Antibacterial protein complex

Abstract

The present invention relates to protein bacteriocins (PBs) as therapeutic agents, specifically protein complexes comprising two or more PB molecules associated with a protein scaffold, wherein the protein scaffold comprises a cognate immunity protein domain for each PB effector moiety. In particular, the present invention provides an antibacterial protein complex comprising: (a) a first PB molecule and a second PB molecule; and (b) an immunity protein scaffold comprising a first immunity protein domain and a second immunity protein domain, wherein the first and second immunity protein domains are non-covalently bound to the first and second PB molecules, respectively.

Description

[Technical Field]

[0001] This application claims priority to UK Patent Application No. 2301639.7, filed on 06 Feb. 2023, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] The present invention relates to bacteriocins as therapeutic agents, particularly protein complexes comprising two or more bacteriocin molecules associated with a protein scaffold, the protein scaffold containing the cognate immunity protein domain for each bacteriocin effector moiety. The complexes of the invention can provide a variety of advantages, including increased cell-killing activity, reduced incidence of resistance, enhanced strain range, and increased production and purification efficiency. [Background technology]

[0003] For Gram-negative pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli, therapeutic options are often limited due to horizontal acquisition of antibiotic resistance determinants and the presence of a highly impermeable outer membrane that severely limits the effectiveness of many classes of antibiotics.

[0004] Therefore, with few antibiotics active against these difficult-to-treat bacteria available in recent decades, there is a pressing need to explore alternative strategies for antibiotic development and strengthen the development pipeline.

[0005] An alternative strategy for discovering effective antibiotics is to develop potent narrow-spectrum antibiotics, which are produced by many bacteria due to intraspecies competition. Protein bacteriocins (PBs) are a family of multidomain protein antibiotics, including S-type pyocins, klebicins, colicins, cloacins, salmosins, and pesticins, produced by Pseudomonas aeruginosa, Klebsiella species (e.g., K. pneumoniae), Escherichia coli, Enterobacter cloacae, Salmonella species (e.g., Salmonella enterica), and Yersinia pestis, respectively. PBs have evolved to efficiently cross Gram-negative outer membranes by parasitizing existing nutrient uptake pathways. The cellular targets of PBs are highly conserved and they have cytotoxic activity, which most commonly takes the form of enzymatic activity (generally nucleases or enzymes that degrade peptidoglycan precursor molecules and thus inhibit cell wall synthesis), or pore-forming activity that targets the cytoplasmic membrane.

[0006] However, while PBs are active against clinically relevant strains, each bacteriocin tends to target a limited number of strains of a particular species. To achieve a therapeutically relevant strain range, it may therefore be desirable to use two or three different bacteriocins together. Creating a pharmaceutical-grade cocktail of protein bacteriocins presents many challenges, including high purification costs. Summary of the Invention [Means for solving the problem]

[0007] The present invention: (a) a first protein bacteriocin (PB) molecule and a second PB molecule; and (b) an immunity protein scaffold comprising a first immunity protein domain and a second immunity protein domain; an antibacterial protein complex comprising: An antibacterial protein complex is provided in which the first and second immunity protein domains are non-covalently bound to the first and second PB molecules, respectively.

[0008] Although their three-dimensional structures vary, PBs share a characteristic multidomain architecture, generally comprising an effector (or cytotoxic) moiety at the C-terminus and a cell-targeting moiety generally extending from the N-terminus to the effector domain, as discussed in more detail below. They are often produced by the same Gram-negative bacteria species on which the bacteriocin acts. Organisms expressing PBs also express the same immunity proteins, which bind to the bacteriocin (typically the effector moiety of the bacteriocin) with extremely high affinity (approximately fM), thereby preventing the bacteriocin from killing the host. Examples of species-specific PBs and their targets include colicins (Escherichia coli), S-type pyocins (Pseudomonas aeruginosa), klevicins (Klebsiella species, e.g., K. pneumoniae), cloacins (Enterobacter cloacae), salmonocins (Salmonella species, e.g., Salmonella enterica), and pesticins (Yersinia pestis). Thus, each bacteriocin generally comprises a cell-targeting moiety and an effector moiety, via which it binds to a corresponding immunity protein domain of the immunity protein scaffold.

[0009] The conjugates of the present invention utilize an immunity protein scaffold containing multiple immunity protein domains. This configuration allows a single conjugate to contain two or more bacteriocin molecules, which are delivered to the target cell surface in close physical proximity to one another. Interaction of the bacteriocin molecule with its cognate receptor on the surface of the target bacterium generally causes the bacteriocin to dissociate from the immunity protein scaffold. As described in detail below, the inventors have discovered that such multivalent conjugates (i.e., conjugates containing multiple bacteriocin molecules) offer numerous advantages over corresponding single bacteriocins, including ease of preparation and purification, increased microbial killing activity, and reduced development of resistance.

[0010] The immunity protein scaffold comprises multiple immunity protein domains coupled to one another. The immunity protein scaffold can contain many immunity protein domains, for example, 2, 3, 4, 5, or even more. When fully occupied, the antibacterial protein complexes will therefore contain the same number of bacteriocin molecules, with each bacteriocin non-covalently associated with a corresponding immunity protein domain of the immunity protein scaffold via its effector moiety. (However, it will be understood that any population of such complexes will necessarily contain some complexes that are not fully occupied.) Two or three immunity protein domains may be preferred, such that the antibacterial protein complexes contain two or three bacteriocin molecules, respectively. Thus, the immunity protein scaffold may include a third immunity protein domain, and the antibacterial protein complexes may include a third bacteriocin molecule non-covalently bound to the third immunity protein domain.

[0011] For the avoidance of doubt, it will be understood that the immunity protein scaffold and each bacteriocin molecule are separate molecules, associated by non-covalent interactions between the immunity protein domain and the effector portion of the bacteriocin molecule, which may be substantially the only interactions between the various components of the complex.

[0012] The scaffold may contain multiple (two or more) repeats of the same immunity protein domain. For example, all of the immunity protein domains may be identical. Thus, the complex will contain multiple copies of the same bacteriocin (or at least multiple bacteriocins each with the same effector moiety). Generally, however, the scaffold contains two or more different immunity protein domains. Thus, the complex contains bacteriocin molecules with two or more different effector moieties. The scaffold may contain two, three, four, or five different immunity protein domains, or even more. In some embodiments, each of the immunity protein domain components in a given scaffold molecule is different from each of the other immunity protein domain components. Two or three different immunity protein domains may be preferred, so that the antibacterial protein complex contains bacteriocin molecules each with two or three different effector moieties.

[0013] The immunity protein scaffold is generally a fusion protein, ie, a single peptide chain containing the relevant immunity protein domains, optionally separated by a linker peptide.

[0014] The immunity protein scaffold may further comprise a heterologous moiety, i.e., a moiety that is neither an immunity protein domain nor a linker peptide. In some embodiments, the heterologous moiety may comprise a cytotoxic domain, such that the immunity protein scaffold itself is an additional toxin in addition to its associated PB. The heterologous moiety may comprise a cytotoxic domain and a moiety capable of mediating translocation across the target cell outer membrane, such as a protein bacteriocin targeting moiety. In preferred embodiments, the heterologous moiety is an M-type bacteriocin, such as colicin M (ColM), KpneM, KpneM2, KvarM, PaeM1, or PaeM4, or a functional variant thereof. In such embodiments, the M-type bacteriocin is typically located at the N-terminus of the scaffold molecule, and the immunity protein domain is located at the C-terminus of the M-type bacteriocin. A suitable linker will typically be present between the M-type bacteriocin and the first immunity protein domain.

[0015] The effector moiety is generally an enzymatic effector moiety. The enzymatic effector moiety generally has nuclease activity. The nuclease can be, for example, a DNase (capable of degrading DNA) or an RNase (capable of degrading RNA). The RNase can be, for example, an rRNase (active against ribosomal RNA) or a tRNase (active against transfer RNA).

[0016] Thus, within a given complex, each bacteriocin generally has an enzymatic effector moiety with nuclease activity. The effector moieties of the bacteriocins can be the same or different. Thus, a complex may contain two or more bacteriocins with the same effector moiety. For example, all of the bacteriocins in a complex may have the same effector moiety. Alternatively, a complex may contain two or more bacteriocins with different enzymatic effector moieties. For example, all of the bacteriocins in a complex may have different effector moieties. Even if the effector moieties are different, they may have the same enzymatic activity, such as DNase or RNase (which may be rRNase or tRNase). Thus, a complex may contain two bacteriocins with different effector moieties, but the effector moieties have the same activity. Alternatively, some or all of the effector moieties may have different enzymatic activities. In some embodiments, each of the effector moieties in a given complex is an enzymatic effector moiety, and each of the effector moieties may have a different enzymatic activity. Examples of suitable combinations include: - effector moieties with DNase activity and effector moieties with RNase activity (e.g. rRNase or tRNase); - an effector moiety having rRNase activity and an effector moiety having tRNase activity; - an effector moiety having DNase activity, an effector moiety having rRNase activity, and an effector moiety having tRNase activity There is.

[0017] Independently, a complex may include bacteriocins that all have the same targeting moiety, or that have two or more different targeting moieties, hi some embodiments, each of the bacteriocins in a complex has a different targeting moiety.

[0018] It may be desirable for all of the bacteriocins in a complex to have targeting moieties specific for the same species or strain of bacteria, yet the bacteriocins may bind to two or more different receptors (e.g., each targeting moiety binds to a different receptor) or use two or more different translocation portals (e.g., each targeting moiety uses a different translocation portal).

[0019] However, it may be desirable for two or more bacteriocins in a given complex to each have a targeting moiety specific for a different species or strain, such that a single complex has activity against more than one species or strain of bacteria. Thus, it is possible to modulate the spectrum of activity of a given complex by appropriate selection of the targeting moieties for the bacteriocins present in the complex.

[0020] Bacteriocin molecules comprise an effector (or cytotoxic) portion and a targeting portion. Within a given bacteriocin molecule, the effector and targeting domains may be derived from the same wild-type bacteriocin. Alternatively, the bacteriocin may be a chimera comprising effector and targeting portions derived from different wild-type bacteriocins. It will be understood that the present invention is not limited to the use of wild-type bacteriocin sequences. Modified or engineered bacteriocin sequences may also be used, whether in their effector portion, targeting portion, or both.

[0021] Illustrative examples of the conjugates of the present invention are: the first PB containing the colicin E9 effector moiety; a second PB comprising a colicin D effector moiety; and It comprises an immunity protein scaffold comprising a colicin E9 immunity protein domain ("Im9") and a colicin D immunity protein domain ("ImD") (together designated "Im9-ImD").

[0022] The first PB may be colicin E9 (i.e., also includes a colicin E9 targeting moiety). The second PB may be colicin D (i.e., also includes a colicin D targeting moiety). Alternatively, one or both may be chimeric PBs, comprising a targeting moiety from a different PB than its effector moiety.

[0023] Further examples of the conjugates of the present invention include: the first PB containing the colicin E9 effector moiety; a second PB containing the colicin E3 effector moiety; a third PB comprising a colicin D effector moiety; and It comprises an immunity protein scaffold comprising a colicin E9 immunity protein domain ("Im9"), a colicin E3 immunity protein domain ("Im3"), and a colicin D immunity protein domain ("ImD") (together designated "Im9-Im3-ImD").

[0024] The first PB can be colicin E9 (i.e., also includes a colicin E9 targeting moiety). The second PB can be colicin E3 (i.e., also includes a colicin E3 targeting moiety). The third PB can be colicin D (i.e., also includes a colicin D targeting moiety).

[0025] Alternatively, one, two or all three PBs may be chimeric PBs, comprising a targeting moiety derived from a different PB than its effector moiety. For example, independently: The first PB may be a chimeric PB comprising a KlebC targeting moiety and a colicin E9 effector moiety ("KlebC-E9"); The second PB may be a second chimeric PB comprising a CloDF13 targeting moiety and a colicin E3 effector moiety ("CloDF13-E3"); The third PB may be a chimeric PB (designated "KlebG-D") that comprises a KlebG targeting moiety and a colicin D effector moiety.

[0026] The present invention further provides an antibacterial method comprising contacting a bacterium or population of bacteria with an antibacterial complex described herein. The invention further provides the antibacterial conjugates described herein for use in a method of medical treatment, for example for use in the prevention or treatment of a bacterial infection, a symptom caused by or associated with a bacterial infection.

[0027] The invention further provides the use of an antibacterial conjugate as described herein in the preparation of a medicament for use in the prevention or treatment of a bacterial infection, a symptom caused by or associated with a bacterial infection.

[0028] The present invention further provides a method for preventing or treating a bacterial infection, a symptom caused by a bacterial infection, or a symptom associated with a bacterial infection, comprising the step of administering to a subject in need thereof an antibacterial complex as described herein.

[0029] The bacterial infection is generally an infection caused by gram-negative bacteria. The present invention: (i) a nucleic acid encoding an immunity protein scaffold, the immunity protein scaffold comprising a first immunity protein domain and a second immunity protein domain; (ii) a nucleic acid encoding at least one bacteriocin, the or each bacteriocin having an effector domain capable of binding to at least one of said immunity protein domains; A host cell comprising: Further provided is a host cell, wherein the host cell is capable of expressing said immunity protein scaffold and said at least one bacteriocin.

[0030] Upon expression of the scaffold and bacteriocin components, an antibacterial complex of the invention can be formed. If the antibacterial complex contains an immunity protein scaffold with only one type of immunity protein domain, and the complex contains only one type of bacteriocin, the cell can encode and express only a single homogeneous bacteriocin.

[0031] Where the antibacterial complex contains at least two different types of bacteriocins (e.g., the immunity protein scaffold contains at least two different immunity protein domains), the cell contains nucleic acids encoding at least two bacteriocins, each having an effector capable of binding to at least one of the immunity protein domains, and is capable of expressing the bacteriocins.

[0032] Preferably, the host cell comprises nucleic acid encoding a bacteriocin having an effector domain capable of binding to each of the immunity protein domains of the immunity protein scaffold and is capable of expressing said bacteriocin.

[0033] So for example, a cell: (i) a nucleic acid encoding an immunity protein scaffold, the immunity protein scaffold comprising a first immunity protein domain and a second immunity protein domain; (ii) first and second nucleic acids encoding, respectively, a first and a second bacteriocin, each bacteriocin having an effector domain capable of binding to a respective one of said immunity protein domains; and The host cell is capable of expressing said immunity protein scaffold and said bacteriocin.

[0034] Additionally, cells: (i) a nucleic acid encoding an immunity protein scaffold, the immunity protein scaffold comprising a first, second and third immunity protein domain; (ii) first, second and third nucleic acids encoding first, second and third bacteriocins, respectively, each bacteriocin having an effector domain capable of binding to a respective one of said immunity protein domains; and The host cell is capable of expressing said immunity protein scaffold and said bacteriocin.

[0035] As discussed above, it will be apparent that the immunity protein scaffold can contain more immunity protein domains as needed, in which case the host cell can encode (and express) a corresponding number of bacteriocins as needed.

[0036] The present invention further provides a method of producing an antibacterial complex, comprising providing a host cell as described and culturing said cell under conditions suitable for expression of the immunity protein scaffold and bacteriocin molecule. The method may further comprise isolating the antibacterial complex and optionally a further step of purification.

[0037] Alternatively, it may not be desirable to express all components of the antibacterial complex in the same host cell. Instead, the components may be expressed in two or more different host cells, each containing nucleic acid encoding one or more individual components and capable of expressing that component. For example, the immunity protein scaffold may be expressed in one host cell and one or more bacteriocin molecules may be expressed in one or more other host cells.

[0038] Accordingly, the present invention further provides a method of producing an antibacterial complex, the method comprising contacting an immunity protein scaffold comprising a first immunity protein domain and a second immunity protein domain with first and second bacteriocin molecules, each having an effector moiety capable of binding to a respective one of the immunity protein domains, to form a complex of the invention.

[0039] In such cases, if a bacteriocin molecule is expressed in a host cell that is sensitive to that bacteriocin and does not express an immunity protein scaffold, the host cell will generally also express the cognate immunity protein for the related bacteriocin. In such cases, the bacteriocin can be dissociated from and isolated from the immunity protein prior to contacting the bacteriocin with the immunity protein scaffold.

[0040] The present invention includes any combination of the described aspects and preferred features unless that combination is clearly unacceptable or explicitly avoided. BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings, in which: FIG. [Brief explanation of the drawings]

[0041] [Figure 1] (a) Schematic diagram of a heterotrimeric complex (two bacteriocin molecules) and (b) heterotetrameric complex (three bacteriocin molecules). The immunity proteins are designated "Im" (Im9, Im3, and ImD). The cognate cytotoxic domains are designated E9, E3, and D. The remaining bacteriocin molecules are simply designated "bacteriocin." (c) Cartoon structure of Im9, Im3, and ImD connected together with a flexible linker represented by a dotted line. (d) Cartoon structure of the Im9-Im3-ImD fusion protein associated with its cognate bacteriocin. [Figure 2]Schematic diagram of the translocation pathway of colicin E9 (Francis et al., 2021) and colicin D across the cell envelope. Colicin E9 binds to BtuB and OmpF in the outer membrane before interacting with TolB in the periplasm, resulting in FtsH-dependent translocation of the C-terminal DNase domain to the cytoplasm. Colicin D crosses the outer membrane through interaction with FepA and interacts with TonB in the periplasm, resulting in FtsH-dependent translocation of the C-terminal tRNase to the cytoplasm (Chauleau et al., 2011). [Figure 3] Cloacin DF13 binds the ferric aerobactin receptor IutA (unpublished) before its unstructured N-terminus passes through the trimeric porin to bind TolB in the periplasm. Klebicin G binds the trimeric porin OmpK35, allowing the N-terminus of Klebicin G to cross the outer membrane and bind TolA. KlebC binds the efflux pump TolC, and the N-terminus of Klebicin G passes through the TolC lumen to interact with TonB in the periplasm. [Figure 4] Figure 1 shows the Superdex 200 Increase 10 / 300GL elution profiles of (i) Im9-ImD + ColE9 + ColD, (ii) Im9-ImD + ColD, (iii) Im9-ImD + ColE9, and (iv) Im9-ImD. The center 0.5 mL of the elution peak for each conjugate was collected for killing assay analysis. [Figure 5A] FIG. 1 shows the killing activity of ColD against BL21(DE3)(btuB-), fepA-BW25113, tolA-BW25113 and tonB-BW25113 E. coli cells. [Figure 5B] FIG. 1 shows the killing activity of ColE9 against BL21(DE3)(btuB-), fepA-BW25113, tolA-BW25113 and tonB-BW25113 E. coli cells. [Figure 5C]FIG. 1 shows the killing activity of [ColE9:Im9-ImD:ColD] against BL21(DE3)(btuB-), fepA-BW25113, tolA-BW25113, and tonB-BW25113 E. coli cells. [Figure 5D] FIG. 1 shows the killing activity of ColE9, ColD, ColE9+ColD, and [ColE9:Im9-ImD:ColD] against tolA-BW25113 E. coli cells. [Figure 6] Growth inhibition of E. coli MG1655 by [ColE9:Im9-ImD:ColD], [Im9-ImD:ColD], and [ColE9:Im9-ImD]. Liquid cultures of E. coli MG1655 were grown in the presence of [ColE9:Im9-ImD:ColD], [Im9-ImD:ColD], or [ColE9:Im9-ImD] and monitored at OD630 nm after 6 hours (top panel) and 24 hours (bottom panel). The complexes were used at concentrations ranging from 5.6 pM to 1 μM. [Figure 7] Nickel affinity purification of colicin E9, Im9-ImD, and colicin D expressed in BL21(DE3) cells from colicin D cloned into pET21a with an additional copy of colicin D cloned into pACYCDuet1 [ColE9:Im9-ImD:ColD]. Protein was eluted from the column with a 0-500 mM imidazole gradient over 10 column volumes, and fractions were analyzed on a 12% SDS-PAGE gel (fractions 18-23 are shown). [Figure 8] 26 / 60 S200 purification of the [ColE9:Im9-ImD:ColD] complex. [ColE9:Im9-ImD:ColD] was purified on a 26 / 60 S200 column equilibrated in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and eluted fractions were analyzed on a 12% SDS-PAGE gel. [Figure 9]This figure shows the activity of the in vivo assembled trimeric [ColE9:Im9-ImD:ColD] complex on a soft agar lawn inoculated with E. coli. Three-fold serial dilutions of [ColE9:Im9-ImD:ColD] were prepared over a concentration range of 100 nM to 137 pM. Five microliters of each dilution was spotted onto a soft agar lawn inoculated with E. coli BW25113, btuB-BL21(DE3), fepA-BW25113, tolA-BW25113, or tonB-BW25113. After overnight incubation at 37°C, bacteriocin activity was observed as a zone of clearance within the bacterial lawn. [Figure 10] The assembled tetrameric complex [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] was purified on a Superdex 200 Increase 10 / 300GL analytical gel filtration column (Cytiva). 12% SDS-PAGE analysis of elution fractions from the peak is shown. [Figure 11] Figure 1 shows the killing activity of the tetrameric complex [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] against SG62, SR3, and SR6 Klebsiella pneumoniae cells, compared with the previously observed activities of KlebC-E9, CloDF13-E3, and KlebG-D against the same strains. [Figure 12]

[0023] Figure 1 shows an illustration of KvarM immunity protein scaffolds and demonstration of cytotoxic activity. [A] Schematic representation of KvarM-Im9-ImD protein scaffold. [B] Schematic representation of KvarM-Im9-Im7-ImD protein scaffold. [C] Cytotoxic activity of wild-type KvarM and KvarM immunity protein scaffolds against K. quasipneumoniae SG96 cells in nutrient broth. [Figure 13] Schematic representation of KvarM immunity protein scaffold fusions complexed with their cognate protein bacteriocin. [Figure 14]Assembly and activity of the trimeric complex [S5E9:Im9-Im7:S5E7] and its activity against Pseudomonas aeruginosa. (A) Schematic diagram of the composition of the ternary complex [S5E9:Im9-Im7:S5E7]. Chimeric pyocins S5E9 and S5E7 consist of the outer membrane transport domain (Tom) and receptor binding (R) domain of pyocin S5, followed by the inner membrane transport domain (TIM) of pyocin G and the DNase domains from colicins E9 and E7, respectively. These chimeric pyocins form a complex with the immunity protein scaffold through the interaction of the E7 and E9 DNase domains with the cognate E7 and E9 immunity protein domains of the scaffold. (B) SDS-PAGE of purified proteins. Free and immunity protein complex forms of S5E9, S5E7, and the [S5E9:Im9-Im7:S5E7] complex were loaded onto a 16% SDS-PAGE gel. In addition to refolded, uncomplexed S5E9 and S5E7 monomeric pyocin samples, bands indicating the presence of multimeric pyocins are shown. (C) Activity of the [S5E9:Im9-Im7:S5E7] complex and component proteins. Three-fold dilutions from a starting concentration of 0.14 mg / mL of each protein or protein complex were spotted onto a growth lawn of P. aeruginosa P8. Clear zones indicate killing of P. aeruginosa. DETAILED DESCRIPTION OF THE INVENTION

[0042] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0043] Although protein bacteriocins are active against clinically relevant strains, each bacteriocin tends to target a limited number of strains of a particular species. To achieve a therapeutically relevant strain range, it may therefore be desirable to use two or three different bacteriocins together. Creating a pharmaceutical-grade cocktail of protein bacteriocins presents many challenges, including high purification costs.

[0044] The complexes of the invention offer many advantages, including the possibility of expressing all components (bacteriocin proteins and immunity protein scaffolds) in a single culture, thus enabling the purification of a single multifunctional protein complex. Even if individual components are expressed in more than one culture, the affinity of the scaffold for the bacteriocin molecule may facilitate the purification of a single, stoichiometrically defined complex from a relatively crude mixture of separate cultures, without the need to purify each component individually.

[0045] The described complexes may also provide functional advantages, e.g., with respect to cell killing and reduced development of resistance. It is believed that the complexes of the invention induce higher levels of cell killing than preparations of the same individual bacteriocins, likely due to the increased avidity effect resulting from the physical association of multiple receptor-binding domains. This phenomenon takes advantage of the fact that bacterial outer membrane proteins tend to associate into clusters.

[0046] Complexes containing different bacteriocins associated with a single immunity protein scaffold ("heterologous" complexes) appear to demonstrate increased cell killing compared to the corresponding individual bacteriocins, even in bacteria lacking one or more components normally required for uptake of one of the bacteriocins, such as outer or inner membrane proteins associated with membrane translocation (referred to herein as "translocation ports"). Without being bound by theory, it is believed that the spatial proximity of different bacteriocin components may enable them to share components of each other's uptake systems in a manner that would not be possible with individual bacteriocins. This is likely facilitated by the clustering of diverse bacterial outer membrane proteins in megaclusters ("Lipids Mediate Supramolecular Outer Membrane Protein Assembly in Bacteria," Webby MN et al., Sci. Adv. 8, eadc9566 (2022); doi:10.1126 / sciadv.adc9566).

[0047] The conjugates of the invention can also be readily adapted to target different species or strains of bacteria. Generally, cytotoxic domains are effective against many types of bacteria and can be readily exchanged between bacteriocin molecules. Strain specificity is primarily determined by the receptor binding and / or translocation domains. Thus, simply exchanging one set of receptor binding and / or translocation domains of a bacteriocin molecule for another can tailor the strain specificity of the conjugate, allowing the same set of cytotoxic domains (and therefore the same immunity protein scaffold) to be used against a wide range of bacterial types.

[0048] The use of a complex containing two or more different bacteriocin molecules may also reduce the likelihood that resistance will emerge to the associated bacteriocin. For example, a single complex may contain two or more bacteriocin molecules that bind to different receptors, use different translocation pathways, and / or have different cytotoxic activities, all of which reduce the chance of resistance emerging. (It will also be understood that a similar effect can be achieved by using a population or "cocktail" containing different complexes, each given complex carrying multiple copies of the same bacteriocin molecule, and the population containing at least two different bacteriocin molecules, e.g., three, four, five, or even more different bacteriocin molecules.) Protein bacteriocin Bacteriocins applicable in the context of the present invention are proteinaceous antimicrobial toxins produced by and effective against Gram-negative bacterial species, designated "protein bacteriocins" (PB).

[0049] Other structurally distinct types of bacteriocins are known but are not included in the definition of "protein bacteriocin." Thus, both granular bacteriocins, such as R-type (rod-shaped) and F-type (mobile and non-contractile) pyocins, are associated with phage tail proteins (derived from P2 and lambda phages, respectively), and are sometimes referred to as "teirosins" or "high molecular weight bacteriocins."

[0050] Peptide-like bacteriocins, sometimes called microcins, are antibiotic peptides generally less than 10 kDa in size and secreted by bacteria, primarily Enterobacteriacea. They can be divided into class I (less than 5 kDa) and class II (5–10 kDa) microcins, and can exhibit various mechanisms of action, including pore formation in bacterial membranes (MccV, MccE492, and MccL), inhibition of aspartyl-tRNA synthetase (MccC), inhibition of DNA gyrase GyrB (MccB17), which leads to double-stranded DNA breaks, inhibition of transcription, and inhibition of cellular respiration via cytochromes (MccJ25), cellular proton channels (MccH47, possibly MccM and MccI), or ATP synthase (MccH47). For a review, see Baquero et al., Front. Microbiol. 2019 October, Volume 10, 2019 (doi:10.3389 / fmicb.2019.02261).

[0051] PBs are thought to be evolutionarily related and share a characteristic multi-domain structure that includes a targeting moiety and an effector (or cytotoxic) moiety. Generally, the targeting moiety is at the N-terminal end of the molecule, and the effector moiety, particularly if it has nuclease activity, is at the C-terminal end.

[0052] PBs include colicins (active against Escherichia coli), S-type pyocins (active against Pseudomonas aeruginosa), klevicins (active against Klebsiella species, e.g., Klebsiella pneumoniae, K. quasipneumoniae, K. oxytoca, K. variicola, and K. aerogenes), cloacins (active against Enterobacter cloacae), salmonocins (active against Salmonella enterica), and pesticins (active against Yersinia pestis). They are sometimes referred to as "colicin-like bacteriocins." For a review, see Behrens et al., Emerging Topics in Life Sciences (2017) 1:65-74 (doi:10.1042 / ETLS20160016).

[0053] The effector moiety may comprise a single, independently folded domain. The targeting moiety may also comprise a single, independently folded domain or may be subdivided into two or more independently folded domains.

[0054] The targeting moiety binds to a receptor on the surface of the target organism (i.e., in the Gram-negative outer membrane) and mediates translocation of the bacteriocin across the outer membrane. For the avoidance of doubt, the term "receptor" is used solely to refer to the molecule on the target organism to which the targeting moiety binds, and should not be taken to imply a cooperative receptor-ligand interaction in the sense that it typically contemplates a pair of molecules expressed by a single organism. The receptor is generally an outer membrane protein, but can be any suitable molecule in the outer membrane, such as lipopolysaccharide (e.g., the common polysaccharide antigen of Pseudomonas aeruginosa, which is thought to function as a receptor for many pyocins, including PyoL1, PyoS2, PyoS3, PyoS5, PyoSD2, and PyoSD3).

[0055] Typically, the targeting moiety determines the species and strain specificity (or tropism) of the bacteriocin. As used herein, when a given targeting moiety (or bacteriocin) is described as "specific for" a particular target organism (e.g., a bacterial strain or species), it simply means that the targeting moiety is generally capable of delivering an associated effector moiety to the relevant species or strain by binding to a receptor on the surface of the target organism and mediating the translocation of the effector moiety so that it can exert its activity against the target organism. It should be noted that a particular bacteriocin may be active against more than one species of bacteria if the receptor and translocation pathway used by the targeting moiety are sufficiently similar between different species. For example, cloacin DF13, despite being a cloacin, is capable of targeting Klebsiella pneumoniae strains (e.g., strain SG62).

[0056] The targeting moieties of most naturally occurring PBs have a characteristic modular structure containing up to four distinct subregions, each of which may represent an individually folded domain or may lack recognizable secondary structure and thus form a flexible region of the molecule. Subregion I, located at the N-terminus, is relatively unstructured and contains a Tol or Ton-binding epitope. Subregion II is a translocator-binding domain. Subregion III is a receptor-binding domain. Subregion IV is an inner membrane translocation domain. In some PBs, the transition port also functions as a receptor, in which case subregions II and III form a single domain. In other cases, the receptor and transition port are different molecules, in which case subregions II and III are usually separate domains. These subregions are generally (but not necessarily) ordered from the N-terminus to the C-terminus (although subregion I is always N-terminal).

[0057] Without wishing to be bound by any particular theory, it is believed that subregions I, II, III and IV may be, at least to some extent, interchangeable between molecules. Within a given complex, the targeting moieties of the bacteriocin molecules can be the same or different. Thus, a complex may contain bacteriocins that all have the same targeting moiety, or that have two or more different targeting moieties. For example, each of the bacteriocins in a complex may have a different targeting moiety.

[0058] It may be desirable for all of the bacteriocins in a complex to have targeting moieties specific for bacteria of the same species or strain. Nevertheless, the bacteriocins may bind to two or more receptors that are different in that strain or species. For example, each targeting moiety may bind to a different receptor. Additionally or alternatively, the targeting moieties may use two or more different translocation portals, such as TolC, a trimeric porin, or a TonB-dependent translocator.

[0059] For example, a complex may include two or more bacteriocins having targeting moieties specific for E. coli, e.g., two or more bacteriocins having different targeting moieties specific for E. coli, e.g., derived from colicins. For example, all of the bacteriocins in the complex may have different targeting moieties specific for E. coli, e.g., derived from colicins, including colicins A, E1, E2, E3, E4, E6, E7, E8, E9, K, M, N, U, B, Ia, Ib, 5, 10, S4, and Y.

[0060] The complex may include two or more bacteriocins having targeting moieties specific for Pseudomonas, e.g., P. aeruginosa, e.g., two or more bacteriocins having different targeting moieties specific for Pseudomonas, e.g., P. aeruginosa, e.g., derived from S-type pyocins. For example, all of the bacteriocins in the complex may have different targeting moieties specific for Pseudomonas, e.g., P. aeruginosa, e.g., derived from S-type pyocins. S-type pyocins include pyocin G, L1, L2, L3, M1, M2, M4, S1, S2, S3, S4, S5, S6, S8, SD1, SD2, SD3, AP41, Sn, SX1, and SX2.

[0061] The complex may include two or more bacteriocins having targeting moieties specific for Klebsiella (e.g., K. pneumoniae, K. quasipneumoniae, K. oxytoca, K. variicola, or K. aerogenes), e.g., two or more bacteriocins having different targeting moieties specific for Klebsiella (e.g., K. pneumoniae, K. quasipneumoniae, K. oxytoca, K. variicola, or K. aerogenes), e.g., derived from a klevicin. For example, all of the bacteriocins in the complex may have different targeting moieties specific for Klebsiella (e.g., K. pneumoniae, K. quasipneumoniae, K. oxytoca, K. variicola, or K. aerogenes), e.g., derived from a klevicin. Klebicins include klebicins C, D, G, KpneA, KaerA, KpneIa, KvarIa, KoxyY, KpneM, KpneM2 and KvarM.

[0062] The complex may include two or more bacteriocins having targeting moieties specific for Enterobacter cloacae, e.g., two or more bacteriocins having different targeting moieties specific for Enterobacter cloacae, e.g., derived from cloacin. For example, all of the bacteriocins in the complex may have different targeting moieties specific for Enterobacter cloacae, e.g., derived from cloacin, e.g., cloacin DF13.

[0063] The complex may include two or more bacteriocins having targeting moieties specific for Salmonella enterica, e.g., two or more bacteriocins having different targeting moieties specific for Salmonella enterica, e.g., derived from Salmosin. For example, all of the bacteriocins in the complex may have different targeting moieties specific for Salmonella enterica, e.g., derived from Salmosin, e.g., SalE1a, SalE1b, SalE2, SalE3, and SalE7.

[0064] The complex may include two or more bacteriocins having targeting moieties specific for Y. pestis, e.g., two or more bacteriocins having different targeting moieties specific for Y. pestis, e.g., derived from pesticins. For example, all of the bacteriocins in the complex may have different targeting moieties specific for Y. pestis, e.g., derived from pesticins, e.g., pesticin 1.

[0065] In other embodiments, it may be desirable for two or more bacteriocins in a given complex to each have a targeting moiety specific for a different bacterial species or strain, to obtain a single complex that is active against more than one species or strain of bacteria, it being understood that the bacteria in question are Gram-negative bacteria.

[0066] For example, a complex may include a bacteriocin having a targeting moiety specific for E. coli (e.g., a colicin targeting moiety), and one or more bacteriocins having targeting moieties specific for one or more of Pseudomonas (e.g., Pseudomonas aeruginosa) (e.g., an S-type pyocin targeting moiety), Klebsiella (e.g., K. pneumoniae, K. quasipneumoniae, K. oxytoca, K. variicola, or K. aerogenes) (e.g., a klebicin targeting moiety), Enterobacter cloacae (e.g., a cloacin targeting moiety), Salmonella enterica (e.g., a salmonocin targeting moiety), or Yersinia pestis (e.g., a pesticin targeting moiety).

[0067] The complex may include a bacteriocin having a targeting moiety (e.g., an S-type pyocin targeting moiety) specific for Pseudomonas (e.g., P. aeruginosa) and one or more bacteriocins having targeting moieties specific for one or more of Escherichia coli (e.g., a colicin targeting moiety), Klebsiella (e.g., K. pneumoniae) (e.g., a klebicin targeting moiety), Enterobacter cloacae (e.g., a cloacin targeting moiety), Salmonella enterica (e.g., a salmonocin targeting moiety), or Yersinia pestis (e.g., a pesticin targeting moiety).

[0068] The complex may include a bacteriocin having a targeting moiety (e.g., a klebicin targeting moiety) specific for Klebsiella (e.g., K. pneumoniae, K. quasipneumoniae, K. oxytoca, K. variicola, or K. aerogenes) and one or more bacteriocins having targeting moieties specific for one or more of Escherichia coli (e.g., a colicin targeting moiety), Pseudomonas (e.g., Pseudomonas aeruginosa) (e.g., an S-type pyocin targeting moiety), Enterobacter cloacae (e.g., a cloacin targeting moiety), Salmonella enterica (e.g., a salmonocin targeting moiety), or Yersinia pestis (e.g., a pesticin targeting moiety).

[0069] The complex may include a bacteriocin having a targeting moiety specific for Enterobacter cloacae (e.g., a cloacin targeting moiety) and one or more bacteriocins having targeting moieties specific for one or more of Escherichia coli (e.g., a colicin targeting moiety), Pseudomonas (e.g., Pseudomonas aeruginosa) (e.g., an S-type pyocin targeting moiety), Klebsiella (e.g., K. pneumoniae, K. quasipneumoniae, K. oxytoca, K. variicola, or K. aerogenes) (e.g., a klebicin targeting moiety), Salmonella enterica (e.g., a salmonocin targeting moiety), or Yersinia pestis (e.g., a pesticin targeting moiety).

[0070] The complex may include a bacteriocin having a targeting moiety specific for Salmonella enterica (e.g., a salmonocin targeting moiety) and one or more bacteriocins having targeting moieties specific for one or more of Escherichia coli (e.g., a colicin targeting moiety), Pseudomonas (e.g., Pseudomonas aeruginosa) (e.g., an S-type pyocin targeting moiety), Klebsiella (e.g., K. pneumoniae, K. quasipneumoniae, K. oxytoca, K. variicola, or K. aerogenes) (e.g., a klebicin targeting moiety), Enterobacter cloacae (e.g., a cloacin targeting moiety), or Yersinia pestis (e.g., a pesticin targeting moiety).

[0071] The complex may include a bacteriocin having a targeting moiety specific for Yersinia pestis (e.g., a pesticin targeting moiety) and one or more bacteriocins having targeting moieties specific for one or more of Escherichia coli (e.g., a colicin targeting moiety), Pseudomonas (e.g., Pseudomonas aeruginosa) (e.g., an S-type pyocin targeting moiety), Klebsiella (e.g., K. pneumoniae, K. quasipneumoniae, K. oxytoca, K. variicola, or K. aerogenes) (e.g., a klebicin targeting moiety), Enterobacter cloacae (e.g., a cloacin targeting moiety), or Salmonella enterica (e.g., a salmonocin targeting moiety).

[0072] As already mentioned, the only effector moieties used in the bacteriocins of the invention are enzymatic effector moieties, generally nucleases, since they generally interact with the cognate immunity protein domain.

[0073] However, any PB-derived targeting moiety can be utilized, regardless of what effector domain is normally associated with it in the corresponding wild-type PB. Pore-forming bacteriocins kill target cells by depolarizing the cytoplasmic membrane. These include pyocin S5, colicins A, E1, K, N, U, B, Ia, Ib, 5, 10, S4, and Y, and klebicins KpneA, KaerA, KpneIa, KvarIa, and KoxyY (see Denkovskiene et al., 2019).

[0074] The enzymatic PB effector moiety can have a variety of activities. As already described, many act as nucleases. Some nuclease effector moieties possess DNase activity, including pyocin G, S1, S2, SD2, S3, and AP41, and colicins E2, E7, E8, and E9. Klebicin G is also thought to be a DNase.

[0075] Some nuclease effector moieties possess RNase activity, such as rRNase or tRNase activity. Nuclease effector moieties with rRNase activity include colicins E3, E4, and E6, klevicin C, and cloacin DF13. Nuclease effector moieties with tRNase activity include pyocin S4, colicins E5 and D, and klevicin D.

[0076] Still other enzymatic effector moieties have different modes of activity, including degradation of peptidoglycan or its precursor molecules, leading to inhibition of cell wall synthesis. These include colicin M, PaeM1, PaeM4 and klevicins KpneM, KpneM2 and KvarM.

[0077] The targets on which effector moieties act tend to be highly conserved throughout the bacterial kingdom, resulting in a given effector moiety being generally active against a broad spectrum of organisms. Thus, species specificity is primarily determined by the targeting portion of the bacteriocin molecule. This allows for relatively free exchange of effector moieties between PBs to generate chimeric bacteriocins. For example, chimeric pyocins containing a targeting portion derived from an S1 or S2 pyocin linked to an effector portion derived from either an E2 or E3 colicin have been demonstrated to retain pseudomonacidal activity (Kageyama et al., 1996). Chimeric bacteriocins are also utilized in the following examples.

[0078] Thus, bacteriocins can comprise any suitable effector moiety with the cognate immunity protein that can be incorporated into the immunity protein scaffold. As already mentioned, these are generally enzymatic (nuclease) effector moieties, which can be combined with any suitable PB-derived targeting moiety.

[0079] Thus, within a given complex, each bacteriocin generally has an enzymatic effector moiety with nuclease activity. The effector moieties of the bacteriocins can be the same or different. Thus, a complex may contain two or more bacteriocins with the same effector moiety. For example, all of the bacteriocins in a complex may have the same effector moiety. Alternatively, a complex may contain two or more bacteriocins with different enzymatic effector moieties. For example, all of the bacteriocins in a complex may have different effector moieties. Even if the effector moieties are different, they may have the same enzymatic activity, such as DNase or RNase (which may be rRNase or tRNase). Thus, a complex may contain two bacteriocins with different effector moieties, but the effector moieties have the same activity. Alternatively, some or all of the effector moieties may have different enzymatic activities. In some embodiments, each of the effector moieties in a given complex is an enzymatic effector moiety, and each of those effector moieties may have a different enzymatic activity. Examples of suitable combinations include: - effector moieties with DNase activity and effector moieties with RNase activity (e.g. rRNase or tRNase); - an effector moiety having rRNase activity and an effector moiety having tRNase activity; - there are effector moieties with DNase activity, effector moieties with rRNase activity and effector moieties with tRNase activity.

[0080] It will be understood from the above discussion that within a given bacteriocin molecule, the effector and targeting domains may be derived from the same wild-type bacteriocin. Alternatively, the bacteriocin may be a chimera, comprising effector and targeting moieties from different bacteriocins. For example, the effector and targeting moieties may be derived from different wild-type bacteriocins, although it will be understood that the invention is not limited to wild-type bacteriocin sequences and that modified or engineered bacteriocin sequences may also be used. They may be modified in their effector moiety, targeting moiety, or both.

[0081] The effector moiety of a bacteriocin for use according to the invention can have the amino acid sequence of a wild-type PB effector moiety or a functional fragment thereof, or can have at least 70% sequence identity to the wild-type effector moiety sequence or a functional fragment thereof, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type effector moiety. Generally, the effector moiety (or a fragment thereof) has the same effector activity (i.e., enzymatic activity, generally a nuclease) as the wild-type effector moiety. The effector moiety or a fragment thereof is also capable of binding to the cognate immunity protein domain of the associated immunity protein scaffold. For example, the effector moiety or a fragment thereof can be capable of binding to the cognate wild-type immunity protein.

[0082] Suitable effects parts include: pyocin G, S1, S2, SD2, S3 and AP41, colicin E2, E7, E8 and E9 and klebicin G (thought to be a DNase), colicins E3, E4 and E6, klebicin C, and cloacin DF13 (thought to be rRNases); and Pyocin S4, colicins E5 and D, and klebicin D (thought to be tRNases) There is an effector section.

[0083] A targeting moiety of a bacteriocin for use according to the invention can have the amino acid sequence of a wild-type PB targeting moiety or a functional fragment thereof (i.e., a fragment capable of binding to an appropriate receptor on a target cell and mediating translocation of an effector moiety into the cytosol of the target cell), or alternatively, it can have at least 70% sequence identity to the wild-type targeting moiety sequence or a functional fragment thereof, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type targeting moiety or a functional fragment thereof. Generally, the targeting moiety binds the same receptor and uses the same translocation portal as the wild-type targeting moiety.

[0084] Suitable targeting moieties include: colicin A, E1, E2, E3, E4, E6, E7, E8, E9, K, M, N, U, B, Ia, Ib, 5, 10, S4 and Y; pyocin G, L1, L2, L3, M1, M2, M4, S1, S2, S3, S4, S5, S6, S8, SD1, SD2, SD3, AP41, Sn, SX1 and SX2; klebicins C, D, G, KpneA, KaerA, KpneIa, KvarIa, KoxyY, KpneM, KpneM2, and KvarM; Cloacin DF13; Salmosins SalE1a, SalE1b, SalE2, SalE3 and SalE7; and Pestisin 1 There is something of origin.

[0085] Examples of chimeric bacteriocins are: a KlebC targeting moiety and a colicin E9 effector moiety (designated "KlebC-E9"); a CloDF13 targeting moiety and a colicin E3 effector moiety (designated "CloDF13-E3"); and It comprises a KlebG targeting moiety and a colicin D effector moiety (designated "KlebG-D").

[0086] These are described in more detail below. The broad-spectrum effectiveness of the effector moieties allows the conjugates of the invention to be readily adapted for use against different bacterial strains or species by simply replacing the targeting moiety while retaining the immunity protein scaffold and the bacteriocin effector moiety. Thus, once a host cell capable of expressing the immunity protein scaffold and one or more cognate bacteriocins is constructed, it is possible to change the target specificity of the conjugates produced by that cell simply by replacing the targeting moiety(s) of the bacteriocin(s) with other targeting moieties specific for one or more different strains or species of bacteria. There is no need to construct entirely new host cells with new immunity protein scaffolds and cognate bacteriocins. Immune Protein Scaffold The immunity protein scaffold comprises multiple (ie, two or more) immunity protein domains, each capable of binding a cognate bacteriocin effector moiety.

[0087] In their natural environment, immunity proteins are single domain proteins. However, it will be understood that the term "immunity protein domain" is used herein to refer to a functional domain of an immunity protein scaffold. Thus, an immunity protein scaffold comprises multiple immunity protein domains, each corresponding to a single individual immunity protein. Each domain may be referred to by the name of the corresponding immunity protein (e.g., as a "colicin E9 immunity protein domain") or by an appropriate abbreviation or shortened form (e.g., "Im9 domain").

[0088] The immunity protein domains in a given scaffold molecule can be the same or different. If the immunity protein scaffold contains multiple copies of the same immunity protein domain, the complex may still contain bacteriocins with two or more different targeting domains, but the bacteriocins will generally have the same effector moiety, or at least be similar enough to be able to bind to the same immunity protein domain. However, if it is desired that the complex contain multiple different bacteriocins, it may be more straightforward to control the stoichiometry by using an immunity protein scaffold with multiple different immunity protein domains.

[0089] Immunity protein domains for use in the immunity protein scaffolds of the present invention can have the amino acid sequence of the wild-type PB immunity protein or a functional fragment thereof, or can have at least 70% sequence identity to the wild-type immunity protein sequence or a functional fragment thereof, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type immunity protein sequence or a functional fragment thereof. Each immunity protein domain (or fragment thereof) is capable of binding to the cognate effector moiety of one of the bacteriocins complexed with the immunity protein scaffold. For example, each immunity protein domain (or fragment thereof) can be capable of binding to the cognate wild-type effector moiety.

[0090] Suitable immunity protein domains include: pyocin G, S1, S2, SD2, S3 and AP41, colicin E2, E7, E8 and E9 and klebicin G, colicin E3, E4 and E6; Klebicin C and cloacin DF13; and those of pyocin S4, colicins E5 and D, and klebicin D; They also provide suitable cognate effector domains, as explained above.

[0091] Examples of immune protein scaffolds are: colicin E9 immunity protein domain (“Im9”) and colicin D immunity protein domain (“ImD”) (together designated “Im9-ImD”); It comprises a colicin E9 immunity protein domain ("Im9"), a colicin E3 immunity protein domain ("Im3") and a colicin D immunity protein domain ("ImD") (together designated "Im9-Im3-ImD").

[0092] It will be understood that "Im9-ImD" will form a complex with PBs having effector domains from colicin E9 and colicin D. "Im9-Im3-ImD" will form a complex with PBs having effector domains from colicin E9, colicin E3 and colicin D. PBs may also be chimeric, having targeting moieties from other PBs, as described elsewhere herein.

[0093] Generally, immunity protein domains bind with very high affinity to their cognate bacteriocin effector moieties. For example, RNase-Im complexes bind with very high affinity, e.g., by stopped-flow fluorescence, e.g., as described in Walker et al. (2003) Biochemistry 42, 4161, e.g., at pH 7 and 25°C. -10 K less than M d , e.g. 10 -10 Under M, 10 -11 Less than M or 10 -12 K less than M d The DNase-Im complex may have, for example, a 10 as measured in terms of the RNase-Im complex or as described in Wallis et al. (1995) Biochemistry 34, 13743-13750. -10 K less than M d , e.g. 10 -10 Under M, 10 -11 Under M, 10-12 Under M, 10 -13 Less than M or 10 -14 K less than M d may have.

[0094] Within a given scaffold, the immune protein domains are covalently coupled to one another. In some embodiments, the domains may be expressed individually or conjugated together, for example, by a chemical linker. However, it is generally more convenient for the immune protein scaffold to be expressed as a fusion protein.

[0095] To generate the immunity protein scaffold as a fusion protein, a nucleic acid expression vector containing the coding sequence for each immunity protein component in one contiguous open reading frame is constructed such that each immunity protein component can be translated as part of the same polypeptide chain.

[0096] Typically, a peptide linker is included between each component to allow each component to freely interact with its respective bacteriocin cytotoxic domain without steric hindrance. Those skilled in the art are fully capable of designing appropriate linkers. Traditionally, such linkers are 10-20 amino acids in length and contain a high proportion of small, hydrophilic amino acid residues (e.g., glycine and serine) to provide the necessary flexibility without compromising the aqueous solubility of the molecule. In some embodiments, it may be desirable to use more rigid linkers, including other residues such as proline and asparagine, to create greater separation between the individual domains. In some embodiments, the linker may have a tendency to form a defined secondary structure, such as an alpha helix. Sequences that adopt such structures are well known. For example, a helical linker may be formed using repeating units of the sequence EAAAK (SEQ ID NO: 1), although other sequences will be familiar to those skilled in the art.

[0097] For example, the immunity protein scaffold described in the Examples below utilizes the linker sequences NINGGPTGIGVS (SEQ ID NO:2) and NGGGNGNSGGGS (SEQ ID NO:3) between the immunity protein domains, both of which are essentially unstructured sequences derived from the N-terminal subregion of colicin E9. A helical linker [(EAAAK)3EL] (SEQ ID NO:4) is used between the M-type bacteriocin (KvarM) and the immunity protein domain. Those skilled in the art will be able to design other suitable linkers as needed.

[0098] The immunity protein scaffold may further comprise heterologous moieties, ie moieties that are not immunity protein domains or linker peptides. The heterologous moiety can be peptidic or non-peptidic, and if the heterologous moiety is peptidic, it can be part of the same fusion protein as the rest of the immunoprotein scaffold, or it can be linked to the immunoprotein scaffold by chemical conjugation.

[0099] For example, the heterologous moiety may act to increase in vivo solubility and / or half-life (e.g., in plasma) and / or bioavailability in a subject compared to a corresponding scaffold (or conjugate) that is otherwise identical but lacks the heterologous moiety. Such modifications are also known to reduce the clearance (e.g., renal clearance) of therapeutic proteins and peptides. Suitable peptidic moieties include immunoglobulin Fc domains. Suitable non-peptidic moieties include polymeric moieties. Polymeric moieties are preferably water-soluble (amphiphilic or hydrophilic), non-toxic, and pharmaceutically inert. Examples include polyethylene glycol (PEG), homo- or copolymers of PEG, monomethyl-substituted polymers of PEG (mPEG), or polyoxyethyleneglycol (POG). See, e.g., Francis et al. (1998), Int. J. Hematology 68:1-18; Zalipsky (1995), Bioconjugate Chem. 6:150-165; and Delgado et al. (1992), Crit. Rev. Therap. Drug Carrier Syst. 9:249-304.

[0100] Other suitable polymeric moieties include polyamino acids, such as polylysine, polyaspartic acid, and polyglutamic acid (see, e.g., Gombotz et al. (1995), Bioconjugate Chem. 6:332-351; Hudecz et al. (1992), Bioconjugate Chem. 3:49-57; Tsukada et al. (1984), J. Natl. Cancer Inst. 73:721-729; and Pratesi et al. (1985), Br. J. Cancer 52:841-848).

[0101] The polymeric portion may be linear or branched. It may have a molecular weight of 500 to 40,000 Da, for example, 500 to 10,000 Da, 1000 to 5000 Da, 10,000 to 20,000 Da, or 20,000 to 40,000 Da.

[0102] Alternatively, the heterologous moiety may comprise a cytotoxic domain (e.g., a protein bacteriocin cytotoxic domain) such that the scaffold itself is an additional toxin in addition to the PB associated with the immunity protein scaffold. The heterologous moiety may further comprise a moiety capable of mediating translocation across the target cell outer membrane, such as a protein bacteriocin targeting moiety. In a preferred embodiment, the heterologous moiety is an M-type bacteriocin, such as colicin M (ColM), KpneM, KpneM2, KvarM, PaeM1, or PaeM4, or a functional variant thereof.

[0103] M-type bacteriocins act by degrading peptidoglycan precursors within the periplasm, leading to cell lysis (Schaller et al., 1982). M-type bacteriocins have been found in a range of different species, including E. coli, Pseudomonas, Pectobacterium, Klebsiella, and Burkholderia (Cherier et al., 2021). Colicin M from E. coli is the best-studied example of this class of bacteriocin. KvarM (see Dekovskiene et al., 2019), a 30.8 kDa protein, binds to the outer membrane ferrichrome receptor FhuA and translocates through the outer membrane into the periplasm of the cell in a process driven by the Ton system. It is of particular interest because it has shown activity against a wide spectrum of Klebsiella strains, including those that are multidrug-resistant, in plate, liquid, and biofilm killing assays.

[0104] KvarM and colicin M are composed of three regions characteristic of bacteriocins: an N-terminal unstructured transition region (Pilsl et al., 1993), a central globular compartment that interacts with the outer membrane receptor FhuA, and a C-terminal catalytic region that hydrolyzes lipid II precursors (Sham et al., 2014). However, their relatively small size and compact folding mean that they do not form independently folded domains, and attempts to shorten these molecules commonly result in protein misfolding (Barreteau et al., 2010).

[0105] When the immunity protein scaffold comprises an M-type bacteriocin, the M-type bacteriocin is typically located at the N-terminus of the scaffold molecule, and the immunity protein domain is located at the C-terminus of the M-type bacteriocin. A suitable linker will typically be present between the M-type bacteriocin and the first immunity protein domain. It may be desirable to have a rigid linker between the M-type bacteriocin and the first immunity protein domain, such as a linker with an alpha-helical secondary structure, as illustrated in the examples below.

[0106] Incorporation of an M-type bacteriocin into the immunity protein scaffold has the potential to increase the receptor-binding capacity and cytotoxic activity of the complex as a whole. The resulting complex can bind to the receptor for the M-type bacteriocin and to the receptor targeted by the PB component of the complex (which is noncovalently associated with the immunity protein domain), thus further increasing the potential avidity of interaction with the target cell surface and being cytotoxic to bacteria susceptible to the action of M-type bacteriocins. host cell The present invention extends to host cells capable of expressing the immunity protein scaffold.

[0107] Thus, the present invention provides a host cell comprising a nucleic acid encoding an immunity protein scaffold, an immunity protein scaffold comprising a first immunity protein domain and a second immunity protein domain, wherein the host cell is capable of expressing said immunity protein scaffold.

[0108] An advantage of the present invention is that the host cell can also express the PB components, allowing the production of the complete antibacterial complex with the correct stoichiometry from a single cell, thus minimizing production and purification costs.

[0109] Thus, the present invention provides: (i) a nucleic acid encoding an immunity protein scaffold, the immunity protein scaffold comprising at least a first immunity protein domain and a second immunity protein domain; (ii) a nucleic acid encoding at least one bacteriocin, the or each bacteriocin having an effector domain capable of binding to at least one of said immunity protein domains; Including, Further provided is a host cell, wherein the host cell is capable of expressing said immunity protein scaffold and said at least one bacteriocin.

[0110] If the immunity protein scaffold contains only one type of immunity protein domain and therefore the complex contains only one type of bacteriocin, the cell can encode and express only a single homogeneous bacteriocin. More commonly, the immunity protein scaffold contains at least two different immunity protein domains and the cell comprises nucleic acids encoding at least two bacteriocins, each having an effector capable of binding to at least one of the immunity protein domains, resulting in an antibacterial complex containing at least two different types of bacteriocins.

[0111] Thus, the host cell comprises nucleic acid encoding each of the respective bacteriocins. The immunity protein scaffold and nucleic acid encoding the bacteriocin or bacteriocins are generally provided as part of one or more nucleic acid expression constructs or vectors. Thus, the nucleic acids may be provided in a single vector or in two or more separate vectors.

[0112] Those skilled in the art will be able to design appropriate nucleic acid expression constructs or vectors to obtain expression of the immunity protein backbone, and further, if necessary, the bacteriocin molecule. Generally, such vectors contain appropriate transcriptional and translational regulatory sequences operably linked to the sequence encoding the desired protein to enable transcription and translation of the protein by the host cell. The vector may also contain other sequences, such as selectable marker genes, as needed, depending on the particular host cell. The vector may be intended to be integrated into the host cell chromosome, or may exist and replicate independently of the host chromosome as an episome, e.g., a plasmid.

[0113] It will be appreciated that because the immunity protein scaffold and each bacteriocin molecule in the final complex are separate molecules and are associated by non-covalent interactions between the immunity protein domain and the effector portion of the bacteriocin molecule, they will generally be expressed as separate molecules in the host cell.

[0114] The host cell is typically a bacterial host cell, such as an E. coli host cell. As discussed above, the specificity of a PB is determined by its targeting moiety. Therefore, an antibacterial conjugate can be adapted for use against different bacterial strains or species by simply exchanging the targeting moiety of the PB component while retaining the immunity protein scaffold and the PB effector moiety. This can be easily achieved by modifying existing host cells; it is not necessary to construct entirely new host cells with a new immunity protein scaffold and the same bacteriocin.

[0115] Therefore, a nucleic acid or expression construct encoding a given PB can be designed to facilitate the exchange of targeting moieties. For example, an appropriate restriction site can be created between the sequence encoding the targeting moiety and the sequence encoding the effector moiety. An additional restriction site can be created at the opposite end of the targeting moiety coding sequence, for example, 5' to the start of the PB open reading frame.

[0116] The present invention further provides a method of producing an antibacterial complex, comprising providing a host cell as described and culturing said cell under conditions suitable for expression of the immunity protein scaffold and bacteriocin molecule. The method may further comprise isolating the antibacterial complex and optionally a further step of purification.

[0117] Alternatively, it may not be desirable to express all components of the antibacterial complex in the same host cell. Instead, the components may be expressed in two or more different host cells, each containing nucleic acid encoding one or more individual components and capable of expressing that component. For example, the immunity protein scaffold may be expressed in one host cell and one or more bacteriocin molecules may be expressed in one or more other host cells.

[0118] Accordingly, the present invention further provides a method of producing an antibacterial complex, the method comprising contacting an immunity protein scaffold comprising a first immunity protein domain and a second immunity protein domain with first and second bacteriocin molecules, each having an effector moiety capable of binding to a respective one of the immunity protein domains, to form a complex of the invention.

[0119] In such cases, if the bacteriocin molecule is expressed in a host cell that is sensitive to that bacteriocin and the immunity protein scaffold is not expressed, the host cell will generally also express the cognate immunity protein for the related bacteriocin. In such cases, the bacteriocin can be dissociated from and isolated from the immunity protein prior to contacting the bacteriocin with the immunity protein scaffold. Treatment targets and symptoms The conjugates and methods of the invention are particularly suitable for the prevention and / or treatment of bacterial infections caused by Escherichia coli, Pseudomonas (particularly Pseudomonas aeruginosa), Klebsiella (e.g. Klebsiella pneumoniae), Enterobacter cloacae, Salmonella (e.g. Salmonella enterica) and Yersinia pestis, Gram-negative bacterial infections in general, and symptoms resulting from or associated with such infections.

[0120] As discussed above, the strain specificity of any given bacteriocin is generally determined by its targeting moiety. Thus, treatment of infection with or symptoms associated with a given bacterial species or strain will generally utilize a conjugate comprising at least one bacteriocin bearing a targeting moiety specific for that species or strain.

[0121] The infection may be acute or chronic. For example, Pseudomonas aeruginosa infections of the lower respiratory tract are particularly common in patients with cystic fibrosis (where it represents a leading cause of death) and chronic obstructive pulmonary disease (COPD). Other patients with airway dysfunction and / or immune dysfunction may also be susceptible to infection, including patients with congestive heart failure, AIDS patients, and patients taking immunosuppressive drugs or other immunosuppressive therapies for, for example, cancer (especially chemotherapy), rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, sarcoidosis, focal glomerulosclerosis, Crohn's disease, Behçet's disease, pemphigus, ulcerative colitis, etc.

[0122] Acute conditions associated with or caused by Pseudomonas infection include community-acquired pneumonia and hospital-acquired infections such as ventilator-associated pneumonia and hospital-acquired pneumonia. Thus, when treating a Pseudomonas (e.g., P. aeruginosa) infection or symptoms associated therewith, it is generally desirable to use a conjugate comprising at least one bacteriocin having a targeting moiety specific for Pseudomonas (e.g., P. aeruginosa), such as an S-type pyocin targeting moiety. As discussed elsewhere herein, it may be desirable to use a conjugate having at least two such bacteriocins, or a conjugate containing only such bacteriocins.

[0123] E. coli infections are associated with a variety of conditions. Pathogenic adherent invasive E. coli (AIEC), which has the ability to form biofilms and invade and replicate within host cells, can be particularly serious. For example, aberrant colonization of the ileal mucosa by AIEC is thought to be a factor in the development of Crohn's disease. E. coli is also commonly associated with urinary tract infections and other conditions.

[0124] When treating E. coli infection or symptoms associated therewith, it is generally desirable to use a conjugate comprising at least one bacteriocin having a targeting moiety specific for E. coli, e.g., a colicin targeting moiety. As discussed elsewhere herein, it may be desirable to use a conjugate having at least two such bacteriocins, or a conjugate containing only such bacteriocins.

[0125] Infection with Klebsiella pneumoniae may be associated with, for example, sepsis or bronchitis or pneumonia (often in the form of bronchopneumonia). Affected patients tend to develop lung abscesses, cavitation, empyema, or pleural adhesions.

[0126] When treating a Klebsiella pneumoniae infection or associated symptoms, it is generally desirable to use a conjugate comprising at least one bacteriocin having a targeting moiety specific for Klebsiella pneumoniae, e.g., a klebicin targeting moiety. As discussed elsewhere herein, it may be desirable to use a conjugate having at least two such bacteriocins, or a conjugate containing only such bacteriocins.

[0127] Similarly, when treating infections caused by or symptoms associated with other bacterial species, such as Salmonella enterica, Enterobacter cloacae, or Yersinia pestis, it will generally be desirable to use a conjugate comprising at least one bacteriocin having a targeting moiety specific for that bacterial species or strain, e.g., a salmonocin, cloacin, or pesticin targeting moiety as appropriate. As discussed elsewhere herein, it may be desirable to use a conjugate having at least two such bacteriocins, or a conjugate containing only such a bacteriocin.

[0128] Generally, the subject to be treated is a mammal. The subject is generally a human, but may also be any other primate (great ape, Old World monkey, or New World monkey) or a domestic, laboratory, or livestock animal such as a mouse, rat, guinea pig, lagomorph (e.g., rabbit), cat, dog, pig, cow, horse, sheep, or goat. Pharmaceutical Composition The conjugates described herein can be formulated into pharmaceutical compositions. These compositions may contain, in addition to one of the above substances, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material may depend on the route of administration, such as oral, intravenous, cutaneous or subcutaneous, pulmonary, intramuscular, intraperitoneal, or topical application. Oral, intravenous, or pulmonary routes may be preferred.

[0129] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder, or liquid form. Tablets may contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline, glucose or other sugar solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may also be included.

[0130] For intravenous, cutaneous or subcutaneous injection or injection at the site of pain, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity and stability.Those skilled in the art can easily prepare suitable solutions using isotonic media such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.If necessary, preservatives, stabilizers, buffers, antioxidants and / or other additives may be included.

[0131] The term "pulmonary administration" is intended to encompass any suitable delivery method by which an active agent is delivered to the lungs via the respiratory tract. The most common methods of pulmonary administration are oral and / or nasal inhalation.

[0132] The conjugates of the invention can be formulated in any suitable manner for pulmonary administration, such as in liquid or solid (generally powder) form. The formulation can be delivered by any suitable mechanism or delivery device, including an inhaler (e.g., a metered dose inhaler, a dry powder inhaler), a nebulizer (e.g., an ultrasonic nebulizer, a jet nebulizer, a vibrating mesh nebulizer), etc.

[0133] Accordingly, the present invention further provides a device for pulmonary administration of a therapeutic composition to a subject, the composition comprising an S-type pyocin as described elsewhere herein. The device may be an inhaler (e.g., a metered-dose inhaler, a dry powder inhaler) or a nebulizer (e.g., an ultrasonic nebulizer, a jet nebulizer, a vibrating mesh nebulizer).

[0134] Liquid compositions generally include an aqueous carrier such as water or saline. Dextrose or other sugar solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may also be included.

[0135] Emulsion and nanoparticle encapsulation, both of which utilize lipids, can be used. Solid (e.g., powder) preparations can utilize carriers such as sugars, cyclodextrins, etc. They can be prepared by any suitable method, including spray drying, spray freeze drying, solvent precipitation, jet milling, etc.

[0136] Administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (in some cases, prevention may be considered therapy), which is sufficient to show benefit to the individual. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescribing treatment, e.g., determining dosage, etc., is the responsibility of general practitioners and other medical doctors, and will generally take into account the disorder being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to medical practitioners. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical textbooks, e.g., Remington's Pharmaceutical Sciences, 20th ed., 2000, pub. Lippincott, Williams and Wilkins; and Handbook of Pharmaceutical Excipients, 2nd ed., 1994. array The following sequences are utilized in the examples below. Deviations from the corresponding wild-type sequences generally result from the incorporation of appropriate restriction sites to facilitate exchange of targeting moieties. It will be understood that either wild-type or variant sequences (or further modifications) may be used in the context of the present invention. ColE9: amino acid sequence (E9 DNase domain is underlined)

[0137] [ka]

[0138] [In the wild-type sequence, the last residue of the targeting moiety is D (not A) and the first residue of the effector moiety is K (not M)] ColE9 targeting moiety: amino acid sequence

[0139] [ka]

[0140] [In the wild-type sequence, the last residue of the targeting moiety is D (not A)] ColE9 effector moiety (DNase): amino acid sequence

[0141] [ka]

[0142] [In the wild-type sequence, the first residue of the effector moiety is K (not M)] ColE9: coding sequence (E9 DNase domain underlined, NcoI site in bold)

[0143] [ka]

[0144] ColE9 immunity protein ("Im9"): amino acid sequence

[0145] [ka]

[0146] ColE9 immunity protein ("Im9") with a C-terminal LE(His)6 tag: amino acid sequence (The C-terminal LE(His)6 tag is underlined.)

[0147] [ka]

[0148] ColE9 immunity protein ("Im9") with a C-terminal LE(His6) tag: coding sequence (C-terminal LE(His)6 tag is underlined, XhoI site is bold)

[0149] [ka]

[0150] ColD: amino acid sequence (tRNase domain is underlined)

[0151] [ka]

[0152] [In the wild-type sequence, the first two residues of the effector moiety are VY (not MD)] ColD targeting moiety: amino acid sequence

[0153] [ka]

[0154] ColD effector moiety: amino acid sequence

[0155] [ka]

[0156] [In the wild-type sequence, the first two residues of the effector moiety are VY (not MD)] ColD: coding sequence (tRNase domain underlined, NcoI site in bold)

[0157] [ka]

[0158] ColD immunity protein ("ImD"): amino acid sequence

[0159] [ka]

[0160] ColD immunity protein ("ImD") with a C-terminal LE(His6) tag: amino acid sequence (LE(His6) tag is underlined)

[0161] [ka]

[0162] Coding sequence of ColD immunity protein ("ImD") with a C-terminal His6 tag (LE(His6) tag is underlined, XhoI cloning site is bold)

[0163] [ka]

[0164] KlebC targeting moiety: amino acid sequence

[0165] [ka]

[0166] KlebC-E9 chimera (KlebC targeting portion; E9 effector portion): amino acid sequence (The E9 effector moiety (DNase) is underlined.)

[0167] [ka]

[0168] KlebC-E9 chimera: coding sequence (The E9 effector moiety (DNase) is underlined.)

[0169] [ka]

[0170] CloDF13 targeting moiety: amino acid sequence

[0171] [ka]

[0172] ColE3 effector portion (rRNase): amino acid sequence

[0173] [ka]

[0174] ColE3 immunity protein ("Im3"): amino acid sequence

[0175] [ka]

[0176] KvarM: amino acid sequence

[0177] [ka]

[0178] CloDF13-E3 chimera (CloDF13 targeting portion; ColE3 effector portion): amino acid sequence (The ColE3 effector moiety (rRNase) is underlined.)

[0179] [ka]

[0180] CloDF13-E3 chimera (CloDF13 targeting portion; E3 effector portion): coding sequence (The E3 effector moiety (rRNase) is underlined.)

[0181] [ka]

[0182] KlebG targeting moiety: amino acid sequence

[0183] [ka]

[0184] KlebG-D chimera (KlebG targeting portion; ColD effector portion (tRNase)): amino acid sequence (ColD effector part is underlined)

[0185] [ka]

[0186] KlebG-D chimera (KlebG targeting portion; ColD effector portion (tRNase)): coding sequence (ColD effector part is underlined)

[0187] [ka]

[0188] Im9-ImD immunity protein backbone: amino acid sequence (Im9 underlined, ImD double underlined)

[0189] [ka]

[0190] Im9-ImD immunity protein scaffold with a C-terminal LE(His6) tag: amino acid sequence (Im9 underlined, ImD double underlined, LE(His6) tag dashed underline).

[0191] [ka]

[0192] Im9-ImD immunity protein scaffold with a C-terminal LE(His6) tag: coding sequence (Im9 underlined, ImD double underlined, XhoI cloning site in bold, LE(His6) tag in dashed underline).

[0193] [ka]

[0194] Im9-Im3-ImD immunity protein backbone: amino acid sequence (Im9 underlined, Im3 double underlined, ImD dashed underlined)

[0195] [ka]

[0196] Im9-Im3-ImD immunity protein scaffold with a C-terminal LE(His6) tag: amino acid sequence (Im9 underlined, Im3 double underlined, ImD dashed underlined).

[0197] [ka]

[0198] Im9-Im3-ImD immunity protein scaffold with a C-terminal LE(His6) tag: coding sequence (Im9 underlined, Im3 double underlined, ImD dashed underlined).

[0199] [ka]

[0200] KvarM-Im9-ImD immunity protein scaffold with a C-terminal LE(His6) tag: amino acid sequence (KvarM in italics, Im9 underlined, ImD double underlined, LE(His6) tag dashed underline).

[0201] [ka]

[0202] KvarM-Im9-ImD immunity protein scaffold with a C-terminal LE(His6) tag: coding sequence (KvarM in italics, Im9 underlined, ImD double underlined, XhoI cloning site in bold, LE(His6) tag dashed underline).

[0203] [ka]

[0204] KvarM-Im9-Im7-ImD immunity protein scaffold with a C-terminal LE(His6) tag: amino acid sequence (KvarM in italics, Im9 underlined, Im7 double underlined, ImD dashed underline).

[0205] [ka]

[0206] KvarM-Im9-Im7-ImD immunity protein scaffold with a C-terminal LE(His6) tag: coding sequence (KvarM in italics, Im9 underlined, Im7 double underlined, ImD dashed underline).

[0207] [ka]

[0208] Im97 immunity protein scaffold with a C-terminal LE(His6) tag: amino acid sequence (Im9 underlined, Im7 double underlined, LE(His6) dashed underlined)

[0209] [ka]

[0210] S5E9 chimera: amino acid sequence (Pyocin S5 outer membrane translocation domain in italics; pyocin S5 receptor-binding domain underlined; pyocin G inner membrane translocation domain double underlined; colicin E9 DNase domain dashed underlined)

[0211] [ka]

[0212] S5E7 chimera: amino acid sequence (Pyocin S5 outer membrane translocation domain in italics; pyocin S5 receptor-binding domain underlined; pyocin G inner membrane translocation domain double underlined; colicin E7 DNase domain dashed underlined)

[0213] [ka]

[0214] ColE7 immunity protein ("Im7") with a C-terminal LE(His6) tag: amino acid sequence (LE(His6) tag is underlined)

[0215] [ka] [Example]

[0216] To illustrate the versatility and applicability of the described approach, we constructed heterotrimeric complexes (containing two bacteriocin molecules and a cognate bivalent immunity protein scaffold, as described in Example 1) and heterotetrameric complexes (containing three bacteriocin molecules and a cognate trivalent immunity protein scaffold, as described in Example 2). These complexes are illustrated in Figure 1.

[0217] Example 1 Components of the trimeric complex The bacteriocin components of the trimeric complex are colicin E9 (ColE9) and colicin D (ColD). The immunity protein scaffold is a fusion protein containing the cognate immunity proteins "Im9" and "ImD" separated by a flexible linker peptide. The scaffold is referred to by the designation "Im9-ImD." Complexes of the scaffold with ColE9, ColD, or both are represented by the shorthand notation: [ColE9:Im9-ImD]; [ColE9:Im9-ImD:ColD]; and [Im9-ImD:ColD] are referenced respectively.

[0218] Colicin E9 and colicin E3 are Group A bacteriocins that bind their essentially unstructured N-termini with nanomolar affinity to the vitamin B12 receptor, BtuB, before passing through trimeric porins such as OmpF in the outer membrane. This essentially unstructured N-terminus contains a TolB box that binds TolB, part of the activated Tol-Pal complex that spans the periplasm. Parasitism of the Tol-Pal system results in the abandonment of immunity proteins and the translocation of the cytotoxic C-terminal domain into the cytoplasm. Colicin E9 and colicin E3 are highly conserved except for their cytotoxic domains, which are DNases in E9 and rRNases in E3.

[0219] Colicin D is a group B bacteriocin that binds with high affinity to the ferric enterobactin receptor, FepA. Colicin D passes through FepA and interacts with TonB of the Ton system within the periplasm, leading to the release of immunity proteins and translocation of the C-terminal tRNase domain to the cytoplasm. The uptake pathways of colicin E9 and colicin D are summarized in Figure 2.

[0220] In vitro assembly of the trimeric complex Individual bacteriocins were expressed in E. coli BL21(DE3) as heterodimeric complexes with their respective immunity proteins. The immunity proteins were engineered to carry a His6 tag at their C-terminus. Cell pellets were lysed by sonication, clarified by centrifugation, and loaded onto a 5 mL HisTrap HP column. In each case, the free bacteriocins were eluted from the nickel affinity column using 6 M guanidinium hydrochloride. The eluted bacteriocins were refolded by dialysis in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and purified by gel filtration through a 26 / 60 S200 column equilibrated in the same buffer. Immunity protein scaffolds Im9-ImD, which also carry a C-terminal His6 tag, were individually expressed and purified by nickel affinity chromatography (eluted with imidazole), followed by gel filtration through a 26 / 60 S200 column equilibrated with 25 mM Tris-HCl, pH 7.5.

[0221] (i) 500 μL samples of 20 μM Im9-ImD + 40 μM ColE9 + 40 μM ColD, 20 μM Im9-ImD + 40 μM ColD, 20 μM Im9-ImD + 40 μM ColE9, and 20 μM Im9-ImD were prepared in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and purified on a Superdex 200 Increase 10 / 300GL analytical gel filtration column (Cytiva) equilibrated in the same buffer.

[0222] Good baseline separation was observed between ColE9:Im9-ImD:ColD and free colicin, which is essential to ensure that any observed killing activity is due to the heterotrimeric complex and not contaminating free colicin (Fig. 4).

[0223] Cell killing by trimeric complexes Fifteen milliliters of LB-0.7% agar was inoculated with 200 μL of either the tolA-BW25113, BL21(DE3)(btuB-), tonB-BW25113, or fepA-BW25113 culture, which was then layered onto an LB-1.5% agar plate. Serial dilutions of ColD, ColE9, and [ColE9:Im9-ImD:ColD] were prepared over a concentration range of 100 nM to 137 pM, and 3 μL of each dilution was spotted onto each inoculated agar plate. Plates were grown overnight at 37°C, and bacteriocin activity was observed as a zone of clearance within the bacterial lawn. The results are shown in Figures 5A-5C. In a similar experiment, ColD, ColE9, a mixture of ColE9 and ColD, and serial dilutions of [ColE9:Im9-ImD:ColD] were spotted onto a lawn of tolA-BW25113. The results are shown in Figure 5D.

[0224] As expected, ColD was inactive against tonB- or fepA- cells, and ColE9 was inactive against tolA- or btuB- cells. The heterotrimer [ColE9:Im9-ImD:ColD] retained activity against all strains tested, indicating that both ColE9 and ColD are active within the complex. Both ColD and ColE9 were also tested as complexes with the Im9-ImD fusion protein ([ColE9:Im9-ImD] and [Im9-ImD:ColD]), which had no negative effect on the activity of either ColD or ColE9 alone (not shown).

[0225] Interestingly, the activity of ColD against tolA cells was impaired, indicating that the level of FepA expression in these cells was reduced. However, [ColE9:Im9-ImD:ColD] showed better activity against tolA cells than either ColD alone or the ColE9 + ColD mixture, suggesting that although ColE9 cannot enter and kill cells, the presence of the ColE9 receptor-binding domain may anchor the heterotrimeric complex to the cell surface and support the FepA-dependent activity of the ColD moiety.

[0226] Trimeric complexes circumvent the development of bacterial resistance In addition to increasing strain coverage, combining multiple bacteriocins that target independent uptake pathways significantly reduces the chance of resistance emerging in cells susceptible to two or more bacteriocin components. Resistance requires simultaneous mutation of multiple components to affect both uptake pathways. In vitro, when grown on rich media, nutrient receptors are largely unaffected, making resistant mutants even more likely to prevail than in vivo. Bacteriocin receptors are often virulence factors and are upregulated during infection, and their loss affects the colonization ability of bacteria.

[0227] Three-fold serial dilutions of [ColE9:Im9-ImD:ColD], [Im9-ImD:ColD], and [ColE9:Im9-ImD] were prepared in LB in 96-well plates from 1 μM to 5.6 pM. An overnight culture of MG1655 E. coli was used to inoculate each well of the plate at a 1 / 125 dilution. Cultures were grown at 37°C with shaking at 190 rpm, and OD was measured after 6 and 24 hours (Figure 6).

[0228] After 6 h, 460 pM, 460 pM, and 37 nM were sufficient to inhibit growth of [ColE9:Im9-ImD:ColD], [ColE9:Im9-ImD], and [Im9-ImD:ColD], respectively. However, after 24 h, 1.4 nM, 1 μM, or 0.33 μM of [ColE9:Im9-ImD:ColD], [ColE9:Im9-ImD], and [Im9-ImD:ColD], respectively, were required to prevent growth. This likely reflects the dominance of overnight ColE9- or ColD-treated cultures by resistant mutants. To test this, the frequency of resistant mutants was calculated by plating serial dilutions of an overnight culture of MG1655 onto plates containing 25 nM [ColE9:Im9-ImD:ColD], 25 nM [ColE9:Im9-ImD], 50 nM [Im9-ImD:ColD], or no bacteriocin. The original culture contained 1.8 × 10 9 cfu / mL, the cultures were reduced to 775 cfu / mL by 50 nM [Im9-ImD:ColD] and 5750 cfu / mL by 25 nM [ColE9:Im9-ImD], with no detectable colonies in the samples inoculated onto 25 nM [ColE9:Im9-ImD:ColD].

[0229] In an attempt to determine the value of [ColE9:Im9-ImD:ColD], 1.75 × 10 10 Five mL overnight cultures of MG1655 E. coli containing cfu were treated with 25 nM [ColE9:Im9-ImD:ColD], and all samples were plated and grown overnight at 37°C. No colonies were obtained. The frequencies of resistant mutants were 1 / (3.0 × 10) for [ColE9:Im9-ImD] and [Im9-ImD:ColD], respectively. 5 ) and 1 / (2.3×10 6 ) for the resistance to [ColE9:Im9-ImD:ColD], which requires mutations in both the ColE9 and ColD uptake pathways, the resistant mutant frequency was consistent with the experimentally observed non-resistant mutants, and the resistance frequency to the individual components was [1 / (6.9 × 10 11)] is a product of

[0230] In vivo expression of the trimeric complex The genes encoding colicin E9, Im9-ImD, and colicin D were cloned into pET21a and transformed into E. coli BL21(DE3). Expression and purification revealed only colicin E9 and Im9-ImD. A second copy of the colicin D gene was cloned into pACYCDuet1, which was cotransformed into BL21(DE3) cells along with colicin E9, Im9-ImD, and colicin D cloned into pET21a. The expressed proteins were first purified using the His tag at the C-terminus of Im9-ImD and colicin D, colicin E9, and Im9-ImD, which was evident in the nickel column elution (Figure 7).

[0231] The [ColE9:Im9-ImD:ColD] complex was further purified on a 26 / 60 S200 gel filtration column to separate the intact complex from the [ColE9:Im9-ImD] and [Im9-ImD:ColD] subcomplexes. The initial elution peak (110 mL, fractions A1–A5) corresponded to aggregated material eluting within the void volume of the column. The second elution peak (145 mL, fractions A11–B2) was the [ColE9:Im9-ImD:ColD] complex. The third elution peak (174 mL) was a mixture of [ColE9:Im9-ImD] and [Im9-ImD:ColD]. SDS-PAGE analysis of representative fractions is shown in Figure 8. Initial purification of [ColE9:Im9-ImD:ColD] gave a yield of approximately 10 mg per liter of culture.

[0232] The activity of purified [ColE9:Im9-ImD:ColD] was tested against E. coli BW25113, btuB-BL21(DE3), fepA-BW25113, tolA-BW25113, and tonB-BW25113 over a concentration range of 100 nM to 137 pM. Similar to the in vitro assembled [ColE9:Im9-ImD:ColD], the in vivo assembled complex was active against E. coli strains lacking either the colicin E9 or colicin D uptake pathway components (Figure 9).

[0233] Example 2 Components of the tetrameric complex The bacteriocin components of the tetrameric complex are chimeric proteins containing the targeting portions (receptor binding and translocation domains) of klebicin C (KlebC), cloacin DF13 (CloDF13), and klebicin G (KlebG) linked to the effector portions (cytotoxic domains) of colicin E9, colicin E3, and colicin D, respectively. The resulting chimeric proteins are designated KlebC-E9, CloDF13-E3, and KlebG-D.

[0234] The immunity protein scaffold is a fusion protein containing its cognate immunity proteins for ColE9 ("Im9"), ColE3 ("Im3"), and ColD ("ImD") separated by a flexible linker peptide. The scaffold is referred to by the designation "Im9-Im3-ImD." The complex of the scaffold and three chimeric bacteriocins is referred to by the shorthand notation [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D].

[0235] The protein-protein interactions involved in the uptake of cloacin DF13 ( Krone et al., 1983 ; Thomas and Valvano, 1993 ; Wooldridge and Williams, 1991 ), klevicin G (unpublished), and klevicin C ( Housden et al., 2021 ) are summarized in Figure 3 .

[0236] In vitro assembly of the tetrameric complex The chimeric bacteriocins were expressed in E. coli BL21(DE3) as heterodimeric complexes with immunity proteins for each cytotoxic domain (Im9, Im3, and ImD). The immunity proteins were engineered to carry a His6 tag at their C-terminus. Cell pellets were lysed by sonication, clarified by centrifugation, and loaded onto a 5 mL HisTrap HP column. In each case, the free bacteriocin was eluted from the nickel affinity column using 6 M guanidinium hydrochloride. The eluted bacteriocin was refolded by dialysis in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and purified by gel filtration on a 26 / 60 S200 column equilibrated in the same buffer. The immunity protein scaffold Im9-Im3-ImD, which also carries a C-terminal His6 tag, was expressed separately and purified by nickel affinity chromatography (eluted with imidazole), followed by gel filtration on a 26 / 60 S200 column equilibrated with 25 mM Tris-HCl, pH 7.5.

[0237] A 500 μL sample containing 20 μM Im9-Im3-ImD, 30 μM KlebC-E9, 30 μM CloDF13-E3, and 30 μM KlebG-D prepared in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl was purified on a Superdex 200 Increase 10 / 300GL analytical gel filtration column (Cytiva) equilibrated in the same buffer. SDS-PAGE analysis of representative fractions is shown in Figure 10.

[0238] Microbial killing activity of the tetrameric complex Serial dilutions of the tetrameric complex [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] were prepared at concentrations ranging from 6.1 μM to 25 nM. Five-μL spots were applied to soft agar lawns of nutrient broth (Merck) inoculated with SG62, SR3, or SR6 Klebsiella pneumoniae. Plates were grown overnight at 37°C, and bacteriocin activity was observed as a zone of clearance within the bacterial lawn (Figure 11).

[0239] The activities of KlebC-E9 against SR3, CloDF13-E3 against SG62, and KlebG-D against SR6 are retained in the tetrameric [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] complex. Furthermore, the activity of the tetrameric [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] complex against SR3 is enhanced compared to the individual bacteriocin components, demonstrating that increased receptor affinity by multiple targeting moieties contributes to the killing activity of the complex.

[0240] Example 3 We designed immunity protein scaffolds incorporating the M-type bacteriocin KvarM. The first contained KvarM linked to two immunity protein domains, Im9 and ImD (KvarM-Im9-ImD), and the second contained KvarM linked to three immunity protein domains, Im9, Im7, and ImD (KvarM-Im9-Im7-ImD). In both of these scaffolds, KvarM (at the N-terminal end of the fusion protein) is linked to Im9 using a rigid helical linker [(EAAAK)3EL], and the immunity protein domains are linked by an essentially unstructured sequence derived from the N-terminal subregion of colicin E9. A His6 tag used for nickel affinity purification was added to the C-terminal end of the fusion protein (after the last immunity domain). The scaffolds are illustrated schematically in Figures 12 [A] and 12 [B].

[0241] KvarM-Im9-ImD and KvarM-Im9-Im7-ImD were expressed in Escherichia coli BL21(DE3) cells and purified by nickel affinity chromatography followed by gel filtration. Serial dilutions of wild-type KvarM, KvarM-Im9-ImD, and KvarM-Im9-Im7-ImD, ranging in concentration from 2.5 μM to 4.9 nM, were spotted onto soft agar lawns of Klebsiella quasipneumoniae SG96 in nutrient broth. Once dry, plates were incubated overnight at 37°C; a zone of clearance indicated bacteriocin-mediated killing.

[0242] Both KvarM immunity protein scaffolds exhibited cytotoxic activity against K. quasipneumoniae SG96 cells, with KvarM-Im9-ImD exhibiting a zone of clearance at concentrations between 2.5 μM and 156 nM, and killing of KvarM-Im9-ImD occurred at concentrations up to 312 nM (Figure 12[C]). This demonstrated that the KvarM immunity protein scaffold can bind to KvarM's outer membrane receptor, FhuA, and that covalent fusion of two and three immunity protein domains to the C-terminus of the KvarM cytotoxic region does not abolish its enzymatic peptidoglycan precursor degradation activity. Interestingly, these results suggest that upon binding of the nuclease bacteriocin to the target cell surface receptor, the immunity protein scaffold generally dissociates from the complexed nuclease bacteriocin and is translocated into the periplasm of the target cell.

[0243] Similar to the other immunity protein scaffolds described herein, KvarM immunity protein scaffold fusions can be complexed with protein bacteriocins having effector moieties (cytotoxic domains) cognate to the immunity protein domains, as illustrated schematically in Figure 13. The addition of KvarM increases the receptor binding and cytotoxic potency of the complex, as the KvarM component provides binding activity for its own receptor (in addition to the receptors recognized by the other PB components of the complex) and cytotoxic activity against strains susceptible to KvarM.

[0244] Example 4 Components of the trimeric complex The bacteriocin component of the complex is a chimeric protein based on the pore-forming pyocin S5. These chimeric pyocins were constructed by replacing the pore-forming cytotoxic domain of pyocin S5 with a fusion of the inner membrane transport domain of pyocin G and the DNase-type cytotoxic domain of either colicin E9 or E7. Thus, the chimeric pyocins contain the outer membrane transport domain (T) of pyocin S5. om ) and receptor binding (R) domain, followed by the inner membrane transport domain of pyocin G (TIM ), and the DNase domain of either colicin E9 or E7. The resulting chimeric proteins are designated S5E9 and S5E7, respectively.

[0245] The immunity protein scaffold is a fusion protein containing the colicin E9 and E7 immunity proteins (Im9 and Im7, respectively), linked by a 7-amino acid linker with the sequence SASGSAS. The scaffold is referred to by the designation "Im97" (but may also be designated "Im9-Im7"), and the trimeric complex is referred to by the shorthand notation "S5E9-Im97-S5E7" (equivalent to "[S5E9:Im9-Im7:S5E7]"). The scaffold and chimeric pyocin are illustrated schematically in Figure 14A.

[0246] In vitro assembly of the trimeric complex The proteins were expressed in E. coli BL21(DE3) and purified by nickel affinity chromatography. The immunity protein scaffold Im97 was isolated by nickel affinity chromatography based on its C-terminal His6 tag and further purified by gel filtration (Superdex 75). Chimeric pyocins S5E9 and S5E7 were coexpressed with their cognate immunity proteins Im9 and Im7, respectively. The pyocin-immunity protein complexes were isolated by nickel affinity chromatography based on the C-terminal His6 tag on the immunity protein and further purified by gel filtration (Superdex S200). To prepare uncomplexed pyocins, the purified pyocin-immunity protein complexes were loaded onto a nickel affinity column, and the pyocins were eluted with 6 M guanidine hydrochloride. Application of guanidine hydrochloride unfolded the proteins, allowing the pyocins to dissociate from the immunity proteins that remained bound to the column. The pyocin was refolded by dialysis in 50 mM Tris, 200 mM NaCl pH 7.5.

[0247] To construct and isolate the trimeric complex scaffold Im97 and chimeric pyocins S5E9 and S5E7, the protein components were mixed in a 1:4:4 ratio in 50 mM Tris, 200 mM NaCl, pH 7.5, respectively, and incubated at room temperature for 1 hour. The complex was isolated by nickel affinity chromatography and eluted with imidazole. Further purification of the complex was performed by gel filtration (Superdex S200) to remove aggregated proteins, thereby obtaining a monodisperse S5E9-Im97-S5E7 complex.

[0248] SDS PAGE was performed to demonstrate the purity and integrity of the S5E9-Im97-S5E7 complex and the pyocin components therein in free and immune protein complex form (Figure 14B). All proteins were shown to be highly purified with little evidence of degradation. However, multimeric pyocins were observed in samples of refolded, uncomplexed pyocins S5E9 and S5E7.

[0249] Cell killing by trimeric complexes The activity of the S5E9-Im97-S5E7 trimeric complex and the individual chimeric pyocins (in their free and immunity protein complex forms) against P. aeruginosa is shown in Figure 14C.

[0250] conclusion The generation of the Im9-ImD fusion protein enabled the in vitro assembly of a heterotrimeric [ColE9:Im9-ImD:ColD] complex, which retains all the activity of the bacteriocin complex. Furthermore, successful coexpression of the components of the trimeric [ColE9:Im9-ImD:ColD] complex in E. coli enabled the purification of the assembled complex, thereby simplifying the production and purification process.

[0251] A heterotetrameric complex [Im9-Im3-ImD:KlebC-E9:CloDF13-E3:KlebG-D] was assembled in vitro and retained the activity of the three protein bacteriocin components.

[0252] We further showed that heterotrimeric complexes containing chimeric pyocins are effective against P. aeruginosa. In the context of the complexes described herein, this construct also demonstrates that targeting moieties from pore-forming PBs can be combined with nuclease effector moieties from other PBs. *** The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, and presented in terms of a particular form, or means for performing a disclosed function, or a method or process for obtaining a disclosed result, may be utilized individually or in any combination of such features as appropriate to realize the invention in diverse forms thereof.

[0253] While the present invention has been described in conjunction with the exemplary embodiments set forth above, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various modifications to the described embodiments may be made without departing from the spirit and scope of the invention.

[0254] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of improving the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0255] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Unless the context otherwise requires, throughout this specification, including the claims which follow, the words "comprise" and "include," and variations such as "comprises," "comprising," and "including," will be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0256] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that the particular value forms another embodiment by use of the antecedent "about." The term "about" with respect to numerical values ​​is arbitrary and means, for example, + / - 10%.

[0257] References A number of publications have been cited above in order to more fully describe and disclose the present invention and the state of the art to which it pertains. Full citations for these references are provided below. Each of these references is incorporated herein in its entirety.

[0258] [Table 1]

[0259] For standard molecular biology techniques, see Sambrook, J., Russell, DW Molecular Cloning, A Laboratory Manual. 3rd ed., 2001, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Claims

1. (a) a first protein bacteriocin (PB) molecule and a second PB molecule; and (b) an immunity protein scaffold comprising a first immunity protein domain and a second immunity protein domain; an antibacterial protein complex comprising: An antibacterial protein complex, wherein said first and second immunity protein domains are non-covalently bound to said first and second PB molecules, respectively.

2. 2. The antibacterial conjugate of claim 1, wherein each bacteriocin comprises a cell-targeting moiety and an effector moiety, and binds to a corresponding immunity protein domain via the effector moiety.

3. 3. The antibacterial conjugate of claim 1 or 2, wherein the immunity protein scaffold comprises a third immunity protein domain, and the conjugate comprises a third bacteriocin molecule non-covalently bound to the third immunity protein domain.

4. The antibacterial conjugate of any one of claims 1 to 3, wherein the immunity protein scaffold comprises two or more repeats of the same immunity protein domain, eg each of the immunity protein domains is identical.

5. 5. The antibacterial conjugate of any one of claims 1 to 4, wherein the immunity protein scaffold contains two or more different immunity protein domains, e.g., each immunity protein domain is different from each of the other immunity protein domain components.

6. The antibacterial conjugate of any one of claims 1 to 5, wherein the immunity protein scaffold is a fusion protein.

7. 7. The antibacterial conjugate of claim 6, wherein the immunity protein scaffold fusion protein further comprises an M-type bacteriocin moiety, such as colicin M (ColM), KpneM, KpneM2, KvarM, PaeM1, or PaeM4.

8. 8. An antibacterial conjugate according to any one of claims 1 to 7, comprising a bacteriocin having two or more different effector moieties, for example wherein the different effector moieties have different enzymatic activities.

9. 9. The antibacterial conjugate of claim 8, wherein each effector moiety has a different enzymatic activity.

10. 10. The antibacterial complex of any one of claims 1 to 9, comprising bacteriocins with two or more different targeting moieties, such as each of the bacteriocins having a different targeting moiety.

11. 11. The antibacterial complex of claim 10, wherein each bacteriocin has a targeting moiety specific for bacteria of the same species or strain.

12. 12. The antibacterial conjugate of claim 10 or 11, wherein the targeting moiety binds to two or more different receptors or uses two or more different entry ports.

13. 13. The antibacterial conjugate of claim 12, wherein each targeting moiety binds to a different receptor and / or each targeting moiety uses a different entry port.

14. 14. The antibacterial complex of claim 13, comprising a bacteriocin having targeting moieties specific for two or more different species or strains of bacteria.

15. An antibacterial method comprising contacting a bacterium or a population of bacteria with an antibacterial complex according to any one of claims 1 to 14.

16. An antibacterial complex according to any one of claims 1 to 14 for use in a method of medical treatment.

17. An antibacterial complex according to any one of claims 1 to 14 for use in the prevention or treatment of a bacterial infection, a symptom caused by a bacterial infection or a symptom associated with a bacterial infection.

18. (i) a nucleic acid encoding an immunity protein scaffold, said immunity protein scaffold comprising a first immunity protein domain and a second immunity protein domain; (ii) a nucleic acid encoding at least one bacteriocin, the or each bacteriocin having an effector domain capable of binding to at least one of said immunity protein domains; A host cell comprising: A host cell, wherein said host cell is capable of expressing said immunity protein scaffold and said bacteriocin.

19. the cell comprises first and second nucleic acids, the first and second nucleic acids encoding first and second bacteriocins, respectively, each bacteriocin having an effector domain capable of binding to a respective one of the immunity protein domains; the host cell is capable of expressing the immunity protein scaffold and the bacteriocin; 19. The host cell of claim 18.

20. (i) a nucleic acid encoding an immunity protein scaffold, said immunity protein scaffold comprising a first, second and third immunity protein domain; (ii) first, second and third nucleic acids encoding first, second and third bacteriocins, respectively, each bacteriocin having an effector domain capable of binding to a respective one of said immunity protein domains; A host cell comprising: the host cell is capable of expressing the immunity protein scaffold and the bacteriocin; 20. A host cell according to claim 18 or 19.

21. 21. A method for producing an antibacterial complex, comprising providing a host cell according to any one of claims 18 to 20, and culturing said cell under conditions suitable for expression of said immunity protein scaffold and bacteriocin molecules, and optionally further comprising isolating the antibacterial complex.

22. 1. A method of producing an antibacterial complex, the method comprising contacting an immunity protein scaffold comprising a first immunity protein domain and a second immunity protein domain with first and second bacteriocin molecules, each having an effector moiety capable of binding to a respective one of the immunity protein domains, to form an antibacterial complex.

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