, in one embodiment, the present invention provides one or more vectors comprising the polynucleotides of the present invention. The vector may be a cloning vector or an expression vector. A suitable vector may be any vector capable of carrying a sufficient amount of genetic information and allowing expression of the encoded polypeptide(s).

[0065] The polynucleotides, expression cassettes, or vectors of the present invention can be introduced into host cells, for example, by transfection. Accordingly, the present invention also provides host cells containing one or more polynucleotides, expression cassettes, or vectors of the present invention. The polynucleotides, expression cassettes, or vectors can be transiently or permanently introduced into host cells to allow the expression of polypeptides. 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 typical prokaryotic cells such as bacterial cells. Suitable host cells can be easily identified by those skilled in the art.

[0066] The invention also provides a method for producing an antimicrobial protein complex of the invention, comprising culturing a host cell containing one or more vectors, expression cassettes, or polynucleotides of the invention under conditions suitable for expression of the polypeptide, which can then be isolated from the culture.

[0067] General methods for constructing vectors or expression cassettes, transfection methods, and culture methods are well known to those skilled in the art. For reference, see "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed.), Wiley Interscience, New York, and the Maniatis Manual, published by Cold Spring Harbor Publishing.

[0068] Products, compositions and uses 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 where the antimicrobial properties of the complexes of the present invention are useful. Examples of compositions containing the antimicrobial protein complexes of the present invention include antimicrobial surface sprays, wound cleansers, and medical lubricants. Other examples of products that may usefully contain the antimicrobial protein complexes of the present invention include food preservatives and animal feeds.

[0069] Pharmaceutical Compositions and Methods of Administration In some embodiments, the present invention relates to a pharmaceutical composition. The composition comprises the antimicrobial protein complex of the present invention. The composition typically further comprises at least one pharmaceutically acceptable excipient, carrier, diluent, buffer, stabilizer, preservative, adjuvant, or other material known to those skilled in the art. Such materials are preferably non-toxic and preferably do not interfere with the pharmacological activity of the active ingredient(s). The pharmaceutical carrier or diluent may be, for example, a water-containing solution. The exact nature of the carrier or other material may depend on the route of administration, for example, oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intradermal, or intraperitoneal routes.

[0070] "Pharmaceutically acceptable carriers" are typically large, slowly metabolized macromolecules such as proteins, sugars, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoleetti et al., 2001, Vaccine, 19:2118), trehalose (WO 00 / 56365), lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those skilled in the art. Pharmaceutical compositions may also contain diluents such as water, saline, glycerol, etc. Additionally, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, etc. may be present. Sterile, pyrogen-free, phosphate-buffered saline is a typical carrier (Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472).

[0071] The pharmaceutical composition of the present disclosure can be in a lyophilized state or in an aqueous form, i.e., in the form of a solution or suspension.This type of liquid formulation is ideal for injection, since it does not need to be reconstituted with an aqueous medium and can be administered directly from the packaged form.The pharmaceutical composition can be provided in a vial or in a pre-filled syringe.The syringe can contain a single dose, and the vial can contain a single dose or multiple doses.

[0072] The liquid formulation of the present disclosure is also suitable for reconstitution of other lyophilized pharmaceuticals.When the pharmaceutical composition is used for such improvised reconstitution, the present invention provides a kit, which may include two vials, or may include one pre-filled syringe and one vial, and the contents of the syringe are used to reconstitute the contents of the vial before injection.

[0073] The pharmaceutical compositions of the present disclosure, particularly when packaged in a multi-dose format, may contain antimicrobial agents as preservatives. Examples include 2-phenoxyethanol or parabens (methyl, ethyl, or propyl parabens). Any preservatives are preferably present at low levels.

[0074] Pharmaceutical compositions of the present disclosure may include surfactants such as, for example, Tween (polysorbate), DMSO (dimethyl sulfoxide), DMF (dimethyl formamide), etc. Surfactants are generally present at low levels, e.g., less than 0.01%, although higher levels, e.g., 0.01-50%, may also be used.

[0075] Pharmaceutical compositions of the present disclosure may contain sodium salts (e.g., sodium chloride) and free phosphate ions (e.g., through the use of a phosphate buffer) in solution. In certain embodiments, the pharmaceutical composition may be encapsulated in a suitable vehicle, for example, to enhance stability. As will be understood by those skilled in the art, various vehicles are suitable for delivering the pharmaceutical compositions of the present disclosure. Non-limiting examples of suitable structured fluid delivery systems include nanoparticles, liposomes, microemulsions, micelles, dendrimers, and other phospholipid-containing systems. Methods for incorporating pharmaceutical compositions into delivery vehicles are known in the art.

[0076] Examples of suitable compositions and administration methods are described in Esseku and Adeyeye (2011) and Van den Mooter G. (2006). Further examples of the above-mentioned techniques and protocols are described in Remington's Pharmaceutical Sciences, 20th Edition, 2000, published by Lippincott, Williams & Wilkins.

[0077] Routes of administration include, but are not limited to, nasal, oral, subcutaneous, intradermal, and intramuscular. Subcutaneous administration can be by injection, for example, into the abdomen, lateral and anterior aspects of the upper arms, thighs, dorsal scapular region, or upper back gluteal region.

[0078] The composition of the present disclosure can be administered in one or more doses and / or by multiple administration routes.For example, such other routes include subcutaneous, intravenous, intravascular, intraarterial, intraperitoneal, intraspinal, intratracheal, intraventricular, intralobar, intramedullary, intrapulmonary, and intravaginal.Depending on the desired treatment period, the composition of the present disclosure can be administered one or more times, or intermittently.

[0079] The solid dosage form for oral administration includes capsules, tablets, caplets, pills, powders, pellets and granules.In such solid dosage forms, active ingredients are usually combined with one or more pharmaceutically acceptable excipients, examples of which are described above in detail.Oral preparations can also be administered as aqueous suspensions, elixirs or syrups.In these cases, active ingredients can be combined with various sweeteners or flavorings, coloring agents, and optionally emulsifiers and / or suspending agents, as well as diluents such as water, ethanol, glycerin and their combinations.

[0080] One or more compositions of the present disclosure may be administered, or the methods and uses for treatment according to the present disclosure may be practiced alone or in combination with other pharmacological compositions or treatments, for example in combination with antibiotics.

[0081] Also included within the scope of the invention are kits comprising the antimicrobial protein complexes of the invention and instructions for use, e.g., in any of the methods of the invention. The kits may further include one or more additional reagents, such as additional therapeutic or prophylactic agents.

[0082] Treatment method The antimicrobial protein complexes of the present invention can be used in methods of treating the human or animal body through therapy. As used herein, the term "treatment" includes therapeutic and prophylactic treatment (although prevention may be considered therapeutic / treatment). Administration is typically a "prophylactically effective amount" or a "therapeutically effective amount," which is an amount sufficient to elicit a clinical response or demonstrate clinical benefit to the individual. For example, treatment can prevent, delay, or shorten the onset of an infection, or disease or condition, ameliorate one or more symptoms, induce or prolong remission, delay recurrence or relapse, or reduce the bacterial load of an infection. Polypeptides can be used in methods of treating or preventing bacterial infections in subjects, or diseases or complications associated therewith, such as sepsis, pneumonia, wound infections, medical device infections (such as catheters), or biofilms. Polypeptides can also be used to alleviate or ameliorate any conditions, symptoms, or side effects associated with antibiotic use, e.g., as described herein, for example, when the polypeptides are used in combination with reduced antibiotic dosages, concentrations, or frequency of administration.

[0083] The method typically involves administering an antimicrobial protein complex (in a therapeutically effective amount) to a subject in need thereof. In some cases, the methods and uses of the present invention may result in a reduction in bacterial load of, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% compared to before treatment. Methods for determining bacterial load are well known in the art and include, for example, infection assays.

[0084] In some cases, the antimicrobial protein complex may be administered in combination with the administration of an antibiotic. In some cases, the polypeptide is for use in a method of treating a subject, the method comprising administering the antimicrobial protein complex to the subject and administering an antibiotic to the subject. The polypeptide and antibiotic may be administered simultaneously, for example, from a pharmaceutical composition containing both agents, or sequentially in either order within an effective time frame, for example, within one week, or within 5, 4, 3, 2, or 1 day(s), or within 20, 15, 12, 10, 8, 6, 5, 4, 3, 2, or 1 hour(s), or within 30, 20, 10, or 5 minutes.

[0085] The bacterial infection is a Gram-negative bacterial infection, such as an infection caused by bacteria of the Gammaproteobacteria class, or the Enterobacteriales order, or the Enterobacteriaceae, Pseudomonas, Acinetobacter, or Yersiniaceae families. Examples include pathogenic Salmonella, Escherichia coli, Shigella, Yersinia, and Klebsiella bacteria. As described herein, one or more of the receptor-binding domains and / or bacterial targeting sites of the complex can bind to a ligand or portion thereof on the surface of infection-causing bacteria, e.g., at least 10%, 20%, 30%, 40%, 0%, 60%, 70%, 80%, or 90% of the bacteria causing the infection. Most typically, at least 50% of the target bacteria express the ligand on their surface and / or are susceptible to the antibacterial activity of the complex.

[0086] When the conjugate is administered in combination with an antibiotic, the antibiotic is preferably one that is active against gram-negative bacteria. The antibiotic may be selected depending on the type of subject (e.g., a human subject), or depending on the particular application (e.g., treatment of wound infection), or depending on the particular mode of administration described herein.

[0087] "Subject" refers to an animal, a plant, a single-cell organism, or a cell culture. For example, the term "subject" is intended to include organisms, such as prokaryotes and eukaryotes, that are susceptible to or suffering from a bacterial infection, such as a gram-negative bacterial infection. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and genetically modified non-human animals. Most typically, the subject is a human, for example, a human suffering from, at risk of, or susceptible to an infection caused by a gram-negative bacteria. The bacterial infection to be treated or prevented may be systemic or local, or concentrated or limited to a specific organ or tissue.

[0088] The present invention also relates to a method for formulating a pharmaceutical composition for treating a bacterial infection or a disease or complication associated therewith, e.g., as described herein, which method comprises mixing an antimicrobial protein complex of the present invention with an acceptable carrier to prepare a composition.

[0089] The present invention also relates to the use of a polypeptide of the present invention in the manufacture of a medicament for the treatment of a bacterial infection, or a disease or complication associated therewith, for example as described herein.

[0090] Dosage and Administration The dosage of the antibacterial protein complex to be administered may depend on many factors, including the activity of the infection to be treated; the activity of the specific complex of the present invention; the nature and activity, if any, of the antibiotic paired with the polypeptide of the present invention, and the combined effect of such a combination. The dosage may also vary depending on parameters related to the treated subject, such as age, weight, and physical condition; the route of administration; and the required regimen. The optimal dosage can be determined by conducting preliminary efficacy experiments in vitro and in vivo. A physician can determine the necessary route of administration and dosage for a particular individual.

[0091] For the polypeptides disclosed herein, therapeutically effective doses can be initially estimated using cell culture assays or animal models, usually mice, rabbits, dogs, or pigs. Animal models can also be used to achieve a desired concentration range and route of administration. The information obtained can then be used to determine effective doses and routes of administration in other subjects, such as humans. Dosage and administration can be further adjusted to ensure sufficient levels of the active ingredient or to maintain the desired effect. Other factors to consider include the severity of the condition, the patient's age, weight, and sex; diet; desired duration of treatment; method of administration; time and frequency of administration; drug combinations; reaction sensitivities; tolerance / response to treatment; and the judgment of the treating physician.

[0092] Proteins and protein complexes are typically administered in the range of 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, and more typically 5 to 50 μg for other routes. Generally, each dose is expected to contain 0.01 to 3 mg. Optimal amounts for a particular treatment can be ascertained by trials involving observation of clinical response in subjects.

[0093] Administration of the antimicrobial protein complex or pharmaceutical composition may be local, ie, applied directly to the desired site of action (eg, directly to a wound), or systemic. Preventing, disrupting, or eradicating biofilms The antimicrobial protein complexes of the present invention are also useful in compositions and methods for preventing, disrupting, or eradicating bacterial biofilms (e.g., Gram-negative bacterial biofilms, such as those caused by Klebsiella pneumoniae). Bacterial biofilms are clusters of bacteria attached to surfaces and / or each other and embedded in a self-produced 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 as biofilms, is one of the major causes of hospital-acquired infections, and is highly resistant to antibiotics. Therefore, the polypeptides of the present invention can be used in methods for preventing, disrupting, or eradicating bacterial biofilms, and such methods are provided by the present invention.

[0094] The method may include contacting a surface (e.g., a surface of biological or non-biological origin) with a composition comprising an antimicrobial protein complex of the invention. The surface may be contacted such that biofilm is prevented, disrupted, reduced, or eradicated. The antimicrobial protein complex of the invention may be used in combination with an antibiotic, as disclosed herein.

[0095] The surface may be a solid biological surface, such as a biological surface such as skin. Alternatively, the surface may be a non-biological surface, such as the surface of a medical device. Examples of such medical devices include contact lenses; drug pumps; implants, such as dental implants, cardiac implants such as pacemakers, artificial heart valves, ventricular assist devices, synthetic vascular grafts, and stents; catheters, such as peritoneal dialysis catheters, indwelling catheters for hemodialysis, and indwelling catheters for chronic administration of chemotherapy (Hickman catheters), urinary catheters, and prosthetic devices such as urinary prostheses, prosthetic / artificial joints (e.g., hip joints); orthopedic materials; and tracheal tubes and ventilator tubes.

[0096] In some embodiments of the treatment methods described herein, the subject may be suffering from a biofilm-associated Gram-negative bacterial infection, including tonsillitis, osteomyelitis, bacterial endocarditis, sinusitis, corneal infection, urinary tract infection, biliary tract infection, infectious kidney stones, urethritis, prostatitis, middle ear infection, dental plaque formation, gingivitis, periodontitis, cystic fibrosis, wound infections, particularly those associated with diabetes, and infections of medical devices, such as catheter infections and infections of artificial joints and heart valves.

[0097] Products with antibacterial surfaces The present inventors have demonstrated that a nuclease bacteriocin:immunity protein complex can be bound to a solid surface (e.g., Sepharose beads) via the immunity polypeptide and still maintain its antibacterial properties (see Example 6 herein). The immunity polypeptide remains bound to the C-domain of the nuclease bacteriocin prior to translocation of the bacteriocin, allowing the bacteriocin to still translocate across the bacterial outer membrane and be cytotoxic to bacterial cells. Thus, the antibacterial protein complex of the present invention can be attached to or coated on an article to confer an antibacterial surface to the article. For example, the C-terminal cysteine of the immunity polypeptide can be attached to the surface of a suitable article, e.g., via a maleimide bond. The present invention also provides a method for conferring an antibacterial surface to an article, and an article having such an antibacterial surface. In some embodiments, the method comprises providing an antibacterial protein complex comprising an immunity polypeptide and a bacteriocin nuclease protein, and conjugating the antibacterial protein complex to the article surface. In another embodiment, a method of providing an article having an antimicrobial surface comprises binding an immunity polypeptide to the surface of the article; and binding a protein nuclease bacteriocin to the surface-bound immunity polypeptide.

[0098] A further advantage of these embodiments of the invention is that once the complex is bound via the immunity protein, if the antimicrobial properties of the surface are diminished, i.e., due to loss of the nuclease bacteriocin polypeptide (due to translocation to contacting bacteria or for other reasons), the surface can be replenished by contacting the surface with a new nuclease bacteriocin polypeptide, which will bind to the active immunity polypeptide on the surface and provide renewed antimicrobial activity. Thus, the invention also provides a method of replenishing the antimicrobial activity of an antimicrobial surface of a product, comprising providing a surface bound to an immunity protein and contacting the surface with (a composition comprising) a nuclease bacteriocin, wherein (the C domain of) the nuclease bacteriocin binds to the immunity protein.

[0099] The present invention also provides an article having an antimicrobial surface, the surface comprising an antimicrobial protein complex comprising an immunity polypeptide and a bacteriocin nuclease protein. In some embodiments, the article is any described herein.

[0100] In some embodiments, the immunity polypeptide, nuclease bacteriocin, or antimicrobial protein complex is any described herein. Medical Devices and Related Products The present invention also provides medical devices incorporating the antimicrobial protein complexes of the present invention. For example, the polypeptides can be coated on the surface of the device or attached (covalently or non-covalently) (e.g., as described herein), or the device or its surface can be impregnated with the antimicrobial protein complexes. The method of incorporation varies depending on the type and use of the device and can be determined by those skilled in the art. The incorporation of the antimicrobial protein complexes can be with or without antibiotics and can prevent or reduce the risk of bacterial infection, such as biofilm formation.

[0101] Examples of medical devices that may incorporate the polypeptides of the invention include 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, pacemaker leads, urinary catheters, wound dressings, medical sheets, endotracheal tubes, tracheostomy tubes, and surgical repair structures and meshes, wound dressings, sutures, sterile packaging, or any of the medical devices listed above.

[0102] Additional definitions Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art of this application. The terms used in the description of this application are intended to describe particular embodiments only and are not intended to limit the scope of this application. The term "and / or" as used in this application includes any and all combinations of one or more of the associated listed items.

[0103] Numerical ranges are inclusive of the numbers defining that range. Accordingly, every numerical range disclosed herein is intended to include every narrower numerical range that falls within that broader numerical range, as if those narrower numerical ranges were all expressly written herein. Also, every maximum (or minimum) numerical limitation disclosed herein is intended to include every lower (or higher) numerical limitation, as if those lower (or higher) numerical limitations were expressly written herein.

[0104] The term "about" as used herein means, in quantitative terms, plus or minus 5%, in another embodiment plus or minus 10%, in another embodiment plus or minus 15%, and in another embodiment plus or minus 20%.

[0105] "Antibiotic" and variations thereof refer to a metabolite or intermediate in a metabolic pathway that kills or arrests the growth of at least one microbial cell. Some antibiotics are produced by microbial cells, such as bacteria. Some antibiotics are chemically synthesized. It is understood that a bacteriocin differs from an antibiotic at least in that a bacteriocin refers to a gene product (which in some embodiments undergoes further post-translational modification) or a synthetic analog thereof, whereas an antibiotic refers to an intermediate or product of a metabolic pathway or a synthetic analog thereof.

[0106] "In combination with" means that two or more agents are administered to a subject simultaneously, in a mixture, as single agents, or sequentially, in any order, as single agents. [Example]

[0107] Example 1 The present invention is demonstrated using Im9 fused to the R domains of three different PBs, two targeting Escherichia coli (ColE9 and ColB) and one targeting Klebsiella quasipneumoniae (CloDF13). Below, these constructs are referred to as Im9-ColE9, respectively. R , Im9-ColB TR , and Im9-CloDF13 R It is written as follows.

[0108] Colicin E9 binds to the vitamin B12 transporter BtuB (receptor) before passing its N-terminus through the trimeric porin OmpF (translocator), enabling it to interact with TolB in the periplasm. TolB is a component of the energy-supplying transperiplasmic Tol-Pal system. Binding to TolB enables colicin E9 to translocate across the E. coli cell envelope in an energy-dependent manner, delivering its cytotoxic DNase domain to the cytoplasm.

[0109] Colicin B binds to the TonB-dependent receptor of the ferric enterobactin receptor FepA (receptor and translocator) and passes directly through FepA in a TonB-dependent manner ( Cohen-Khait et al., 2021 ; Hilsenbeck et al., 2004 ). From the periplasm, colicin B inserts a depolarizing pore into the inner membrane.

[0110] BL21(DE3) cells are resistant to colicin E9 because they do not express BtuB due to a frameshift in the btuB gene. TR The fusion protein was designed by linking ColB, which lacks the C-terminal cytotoxic domain, to the C-terminus of Im9. Despite the fusion of the N-terminal His tag and the C-terminal 37 kDa fragment, Im9-ColB TR still bound to colicin E9.

[0111] colicin E9, Im9-ColB TR , and Im9-ColB TR colicin E9 complexed with Im9-ColB TR Serial dilutions of colicin E9 and Im9-ColB were prepared over a concentration range of 1 μM to 5.7 pM. Five μl of each dilution was spotted onto a soft agar lawn inoculated with BL21(DE3) cells. The plates were grown overnight at 37°C, and a clearance zone was formed due to the activity of the bacteriocin, as shown in Figure 3. TRAlthough neither of these compounds exhibits activity against BL21(DE3), when they form a complex, killing is observed up to a concentration of at least 50 pM.

[0112] To confirm that the R-domain in colicin E9 does not affect killing, residues 317 to 448 corresponding to the R-domain were deleted (colicin E9 ΔR ) Colicin E9 against soft agar lawns inoculated with NEB5α cells. ΔR , Im9-ColB TR and colicin E9 ΔR :Im9-ColB TR The activity of the complex was tested in the concentration range of 1 μM to 5.7 pM, as shown in Figure 4. Again, the activity of the complex (colicin E9 ΔR :Im9-ColB TR ) was active as a toxin.

[0113] Example 2 The use of Im9-R fusions to target bacteriocins was demonstrated using Im9 linked to the R domain of colicin E9 (Im9-ColE9) R ) was further explored by constructing a

[0114] Colicin E9 on soft agar lawns inoculated with NEB5α cells ΔR , Im9-ColE9 R , and colicin E9 ΔR :Im9-ColE9 R The activity of the complex was tested in the concentration range of 1 μM to 5.7 pM, as shown in Figure 6. ΔR :Im9-ColE9 R Only the complex was active as a toxin.

[0115] Example 3 As shown in Figure 7, the R-domain of cloacin DF13 (residues 301 to 460) was fused to Im9 to form Im9-CloDF13. R was produced.

[0116] Colicin E9:Im9-CloDF13 RThe complex was inactive against Klebsiella quasipneumoniae M1-977, but was active against Escherichia coli expressing the CloDF13 receptor IutA cloned from Klebsiella quasipneumoniae. Therefore, the lack of killing of Klebsiella quasipneumoniae is likely due to the inability of colicin E9 to interact with Klebsiella or the porin of Klebsiella TolB. To test this, the receptor-binding domain (residues 324–459) was deleted from cloacin DF13-E9 (a chimera containing the DNase domain of E9), resulting in CloDF13-E9. ΔR Im9-CloDF13 was prepared. R CloDF13-E9 in the presence and absence of ΔR The killing ability of against Klebsiella quasipneumoniae was tested in the concentration range of 10 μM to 57 pM, as shown in Figure 8.

[0117] Deletion of the R domain of cloacin DF13 renders it inactive against Klebsiella quasipneumoniae M1-977, which is sensitive to intact cloacin DF13. Addition of the R domain as an Im9 fusion restores activity.

[0118] Example 4 The cytotoxic activity of KlebC-E9 is limited by its slow binding kinetics to its receptor, TolC (Housden et al., 2021). Here, we first show that Im-R fusions, specifically Im9-CloDF13 and IutA, can enhance killing efficiency by binding to a different outer membrane receptor (Figure 9). This is demonstrated by binding of KlebC-E9:Im9-CloDF13 to IutA at concentrations ranging from 10 μM to 41 nM. RSerial dilutions of KlebC-E9 were prepared and tested by spotting onto nutrient broth (Merck) soft agar lawns inoculated with SR3, SR68 Klebsiella pneumoniae, or SG96 Klebsiella quasipneumoniae. These data are compared with equivalent experiments previously performed with KlebC-E9, as shown in Figure 9, and KlebC-E9:Im9-CloDF13. R Enhanced killing by the construct is shown.

[0119] Example 5 The above examples demonstrate for the first time that the PB R-domain does not have to reside within the bacteriocin molecule but can be fused to the Im of a 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, essentially greatly expanding the strain selectivity of the PB. A previous criticism of PBs as protein antibacterial agents has been that their strain coverage is sometimes limited. This development addresses this issue.

[0120] Im9 is ColE9 R and ColB TR and Im9-ColE9 R -ColB TR As ColE9 ΔR was complexed with ColE9 ΔR :Im9-ColE9 R -ColB TR The complex was active against both E. coli fepA-BW25113, which lacks the colicin B receptor, and BL21(DE3), which lacks the colicin E9 receptor BtuB (Figure 10). Thus, the receptor-binding domains linked in tandem to the immunity protein are both functional. Therefore, the specificity of bacterial strains can be expanded by including multiple receptor-binding domains linked to the immunity protein.

[0121] Example 6 - Im-surface functionalization The ColE9:Im9 complex was bound to Sepharose 4B beads via covalent attachment of the Im9 polypeptide of the complex with a C-terminal cysteine residue. The C-terminal cysteine of Im was covalently attached to the Sepharose beads via a maleimide bond. The beads were added to a lawn of E. coli JM83 cells. ColE9:Im9 showed activity against E. coli. Sepharose 4B activated and blocked with cysteine was used as a negative control. See Figure 11.

[0122] Example 7 – Promiscuous binding between heterologous outer membrane proteins (OMPs) This study demonstrates that targeting one OMP / bacterial surface ligand with a bacteriocin R domain can also provide access to other OMPs, which can then be targeted / bound by additional R domains and the necessary translocators. Therefore, it is possible to use the receptor-binding domain of one bacteriocin to deliver bacteriocins with different native receptor-binding domains. It is also possible to use different bacterial-binding moieties to target different surface ligands instead of the native bacteriocin receptor-binding domain bound to an immunity protein.

[0123] Beta-barrel outer membrane proteins (OMPs) are thought to assemble into heterogeneous supramolecular assemblies that confer functionality to the impermeable outer membrane (OM) of Gram-negative bacteria (Rassam et al. (2015) Nature 523, 333-336). However, the molecular basis of OM organization has remained unclear. As presented herein, a foundation for such organization was established by investigating OMP organization using photoactivated crosslinking to the OM of Escherichia coli, simulations, and biochemical and biophysical analyses. A photoactivatable crosslinking strategy was devised to capture OMP near-contacts in live E. coli cells, identify crosslinked species, reconstitute higher-order OMP complexes in vitro, and incorporate binding principles derived from these data into molecular dynamics and coarse-grained simulations. This approach revealed that asymmetric lipids, which form an effective impermeable barrier to the OM, mediate promiscuous OMP interactions and act as adhesives to stabilize the OMP network across the bacterial surface.

[0124] A molecular simulation model of supramolecular OMP islands, constrained by crosslinking, native MS, fluorescence microscopy, and AFM data, was developed to understand how OMP-lipid-OMP complexes lead to higher-order OMP assemblies (Figure 12). This model is based on six principles / assumptions. First, all OMPs are surrounded by a shell composed of asymmetric outer membrane lipids. Second, OMPs do not reside within a sea of LPS but primarily associate with other OMPs through interfacial lipids (Figure 12A). Third, networks formed by the abundant porin OmpF / C dominate the structure of the OMP. Consequently, less abundant OMPs, such as TBDT and LptD, reside within these networks (Figure 12C). Fourth, it has been suggested that "guest" OMPs not only reside within porin-rich regions but also associate with these porins via covalent cyclic lipids, presumably exploiting the porin's three-fold symmetry (Figure 12B). Fifth, previous AFM data have shown that the imperfect hexagonal arrays formed by OmpF in OMs are often interspersed with small triangular OMP arrays. Our supramolecular model takes into account two aspects of these geometries: the distance between the OmpF-OmpF centers of mass (approximately 80-90 Å) and the internal angle of the triangle (approximately 57-63° in our model). Sixth, OMP clusters are highly diverse in their components and likely contain both monomeric and trimeric OMPs.

[0125] The OMP island in E. coli is estimated to be approximately 300–500 nm in diameter. To reduce the complexity of the simulated system, the model is set to a diameter of 200 nm, which is the size of BamA clusters observed by super-resolution fluorescence microscopy. This resulted in an island containing 208 OMPs within a bilayer consisting of up to 16,631 LPS molecules and 56,304 PL molecules. Typically, a trimeric OmpF is surrounded by up to 20 LPS molecules, while a monomeric β-barrel is surrounded by 12–15 LPS molecules. Assuming these ratios are reasonable estimates of the E. coli OM structure, this means that LPS outnumbers OMPs by nearly two orders of magnitude.

[0126] Simulated OMP islands (SOIs) were used to investigate two aspects of this supramolecular assembly: its packing and internal mobility. Long-term simulations showed that interfacial PL moved within the islands, consistent with experimental single-particle tracking data demonstrating that lipoylated mCherry within the OM is diffusive. In contrast, LPS molecules associated with OMPs barely moved, again consistent with previous experimental data. As a result, no new LPS-LPS or OMP-LPS interactions were formed or disrupted during the simulations. The original OMP island hypothesis assumed that OMP-OMP interactions would predominate within the OM to promote OMP clustering, but in this study, only 1 / 36 of the BPA mutants identified direct OMP-OMP contacts. Using SOIs, we investigated why such direct contacts were rare. Interfacial LPS and PL were manually removed from the system, leaving "holes" in the OM. After 2 microseconds of simulation, some OMPs moved into positions where they directly interacted with each other, but the "hole" between them remained the size of the antibiotic vancomycin. Thus, in the absence of LPS-mediated interactions, OMP-OMP packing was insufficient, potentially compromising the membrane's barrier function.

[0127] The cross-linking data suggest that although both lipids in the OMP mediate promiscuous binding between OMPs, LPS is more effective. To understand why, we performed extended simulations of a 48-OMP cluster and compared the results when the OMPs were present in either symmetric PL / PL or asymmetric PL / LPS membranes. OMPs and PLs exhibited enhanced mobility in the symmetric membrane, whereas, as in previous simulations, OMPs and LPS remained stationary in the asymmetric membrane. As a result, the triangular and hexagonal lattice arrangement of the OMPs was quickly lost in the symmetric PL bilayer. A closer look at the OMP-lipid lifetimes in the two simulations revealed that LPS interacted with the OMP for 97.3% of the simulation, with roughly equal contributions from the three parts of the molecule (six lipid tails, head group, and glycan). In contrast, the PL interaction persisted for only 5.6% of the simulation, and the transient nature of this interaction is a result of its smaller size and reduced propensity for hydrophobic, polar, and electrostatic interactions with the OMP. Thus, SOI explains why BPA-mediated cross-linking to LPS stabilizes interconnections between OMPs more effectively than PL.

[0128] It is generally accepted that Gram-negative bacteria evolved LPS in the outer leaflet of the OM because its dual hydrophobic / hydrophilic nature serves as an effective permeability barrier to molecules of either polarity. This study suggests that a further evolutionary driving force is a stronger tendency for LPS to stabilize interactions between neighboring OMPs, thereby reducing membrane mobility and maintaining the lattice-like supramolecular structure of the OM, thereby preserving its integrity.

[0129] This example demonstrates that the fundamental organizational unit of the Gram-negative OM is the noncovalent OMP-lipid-OMP complex. These units are components of larger OMP islands, which contain low concentrations of monomeric β-barrel OMPs housed within an extensive network formed by trimeric porins. The resulting heterogeneous structure contains functionally diverse OMPs, including LptD and BamA. As a result, the molecules produced by these biosynthetic machinery, LPS and OMPs, respectively, diffuse only short distances to be incorporated into the expanding OM, circumventing the diffusion-limited problem. Lipid-mediated OMP complexes are not very stable on their own and are easily dissociated by detergents. However, when extended across the entire bacterial surface, they constitute a powerful cell envelope stabilization mechanism that may also contribute to OM load tolerance (29,30). For example, in a 200 nm × 200 nm OMP island model, there are 1,418 OMP-LPS-OMP contacts. This island occupies approximately 0.06% of the OM surface area of a typical E. coli cell. Thus, in total, over 2 million LPS-mediated crosslinks interconnect the OMPs of the OM, with additional stabilization by divalent metal ions bridging adjacent LPS molecules.

[0130] Thus, this latest cross-linking and simulation data demonstrate that OMPs assemble at the bacterial surface and that these clusters are heterogeneous with respect to the OMPs within them. Using the antimicrobial complexes of the present invention as a means of delivering PBs to kill bacteria accomplishes two important results: first, it minimizes the risk of depleting the surface of OMPs specific for any one R-domain. Second, it exploits the affinity resulting from multiple binding events of different assembled OMPs.

[0131] Example 8 – KvarM-Im9 fusion M-type bacteriocins kill target cells by degrading peptidoglycan precursors in the periplasm, leading to cell lysis (Schaller et al., 1982). They have been discovered in various species, including Escherichia coli, Pseudomonas, Pectobacterium, Klebsiella, and Bulkholderia (Cherier et al., 2021). Colicin M from Escherichia coli is the most studied example of this class of bacteriocin. KvarM is a novel bacteriocin identified by Dekovskiene et al. from Klebsiella varicola by its homology to colicin M. It is a 30.8 kDa protein that binds to the outer membrane ferrichrome receptor FhuA and translocates across the cell's outer membrane into the periplasm in a process driven by the Ton system. It is of particular interest as it has been shown to be active against a wide range of strains of the genus Klebsiella, including strains that are multidrug resistant in plate, liquid, and biofilm killing assays.

[0132] KvarM and colicin M contain three regions characteristic of bacteriocins: an N-terminal unstructured translocation domain (Pilsl et al., 1993), a central globular section that interacts with the outer membrane receptor FhuA, and a C-terminal catalytic domain that hydrolyzes lipid II precursors (Sham et al., 2014). However, due to the relatively small size and compact folding of the molecules, these regions do not form independent folding domains, and attempts to shorten the molecules result in misfolded proteins (Barreteau et al., 2010).

[0133] result KvarM retains cytotoxic activity in C-terminal fusions To facilitate fluorescent labeling for cell labeling assays, a cysteine residue was added to the C-terminus of KvarM (KvarM-cys), followed immediately by a His6 tag. Expression in E. coli BL21(DE3) cells yielded large amounts of protein. At this high concentration, the added cysteine residue was able to form an intermolecular disulfide bond, resulting in the formation of a dimeric KvarM in which the C-termini of two molecules were covalently fused (Figure 13[A]). Gel filtration was used to separate the monomeric and dimeric KvarM-cys species (Figure 13[B]), and the presence of the disulfide was confirmed by electrophoresis of the protein on a non-reducing SDS-PAGE gel (which kept the disulfide bond intact) (Figure 13[C]).

[0134] Serial dilutions of wild-type KvarM, monomeric, and dimeric KvarM-cys, ranging in concentration from 10 μM to 169 pM, were spotted onto soft agar lawns of Klebsiella quasipneumoniae SG96. After drying, plates were incubated overnight at 37°C to reveal zones of clearance due to bacteriocin-mediated killing.

[0135] Surprisingly, dimeric KvarM-cys, monomeric KvarM-cys, and wild-type KvarM all exhibited similar killing efficiencies, with killing observed at concentrations ranging from 10 μM to 14 nM (Fig. 14). This result suggests that KvarM, and by extension other homologous M-type bacteriocins, may be able to maintain receptor binding, translocation, and significant cytotoxic activity even with large C-terminal fusions.

[0136] KvarM-Im9 fusion retains killing activity The observation that KvarM can maintain activity as a C-terminal fusion inspired the design of a KvarM-immunity protein fusion. Im9 (which confers immunity to the E9 DNAse domain) was fused to the C-terminus of KvarM, with the two proteins separated by two residues (leucine and glutamic acid from a restriction cloning lesion) (sequence shown in Figure 15; SEQ ID NO: 23). A His6 tag used for nickel affinity purification was located at the C-terminus of Im9.

[0137] Serial dilutions of wild-type KvarM and KvarM-Im9, ranging from 10 μM to 169 pM, were spotted onto soft agar lawns inoculated with Klebsiella quasipneumoniae SG96. KvarM-Im9 exhibited a zone of clearance down to 41 nM, a concentration range comparable to that of wild-type KvarM. Under the same conditions, KvarM-Im9 exhibited a clear bactericidal zone down to 14 nM and an equivocal zone down to 0.5 nM (Figure 16). This indicates that KvarM-Im9 retains the ability to bind to its receptor, FhuA. However, the zone of clearance produced by KvarM-Im9 was equivocal, suggesting impaired cytotoxic activity compared to wild-type KvarM. This example demonstrates that Im9, which normally dissociates from colicin E9 upon binding to its outer membrane receptor and prior to cellular uptake (Vankemmelbeke et al., 2009), can be translocated to the periplasm of target cells.

[0138] KvarM-Im9 forms a complex with KlebC-E9 Klebsiella coli bacteriocin C binds and translocates into cells via the outer membrane protein TolC, a method powered by the Ton system (Housden et al., 2021). The KlebC-E9 hybrid (sequence shown in Figure 17, SEQ ID NO: 23) kills target cells via the DNAse activity of the colicin E9 cytotoxic domain. Im9 and the E9 DNAse domain form a high-affinity complex that dissociates upon binding of the bacteriocin to receptors on the target cell surface (Vankemmelbeke et al., 2009). The Im9:E9 DNAse interaction can be exploited to form complexes 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 binds via the receptor-binding domain of KvarM, and TolC binds via the receptor-binding domain of KlebC. This complex should also have dual cytotoxic activity, utilizing both the peptidoglycan precursor-degrading activity of the C-terminal cytotoxic domain of KvarM and the DNAse activity of the KlebC-E9 hybrid to kill target cells.

[0139] KlebC-E9 was mixed with a 1.5-fold molar excess of KvarM-Im9 and applied to a gel filtration column (Fig. 18[A]). Fractions from both peaks in the SEC profile were subjected to SDS-PAGE (Fig. 18[B]). Both KvarM-Im9 and KlebC-E9 bands were observed in the fractions from the first peak, indicating the formation of a complex. The KvarM Im9:KlebC-E9 complex exhibits enhanced killing of Klebsiella strains SG96 and SR3. Serial dilutions ranging from 6 μM to 0.3 nM of KlebC-E9, KvarM-Im9, and the KvarM-Im9:KlebC-E9 complex were prepared, and 5 μl was spotted onto soft agar lawns inoculated with K. pneumoniae SR3 or K. quasipneumoniae SG96.

[0140] The KvarM-Im9:KlebC-E9 complex demonstrated enhanced killing against both K. pneumoniae SR3 and K. quassi-pneumoniae SG96. In K. pneumoniae SR3 (Figure 19[A]), the complex exhibited a clearance zone at concentrations as low as 0.9 nM, three orders of magnitude lower than the lowest concentration at which KlebC-E9 killing was observed. Because the rate-limiting step in the cytotoxic activity of Klebicin C is its slow binding to the outer membrane receptor TolC (Housden et al., 2021), the complex's ability to also bind to the outer membrane receptor FhuA concentrates KlebC-E9 on the target cell surface, enhancing killing at low concentrations. Similarly, enhanced killing was observed in K. quassi-pneumoniae SG96. KlebC-E9 showed a clear zone of inhibition up to 2 nM (although a vague zone was observed up to 0.3 nM), whereas the complex showed a clear zone of inhibition at all concentrations tested. The KvarM-Im9 spot also produced a clear zone of clearance up to 74 nM, demonstrating both direct binding of the complex via FhuA and maintenance of its translocation and cytotoxic activity.

[0141] conclusion These results demonstrate that M-type bacteriocins can maintain both receptor-binding and translocation activities when fused to a nuclease bacteriocin immunity protein at their C-terminus. Due to the strong interaction between the fused immunity protein and the nuclease bacteriocin, the M-type bacteriocin-immunity fusion can form a complex with the nuclease bacteriocin. This complex possesses both the receptor-binding and cytotoxic activities of the component bacteriocins, resulting in improved killing efficiency and broader strain coverage.

[0142] Example 9 – Different KvarM-Im9 linkers Three KvarM-Im9 fusions were generated (Figure 20): KvarM-Im9, in which KvarM and Im9 are linked by two amino acids (EL) (Figure 15; SEQ ID NO: 23); KvarM-GS5-Im9, in which KvarM and Im9 are linked by a flexible linker consisting of five alternating glycine and serine residues followed by E and L residues (Figure 22; SEQ ID NO: 25); and KvarM-helix-Im9, in which KvarM and Im9 are linked by a rigid helix linker consisting of three repeats of five residues (EAAAK) followed by E and L residues (Figure 23; SEQ ID NO: 27). All three KvarM-Im9 fusions retain cytotoxic activity but demonstrate reduced killing efficiency compared to wild-type KvarM (Figure 21).

[0143] The observed decrease in killing efficiency of the KvarM-Im9 fusion may be due to steric hindrance, where the presence of the immunity protein may interfere with the hydrolysis of peptidoglycan precursors by the C-terminal cytotoxic domain of KvarM. AlphaFold2 structural predictions of these fusions were generated to compare the proximity of the fused immunity protein to the C-terminal active site of KvarM (Figure 20). In the KvarM-Im9 fusion, where the two proteins are linked by a two-amino acid residue (EL), the immunity protein is in close proximity to the active site of KvarM. The addition of the flexible glycine-serine linker may have intensified the steric clash, and Im9 appears to be more strongly attracted to the cytotoxic domain of KvarM. On the other hand, the rigid helical linker ((EAAAK)3EL) appears to distance the immunity protein from the C-terminal cytotoxic domain of KvarM, suggesting that this fusion is less likely to interfere with catalysis.

[0144] These structural predictions are consistent with the experimental results (Figure 21), which suggest that KvarM-helix-Im9 retains the highest killing activity (characterized by a distinct zone of killing activity), followed by KvarM-Im9, and that KvarM-GS5-Im9 has the lowest activity.

[0145] array SEQ ID NO:1 - Amino acid sequence of colicin E9 (ColE9). * SEQ ID NO: 2 - ColE9 lacking residues 317-448 corresponding to the R domain (ColE9 ΔR ) amino acid sequence. MSGGDGRGGHNTGAHSTSGNINGGPTGIGVSGGASDGSGWSSENNPWGGGSGSGIHWGGGSGRGNGGGNSGGGSGTGGNLSAVAAPVAFGFPALSTPGAGGLAVSISASELSAAIAIAGIIAKLKKVNLKFTPFGVVLSSLIPSEIAKDDPNMMSKIVTSLPADDITESPVSSLPLDKATVNVNVRVVDDVKDERQNISVVSSGVPMSVPVVDAKPTERPGVFTASIP GAPVLNISVNNSTPAVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHPAMESKRNKPGKATGKGKPVGDKW LDDAGKDSGAPIPDRIADKLRDKEFKSFDDFRKAVWEEVSKDPELSKNLNPSNKSSVSKGYSPFTPKNQQVGGRKVYELHHDKPISQGGEVYDMDNIRVTTPKRHIDIHRGK* SEQ ID NO:3 - Amino acid sequence of cloacin DF13 (CloDF13). * SEQ ID NO: 4 - Amino acid sequence of the cloacin DF13-E9 chimera, in which the C domain of CloDF13 is replaced with the C domain of E9 (CloDF13-E9). * SEQ ID NO: 5 - Amino acid sequence of cloacin DF13-E9 (CloDF13-E9) with deletion of residues 324 to 459 corresponding to the R domain ΔR ). MSGGDGRGPGNSGLGHNGGQASGNVNGTSGKGGPSSGGGTDPNSGPGWGTTHTPNGDIHNYNPGEFGNGGSKPGGNGGNSGNHSGSSGGGQSSATAMAFGLPALATPGAEGLAL SVSGDALSAAVADVLAALKGPFKFGLWGIAIYGVLPSEIAKDDPNMMSKIMTSLPADTVTETPVSTLPLEQATVRVRQRVVDVVKDERQHIAVVAGRPMSVPVVDAKPTKRPGVF SVSIPGLPSLQVSVPKGVPAAKAPPKGIVAEKGDSRPAGFTAGGNSREAVIRFPKETGQKPVYVSVTDVLTPAQVKQRQEEEKRRQQAWDAAHPAMESKRNKPGKATGKGKPVGD KWLDDAGKDSGAPIPDRIADKLRDKEFKSFDDFRKAVWEEVSKDPELSKNLNPSNKSSVSKGYSPFTPKNQQVGGRKVYELHHDKPISQGGEVYDMDNIRVTTPKRHIDIHRGK* SEQ ID NO: 6 - Amino acid sequence of Klebsiella pneumoniae KlebC. * SEQ ID NO:7—amino acid sequence of a Klebsiella pneumoniae KlebC-E9 chimera in which the C domain of KlebC is replaced with the C domain of E9 (KlebC-E9). * SEQ ID NO:8 - Amino acid sequence of Im9. MELKHSISDYTEAEFLQLVTTICNADTTSSEEELVKLVTHFEEMTEHPSGSDLIYYPKEGDDDSPSGIVNTVKQWRAANGKSGFKQG SEQ ID NO:9 - Amino acid sequence of the R domain of CloDF13. PAQVKQRQEEEKRRQQAWDAAHPEEGLKREYDKAKAELDAEDKNIATLNGRITSTEKTIPGARTAVQEADKKVKEAEANKDDFVTYNPPHEYGSGWQDQVRYLDKDIQNQNEKLKAAQASLNAMNESLSRDKAALSGAMESRKQKEKKAKDAENKLNEEA SEQ ID NO: 10 - Amino acid sequence of the TR domain of ColB. MSDNEGSVPTEGIDYGDTMVVWPSTGRIPGGDVKPGGSSGLAPSMPPGWGDYSPQGIALVQSVLFPGIIRRIILDKELEEGDWSGWSVSVHSPWGNEKVSAARTVLENGLRGGLPEPSRPAAVSFARLEPASGNEQKIIRLMVTQQLEQVTDIPASQLPAAGNNVPVKY RLTDLMQNGTQYMAIIGGIPMTVPVVDAVPVPDRSRPGTNIKDVYSAPVSPNLPDLVLSVGQMNTPVRSNPEIQEDGVISETGNYVEAGYTMSSNNHDVIVRFPEGSGVSPLYISAVEILDSNSLSQRQEAENNAKDDFRVKKEQENDEKTVLTKTSEVIISVGDKVGEY SEQ ID NO: 11 - Amino acid sequence of the R domain of ColE9. PDQVKQRQDEENRRQQEWDATHPVEAAERNYERARAELNQANEDVARNQERQAKAVQVYNSRKSELDAANKTLADAIAEIKQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAFDAAAKEKSDADAALSAAQERRKQKENKEKDAKDKLAMESKRNK SEQ ID NO: 12 - ColE9 R -ColB TR Amino acid sequence of. PDQVKQRQDEENRRQQEWDATHPVEAAERNYERARAELNQANEDVARNQERQAKAVQVYNSRKSELDAANKTLADAIAEIKQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAFDAAAKE KSDADAALSAAQERRKQKENKEKDAKDKLAMESKRNKELSDNEGSVPTEGIDYGDTMVVWPSTGRIPGGDVKPGGSSGLAPSMPPGWGDYSPQGIALVQSVLFPGIIRRIILDKELEEGDWSGWSV SVHSPWGNEKVSAARTVLENGLRGGLPEPSRPAAVSFARLEPASGNEQKIIRLMVTQQLEQVTDIPASQLPAAGNNVPVKYRLTDLMQNGTQYMAIIGGIPMTVPVVDAVPVPDRSRPGTNIKDV YSAPVSPNLPDLVLSVGQMNTPVRSNPEIQEDGVISETGNYVEAGYTMSSNNHDVIVRFPEGSGVSPLYISAVEILDSNSLSQRQEAENNAKDDFRVKKEQENDEKTVLTKTSEVIISVGDKVGEY SEQ ID NO: 13 - Im9-ColE9 R Amino acid sequence of. MKHHHHHHNMELKHSISDYTEAEFLQLVTTICNADTTSSEEELVKLVTHFEEMTEHPSGSDLIYYPKEGDDDSPSGIVNTVKQWRAANGKSGFKQIEGRHMPDQVKQRQDEENRRQQEWDATHPVEAAERNY ERARAELNQANEDVARNQERQAKAVQVYNSRKSELDAANKTLADAIAEIKQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAFDAAAKEKSDADAALSAAQERRKQKENKEKDAKDKLAMESKRNK* SEQ ID NO: 14 - Im9-ColB TR Amino acid sequence of. MKHHHHHHHNMELKHSISDYTEAEFLQLVTTICNADTSSEEELVKLVTHFEEMTEHPSGSDLIYYPKEGDDDSPSGIVNTVKQWRAANGKSGFKQIEGRHMSDNEGSVPTEGID YGDTMVVWPSTGRIPGGDVKPGGSSGLAPSMPPGWGDYSPQGIALVQSVLFPGIIRRIILDKELEEGDWSGWSVSVHSPWGNEKVSAARTVLENGLRGGLPEPSRPAAVSFARL EPASGNEQKIIRLMVTQQLEQVTDIPASQLPAAGNNVPVKYRLTDLMQNGTQYMAIIGGIPMTVPVVDAVPVPDRSRPGTNIKDVYSAPVSPNLPDLVLSVGQMNTPVRSNPEI QEDGVISETGNYVEAGYTMSSNNHDVIVRFPEGSGVSPLYISAVEILDSNSLSQRQEAENNAKDDFRVKKEQENDEKTVLTKTSEVIISVGDKVGEYELMRPRVPTCRPAAKLN* SEQ ID NO: 15 - Im9-CloDF13 R Amino acid sequence of. MKHHHHHHHNMELKHSISDYTEAEFLQLVTTICNADTSSEEELVKLVTHFEEMTEHPSGSDLIYYPKEGDDDSPSGIVNTVKQWRAANGKSGFKQIEGRHMPAQVKQRQEEEKRRQQAWDAAHPEEGLKRE YDKAKAELDAEDKNIATLNGRITSTEKTIPGARTAVQEADKKVKEAEAANKDDFVTYNPPHEYGSGWQDQVRYLDKDIQNQNEKLKAAQASLNAMNESLSRDKAALSGAMESRKQKEKKAKDAENKLNEEA* SEQ ID NO: 16 - Im9-ColE9 R -ColB TR Amino acid sequence of. * SEQ ID NO: 17 - Polynucleotide sequence encoding ColE9. SEQ ID NO: 18 - Polynucleotide sequence encoding ColB. SEQ ID NO:19 - Polynucleotide sequence encoding CloDF13-E9. SEQ ID NO: 20 - Polynucleotide sequence encoding KlebC-E9. SEQ ID NO:21 - Polynucleotide sequence encoding Im9. ATGGAACTGAAGCATAGCATTAGTGATTATACAGAAGCTGAATTTTTACAACTTGTAACAACAATTTGTAATGCGGACACTTCCAGTGAAGAAGAACTGGTTAAATTGGTTACACACTTTGAGGAAATGA CTGAGCACCCTAGTGGTAGTGATTTAATATATTACCCAAAAGAAGGTGATGATGACTCACCTTCAGGTATTGTAAACACAGTAAAACAATGGCGAGCCGCTAACGGTAAGTCAGGATTTAAACAGGGCTAA SEQ ID NO: 22 - KvarM-Im9 DNA sequence SEQ ID NO: 23 - KvarM-Im9 amino acid sequence MSDTMIVVATPTPGFSYASGLTYGGGAFAGAPANGPSEGQIFFQTVLPAYQSPNLCIGQLAWMTDYINKNGVGNPKTWEVISQNVLIFCSADTALVLNPRIAVYDGFHKTKWAPAKFNFKTQSQEKFSGNVTTPIAAFGHYLWGEGKPRTVDLSSVGLKIQANQIDPVMIAVKNNAAGTYQISGNF NRNTFIDGDIPGLYLGNITMKTEGTLKIDAKGNWNYNGVVRAFNDTYDANPSTHRSKSAEDLTTLLRLTQGTPYEIRIPGELKVSGSGKKELMELKHSISDYTEAEFLQLVTTICNADTSSEEELVKLVTHFEEMTEHPSGSDLIYYPKEGDDDSPSGIVNTVKQWRAANGKSGFKQGLEHHHHHH* SEQ ID NO: 24 - KvarM-GS5- Im9 DNA sequence SEQ ID NO: 25 - KvarM-GS5-Im9 amino acid sequence MSDTMIVVATPTPGFSYASGLTYGGGAFAGAPANGPSEGQIFFQTVLPAYQSPNLCIGQLAWMTDYINKNGVGNPKTWEVISQNVLIFCSADTALVLNPRIAVYDGFHKTKWAPAKFNFKTQSQEKFSGNVTTPIAAFGHYLWGEGKPRTVDLSSVGLKIQANQIDPVMIAVKNNAAGTYQISGNFNRN TFIDGDIPGLYLGNITMKTEGTLKIDAKGNWNYNGVVRAFNDTYDANPSTHRSKSAEDLTTLLRLTQGTPYEIRIPGELKVSGSGKKGSGSGELMELKHSISDYTEAEFLQLVTTICNADTSSEEELVKLVTHFEEMTEHPSGSDLIYYPKEGDDDSPSGIVNTVKQWRAANGKSGFKQGLEHHHHHH* SEQ ID NO: 26 - KvarM-helix-Im9 DNA sequence SEQ ID NO: 27 - KvarM-helix-Im9 amino acid sequence MSDTMIVVATPTPGFSYASGLTYGGGAFAGAPANGPSEGQIFFQTVLPAYQSPNLCIGQLAWMTDYINKNGVGNPKTWEVISQNVLIFCSADTALVLNPRIAVYDGFHKTKWAPAKFNFKTQSQEKFSGNVTTPIAAFGHYLWGEGKPRTVDLSSVGLKIQANQIDPVMIAVKNNAAGTYQISGNFNRNTFIDG DIPGLYLGNITMKTEGTLKIDAKGNWNYNGVVRAFNDTYDANPSTHRSKSAEDLTTLLRLTQGTPYEIRIPGELKVSGSGKKEAAAKEAAAAKEAAAKELMELKHSISDYTEAEFLQLVTTICNADTSSEEELVKLVTHFEEMTEHPSGSDLIYYPKEGDDDSPSGIVNTVKQWRAANGKSGFKQGLEHHHHHH* SEQ ID NO: 28 - KvarM amino acid sequence MSDTMIVVATPTPGFSYASGLTYGGGAFAGAPANGPSEGQIFFQTVLPAYQSPNLCIGQLAWMTDYINKNGVGNPKTWEVISQNVLIFCSADTALVLNPRIAVYDGFHKTKWAPAKFNFKTQSQEKFSGNVTTPIAAF GHYLWGEGKPRTVDLSSVGLKIQANQIDPVMIAVKNNAAGTYQISGNFNRNTFIDGDIPGLYLGNITMKTEGTLKIDAKGNWNYNGVVRAFNDTYDANPSTHRSKSAEDLTTLLRLTQGTPYEIRIPGELKVSGSGKK

Claims

1. An antimicrobial protein complex, (i) Chimeric polypeptides comprising an immune polypeptide fused with a first bacterial binding site; and (ii) Nuclease bacteriocin polypeptide, (a) Translocation domain, (b) Nuclease domain, and optionally (c) Second bacterial binding site, which is the receptor binding domain Nuclease bacteriocin polypeptides containing; Includes, The immune polypeptide forms a complex with the nuclease bacteriocin polypeptide; The first bacterial binding portion binds to a ligand on the surface of Gram-negative bacteria. Antimicrobial protein complex.

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

3. (a) The receptor-binding domain of the second bacterial-binding portion and the receptor-binding domain of the first bacterial-binding portion are different, and / or (b) The receptor-binding domain of the second bacterial binding portion and the first bacterial binding portion bind to different Gram-negative bacterial surface ligands and / or different Gram-negative bacterial strains. The antimicrobial protein complex according to claim 1.

4. The chimeric polypeptide comprises a plurality of bacterial binding sites, and optionally, one or more of the bacterial binding sites of the chimeric polypeptide, or each of them, is a bacteriocin receptor binding domain. Furthermore, optional, (a) Each bacterial binding portion of the chimeric polypeptide binds to a different bacterial surface ligand and / or a different bacterial strain, and / or (b) Each bacterial binding portion of the chimeric polypeptide binds to a different ligand and / or bacterial strain than the receptor-binding domain of the nuclease bacteriocin polypeptide. The antimicrobial protein complex according to claim 1.

5. The first bacterial binding portion of the chimeric polypeptide (a) a nanobody and / or (b) Contains an M-type bacteriocin, and optionally the M-type bacteriocin is KvarM. The antimicrobial protein complex according to claim 1.

6. An antimicrobial composition comprising an antimicrobial protein complex according to any one of claims 1 to 5.

7. A pharmaceutical composition comprising the antimicrobial protein complex according to any one of claims 1 to 5, for use in a method of treating the body of a human or animal by therapeutic means.

8. A pharmaceutical composition comprising an antimicrobial protein complex according to any one of claims 1 to 5 as an active ingredient for the treatment or prevention of bacterial infection.

9. A medical device comprising an antimicrobial protein complex according to any one of claims 1 to 5.

10. One or more polynucleotides encoding the antimicrobial protein complex described in claim 1.

11. One or more vectors comprising the polynucleotide described in claim 10.

12. A host cell comprising the vector according to claim 11.

13. A method for producing an antimicrobial protein complex according to any one of claims 1 to 5, comprising the steps of culturing the host cells according to claim 12, and isolating the antimicrobial protein complex from the culture.

14. A product having an antimicrobial surface, wherein the surface contains an antimicrobial protein complex according to any one of claims 1 to 5.

15. A method for providing a product having an antibacterial surface, the following: (I) (a) A step of providing an antimicrobial protein complex according to any one of claims 1 to 5, and (b) A step of binding an antimicrobial protein complex to the surface of the product, or (II) (a) A step of binding a chimeric polypeptide containing an immune polypeptide fused to a first bacterial binding portion to the surface of a product, wherein the first bacterial binding portion binds to a ligand on the surface of a bacterium, and (b) A step of attaching a protein nuclease bacteriocin to an immune polypeptide bound to the surface. Methods that include...

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