Chimeric bacteriocins and methods for controlling Pseudomonas spp.

Abstract

The present invention provides antibacterial proteins (bacteriocins) for controlling Pseudomonas, preferably Pseudomonas aeruginosa, and nucleic acid molecules encoding the bacteriocins. The present invention also relates to compositions, in particular pharmaceutical compositions, comprising the bacteriocins or combinations of bacteriocins. The present invention further relates to the bacteriocins or compositions for use in therapy.

Description

[Technical field]

[0001] The present invention relates to antibacterial proteins (bacteriocins) for controlling Pseudomonas, preferably Pseudomonas aeruginosa, and to nucleic acid molecules encoding the bacteriocins. The present invention also relates to compositions, in particular pharmaceutical compositions, comprising the bacteriocins or combinations of bacteriocins. The present invention further relates to the bacteriocins or compositions for use in therapy, preferably for the treatment of lung infections or keratitis. The present invention further relates to a method for treating or preventing bacterial infections in mammals, preferably humans. Furthermore, the present invention relates to nucleic acid molecules comprising a nucleic acid sequence encoding a protein or polypeptide according to the present invention. [Background technology]

[0002] Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes both acute and chronic infections. P. aeruginosa contains a large reservoir of virulence factors and antibiotic resistance determinants within its genome, conferring remarkable metabolic flexibility and the ability to adapt to multiple conditions, including the host immune response. 1 Furthermore, rapid emergence of resistance to previously effective antimicrobial agents, such as fluoroquinolones, aminoglycosides, and polymyxins, has been observed. 2 This is of particular concern with respect to conditions resulting from biofilm colonization of the lungs, urinary tract infections, and ear and eye infections in cystic fibrosis patients. The involvement of P. aeruginosa in a wide range of biofilm-associated infections frequently results in treatment failure. 3 Therefore, new antibacterial agents are urgently needed.

[0003] The potential use of bacteriocins as new generation antibacterial agents has been previously proposed. 4P. aeruginosa produces pyocins, which are colicin-type bacteriocins that belong to different classes: deoxyribonucleases, ribonucleases, pore-forming proteins, peptidoglycan synthesis-blocking proteins, lectin-like proteins, and bacteriophage tail-like protein complexes. 5、6 Several studies have described the use of natural pyocins for the effective treatment of P. aeruginosa infections in various animal models. 7~12 . The main obstacle in the clinical development of bacteriocin antibacterial drugs is the proteinaceous nature of the bacteriocins and their limited spectrum of activity. Being proteins, colicin-type bacteriocins, when used intravenously, are expected to present the same challenges observed in the clinical development of bacteriophage lysins, namely, short half-life in serum, immunogenicity, and reduced activity. 13、14 These issues may be less relevant in the development of topical, oral, or inhaled antimicrobial agents.

[0004] Previously, we successfully expressed six different pyocins in a plant transient expression system. 11 S5, the only P. aeruginosa pore-forming pyocin known so far, was found to be active against 40% of the clinical isolates tested. S5 showed superior activity compared to all other pyocins in reducing bacterial numbers in liquid culture and biofilm assays, and was also the most effective in protecting Galleria mellonella larvae from mortality due to P. aeruginosa infection. 11 . Autoinhibition of S5-producing strains was prevented by coexpression of immunity genes downstream of the bacteriocin genes. Lethality due to pore formation by pyocin S5 was transiently delayed along the secretory pathway via the membrane-integrated immunity protein consisting of three transmembrane helices (TMH). 15 . A general problem with bacteriocins is their limited spectrum of activity, i.e. rather high specificity for a target strain or species of Pseudomonas or for a strain of Pseudomonas aeruginosa, which is problematic when the pathogenic agent causing the infection is not known or has not yet been identified.

[0005] Thus, one object of the present invention is to provide a bacteriocin that is active against Pseudomonas for the control of Pseudomonas. A further object is to provide a bacteriocin that has broad spectrum activity against multiple strains of Pseudomonas. A further object is to provide a treatment for infections caused by Pseudomonas, and in particular by Pseudomonas aeruginosa. Summary of the Invention

[0006] These objectives are achieved by: 1) The following groups (ai) to (di): (ai) a segment of the amino acid sequence of SEQ ID NO: 1 (S5 translocation domain + receptor binding domain), or (bi) a segment having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1; or (ci) a segment having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO:1; or (di) a segment having a substitution, addition, insertion, and / or deletion of 1 to 60, preferably 1 to 45, more preferably 1 to 30, and most preferably 1 to 15 amino acid residues compared to the amino acid sequence of SEQ ID NO: 1; and preferably adjacent thereto, a first polypeptide segment selected from the group (ai)' to (d-iii)' below: (ai)' a segment of the amino acid sequence of SEQ ID NO: 3 (the cytotoxic domain of PmnH), or (a-ii)' a segment of the amino acid sequence of SEQ ID NO: 7 (the cytotoxic domain of Pflu095), or (a-iii)' a segment of the amino acid sequence of SEQ ID NO: 11 (the cytotoxic domain of Pflu373), or (bi)' a segment having at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3; or (b-ii)' a segment having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7; or (b-iii)' a segment having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11; or (ci)' a segment having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably up to 98% sequence similarity to the amino acid sequence of SEQ ID NO: 3; or (c-ii)' a segment having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably up to 98% sequence similarity to the amino acid sequence of SEQ ID NO: 7; or (c-iii)' a segment having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably up to 98% sequence similarity to the amino acid sequence of SEQ ID NO: 11; or (di)' a segment having a substitution, addition, insertion, and / or deletion of 1 to 38, preferably 1 to 28, more preferably 1 to 19, and most preferably 1 to 10 amino acid residues compared to the amino acid sequence of SEQ ID NO: 3, or (d-ii)' a segment having a substitution, addition, insertion, and / or deletion of 1 to 38, preferably 1 to 28, more preferably 1 to 19, and most preferably 1 to 10 amino acid residues compared to the amino acid sequence of SEQ ID NO: 7, or (d-iii)' A segment having a substitution, addition, insertion, and / or deletion of 1 to 38, preferably 1 to 28, more preferably 1 to 19, and most preferably 1 to 10 amino acid residues compared to the amino acid sequence of SEQ ID NO: 11. (Chimeric) antimicrobial proteins comprising a polypeptide comprising a second polypeptide segment selected from:

[0007] 2) (Ai) the amino acid sequence of SEQ ID NO:5 (the entire sequence of the S5-PmnH protein); or (A-ii) the amino acid sequence of SEQ ID NO: 9 (the entire sequence of the S5-Pflu095 protein), or (A-iii) the amino acid sequence of SEQ ID NO: 13 (the entire sequence of S5-Pflu373 protein); or (Bi) an amino acid sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 5; or (B-ii) an amino acid sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 9; or (B-iii) an amino acid sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13; or (Ci) an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO:5; or (C-ii) an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO: 9; or (C-iii) an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO: 13; or (Di) an amino acid sequence having a substitution, addition, insertion, and / or deletion of 1 to 100, preferably 1 to 75, more preferably 1 to 50, even more preferably 1 to 25, and most preferably 1 to 12 amino acid residues relative to the amino acid sequence of SEQ ID NO: 5, or (D-ii) an amino acid sequence having a substitution, addition, insertion, and / or deletion of 1 to 100, preferably 1 to 75, more preferably 1 to 50, even more preferably 1 to 25, and most preferably 1 to 12 amino acid residues relative to the amino acid sequence of SEQ ID NO: 9, or (D-iii) An amino acid sequence having a substitution, addition, insertion, and / or deletion of 1 to 100, preferably 1 to 75, more preferably 1 to 50, even more preferably 1 to 25, and most preferably 1 to 12 amino acid residues relative to the amino acid sequence of SEQ ID NO: 13. A (chimeric) antibacterial protein comprising or consisting of a polypeptide comprising:

[0008] 3) The antibacterial protein according to item 1, wherein the first segment is N-terminal to the second segment in the amino acid sequence of the antibiotic protein.

[0009] 4) A composition comprising the protein according to any one of items 1 to 3.

[0010] 5) The composition according to item 4, further comprising a second bacteriocin according to any one of items 1 to 3. 6) A pharmaceutical composition comprising an antibacterial protein as defined in any one of items 1 to 3, or a composition as defined in item 4 or 5, and one or more pharma- ceutically acceptable carriers or excipients. 7) The pharmaceutical composition according to item 6, which is a sterile aqueous solution or a solid formulation comprising the antibacterial protein, preferably the solid formulation is a powder suitable for reconstitution in an aqueous liquid medium or a solid formulation suitable for administration as an aerosol. 8) The antibacterial protein according to any one of items 1 to 3 or the composition according to any one of items 4 to 7 for use in therapy. 9) The antibacterial protein according to any one of items 1 to 3 or the composition according to any one of items 4 to 7 for use in the treatment or prevention of a bacterial infection, preferably a bacterial infection caused by the genus Pseudomonas, more preferably a bacterial infection caused by Pseudomonas aeruginosa.

[0011] 10) The antibacterial protein according to any one of items 1 to 3 or the composition according to any one of items 4 to 7 for use in therapy, preferably for the treatment of lung infections or keratitis. 11) The antibacterial protein according to any one of items 1 to 3 or the composition according to any one of items 4 to 7 for use in the treatment of pulmonary infections, by administration to the lungs of a mammal, as a solid or liquid aerosol; or for the treatment of keratitis, by administration in the form of an aqueous solution to the eye of an affected mammal. 12) A method for treating or preventing a bacterial infection in a mammal, comprising the step of administering to a mammal in need of such treatment or prevention an antibacterial protein defined in any one of items 1 to 3, or a composition according to any one of items 4 to 6, or a pharmaceutical composition according to item 7. 13) The method according to item 12, wherein the method is for treating bacterial pulmonary infections by administration to the lungs of a mammal as a solid or liquid aerosol; or for treating bacterial keratitis by administration to the affected eye of a mammal in the form of an aqueous solution. 14) A nucleic acid molecule comprising a nucleic acid sequence encoding the protein according to any one of items 1 to 3.

[0012] 15) The nucleic acid molecule according to item 14, wherein the nucleic acid sequence comprises a polynucleotide set forth in any one of SEQ ID NOs: 6, 10, or 14. 16) A bacterial or eukaryotic cell comprising a nucleic acid molecule according to items 14 or 15. The present inventors have investigated ways to broaden the spectrum of activity of anti-Pseudomonas bacteriocins, such as pyocin S5. The present inventors have surprisingly found that the spectrum of activity of pyocin S5 can be broadened by modifying its killing domain so that it is not recognized by the immunity proteins of the target pathogen. Other Pseudomonas species (notably P. fluorescens) contain various genes within their genomes that code for pore-forming bacteriocin-like proteins. 6、16 By mining the GenBank database, we retrieved several sequences of putative pore-forming bacteriocins from different Pseudomonas species and selected six colicin E1-like and colicin A-like proteins. We then used the cytotoxic pore-forming domains of those bacteriocins to fuse them with the receptor-binding and translocation domains of pyocin S5 to construct chimeric proteins. We tested the activity of the resulting chimeric bacteriocins in vitro and were surprisingly able to identify bacteriocins with high cytotoxic activity and broad specificity against Pseudomonas strains. Using topical and inhalation administration of the chimeric bacteriocins of the invention, we found that the identified chimeric bacteriocins were active in different animal disease models, P. aeruginosa keratitis and a lung colonization mouse model. [Brief description of the drawings]

[0013] [Figure 1]Diagram of sequence analysis of the pore-forming domain of a putative Pseudomonas bacteriocin (pfam01024). A. ClustalW amino acid sequence alignment of Pseudomonas bacteriocins with pyocin S5. Sequences shown, from top to bottom, are SEQ ID NOs: 3, 19, 23, 15, 11, 7, and 39, respectively. B. Neighbor-Joining tree alignment of known E. coli, Klebsiella bacteriocins, pyocin S5, PmnH, and the putative Pseudomonas fluorescens and P. putida bacteriocins. Col28B(CAA44310.1), ColE1(AAA87379.1), Col10(CAA57998.1), Col5(CAA61102.1), ColIb(AAA23188.1), Col Ia(WP_001283344.1), ColN(P08083.1), ColA(P04480.1), ColU(CAA72509.1), ColB(P05819.3), ColY(AAF8268 3.1), KpneA (SAV78255.1), KvarIa (KDL88409), Pyocin S5 (WP_003115311), PmnH (EIK72868.1), Pflu618 (WP_0341 55618), Ppu259(WP_098964259), Pflu794(WP_081041794), Pflu373(WP_014717373), Pflu095(WP_016979095). [Diagram 2] SDS-PAGE Coomassie staining of purified chimeric pyocins. Lane 1: PageRuler Prestained Protein Ladder (Thermo Fisher Scientific), lane 2: S5 (0.9 mg / ml), lane 3: S5-PmnH (0.9 mg / ml), lane 4: S5-Pflu095 (0.9 mg / ml), lane 5: S5-Pflu373 (0.9 mg / ml), lane 6: S5-Pflu794 (0.9 mg / ml), lane 7: S5-Pflu618 (0.9 mg / ml), lane 8: S5-Pput259 (1.4 mg / ml). 4 μl protein per lane. [Diagram 3]Activity of pyocin S5 and chimeric pyocins against pyocin S5-producing P. aeruginosa strains. 0.3, 3, and 30 μg of S5 and the chimeric bacteriocins were spotted onto 6 mm Whatman discs placed on CAA agar lawns of different P. aeruginosa strains and incubated overnight. [Figure 4] CFU counts in ex vivo pig corneas infected with P. aeruginosa ATCC 19660 or PAO1 and treated with S5-PmnH. 3x104 CFU of P. aeruginosa ATCC 19660 or 0.4x104 CFU of P. aeruginosa PAO1 were applied to the cornea and incubated at 37°C for 16-20 hours. 5μg of S5-PmnH was then applied to the cornea and incubated for an additional 16-20 hours. Statistical significance of quantitative data was analyzed by one-way repeated measures analysis of variance and Bonferroni correction for multiple comparisons using GraphPad Prism software. Means are indicated by horizontal bars. ***P≦0.001, ****P≦0.0001 vs. vehicle-treated mice. [Diagram 5]Illustrative diagram of mouse corneal infection with cytotoxic strain ATCC 19660 and treatment with S5-PmnH or tobramycin. A. CFU counts and clinical scores in mouse eyes when treatment was started 30 min after infection. B. CFU counts and clinical scores in mouse eyes when treatment was started 6 h after infection. C. Hematoxylin-eosin staining of corneal sections. Left panel - Pyocin treatment started 30 min after infection. Non-infected eyes - no microscopic aberrations are observed. Infected control eyes: day 1 - significant corneal inflammation, days 3 and 5 - histology was not possible due to destruction of ocular structures. Slight edema in the corneal stroma is observed in all infected eyes treated with S5-PmnH and tobramycin. Right panel - Pyocin treatment started 6 h after infection. Non-infected eyes: no significant corneal abnormalities. Infected untreated eyes: day 1 - thinning of corneal epithelium, thickening of stroma, acute inflammation, days 3 and 5 - acute suppurative inflammation of the cornea. Infected eyes treated with S5-PmnH: day 1 - acute inflammation of the cornea, days 3 and 5 - no significant abnormalities, slight edema of the corneal stroma. Infected eyes treated with tobramycin: days 3 and 5 - slight thickening of the epithelium. Statistical significance of quantitative data was analyzed by two-way repeated measures analysis of variance and Dunnett's correction for multiple comparisons using GraphPad Prism software. Means are indicated by horizontal bars. ***P≦0.001, ****P≦0.0001 vs. vehicle-treated mice. [Figure 6]FIG. 1. Mouse corneal infection with invasive strain PAO1 and treatment with S5-PmnH or tobramycin. Corneas of left eyes of mice were infected with 4x106 CFU of P. aeruginosa strain PAO1. Treatment with 20 μg S5-PmnH or 140 μg tobramycin was started 6 hours after infection and applied twice daily. A. CFU counts in mouse corneas at 1, 3, and 5 dpi (days post infection). B. Corneal clinical scores at 1, 3, and 5 days of the experiment. C. Hematoxylin-eosin staining of corneal sections. Non-infected eyes: no significant abnormalities, slight edema in the corneal stroma observed in most samples. Infected S5-PmnH treated eyes: days 1 and 3 - acute inflammation of the cornea, day 5 - no abnormalities. Infected tobramycin-treated eyes: day 1 - acute inflammation of the cornea, localized corneal lesions, day 3 - thinning of the corneal epithelium, significant edema of the corneal stroma; day 5 - localized inflammation of the cornea, thinning and degeneration of the corneal epithelium. Statistical significance of quantitative data was analyzed by two-way repeated measures analysis of variance and Dunnett's correction for multiple comparisons using GraphPad Prism software. Means are indicated by horizontal bars. **P≦0.01, ***P≦0.001, ****P≦0.0001 vs. vehicle-treated mice. Not significant (ns) P>0.05. [Figure 7] Scatter plots of terminal lung burden following IN infection with Pseudomonas aeruginosa ATCC 27853. Data from culture burdens were analyzed in StatsDirect (v.3.3.3) using appropriate nonparametric statistical models (Kruskal-Wallis with Conover-Inman for all pairwise comparisons between groups). The geometric mean burden for each treatment is indicated by the horizontal bars. *P≦0.05, **P≦0.0005, ***P≦0.0001 compared to vehicle control. LOD=limit of detection. [Figure 8]PCR amplification of S5 killing and S5 immunity genes and fptA from genomic DNA of 25 P. aeruginosa isolates. Sequence-specific primers were designed to amplify a 1500 bp fragment of S5K, a 330 bp fragment of S5I, and a 556 bp fragment of fptA. FptA was amplified in all strains tested, and S5K and S5I were amplified in strains PA14, PAO1, HP6, HP7, ATCC 19660, and NCTC 13921. K-negative control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The bacteriocins of the present invention are colicin-type antibiotic proteins. Colicins, in the case of E. coli colicins, are plasmid-encoded cytotoxins synthesized by E. coli, secreted into the medium, and kill susceptible strains of E. coli. Colicins are classified into different cytotoxic classes according to their mechanism of killing susceptible bacterial strains: - pore-forming colicins that kill cells by causing membrane depolarization, e.g., ColA, ColE1, ColN, ColK, ColIa, ColIb, and ColD, - RNase colicin, - DNase colicin, and - Inhibitors of cell wall synthesis, e.g. ColM may belong to.

[0015] Colicins, as well as bacteriocins of the invention, contain three domains along their amino acid sequence, from N-terminus to C-terminus, called the receptor binding domain, the translocation domain, and the killing or cytotoxic domain (Cascales et al., Colicin Biology, Microbiology and Molecular Biology Reviews, Vol. 71, (2007) 158-229). The receptor binding domain allows the colicin to bind to a receptor on the outer membrane of the bacterial cell. The translocation domain binds to a translocation protein of the target bacterial cell, allowing translocation to the inside of the target cell. The bacteriocins of the invention are pore-forming bacteriocins. They exert their cytotoxic activity by pore formation in the intracellular membrane of the target cell. Thus, their target compartment in the target cell is the periplasm of the target cell. The translocation domain of the bacteriocins of the invention allows the bacteriocin to translocate into the periplasm of the target pathogen cell.

[0016] The antimicrobial protein of the invention (also referred to herein as "bacteriocin" or "pyocin") is a pore-forming bacteriocin. It comprises a polypeptide comprising a first polypeptide segment (referred to herein simply as "first segment") and a generally adjacent second polypeptide segment (referred to herein simply as "second segment"). The first segment comprises (N- to C-terminus) a receptor binding domain and a translocation domain. The second segment comprises a pore-forming cytotoxic (or killing) domain. The bacteriocin of the invention is sometimes referred to herein as a "chimeric" or "chimeric pyocin" because it comprises segments that may be or are derived from different origins, in particular bacteriocins from different Pseudomonas species.

[0017] A polypeptide segment (or simply "segment") herein refers to a number of consecutive amino acid residues of a polypeptide or protein having a greater number of amino acid residues than the segment. Thus, a segment is a portion of a polypeptide or protein. The term "polypeptide" refers to a polypeptide molecule that is not limited with respect to the number of amino acid residues of the polypeptide (in contrast to a fragment, which is a portion or part of a molecule). The term protein encompasses polypeptides, and combinations or complexes of two or more polypeptides, which may contain further components, such as metal ions complexed to the polypeptides. In general, proteins may be monomers, homo-oligomers or hetero-oligomers (e.g. dimers), and may contain further components, such as metal ions complexed to one or more of the polypeptides. The term "amino acid sequence" refers to the sequence of amino acid residues of a protein, polypeptide, or segment; this term refers to sequence information that is structural information of the protein, polypeptide, or segment.

[0018] The first segment of the bacteriocin of the invention is selected from the group (ai) to (di) below: (ai) a segment of the amino acid sequence of SEQ ID NO:1, or (bi) a segment having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1; or (ci) a segment having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO:1; or (di) a segment having a substitution, addition, insertion, and / or deletion of 1 to 60, preferably 1 to 45, more preferably 1 to 30, and most preferably 1 to 15 amino acid residues compared to the amino acid sequence of SEQ ID NO: 1; It may be any one selected from the above.

[0019] The second segment of the bacteriocin of the invention is selected from the group (ai)' to (d-iii)' below: (ai)' a segment of the amino acid sequence of SEQ ID NO: 3 (the killing domain of PmnH), or (a-ii)' a segment of the amino acid sequence of SEQ ID NO: 7 (the killing domain of Pflu095), or (a-iii)' a segment of the amino acid sequence of SEQ ID NO: 11 (the killing domain of Pflu373), or (bi)' a segment having at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3; or (b-ii)' a fragment having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7; or (b-iii)' a segment having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11; or (ci)' a segment having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably up to 98% sequence similarity to the amino acid sequence of SEQ ID NO: 3; or (c-ii)' a segment having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably up to 98% sequence similarity to the amino acid sequence of SEQ ID NO: 7; or (c-iii)' a segment having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably up to 98% sequence similarity to the amino acid sequence of SEQ ID NO: 11; or (di)' a segment having a substitution, addition, insertion, and / or deletion of 1 to 38, preferably 1 to 28, more preferably 1 to 19, and most preferably 1 to 10 amino acid residues compared to the amino acid sequence of SEQ ID NO: 3, or (d-ii)' a segment having a substitution, addition, insertion, and / or deletion of 1 to 38, preferably 1 to 28, more preferably 1 to 19, and most preferably 1 to 10 amino acid residues compared to the amino acid sequence of SEQ ID NO: 7, or (d-iii)' A segment having a substitution, addition, insertion, and / or deletion of 1 to 38, preferably 1 to 28, more preferably 1 to 19, and most preferably 1 to 10 amino acid residues compared to the amino acid sequence of SEQ ID NO: 11. It may be any one selected from the above.

[0020] The expression "a segment of an amino acid sequence of SEQ ID NO: z", where z means any one of the mentioned SEQ ID NOs, means that the amino acid sequence of the segment is the amino acid sequence of SEQ ID NO: z, i.e. the entire amino acid sequence of SEQ ID NO: z. The expression "a segment having at least ... sequence identity / similarity to an amino acid sequence of SEQ ID NO: z", respectively, means that the amino acid sequence of the segment has at least the indicated amino acid sequence identity or similarity to the entire amino acid sequence of SEQ ID NO: z, preferably at least the number of amino acid residues of the mentioned SEQ ID NO. The SEQ ID NO for which a segment or an amino acid sequence is defined is generally referred to herein as a reference sequence.

[0021] As used herein, determination of sequence identity and similarity is performed using Align Sequences Protein BLAST (BLASTP 2.6.1+) (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.).

[0022] When a polypeptide or segment is defined by a number or number range of substitutions, additions, insertions, and / or deletions of amino acid residues, the substitutions, additions, insertions, or deletions of amino acid residues may be combined, but a given number or number range refers to the sum of all substitutions, additions, insertions, and deletions of amino acid residues. Among the substitutions, additions, insertions, and deletions of amino acid residues, the substitutions, additions, and deletions of amino acids are preferred. The term "insertion" refers to the insertion of an amino acid residue within the amino acid sequence of the reference sequence, i.e., excluding additions at the C-terminus or N-terminus of the reference sequence. The term addition refers to the addition of an amino acid residue at the C-terminus or N-terminus of the amino acid sequence of the reference sequence. The deletion may be a deletion of a terminal amino acid residue or an internal amino acid residue of the reference sequence. In a further embodiment herein, when a polypeptide or segment is defined by a number or range of amino acid residue substitutions, additions, insertions, and / or deletions relative to a reference sequence, the polypeptide or segment may have one to several amino acid residue substitutions, additions, insertions, or deletions relative to the amino acid sequence of the indicated segment.

[0023] In the bacteriocin of the invention, the first segment is preferably N-terminal to the second segment in the amino acid sequence of the bacteriocin. In any item (xy)' herein, where x represents any one of a, b, c, or d, and y represents any Roman numeral i to iii, the dash' denotes a killing domain or killing segment. Item (xy) lacking a dash denotes a segment comprising a receptor binding domain and a translocation domain. Among items (a) to (d), items (a), (b) and (d) are preferred, with items (a) and (d) being more preferred. Similarly, among items (a)' to (d)', items (a)', (b)' and (d)' are preferred, with items (a)' and (d)' being more preferred. The same applies analogously to combinations of items (a) to (d) and (a)' to (d)', with combinations of the same letters of a to d being preferred. When the protein of the invention comprises a first segment as defined above and a second segment as defined above, any first segment as defined above may be combined with any second segment as defined above. In one embodiment, the binding and translocation segments of any of items (a) to (d) are combined with the catalytic domain or segment of any of items (a)' to (d)', respectively.

[0024] In a first general embodiment, a first segment according to any one of items (a) to (d) is combined with a second segment according to any one of items (xi)', i.e. the killing domain of PmnH or a derivative thereof, as defined in any one of items (xi)'. In a second general embodiment, a first segment of any one of items (a) to (d) is combined with a second segment according to any one of items (x-ii)', i.e., the killing domain of Pflu095 or a derivative thereof, as defined in any one of items (x-ii)'. In a third general embodiment, a first segment of any one of items (a) to (d) is combined with a second segment according to any one of items (x-iii)', i.e., the killing domain of Pflu373 or a derivative thereof, as defined in any one of items (x-iii)'.

[0025] The bacteriocin preferably has a minimum inhibitory concentration (MIC) against Pseudomonas aeruginosa PAO1, as determined by the assay of Example 2, that is at most 10-fold, more preferably at most 5-fold, and even more preferably at most 2.5-fold the MIC of S5-Pflu095 of SEQ ID NO:5. The first and second segments may or may not be linked by a peptide linker. Preferably, there is no peptide linker. If a peptide linker is used, it may consist of 1 to 50 amino acid residues, preferably 1 to 30 amino acid residues, more preferably 1 to 10 amino acid residues. The peptide linker may be selected such that in the assay of Example 2 against P. aeruginosa PAO1, its MIC is increased by more than 5-fold, preferably by no more than 2-fold, compared to a bacteriocin lacking the linker but otherwise having the same amino acid sequence. Alternatively, the provisos of the previous paragraph may apply.

[0026] Alternatively, the bacteriocin of the invention comprises: (Ai) the amino acid sequence of SEQ ID NO: 5 (sequence of protein S5-PmnH), or (A-ii) the amino acid sequence of SEQ ID NO: 9 (sequence of protein S5-Pflu095), or (A-iii) the amino acid sequence of SEQ ID NO: 13 (sequence of protein S5-Pflu373), or (Bi) an amino acid sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 5; or (B-ii) an amino acid sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 9; or (B-iii) an amino acid sequence having at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13; or (Ci) an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO:5; or (C-ii) an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO: 9; or (C-iii) an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO: 13; or (Di) an amino acid sequence having a substitution, addition, insertion, and / or deletion of 1 to 100, preferably 1 to 75, more preferably 1 to 50, even more preferably 1 to 25, and most preferably 1 to 12 amino acid residues relative to the amino acid sequence of SEQ ID NO: 5, or (D-ii) an amino acid sequence having a substitution, addition, insertion, and / or deletion of 1 to 100, preferably 1 to 75, more preferably 1 to 50, even more preferably 1 to 25, and most preferably 1 to 12 amino acid residues relative to the amino acid sequence of SEQ ID NO: 9, or (D-iii) An amino acid sequence having a substitution, addition, insertion, and / or deletion of 1 to 100, preferably 1 to 75, more preferably 1 to 50, even more preferably 1 to 25, and most preferably 1 to 12 amino acid residues relative to the amino acid sequence of SEQ ID NO: 13. It may be defined as including a polypeptide comprising or consisting of:

[0027] The expression "amino acid sequence of SEQ ID NO: z" in item (Ay) means that the amino acid sequence of the polypeptide is or comprises the amino acid sequence of the reference sequence (SEQ ID NO: z). The expression "amino acid sequence having at least ... sequence identity / similarity to amino acid sequence of SEQ ID NO: z" in item (By) or (Cy) means that the amino acid sequence of the polypeptide has at least the indicated amino acid sequence identity or similarity to the entire amino acid sequence of SEQ ID NO: z, respectively, and preferably at least the number of amino acid residues of the mentioned SEQ ID NO. In this specification, the determination of sequence identity and similarity is carried out as defined above.

[0028] The expression "an amino acid sequence having substitutions, additions, insertions and / or deletions of amino acid residues ... relative to the amino acid sequence of SEQ ID NO: z" (SEQ ID NO: z represents the reference sequence) in item (Dy) means that the amino acid sequence of the polypeptide has the indicated number or number range of substitutions, additions, insertions and / or deletions of amino acid residues relative to the entire amino acid sequence of the reference sequence. Otherwise, the definitions and preferred embodiments defined above apply analogously.

[0029] In addition to the above definition, the bacteriocin of the invention may comprise further segments at the N-terminus and / or C-terminus, such as purification tags, signal sequences, etc. However, in one embodiment, the bacteriocin polypeptide does not contain further segments, but the bacteriocin polypeptide consists of the above definition. The bacteriocin preferably has a minimum inhibitory concentration (MIC) against Pseudomonas aeruginosa PAO1, as determined by the assay of Example 2, that is at most 10-fold, more preferably at most 5-fold, and even more preferably at most 2.5-fold the MIC of S5-Pflu095 of SEQ ID NO:5. The invention also provides a nucleic acid molecule and a nucleic acid construct, both of which comprise a nucleic acid sequence encoding a bacteriocin according to the invention. The nucleic acid molecule and the nucleic acid construct may comprise a transcription promoter that is preferably active in a cell in which the bacteriocin can be expressed, such as a bacterial cell or a plant cell. The present invention also provides a bacterial or eukaryotic cell comprising a nucleic acid molecule according to the invention. The eukaryotic cell may be a mammalian cell or a plant cell. The plant cell may be a cell of a plant in which a bacteriocin is expressed or can be expressed. Furthermore, a plant is provided comprising a nucleic acid molecule according to the present invention. The plant may be any one of those described below. The present invention also provides a bacterial or eukaryotic cell comprising a protein of the invention, for example a bacterial or eukaryotic cell expressing or having expressed a protein of the invention.

[0030] The invention further provides a composition comprising a (first) bacteriocin of the invention and optionally further components, such as one or more carriers or excipients. The composition may further comprise a second or further bacteriocin of the invention. A second bacteriocin means that it comprises a polypeptide whose amino acid sequence differs from that of the first bacteriocin. Preferably, the second bacteriocin has a killing domain of a different origin than the first bacteriocin. Among the bacteriocins of items (Xi) to (X-iii), where X is selected from A, B, C, D, it means that the bacteriocin of item (Xi) may be combined with a bacteriocin of items (X-ii) or (X-iii), for example.

[0031] Since the bacteriocins of the invention may be produced by expression in a plant or cells thereof, the composition may be a plant material or an extract of a plant material, where the plant material is a plant expressing the bacteriocin, preferably a material from a Nicotiana or edible plant expressing said bacteriocin. An extract of a plant material may be an aqueous solution containing the bacteriocin of the invention and optionally other water-soluble components present or expressed in said plant material, or a dried version of such an aqueous solution. The extract preferably has the water-insoluble components of the plant material removed, for example by filtration or centrifugation. The plant material may be material from a plant selected from the group consisting of spinach, chard, beet root, carrot, sugar beet, leaf beet, amaranth, Nicotiana, and / or the plant material may be one or more leaves, roots, tubers, or seeds, or a crushed, milled, or ground version of said leaves, roots, tubers, or seeds. In a preferred embodiment, particularly for use as a pharmaceutical composition, the bacteriocin is purified to the level required by the respective regulatory provisions for the intended application or use of the bacteriocin or composition of the invention.

[0032] The composition (e.g. said extract from a plant) may be a solid or liquid composition, e.g. a solution or dispersion, containing the bacteriocin of the invention. The liquid composition may be aqueous, e.g. an aqueous solution. The concentration of the bacteriocin(s) in said aqueous dispersion or solution may be 0.0001-10 mg / ml, preferably 0.001-5.0 mg / ml, more preferably 0.005-1.0 mg / ml, most preferably 0.01-0.2 mg / ml. When two or more bacteriocins capable of exerting a cytotoxic effect against Pseudomonas are utilized, their concentrations relate to the total concentration of all such bacteriocins of the invention. The aqueous solution as the composition may contain, in addition to one or more bacteriocins, a buffer. The buffer may be an inorganic acid or an organic acid, and / or a salt thereof. An example of an inorganic acid is phosphoric acid or a salt thereof. Examples of organic acids are HEPES, acetic acid, succinic acid, tartaric acid, malic acid, benzoic acid, cinnamic acid, glycolic acid, lactic acid, citric acid, and ascorbic acid. Preferred organic acids are malic acid, lactic acid, citric acid, and ascorbic acid. The pH of the solution may generally be 6 to 8, preferably 6.5 to 7.5. The pH of the solution containing the buffer may be adjusted using hydrochloric acid or a sodium hydroxide solution.

[0033] Furthermore, the solution may contain an isotonicity agent, such as glycerol, mannitol, sorbitol, a salt, or a combination of two or more of such compounds. The salt is preferably an alkali metal salt. The preferred salt used is sodium chloride. The aqueous solution containing one or more bacteriocins may be a buffered aqueous solution that may contain additional solutes, such as 50-400 mM NaCl, preferably 100-200 mM NaCl. The aqueous solution may further contain a sulfhydryl compound, such as dithiothreitol (DTT), dithioerythritol, thioethanol, or glutathione, preferably DTT. The total concentration of sulfhydryl compounds in the aqueous solution may be 1-50 mM, preferably 2-20 mM, more preferably 4-10 mM.

[0034] Examples of carriers for the composition are solvents, such as water or aqueous buffer (as described above); salts; sugars, such as monosaccharides and disaccharides; sugar alcohols; hydrophilic polymers, such as polysaccharides, polyethylene glycol, or hyaluronic acid (or its sodium salt); and other carriers known from pharmaceutical compositions. Examples of polysaccharides are starch, cellulose, and cellulose derivatives, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose (HPMC), and carmellose. Examples of sugars are glucose, fructose, lactose, sucrose, and maltose. When the composition of the present invention is a solid composition, it may be a powder, for example a lyophilized solid composition obtained by lyophilization of the extract or solution described above. The powder may contain additional solid components, for example those described above for the aqueous solution. Before use, the powder may be reconstituted with a suitable liquid, for example water or a buffer. The solid composition may contain buffers, salts, or other components described above, so that the concentrations described above are achieved upon reconstitution or dissolution. In a preferred embodiment, the composition is a pharmaceutical composition comprising one or more pharma- ceutically acceptable carriers or excipients. Examples of possible carriers and excipients are described above. A preferred pharmaceutical composition is a liquid aqueous solution of one or more bacteriocins. In another embodiment, the composition is a solid formulation suitable for administration to a patient as an aerosol.

[0035] For the treatment of keratitis, the pharmaceutical composition may be an ophthalmic composition comprising a bacteriocin according to the invention. The ophthalmic composition may be a sterile buffer solution of pH 7.0-7.5 with an osmolality of 250-350 mOsm / L, preferably 270-330 mOsm / L. When used, possible buffer solutions for the ophthalmic composition are phosphate, citrate, Tris(trometamol), or sodium bicarbonate, with citrate being preferred. In one embodiment, the buffer solution is phosphate-free, with a phosphate-free citrate-buffered ophthalmic composition being preferred. The concentration of the one or more bacteriocins may be 0.1-10 mg / mL, preferably 0.5-8 mg / mL, more preferably 1.0-5 mg / mL. As a wetting agent, the polysaccharides mentioned above, or sodium hyaluronate may also be included.

[0036] The bacteriocins and compositions of the invention may be used in therapy. They may be used in the treatment or prevention of bacterial infections, preferably bacterial infections caused by Pseudomonas, such as Pseudomonas aeruginosa. In one embodiment, the bacteriocin or composition is used in the treatment of bacterial lung infections or bacterial pneumonia, preferably lung infections or pneumonia caused by Pseudomonas, such as Pseudomonas aeruginosa. In another embodiment, the bacteriocin or composition is used in the treatment of keratitis, preferably bacterial keratitis caused by Pseudomonas, such as Pseudomonas aeruginosa. The invention also provides a method of treating an infection caused by Pseudomonas, e.g. Pseudomonas aeruginosa, in a subject in need thereof, comprising administering to said subject one or more bacteriocins as described above or said composition. Administration is preferably topical, e.g. to the eye in the case of keratitis, or to the lungs in the case of pulmonary infection. The subject may be a human or a mammal, e.g. a domestic animal. A preferred subject is a human. Generally, a liquid or solid pharmaceutical composition containing the bacteriocin and optionally further components as described above is prepared for administration to a mammal. A liquid composition may be an aqueous solution as described above. A solid composition may be a powder containing at least one bacteriocin, e.g. in lyophilized form. For the treatment of keratitis, the bacteriocin or pharmaceutical composition is administered to the affected eye, preferably in the form of an ophthalmic composition as described above. The solution may be instilled into the affected eye. The solution may be administered one drop into the affected eye, three times per day. The duration of treatment may be from one day until the infection is effectively treated, which may be 2-10 days.

[0037] For the treatment of pulmonary infections, the bacteriocin or pharmaceutical composition is administered to the lungs of a subject in need of treatment. The bacteriocin or pharmaceutical composition may be administered to the lungs as a solid or liquid aerosol. Thus, the present invention also provides a pulmonary formulation comprising the bacteriocin of the present invention or a composition according to the present invention. For a review of localized pulmonary delivery of protein therapeutics, see, for example, Bodier-Montagutelli et al., EXPERT OPINION ON DRUG DELIVERY 2018, VOL. 15, NO. 8, 729-736; doi.org / 10.1080 / 17425247.2018.1503251. The formulation may be a dry powder for aerosolization, or a liquid solution for nebulization. The composition may be administered to the subject 1-3 times per day. The duration of treatment may be from 1 day to until the infection is effectively treated, which may be 2-10 days. The dosage may be from 10 μg to 500 μg, preferably from 50 to 200 μg, of the bacteriocin of the invention per administration to a human subject.

[0038] The bacteriocin according to the invention may be produced by known methods of protein expression in standard expression systems. To prevent toxic effects of the bacteriocin on the host organism, co-expression of an immunity protein of the bacteriocin may be beneficial. To produce the bacteriocin, a nucleotide sequence encoding the bacteriocin may be expressed in a suitable host organism. Methods that can be used for the production and purification of proteins of interest are described in the prior art and any such method may be used. For example, an E. coli expression system generally known in the art may be used. When using a eukaryotic expression system, one or more introns may be inserted into the coding sequence of the bacteriocin to prevent toxicity to the bacterial organism used for cloning.

[0039] Particularly efficient expression methods are plant expression systems, which are also known in the prior art. Plant expression systems that can be used for the expression of the bacteriocin according to the invention are described in the examples. A possible way to achieve expression of the nucleotide sequence of interest in plants is the use of a self-replicating (viral) replicon containing a nucleotide sequence encoding the bacteriocin. The coding sequence of the bacteriocin can be codon-optimized for expression in plants or in the specific plant used as the expression host. Plant viral expression systems are described in many publications, for example in WO 2012019660, WO 2008028661, WO 2006003018, WO 2005071090, WO 2005049839, WO 2006012906, WO 02101006, WO 2007137788 or WO 02068664, and many more publications are cited in these documents. Various methods are known for introducing nucleic acid molecules, such as DNA molecules, into plants or plant parts for transient expression. Agrobacterium can be used to transfect plants with nucleic acid molecules (vectors) or nucleic acid constructs by agroinfiltration or spraying of an Agrobacterium suspension. For reference, see International Publication No. WO2012019660, WO2014187571, or WO2013149726.

[0040] In embodiments where strong expression of the bacteriocin as a protein of interest is desired, the nucleic acid construct containing the nucleotide sequence encoding the bacteriocin may encode a viral vector capable of replicating in a plant cell to form its replicon. To be replicative, the viral vector and replicon may contain an origin of replication that can be recognized by a nucleic acid polymerase present in the plant cell, for example, a viral polymerase expressed from the replicon. In the case of an RNA viral vector (called an "RNA replicon"), the replicon may be formed by transcription from a DNA construct under the control of a promoter active in the plant cell after its introduction into the plant cell nucleus. In the case of a DNA replicon, the replicon may be formed by recombination between two recombination sites flanking the viral replicon-encoding sequence in the DNA construct, as described, for example, in WO 00 / 17365 and WO 99 / 22003. When the replicon is encoded by a DNA construct, an RNA replicon is preferred. The use of DNA and RNA viral vectors (DNA or RNA replicons) has been extensively described in the literature for many years. Some examples are the following patent publications: WO2008028661, WO2007137788, WO2006003018, WO2005071090, WO2005049839, WO02097080, WO02088369, WO02068664. Examples of DNA viral vectors are based on geminiviruses. For the present invention, viral vectors or replicons based on plant RNA viruses, in particular those based on single-stranded positive-stranded RNA viruses, may preferably be used. Thus, the viral replicon may be a single-stranded positive-stranded RNA replicon. Examples of such viral vectors are based on tobacco mosaic virus (TMV) and potexvirus X (PVX). By "based on" it is meant that the viral vectors use a replication system, e.g., replicase, and / or other proteins involved in the replication of those viruses. Potexvirus-based viral vectors and expression systems are described in EP 2061890 or WO 2008 / 028661. The methods described in ref. 11 can be used.

[0041] The bacteriocin may be expressed in a multicellular plant or part thereof, particularly in a higher plant or part thereof. Both monocotyledonous and dicotyledonous (crop) plants may be used. Common plants that may be used to express the protein of interest include Nicotiana benthamiana, Nicotiana tabacum, spinach, Brassica campestris, B. juncea, Beta vulgaris, watercress, arugula, mustard, strawberry, Chenopodium capitatum, lettuce, sunflower, cucumber, Chinese cabbage, cabbage, carrot, leek, onion, radish, lettuce, snow peas, cauliflower, broccoli, burdock, turnip, tomato, eggplant, pumpkin, watermelon, prince melon, and melon. Preferred plants are spinach, chard, beetroot, carrot, sugar beet, tobacco, and Nicotiana benthamiana. In one embodiment, plants that do not normally enter the human or animal food chain are used, such as Nicotiana species, e.g., Nicotiana tabacum and Nicotiana benthamiana.

[0042] Generally, the bacteriocin as the protein of interest is expressed in the cytosol of the cells of the plant or plant part. In this case, no signal peptide is added to the protein to direct the protein of interest to a specific compartment. Alternatively, the protein of interest can be expressed in or targeted to the chloroplast of the plant, in which case an N-terminal presequence, commonly called a plastid transit peptide or a chloroplast targeting peptide, is added to the N-terminus or C-terminus, preferably the N-terminus, of the bacteriocin as the protein of interest. The bacteriocin can be co-expressed with an immunity protein.

[0043] In the process of producing a composition comprising at least one bacteriocin, the bacteriocin may be expressed in a first step in a plant or a cell of a plant, such as an edible plant. In a second step, the plant material containing the expressed bacteriocin is collected from the plant that expressed the bacteriocin. The plant material may be, for example, leaves, roots, tubers or seeds, or crushed, milled or ground leaves, roots, tubers or seeds. In step (iii), the bacteriocin is extracted from the plant material using an aqueous buffer. This may include homogenizing the plant material, and non-water soluble materials may be removed by centrifugation or filtration. To produce a bacteriocin solution in an aqueous buffer, the soluble components, including the bacteriocin, are extracted into the aqueous buffer. The aqueous buffer may contain an inorganic or organic acid or a salt thereof and may have a pH as defined above for the aqueous solution as the composition of the invention. Additionally, the aqueous buffer may contain salts and / or sulfhydryl compounds, as also described above for the aqueous solutions as compositions of the invention. If a relatively pure bacteriocin composition is desired, the bacteriocin solution in the aqueous buffer may be further purified by removing undesired components in step (iv) according to known methods of protein purification. The bacteriocin may be purified by removing undesired components in step (iv) according to known methods of protein purification. 11 It can be purified as described in. EXAMPLES

[0044] (Reference Example 1) Bacterial strains and culture Unless otherwise specified, P. aeruginosa strains were prepared by cultivation in Lysogeny (LB) medium (Roth) or Casamino Acids (0.5% Bacto™ Casamino Acids, 5.2 mM K2HPO4, 5 mM MgSO4) medium (BD Bacto) at 37 °C under shaking conditions (200 rpm) and overnight cultures were prepared by inoculation from frozen stocks. The P. aeruginosa strains used in the experiments are listed in Supplementary Table 1.

[0045] [Table 1] TIFF2025512803000002.tif55169

[0046] Example 1 Construction of chimeric pyocins Open reading frames encoding PmnH (P. synxantha EIK72868), Pflu095 (Pseudomonas fluorescens WP_016979095), Pflu373 (Pseudomonas fluorescens WP_014717373), Pflu794 (Pseudomonas fluorescens WP_081041794), Pflu618 (Pseudomonas fluorescens WP_034155618), and Pput259 (P. putida WP_098964259), optimized for expression in N. benthamiana, were synthesized by Thermofisher Scientific (USA). Pyocin S5 synthesis and expression vector construction were as previously described. 11 .

[0047] Chimeric proteins were constructed as follows: The N-terminus of pyocin S5 (coding sequence 1-310 aa containing the receptor binding and translocation domains of this pyocin) was amplified using sequence-specific primers flanked by BsaI recognition sites (Supplementary Table 2). The cytotoxic domains of all non-P. aeruginosa putative bacteriocins were amplified using sequence-specific primers flanked by BsaI recognition sites (Supplementary Table 2). Each killing domain was paired with the S5 fragment, and both fragments were inserted into BsaI-digested, assembled TMV-based MagnICON vector pICH29912. 39 The resulting plasmid was used to transform A. tumefaciens GV3101. The sequence of the chimeric pyocin is shown below.

[0048] [Table 2]

[0049] Expression of chimeric pyocins in plants Nicotiana benthamiana plants were grown in a growth chamber at 25° C. under a photoperiod of 16 h light and 8 h dark. Four to six week old plants were used for vacuum infiltration with recombinant A. tumefaciens.

[0050] Agrobacterium strains were inoculated from frozen stocks into 4 ml of LB medium containing 50 μg / ml rifampicin and 50 μg / ml kanamycin and grown at 28° C. with shaking at 220 rpm. Overnight cultures reached an OD 595 Starting at pH = 1.0, the solution was diluted 1:1000 in tap water and supplemented with 0.05% Silwet L77 (Kurt Obermeier). The Agrobacterium suspension was poured into a desiccator container connected to a vacuum pump. The entire leaf system of the plant was then immersed in the suspension. Agroinfiltration was achieved by applying and releasing a vacuum with the pump (up to a pressure of 200 mbar). Plant leaves were harvested 5-6 days after agroinfiltration.

[0051] Purification of chimeric pyocins produced in plants Frozen leaf tissue was homogenized in liquid nitrogen using a chilled mortar and pestle. The prepared powder was mixed with cold extraction buffer (50 mM Tris, 5 mM sodium acetate, pH 5.0) in a ratio of 1 g plant material to 5 ml buffer. The crude extract was incubated at 20-25 °C for 15-20 min. Cellular debris was removed by centrifugation at 3220 g and 4 °C for 20 min. The pellet was discarded and the supernatant was filtered through a membrane filter (pore size 0.45 μm). The pH of the solution was adjusted to 8.0 by adding ammonium sulfate to 0.60 M. The resulting precipitate was removed by centrifugation at 3220 g and 4 °C for 5 min. The supernatant was taken as total soluble protein and applied for purification in two steps.

[0052] In the first purification step, a chromatography column was packed with Phenyl Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM Tris, 5 mM sodium acetate, 0.60 M (NH4)2SO4, pH 8.0). The protein solution was loaded onto the column and the Phenyl Sepharose-bound protein fraction was eluted by washing with elution buffer (50 mM Tris, 5 mM sodium acetate, 0.30 M (NH4)2SO4, pH 8.0). The collected protein fraction was loaded onto a diafiltrating concentrator (10 kDa) and centrifuged at 3220 g until the volume of the protein solution was reduced 10-fold. The concentrate was then diluted to the primary volume with 50 mM Tris and 5 mM sodium acetate (pH 8.0). This procedure was repeated until the conductivity dropped below 5 mS / cm, and then the protein solution was finally purified using Q Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden). The chromatography medium was pre-equilibrated with cold buffer (50 mM Tris, 5 mM sodium acetate, pH 8.0). The protein solution was loaded onto the column and the Q Sepharose unbound proteins were collected in the flow-through fraction. The collected protein fraction was loaded onto a diafiltration concentrator (10 kDa) and centrifuged at 3220 g until the volume of the protein solution was reduced 10-fold. The concentrate was diluted to the initial volume with phosphate-buffered saline (PBS) buffer. For long-term storage, the protein was lyophilized. Pyocin S5 was previously 11 The product was purified as described in.

[0053] result Identification of putative pore-forming bacteriocin sequences from Pseudomonas. Putative pore-forming bacteriocins from Pseudomonas genus were retrieved from NCBI by BLAST search using the pore-forming domain of pyocin S5 (pfam01024) as a query. After analysis of the BLAST results, we selected six putative pore-forming bacteriocins from different Pseudomonas species: Pflu095 (Pseudomonas fluorescens WP_016979095), Pflu373 (WP_014717373 from strain A506), Pflu794 (WP_081041794 from strain ATCC 17400), Pflu618 (WP_034155618 from strain H16), Pput259 (WP_098964259 from P. putida strain FDAARGOS_376), and PmnH from P. scinxantha strain BG33R.

[0054] Clustal W amino acid sequence alignment of the pore-forming domain showed 32–49% identity with pyocin S5 (Fig. 1A). The amino acid sequence of the pfam01024 domain of the bacteriocin was analyzed in a phylogenetic tree with the pore-forming domains of some described pore-forming colicins, klevicins, and pyocin S5 (Fig. 1B). Two major phylogenetic groups were distinguished: the pore-forming domains of Pflu794, Pflu373, and Pflu095 are most related to pyocin S5 and belong to the group of colE1-like proteins, whereas Pflu618 and Put259 are most related to PmnH and belong to the group of colA-like or colN-like proteins (Fig. 1B).

[0055] Construction and plant expression of chimeric pyocins The pore-forming domains of six selected Pseudomonas putative porins were used to construct chimeric proteins. All chimeric proteins contain the same N-terminus of the first 309 amino acid residues of pyocin S5, including the translocation domain, the FptA-binding domain, and the CPA-binding domain. 17 The S5 fragment was fused to the cytotoxic domain of a putative pore-forming bacteriocin. All six chimeric proteins were efficiently expressed in a N. benthamiana transient expression system and purified by two-step chromatography (Figure 2).

[0056] Example 2 Agar disk diffusion assay and agar dilution method for MIC determination Agar disc diffusion assay Overnight P. aeruginosa cultures grown in CAA medium were analyzed at OD in CAA. 595 = 1.0 and diluted 100x. A sterile cotton swab was briefly dipped into the diluted microbial suspension and excess liquid was removed by pressing it against the container wall. The swab was used to streak the bacteria evenly onto a plate containing growth medium with CAA solid agar (1.5%). A sterile Whatman disk, 6 mm in diameter, was placed on the soft agar and 0.3–30 μg of the chimeric pyocin was spotted onto the paper disk. The plates were incubated overnight at 37 °C and observed for zones of bacteriocin inhibition.

[0057] result A panel of 25 P. aeruginosa strains (from culture collections and clinical isolates; Supplementary Table 1) underwent genomic DNA extraction and PCR analysis using primers specific for pyocin S5, pyocin S5 immunity protein, and FptA receptor-encoding genes. All 25 strains tested positive for the presence of fptA. Six strains (PA14, PAO1, HP6, HP7, ATCC 1960, and NCTC 13921) tested positive for amplification of both pyocin S5 and pyocin S5 immunity protein-encoding genes (Figure 8). All 25 P. aeruginosa strains were then subjected to agar disk diffusion assays by spotting different amounts of purified pyocin S5 (0.3 μg, 3 μg, and 30 μg) onto the lawns. Six P. aeruginosa strains were resistant to pyocin S5. Four of those six strains were pyocin S5 and immunity protein encoding strains-PA14, PAO1, ATCC 1960, and NCTC 13921. Two cystic fibrosis isolates, 12-35708 and 12-29165, were also completely resistant to pyocin S5 despite the absence of S5 or S5 immunity protein encoding genes. Surprisingly, pyocin S5 still had a weak (week) inhibitory effect on the lawns of the remaining two S5 producing strains, clinical isolates HP6 and HP7. Turbid inhibition zones were detected when 3 μg and 30 μg of S5 were spotted onto the lawns of those strains (Table 1, Figure 3).

[0058] We analyzed the activity of the S5 chimeras in an agar disk diffusion assay. Two chimeric proteins, S5-Pflu095 and S5-PmnH, showed a broader activity spectrum compared to pyocin S5. Both chimeras formed inhibition zones on lawns of all six S5-producing strains. The inhibition zones on HP6 and HP7 lawns were significantly larger and clearer than those formed by S5. The chimeric proteins also showed similar activity to S5 with only minor differences against all remaining P. aeruginosa strains (Table 1, Fig. 3). Between the two proteins, S5-PmnH showed slightly better activity and was selected for further experiments. The chimeric bacteriocin S5-Pflu373, like S5-Pflu095, also showed a good activity profile, except that it was less active against one of the S5 producers, ATCC 19660. The S5-Pflu628 and S5-Pput259 chimeras performed less well, with weaker and less broad activity compared to pyocin S5, while S5-Pflu794 showed only weak (week) activity against six test strains (Table 1, Figure 3).

[0059] [Table 3]

[0060] Determination of MIC by agar dilution method Aliquots of molten CAA (1.5% agar) medium (final volume-25 ml) pre-warmed to 51°C were supplemented with 0.1 mg / ml BSA and appropriate amounts of pyocin stock solutions at different concentrations (2-fold reductions). Aliquots were poured into Petri dishes. P. aeruginosa cultures from single colonies were cultured at OD in CAA medium. 595 Grown at 37°C, 200 rpm, until β-K+ / -0.2 and incubated at 10°C in CAA medium. 7 CFU / ml. 1 μL of the bacterial suspension was applied in triplicate on each test plate. Plates were incubated overnight at 37° C. The MIC determined was the pyocin concentration at which no confluent bacterial growth was observed.

[0061] Example 3 The ex vivo pig cornea as a model of bacterial keratitis Preparation of porcine corneas. Pig eyes were obtained from the nearest slaughterhouse. Enucleated eyes were stored at -70°C. Before the start of the experiment, eyes were transferred to 4°C for 1 h and then to room temperature for 1-2 h to allow the eyeballs to thaw completely. The eyeballs were then placed individually in a sterile plastic container and immersed in 2.5% povidone-iodine (Betadine 100 mg / ml; EGIS Pharmaceuticals PLC) for 5 min and then washed twice with sterile PBS. Corneas were excised with a sterile surgical blade #12. The excised corneas were stored in Minimum Essential Medium (MEM) supplemented with non-essential amino acids, L-glutamine (2 mM), penicillin (200 U / ml) + spectinomycin (25 μg / ml) at 4°C for up to 2 weeks until further use.

[0062] Corneal infection and pyocin treatment. P. aeruginosa was grown overnight from a frozen stock. The following morning, the culture was diluted 100-fold in fresh LB medium and the OD 595= 0.6 (approximately 6 hours). Bacteria were harvested by centrifugation and resuspended in PBS. Dissected corneas were placed on agarose-gelatin (0.5% each) solid support in a 6-well culture plate containing 800 μL of MEM with antibiotics (100 U / ml penicillin, 25 μg / ml spectinomycin), and then 3 horizontal and 3 vertical scratches were made using a sterile 25-gauge needle. 3x10 4 CFU of Pseudomonas aeruginosa ATCC 19660 strain or 0.4x10 4 CFU of P. aeruginosa strain PAO1 were applied to the corneas and incubated for 16-20 hours in a CO2 incubator (20% CO2) at 37°C. Five corneas were used for each experimental point. After incubation with P. aeruginosa, the corneas were visually inspected for opacity. Corneas that were clear and had no signs of infection were considered uninfected and were excluded from further testing. 5 μg of S5-PmnH (in 5 μl PBS) or 5 μl PBS were applied to the infected corneas and incubated for an additional 16-20 hours in a CO2 incubator at 37°C.

[0063] Homogenization and CFU counting. Prior to homogenization, corneas were washed three times for 10 min in 50 ml of sterile PBS with occasional agitation. Each cornea was minced into 4 equal parts, making an effort to remove the sclera. Two pieces were placed in a Precellys 24 tissue homogenizer (Bertin technologies) tube CKMix50-7 ml and 2 ml of PBS was added. Homogenization was performed under the following conditions: 6500 rpm for 20 s, 5 cycles, with 3-5 min breaks on ice between cycles. The resulting homogenate was transferred to a 15 ml Falcon tube and spun briefly to pellet larger debris, then the supernatant was collected in a new tube and centrifuged at full speed for 10 min to pellet all bacteria. Bacteria were resuspended in 200 μl of sterile PBS and serially diluted and plated on LB agar plates.

[0064] result Two types of P. aeruginosa strains can be isolated from keratitis cases; the cytotoxic P. aeruginosa strains are primarily responsible for contact lens wear-associated keratitis, whereas the invasive strains are most often responsible for postoperative complications. 18 We aimed to investigate whether both types of strains could be targeted by S5-PmnH in disease models. Cytotoxic and invasive strains could be distinguished by genotyping the effector protein-encoding genes of their type III secretion system (TTSS). Invasive strains were found to harbor both exoS and exoT genes, whereas cytotoxic strains appeared to have lost exoS but presented exoT and exoU genes. 19~21 For our experiments, we used the cytotoxic strain ATCC 19660 (exoT, exoU) and the invasive strain PAO1 (ExoY, exoT, exoS). 22 Both strains are pyocin S5 producers and immune to pyocin S5, but both are sensitive to S5-PmnH.

[0065] S5-PmnH treatment can reduce Pseudomonas aeruginosa bacterial numbers in ex vivo pig corneas We first investigated the feasibility of using S5-PmnH for eradicating corneal colonization of P. aeruginosa in an ex vivo model, dissected porcine corneas, which were colonized with the invasive strain PAO1 or the cytotoxic strain ATCC 19660. The S5-PmnH MICs, determined by agar dilution, were 4 μg / ml for PAO1 and 32 μg / ml for ATCC 19660. To obtain P. aeruginosa colonization, porcine corneas were cultured at 3x10 4 CFU of Pseudomonas aeruginosa ATCC 19660 or 0.4x10 4 CFU of P. aeruginosa PAO1 for 16-20 hours. 5 μg of S5-PmnH was then applied to the cornea and incubated for an additional 16-20 hours. At the end of the experiment, the PAO1 load in untreated corneas averaged 7.6 log 10 CFU / cornea, whereas the burden in S5-PmnH-treated corneas averaged only 10 CFU per cornea, a 6.6 log 10In corneas colonized with ATCC 19660, S5-PmnH treatment reduced CFU numbers by 5.3 log 10 Thus, in ex vivo pig corneas, S5-PmnH efficiently reduced P. aeruginosa colonization by both strains.

[0066] Example 4 Mouse keratitis model animal Equal numbers of both sexes of inbred mice of the C57BL / 6 strain were used in this study. Adult female and male mice aged 2-6 months were obtained from the Vilnius University Vivarium of Laboratory Animals. Mice were fed standard chow and water ad libitum for the duration of the experiment. Mice were housed in individual plastic cages with a 12-h light / dark cycle at 21-23 °C. All regulatory procedures involving live animals were approved by the Lithuanian Biomedical Research Ethics Committee (protocol number B1-442) and were performed in accordance with the OECD European Union legislation (Directive 2010 / 63 / EU). Experiments were performed at the Biological Research Centre of the Lithuanian University of Health Sciences.

[0067] Induction and treatment of Pseudomonas aeruginosa keratitis For P. aeruginosa infection and keratitis induction, mice were anesthetized with an intraperitoneal injection of ketamine:xylazine 90:9 (mg / kg). The cornea of ​​the left eye of each mouse was visualized under a stereomicroscope and three 1 mm scratches were made using a sterile 25-gauge needle. 6Aliquots of 10 μL containing cells of P. aeruginosa ATCC 19660 (cytotoxic strain) or PAO1 ATCC 15692 (invasive strain) were applied to the corneal surface. Depending on the experiment, treatments were started 30 minutes or 6 hours after infection. Aliquots of 10 μL containing 0.14 mg of tobramycin or 20 μg of S5-PmnH (both containing 0.5% hydroxypropylmethylcellulose (HPMC)), or PBS containing 0.5% HPMC, were applied as one drop of material to each eye. HPMC was used as an artificial tear to thicken the tear film and to prolong the presence of the application on the corneal surface. Treatments continued twice daily for 5 days. At 1, 3, and 5 days after infection, mice were euthanized by cervical dislocation and eyes were harvested and homogenized for viable counts.

[0068] Clinical score assessment To monitor disease progression, eyes were examined and photographed at 1, 3, and 5 dpi using a dissecting microscope equipped with a digital camera. At 1, 3, and 5 dpi, an established corneal damage scale was established: 0, pupil partially or completely covered by clear or slight opacity; +1, anterior segment partially or completely covered by slight opacity; +2, pupil partially or completely covered by dense opacity; +3, entire anterior segment covered by dense opacity; and +4, corneal perforation. 40 Disease severity was visually graded using the .

[0069] histopathology Histopathological experiments were performed at the Lithuanian University of Health Sciences, Department of Veterinary Pathobiology of Veterinary Academy. One randomly selected mouse from each experimental group was used for histopathological experiments. Enucleated eyes were preserved in 10% formaldehyde. Eyes were embedded in paraffin, cut into 3 μm-thick sections, deparaffinized, rehydrated, and used to prepare hematoxylin / eosin stained specimens. All samples were observed under an Eclipse TE2000-U microscope (Nikon, Tokyo, Japan).

[0070] result S5-PmnH efficiently kills bacteria and prevents acute disease in a mouse keratitis model Infection with cytopathogenic strain ATCC 19660 To induce keratitis, mice were anesthetized, the cornea of ​​the left eye was scratched with a sterile needle, and Pseudomonas aeruginosa ATCC 19660 (4x10 6 CFU were applied to infected eyes. Treatment with S5-PmnH, tobramycin, or PBS (mock treatment) was initiated 30 min or 6 h post-infection. When treatment was initiated 30 min post-infection, no viable P. aeruginosa was isolated from infected eyes in both S5-PmnH and tobramycin-treated groups at 1, 3, or 5 dpi. In contrast, bacterial burdens in infected, untreated eyes were 6-7 log 10 CFU / cornea (Fig. 5A, left panel).

[0071] Visual inspection of infected, untreated eyes by microscopy revealed signs of acute disease, namely slight to dense opacification of the cornea at 1 dpi, and dense opacification and occasional corneal perforation at 3 dpi. At 5 dpi, all mice had corneal perforation. No signs of disease were observed in the majority of specimens treated with S5-PmnH or tobramycin (Figure 5A, right panel). Histopathological examination of infected and mock-treated eyes revealed significant corneal inflammation at 1 dpi, and ocular structural destruction at 3 and 5 dpi. Only slight edema in the corneal stroma was observed in all PyoS5-PmnH- and tobramycin-treated infected eyes (Figure 5C). Thus, when treatment was initiated almost immediately after 30 min postinfection, both S5-PmnH and tobramycin completely eradicated P. aeruginosa ATCC 19660 and prevented disease.

[0072] This experiment was repeated with a delayed treatment time to allow infection to establish for 6 hours. As in the previous experiment, in the infected mock-treated eyes, the mean CFU load was 6.1-6.6 log 10 In the tobramycin-treated groups of mice, no viable bacteria were isolated at 1, 3, or 5 dpi. In the S5-PmnH-treated group, viable bacteria were isolated from one mouse at 1 dpi (3.6 log 10 CFU / cornea), viable bacteria were isolated from one mouse at 3 dpi (5 log 10CFU / cornea). No viable bacteria were isolated from any of the three mice at 5 dpi (Figure 5B, left panel). Clinical scoring of the infected mock-treated eyes revealed a picture similar to the previous experiment, with corneal opacification beginning at 1 dpi and three of four mice having corneal perforation at 5 dpi. Two S5-PmnH-treated and one tobramycin-treated mouse showed only mild signs of disease at 1 and 3 dpi, and all corneas were completely clear at 5 dpi (Figure 5B, right panel). Histopathological examination of mock-treated eyes revealed thinning of the corneal epithelium, thickening of the stroma, acute inflammation at 1 dpi, and acute suppurative inflammation of the cornea at 3 and 5 dpi. PyoS5-PmnH-treated infected eyes presented signs of acute corneal inflammation at 1 dpi, with only slight edema of the corneal stroma without any significant abnormalities at 3 and 5 dpi. Tobramycin-treated infected eyes showed slight thickening of the epithelium at 3 and 5 dpi (FIG. 5C, right panel). Thus, here again, S5-PmnH treatment was efficient in eradicating P. aeruginosa from infected corneas and in preventing disease establishment and progression.

[0073] Infection with invasive strain PAO1 The efficacy of S5-PmnH for treating corneal infection with invasive P. aeruginosa PAO1 was examined. Infection and treatment procedures were similar to previous experiments, with treatment starting 6 hours after infection. The control group of infected mice suffered severe disease and were euthanized at 3 dpi. At 1 dpi, the bacterial load in the mock-treated group of mice was 6.26 log 10 CFU / cornea. S5-PmnH and tobramycin treatments each reduced the burden by an average of 1.04 log 10 CFU / cornea and 1.42 log 10 At 3 dpi, no viable bacteria were isolated from all three tobramycin-treated mice and from the two S5-PmnH-treated mice. The third S5-PmnH-treated mouse had a 2.38 log 10 In contrast, the challenge in the control group of mice was 6.73 log 10At 5 dpi, no viable bacteria were isolated from two tobramycin-treated mice and one S5-PmnH-treated mouse. One mouse from the tobramycin-treated group had a 2.8 log 10 The remaining two mice from the S5-PmnH treatment group contained 5.14 log 10 and 3.34log 10 CFU / cornea (Fig. 6A, left panel).

[0074] Clinical examination revealed significant disease symptoms compared to the cytotoxic strain ATCC 19660. Mock-treated eyes presented severe disease signs at 1 dpi (clinical scores of 2-3). Some S5-PmnH- and tobramycin-treated eyes presented mild clinical scores (1-2) starting at 1 dpi until the end of the experiment (Figure 6A, right panel). Histological examination of uninfected eyes revealed no significant abnormalities, although slight edema in the corneal stroma was observed in most specimens. Infected S5-PmnH-treated eyes exhibited signs of acute corneal inflammation at 1 dpi and 3 dpi. Infected tobramycin-treated eyes exhibited signs of acute corneal inflammation at 1 dpi, thinning of the corneal epithelium and significant edema of the corneal stroma at 3 dpi, and focal corneal inflammation, thinning and degeneration of the corneal epithelium at 5 days (Figure 6C). In conclusion, S5-PmnH efficiently reduced bacterial load and prevented acute disease, regardless of whether cytotoxic or invasive strains were used for infection. However, despite a lower S5-PmnH MIC for P. aeruginosa PAO1 than for P. aeruginosa ATCC 19660, the chimeric pyocin more efficiently eradicated cytotoxic strains of P. aeruginosa and prevented disease establishment, a similar effect observed for tobramycin.

[0075] Example 5 Mouse lung colonization and treatment animal CD1 male mice were supplied by Charles River (Margate, UK) and were specific pathogen free. Mice were 11-15 g upon receipt at the facility and were allowed to acclimate for at least 7 days. Mice were housed in individually ventilated cages and exposed to HEPA filtered air at all times. Mice had free access to food and water and were provided with aspen chip bedding. Room temperature was 22±1° C., relative humidity was 60%, and maximum background noise was 56 dB. Mice were exposed to a 12-hour light / dark cycle.

[0076] Mouse infection and pyocin treatment P. aeruginosa ATCC 27853 was recovered from long-term storage (-80°C) and grown on cysteine, lactose, and electrolyte-deficient (CLED) agar at 37°C under aerobic conditions for approximately 16-24 hours. A single, well-separated colony was inoculated into 20 ml of Mueller-Hinton broth and grown overnight at 37°C with shaking at 300 rpm. The overnight broth was diluted 1:100 in Mueller-Hinton broth and 100 ml was grown in a baffled flask at 37°C with shaking at 300 rpm for approximately 6 hours until the OD of the broth was approximately 0.6 (approximately mid-log phase). The 20 ml culture was centrifuged at 2465 g for 10 minutes and washed in PBS. The pellet was suspended in PBS and the OD 600 was adjusted to 0.67 (approximately 2.6x10 8 From the suspension, the test inoculum was prepared by appropriate dilution in PBS. The inoculum concentration was confirmed by quantitative culture on Pseudomonas selective agar (PSA). The test inoculum concentration was 1.47x10 7 CFU / ml (5.87x10 5 CFU / mouse). Mice were infected by intranasal (IN) instillation of 40 μl of inoculum suspension divided into 20 μl / nostril under brief inhaled 3% isoflurane anesthesia.

[0077] One hour after infection, six mice were sacrificed to assess pre-treatment lung burden. Treatment was administered IN once (50 μl divided into 25 μl / nostril) one hour after infection. Three treatment groups of six mice each received S5-PmnH (2.5, 25, and 250 μg). A fourth group of mice (n=6) received 200 μg tobramycin (40 mg / ml injection, Hospira UK Ltd, 1:10 dilution). The study was terminated 5 hours after infection for all mice. Clinical condition and body weight were assessed, and mice were immediately euthanized using an overdose of pentobarbitone. After death was confirmed, lungs were removed and homogenized in ice-cold sterile phosphate-buffered saline using a Precellys bead beater. Homogenates were quantitatively plated on PSA agar and incubated at 37°C for 24 h before colonies were counted.

[0078] result S5-PmnH efficiently eradicates lung colonization by Pseudomonas aeruginosa in a mouse model of the disease Mice were infected intranasally (IN) with P. aeruginosa strain ATCC 27853. One hour later, mice were administered S5-PmnH IN once into both nostrils at 2.5, 25, and 250 μg / mouse. Five hours later, mice were euthanized and lung burden of P. aeruginosa ATCC 27853 was assessed.

[0079] Five hours after infection, the P. aeruginosa ATCC 27853 load in mock-treated mice was 1.24x10 7 CFU / g lung tissue, 1.53 log 10 S5-PmnH administered IN at 2.5 μg / mouse increased the lung burden by 2.1 log 10 CFU / g, and a dose of 25 μg / mouse reduced 10 CFU / g, and a dose of 250 μg / mouse reduced CFU by 2.66 log 10 The bacterial load was reduced below equilibrium (pre-treatment) levels in all S5-PmnH treatment groups, with a reduction of 0.58 log 10CFU / g, 0.78 log 10 CFU / g and 1.13 log 10 CFU / g. Improved reduction in burden was observed at higher dose levels of S5-PmnH, but the differences were not statistically significant. Tobramycin given IN once at 200 μg / mouse reduced bacterial burden by 2.75 log compared to vehicle. 10 CFU / g reduction of equilibrium levels of 1.23 logs 10 CFU / g. A large variability was observed in this group compared to S5-PmnH or vehicle treatment (Figure 7).

[0080] Overall Consideration of the Results Attempts to use natural antimicrobial proteins to combat pathogens began some time ago. In this regard, bacteriophage lysins (endolysins), peptidoglycan hydrolases, have been widely exploited and shown to effectively target a number of Gram-positive pathogens. 23 The most advanced of these, the lysin PlySs2 (exebacase), is already in phase III clinical trials. Similar to colicin-like bacteriocins, the modular structure of bacteriophage lysins offers the opportunity to engineer enzymes with altered lytic activity, and several novel active hybrid molecules have been constructed by swapping domains of different lysins. 24 However, combating Gram-negative pathogens is a much bigger challenge, since their outer membrane hampers the access of potentially antibacterial biologics to the periplasm or the cytoplasm and nucleus of the bacterial cell. Several attempts have been made to make endolysins permeable to the outer membrane. For example, endolysins have been engineered by the addition of polycationic or / and hydrophobic / amphipathic peptides to enable them to cross the outer membrane (Artilysin). 25As an alternative approach, attempts have been made to equip colicin-like bacteriocins with the lytic domain of an endolysin, thus enabling the engineered protein to be translocated to the periplasm of Gram-negative bacteria. 26、27 In the first such attempt, the FyuA-binding domain of pesticin, a bacteriocin from Yersinia pestis, was fused to the N-terminus of T4 lysozyme. This hybrid toxin has been shown to kill certain Yersinia and pathogenic E. coli strains and, importantly, is able to evade the pesticin immunity protein (Pim), conferring a distinct advantage over pesticin. 26 In another study, the pyocin S2 domain of P. aeruginosa bacteriocin, responsible for surface receptor binding and outer membrane translocation, was fused to the GN4 lysocin to generate PyS2-GN4 lysocin. PyS2-GN4 induced peptidoglycan cleavage and log-fold killing of P. aeruginosa, efficiently disrupted biofilms, and protected mice from P. aeruginosa challenge in a bacteremia model. 27 However, the utility of this chimeric bacteriocin is limited due to its narrow activity spectrum, since it only targets P. aeruginosa strains carrying the ferripyoverdine receptor FpvAI. These and other previous attempts to engineer novel active molecules for the control of Gram-negative bacteria have always relied on the combination of domains of phylogenetically and / or functionally unrelated proteins. Therefore, we decided to construct a chimeric molecule, a porin-porin type chimera, by swapping functional domains of closely related bacterial species that have the same mechanism for antibacterial activity.

[0081] The only known pore-forming bacteriocin of Pseudomonas aeruginosa was first detected in strain PAO1 28 Pyocin S5 binds to the highly conserved ferripyochelin FptA receptor 29 Although the exact prevalence of S5-producing clinical P. aeruginosa strains is not known, a bacteriocin prevalence study of catheter P. aeruginosa isolates demonstrated that approximately 25% of those strains contained the pyocin S5-encoding gene.30 Therefore, those 25% of catheter isolates would theoretically be resistant to pyocin S5. We tested for the presence of pyocin S5 and S5 immunity protein genes in 25 P. aeruginosa isolates in our collection and found similar results, with 6 strains (or 24%) containing pyocin S5 and immunity protein coding gene sequences. As expected, the presence of pyocin S5 coding genes in those strains correlated with resistance to the antibacterial activity of pyocin S5. In our previous study, we also found that pyocin S5 was active against 40% of P. aeruginosa clinical isolates tested. 11 We therefore speculated that if immunity to S5-producing strains could be overcome by the use of cytotoxic domains other than S5, the spectrum of the chimeras could be significantly broadened, with more than half of the strains being targetable.

[0082] Towards this goal, we constructed six chimeric S5 pyocins in which the pore-forming domain of S5 was replaced by the pore-forming domain of a putative bacteriocin from a Pseudomonas species other than P. aeruginosa. All six chimeric pyocins were efficiently expressed in the Nicotiana benthamiana transient expression system, our system of choice for expression of colicin-like bacteriocins. 11、31 The partially purified chimeric protein was subjected to agar drop test assays on 25 P. aeruginosa strains, six of which were producers of S5, and was insensitive or only slightly sensitive to the porin.

[0083] The chimeric proteins displayed different activity profiles, three of which stood out with a broader range of activity than the parent strain S5, two containing a bacteriocin-like porin fragment encoded by Pseudomonas fluorescens (S5-Pflu095 and S5-Pflu373) and one containing the pore-forming domain from P. scinxantha bacteriocin (S5-PmnH). PmnH has an unusual architecture, since it contains two cytotoxic domains, a colicin M-like domain, as well as a pore-forming domain. So far, only the activity of its pore-forming domain has been demonstrated. 16The chimeric protein S5-PmnH was active against all six S5-producing strains and showed activity comparable to S5 against all remaining P. aeruginosa isolates tested. This chimeric protein was selected for further experiments in mouse models for topical treatment of two unrelated models of disease caused by P. aeruginosa, a keratitis model and a pulmonary infection model.

[0084] Bacterial keratitis mainly affects contact lens wearers, but several other risk factors, such as ocular surface diseases, ocular trauma, or ocular surgery, cannot be ignored. A 5-year evaluation of cases in Dubai Hospital revealed that in 37% of bacterial keratitis cases, the causative agent of the disease was Pseudomonas aeruginosa, although this frequency may vary in different geographical regions. 32、33 Untreated bacterial keratitis usually leads to blindness. Two types of P. aeruginosa strains are found in eyes affected by keratitis: cytotoxic strains, which primarily cause keratitis in contact lens wearers, and invasive strains, which mostly cause disease in postoperative complications. 18 Cytotoxic and invasive strains can be distinguished by genotyping the effector protein-encoding genes of their type III secretion system (TTSS). Invasive strains were found to possess both exoS and exoT genes, whereas cytotoxic strains appeared to have lost exoS but displayed exoT and exoU genes. 19~21 In our experiments, the cytotoxic strain ATCC 19660 (exoT, exoU) and the invasive strain PAO1 (ExoY, exoT, exoS) 22 Both strains were confirmed to be pyocin S5 producers, immune to pyocin S, but sensitive to the chimeric pyocin S5-PmnH.

[0085] The chimeric pyocin S5-PmnH was effective in both the cytotoxic and invasive P. aeruginosa models of ocular infection. In the cytotoxic keratitis model, treatment was initiated 30 minutes after infection and completely eliminated all bacteria 1 day after infection. When treatment was delayed for 6 hours, viable bacteria were isolated from one mouse at 1 dpi and from one mouse at 3 dpi. Visual and histological examination of the eyes of all treated mice in both experiments did not reveal any gross lesions and were not significantly different from uninfected eyes. In contrast, all infected eyes showed very significant keratitis symptoms at 3 and 5 dpi. Thus, S5-PmnH treatment efficiently eradicated P. aeruginosa from corneas infected with the cytotoxic strain P. aeruginosa ATCC 19660 and prevented the establishment and progression of the disease.

[0086] Similar experiments were performed using the invasive P. aeruginosa strain PAO1, and treatment was initiated 6 hours after infection. Although both treatments (S5-PmnH or tobramycin) were less efficient at eradicating the bacteria, disease progression was significantly inhibited in all treated mice. Taken together, these studies clearly demonstrated an effect of the chimeric pyocin in this disease model that was comparable to that of the standard of care antibacterial tobramycin. Pseudomonas aeruginosa is also a frequent cause of pulmonary infections, including hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP). This pathogen has a worsening global trend toward a higher likelihood of exhibiting the MDR phenotype. 34 Furthermore, P. aeruginosa can cause chronic pulmonary infections in patients with cystic fibrosis (CF) and non-CF bronchiectasis. Acquisition of P. aeruginosa is associated with increased morbidity and mortality in patients with CF and is an important factor in the development and progression of CF respiratory disease. 35~37 Patients with CF are at extremely high risk of developing infections with multidrug-resistant (MDR) Pseudomonas aeruginosa due to the frequent and often long-term use of oral, intravenous, and aerosolized antibiotics used to treat the chronic lung disease of CF. 38In our validated mouse lung colonization model, one single dose of chimeric pyocin S5-PmnH reduced the bacterial burden in the lung below equilibrium levels, even at the lowest concentration used, 2.5 μg. The effective results obtained with the chimeric pyocin S5-PmnH used in our study indicate that such bacteriocins can be used to efficiently target bacteria in the lung and surprisingly demonstrate the engineering of bacteriocins with modified or extended spectrum of activity.

[0087] Amino acid and nucleic acid sequences SEQ ID NO: 1: First segment of PyoS5 MSNDNEVPGSMVIVAQGPDDQYAYEVPPIDSAAVAGNMFGDLIQREIYLQKNIYYPVRSIFEQGTKEKKEINKKVSDQVDGLLKQITQGKREATRQERVDVMSAVLHKMESDLEGYKKTFTKGPFIDYEKQSSLSIYEAWVKIWEKNSWEERKK YPFQQLVRDELERAVAYYKQDSLSEAVKVLRQELNKQKALKEKEDLSQLERDYRTRKANLEMKVQSELDQAGSALPPLVSPTPEQWLERATRLVTQAIADKKQLQTTNNTLIKNSPTPLEKQKAIYNGELLVDEIASLQARLVKLNAETTRRRTE

[0088] SEQ ID NO:2: Coding sequence of the first segment of PyoS5 ATGTCCAATGATAACGAAGTGCCAGGTTCAATGGTTATAGTTGCTCAAGGTCCAGATGATCAATACGCTTACGAAGTTCCTCCAATCGATTCTGCTGCTGTTGCTGGAAATATGTTCGGAGATCTTATCCAAAGGGAAATATATCTTCAAAAGAATATCTATTACCCAGTTAGATCTATCTTCGAGCAAGGAACAAAGGAAAAGAAAGAGATTAATAAGAAAGTTTCAGATCAAGTTGATGGACTTTTGAAGCAAATTACACAAGGAAAGAGAGAAGCTACTAGGCAAGAGAGAGTTGATGTTATGTCAGCTGTTCTTCATAAGATGGAATCTGATTTGGAGGGATATAAGAAAACTTTTACAAAGGGTCCTTTTATTGATTACGAAAAGCAATCTTCACTTTCAATCTATGAAGCTTGGGTTAAGATTTGGGAGAAAAATTCTTGGGAGGAGAGAAAGAAATACCCTTTTCAACAACTTGTTAGGGATGAATTGGAGAGAGCTGTTGCTTATTACAAGCAAGATTCTCTTTCAGAAGCTGTTAAAGTTCTTAGGCAAGAGTTGAATAAGCAAAAGGCTTTGAAGGAAAAGGAGGATCTTTCACAATTGGAAAGAGATTACAGGACAAGAAAGGCTAATCTTGAGATGAAGGTTCAATCTGAGTTGGATCAAGCTGGATCAGCTCTTCCACCTTTGGTTTCTCCAACTCCTGAACAATGGCTTGAGAGGGCTACAAGATTGGTTACTCAAGCTATTGCTGATAAGAAACAACTTCAAACTACAAATAACACATTGATTAAGAACTCTCCAACTCCTCTTGAAAAGCAAAAAGCTATCTATAATGGTGAACTTTTGGTTGATGAGATTGCTTCATTGCAAGCTAGGCTTGTTAAATTGAATGCTGAGACTACAAGAAGGAGAACAGAA Sequence number 3: The second segment of PmnH KELTPDEKAAYTDAVSFVSTANEQMLQKYGANLSKVAQDMQAEIAGKKVRSYAEAMATFEKISANPAMKLNALDTQAVVDALNALDKASFADNITRLGKAFGVVGKVVQAEAIREKTVSGFQTGDWKPLMLELEAMAVGTGAGILLATSMAFFFPVFASAAAGVVVVALMMAATAAYFDAAKVDEINNLILN

[0089] SEQ ID NO: 4: Coding sequence of the second segment of PmnH AAGGAACTTACACCAGATGAGAAAGCTGCTTATACTGATGCTGTTTCTTTTGTTTCAACAGCTAATGAGCAAATGTTGCAAAAGTACGGTGCTAATCTTTCAAAAAGTTGCTCAAGATATGCAAGCTGAGATTGCTGGAAAGAAA GTTAGGTCTTATGCTGAAGCTATGGCTACTTTTGAGAAGATTTCAGCTAATCCAGCTATGAAACTTAATGCTTTGGATACACAAGCTGTTGTTGATGCCTTTAATGCTTTGGATAAGGCTTCTTTCGCTGATAACATCACTAGGT TGGGAAAGGCTTTTGGTGTTGTTGGAAAAGTTGTTCAAGCTGAAGCTATTAGAGAGAAGACAGTTTCAGGTTTTCAAACTGGAGATTGGAAACCTTTGATGCTTGAATTGGAGGCTATGGCTGTTGGAACTGGTGCTGGAATTCT TTTGGCTACATCTATGGCTTTCTTTTTCCCAGTTTTTGCTTCAGCTGCTGCTGGAGTTGTTGTTGTTGCTCTTATGATGGCTGCTACAGCTGCTTACTTTGATGCTGCTAAGGTTGATGAAATTAATAACCTTATTTTGAATTAA

[0090] SEQ ID NO:5: S5-PmnH, pyocin S5 portion is underlined MSNDNEVPGSMVIVAQGPDDQYAYEVPPIDSAAVAGNMFGDLIQREIYLQKNIYYPVRSIFEQGTKEKKEINKKVSDQVDGLLKQITQGKREATRQERVDVMSAVLHKMESDLEGYKKTFTKGPFIDYEKQSSLSIYEAWVKIWEKNSWEERKK YPFQQLVRDELERAVAYYKQDSLSEAVKVLRQELNKQKALKEKEDLSQLERDYRTRKANLEMKVQSELDQAGSALPPLVSPTPEQWLERATRLVTQAIADKKQLQTTNNTLIKNSPTPLEKQKAIYNGELLVDEIASLQARLVKLNAETTRRRTEKELTPDEKAAYTDAVSFVSTANEQMLQKYGANLSKVAQDMQAEIAGKKVRSYAEAMATFEKISANPAMKLNALDTQAVVDALNALDKASFADNITRLGKAFGVVGKVVQAEAIREKTVSGFQTGDWKPLMLELEAMAVGTGAGILLATSMAFFFPVFASAAAGVVVVALMMAATAAYFDAAKVDEINNLILN SEQ ID NO:6: S5-PmnH coding sequence, pyocin S5 portion is underlined ATGTCCAATGATAACGAAGTGCCAGGTTCAATGGTTATAGTTGCTCAAGGTCCAGATGATCAATACGCTTACGAAGTTCCTCCAATCGATTCTGCTGCTGTTGCTGGAAATATGTTCGGAGATCTTATCCAAAGGGAAATATATCTTCAAAAGAATATCTATTACCCAGTTAGATCTATCTTCGAGCAAGGAACAAAGGAAAAGAAAGAGATTAATAAGAAAGTTTCAGATCAAGTTGATGGACTTTTGAAGCAAAATTACACAAGGAAAGAGAGAAGCTACTAGGCAAGAGAGAGTTGATGTTATGTCAGCTGTTCTTCATAAGATGGAATCTGATTTGGAGGGATATAAGAAAACTTTTACAAAGGGTCCTTTTATTGATTACGAAAAGCAATCTTCACTTTCAATCTATGAAGCTTGGGTTAAGATTTGGGAGAAAAATTCTTGGGAGGAGAGAAAGAAAT ACCCTTTTCAACAACTTGTTAGGGATGAATTGGAGAGAGCTGTTGCTTATTACAAGCAAGATTCTCTTTCAGAAGCTGTTAAAGTTCTTAGGCAAGAGTTGAATAAGCAAAAGGCTTTGAAGGAAAAGGAGGATCTTTCACAATTGGAAAGAGATTACAGGACAAGAAAGGCTAATCTTGAGATGAAGGTTCAATCTGAGTTGGATCAAGCTGGATCAGCTCTTCCACCTTTGGTTTCTCCAACTCCTGAACAATGGCTTGAGAGGGCTACAAGATTGGTTACTCAAGCTATTGCTGATAAGAAACAACTTCAAACTACAAATAACACATTGATTAAGAACTCTCCAACTCCTCTTGAAAGCAAAAAGCTATCTATAATGGTGAACTTTTGGGTTGATGAGATTGCTTCATTGCAAGCTAGGCTTGTTAAATTGAATGCTGAGACTACAAGAAGGAGAACAGAA AAGGAACTTACACCAGATGAGAAAGCTGCTTATACTGATGCTGTTTCTTTTGTTTCAACAGCTAATGAGCAAATGTTGCAAAAGTACGGTGCTAATCTTTCAAAAAGTTGCTCAAGATATGCAAGCTGAGATTGCTGGAAAGAAA GTTAGGTCTTATGCTGAAGCTATGGCTACTTTTGAGAAGATTTCAGCTAATCCAGCTATGAAACTTAATGCTTTGGATACACAAGCTGTTGTTGATGCCTTTAATGCTTTGGATAAGGCTTCTTTCGCTGATAACATCACTAGGT TGGGAAAGGCTTTTGGTGTTGTTGGAAAAGTTGTTCAAGCTGAAGCTATTAGAGAGAAGACAGTTTCAGGTTTTCAAACTGGAGATTGGAAACCTTTGATGCTTGAATTGGAGGCTATGGCTGTTGGAACTGGTGCTGGAATTCT TTTGGCTACATCTATGGCTTTCTTTTTCCCAGTTTTTGCTTCAGCTGCTGCTGGAGTTGTTGTTGTTGCTCTTATGATGGCTGCTACAGCTGCTTACTTTGATGCTGCTAAGGTTGATGAAATTAATAACCTTATTTTGAATTAA

[0091] SEQ ID NO: 7: The second segment of Pflu095 KDKVRTEVVDIEAAIKFTSDFYAEAGVRFGDSASKLASDLADSAQGKKIRSADEAFKAFDKYKNELDKKFSVKDRAAAAKYIDSIDYEAIGKAATKFSKGLGYVGPVIDAKDSIIEFINSMESGDWKPFFLKLESIALGLAATAFVGIAFGFIATTPMGILAFAFIVAATGAAIDDNFAEKLNKFVSSL SEQ ID NO: 8: Coding sequence of the second segment of Pflu095 AAGGATAAGGTTAGGACTGAGGTGGTGGACATCGAGGCTGCTATTAAGTTCACCAGCGACTTCTATGCTGAGGCTGGTGTTAGGTTCGGCGACTCTGCTTCTAAGCTTGCTAGCGATCTTGCTGATAGCGCCCAGGGTAAGA AAATCAGGTCTGCTGATGAGGCTTTCAAGGCCTTCGACAAGTACAAGAACGAGCTGGACAAGAAGTTCAGCGTCAAGGATAGAGCTGCTGCCGCTAAGTACATCGACAGCATTGATTACGAGGCTATCGGCAAGGCTGCCACC AAGTTCTCTAAAGGTCTTGGTTACGTGGGCCCTGTGATCGATGCTAAGGATAGCATCATCGAGTTCATCAACAGCATGGAAAGCGGCGACTGGAAGCCATTCTTCCTGAAGCTTGAGTCTATCGCTCTGGGTCTTGCTGCTA CTGCTTTCGTGGGTATTGCCTTCGGTTTCATTGCTACTACCCCTATGGGTATCCTGGCCTTCGCTTTTATTGTGGCTGCTACCGGTGCTGCTATCGACGATAATTTCGCTGAGAAGCTGAACAAGTTCGTGTCCAGCCTTTAA

[0092] SEQ ID NO: 9: S5-Pflu095, pyocin S5 portion is underlined MSNDNEVPGSMVIVAQGPDDQYAYEVPPIDSAAVAGNMFGDLIQREIYLQKNIYYPVRSIFEQGTKEKKEKINKKVSDQVDGLLKQITQGKREATRQERVDVMSAVLHKMESDLEGYKKTFTKGPFIDYEKQSSLSIYEAWVKIWEKNSWEERKKYPFQQLVRDELERAVAYYKQDSLSEAVKVLRQELNKQKALKEKEDLSQLERDYRTRKANLEMKVQSELDQAGSALPPLVSPTPEQWLERATRLVTQAIADKKQLQTTNNTLIKNSPTPLEKQKAIYNGELLVDEIASLQARLVKLNAETTRRRTEKDKVRTEVVDIEAAIKFTSDFYAEAGVRFGDSASKLASDLADSAQGKKIRSADEAFKAFDKYKNELDKKFSVKDRAAAAKYIDSIDYEAIGKAATKFSKGLGYVGPVIDAKDSIIEFINSMESGDWKPFFLKLESIALGLAATAFVGIAFGFIATTPMGILAFAFIVAATGAAIDDNFAEKLNKFVSSL

[0093] SEQ ID NO: 10: S5-Pflu095 coding sequence, pyocin S5 portion is underlined ATGTCCAATGATAACGAAGTGCCAGGTTCAATGGTTATAGTTGCTCAAGGTCCAGATGATCAATACGCTTACGAAGTTCCTCCAATCGATTCTGCTGCTGTTGCTGGAAATATGTTCGGAGATCTTATCCAAAGGGAAATATATCTTCAAAAGAATATCTATTACCCAGTTAGATCTATCTTCGAGCAAGGAACAAAGGAAAAGAAAGAGATTAATAAGAAAGTTTCAGATCAAGTTGATGGACTTTTGAAGCAAAATTACACAAGGAAAGAGAGAAGCTACTAGGCAAGAGAGAGTTGATGTTATGTCAGCTGTTCTTCATAAGATGGAATCTGATTTGGAGGGATATAAGAAAACTTTTACAAAGGGTCCTTTTATTGATTACGAAAAGCAATCTTCACTTTCAATCTATGAAGCTTGGGTTAAGATTTGGGAGAAAAATTCTTGGGAGGAGAGAAAGAAAT ACCCTTTTCAACAACTTGTTAGGGATGAATTGGAGAGAGCTGTTGCTTATTACAAGCAAGATTCTCTTTCAGAAGCTGTTAAAGTTCTTAGGCAAGAGTTGAATAAGCAAAAGGCTTTGAAGGAAAAGGAGGATCTTTCACAATTGGAAAGAGATTACAGGACAAGAAAGGCTAATCTTGAGATGAAGGTTCAATCTGAGTTGGATCAAGCTGGATCAGCTCTTCCACCTTTGGTTTCTCCAACTCCTGAACAATGGCTTGAGAGGGCTACAAGATTGGTTACTCAAGCTATTGCTGATAAGAAACAACTTCAAACTACAAATAACACATTGATTAAGAACTCTCCAACTCCTCTTGAAAGCAAAAAGCTATCTATAATGGTGAACTTTTGGGTTGATGAGATTGCTTCATTGCAAGCTAGGCTTGTTAAATTGAATGCTGAGACTACAAGAAGGAGAACAGAA AAGGATAAGGTTAGGACTGAGGTGGTGGACATCGAGGCTGCTATTAAGTTCACCAGCGACTTCTATGCTGAGGCTGGTGTTAGGTTCGGCGACTCTGCTTCTAAGCTTGCTAGCGATCTTGCTGATAGCGCCCAGGGTAAGA AAATCAGGTCTGCTGATGAGGCTTTCAAGGCCTTCGACAAGTACAAGAACGAGCTGGACAAGAAGTTCAGCGTCAAGGATAGAGCTGCTGCCGCTAAGTACATCGACAGCATTGATTACGAGGCTATCGGCAAGGCTGCCACC AAGTTCTCTAAAGGTCTTGGTTACGTGGGCCCTGTGATCGATGCTAAGGATAGCATCATCGAGTTCATCAACAGCATGGAAAGCGGCGACTGGAAGCCATTCTTCCTGAAGCTTGAGTCTATCGCTCTGGGTCTTGCTGCTA CTGCTTTCGTGGGTATTGCCTTCGGTTTCATTGCTACTACCCCTATGGGTATCCTGGCCTTCGCTTTTATTGTGGCTGCTACCGGTGCTGCTATCGACGATAATTTCGCTGAGAAGCTGAACAAGTTCGTGTCCAGCCTTTAA

[0094] SEQ ID NO: 11: The second segment of Pflu373 AEEQAKAEEEIKGAIKFTADFYKEIGEKYGAQMTASATDLAETAKGKTLRSAEEALKAFDQYKDHLDKKFSAADRAAIVNALDSLDRAELAKNLNLFAKGFGYTSKAFDVYDLVEEVKKSYASGDWNNTALKVETLFAGSAATGLIAFAFGVTVSTPVGIVAFALIMALVSAYIDDAHVKQFNDALDAILP SEQ ID NO: 12: Coding sequence of the second segment of Pflu373 GCTGAGGAACAGGCTAAGGCTGAGGAAGAGATTAAGGGCGCTATCAAGTTCACCGCCGACTTCTACAAAGAAATCGGCGAGAAGTACGGCGCTCAGATGACTGCTTCTGCTACTGATCTTGCTGAGACTGCTAAGGGTAAGACC CTTAGGTCAGCAGAAGAGGCTCTGAAGGCTTTCGATCAGTACAAGGATCACCTGGACAAGAAGTTCAGCGCTGCTGATAGGGCTGCTATTGTGAACGCTCTGGATTCTCTTGACAGGGCTGAGCTTGCTAAGAACCTGAACCTG TTCGCTAAAGGCTTCGGCTACACCTCTAAGGCATTCGATGTGTACGATCTGGTGGAAGAGGTGAAGAAAAGCTACGCTAGCGGCGATTGGAACAACACCGCCTTTAAGGTTGAGACTCTGTTCGCTGGTTCTGCTGCTACCGGT TTGATTGCTTTCGCTTTCGGTGTGACTGTGTCTACCCCTGTTGGTATTGTGGCTTTCGCTCTGATTATGGCTCTGGTGAGCGCTTACATCGATGACGCTCATGTGAAGCAGTTCAACGATGCTCTGGATGCTATCCTGCCTTAA

[0095] SEQ ID NO: 13: S5-Pflu373, pyocin S5 portion is underlined MSNDNEVPGSMVIVAQGPDDQYAYEVPPIDSAAVAGNMFGDLIQREIYLQKNIYYPVRSIFEQGTKEKKEKINKKVSDQVDGLLKQITQGKREATRQERVDVMSAVLHKMESDLEGYKKTFTKGPFIDYEKQSSLSIYEAWVKIWEKNSWEERKKYPFQQLVRDELERAVAYYKQDSLSEAVKVLRQELNKQKALKEKEDLSQLERDYRTRKANLEMKVQSELDQAGSALPPLVSPTPEQWLERATRLVTQAIADKKQLQTTNNTLIKNSPTPLEKQKAIYNGELLVDEIASLQARLVKLNAETTRRRTEAEEQAKAEEEIKGAIKFTADFYKEIGEKYGAQMTASATDLAETAKGKTLRSAEEALKAFDQYKDHLDKKFSAADRAAIVNALDSLDRAELAKNLNLFAKGFGYTSKAFDVYDLVEEVKKSYASGDWNNTALKVETLFAGSAATGLIAFAFGVTVSTPVGIVAFALIMALVSAYIDDAHVKQFNDALDAILP

[0096] SEQ ID NO: 14: S5-Pflu373 coding sequence, the pyocin S5 part is underlined ATGTCCAATGATAACGAAGTGCCAGGTTCAATGGTTATAGTTGCTCAAGGTCCAGATGATCAATACGCTTACGAAGTTCCTCCAATCGATTCTGCTGCTGTTGCTGGAAATATGTTCGGAGATCTTATCCAAAGGGAAATATATCTTCAAAAGAATATCTATTACCCAGTTAGATCTATCTTCGAGCAAGGAACAAAGGAAAAGAAAGAGATTAATAAGAAAGTTTCAGATCAAGTTGATGGACTTTTGAAGCAAAATTACACAAGGAAAGAGAGAAGCTACTAGGCAAGAGAGAGTTGATGTTATGTCAGCTGTTCTTCATAAGATGGAATCTGATTTGGAGGGATATAAGAAAACTTTTACAAAGGGTCCTTTTATTGATTACGAAAAGCAATCTTCACTTTCAATCTATGAAGCTTGGGTTAAGATTTGGGAGAAAAATTCTTGGGAGGAGAGAAAGAAAT ACCCTTTTCAACAACTTGTTAGGGATGAATTGGAGAGAGCTGTTGCTTATTACAAGCAAGATTCTCTTTCAGAAGCTGTTAAAGTTCTTAGGCAAGAGTTGAATAAGCAAAAGGCTTTGAAGGAAAAGGAGGATCTTTCACAATTGGAAAGAGATTACAGGACAAGAAAGGCTAATCTTGAGATGAAGGTTCAATCTGAGTTGGATCAAGCTGGATCAGCTCTTCCACCTTTGGTTTCTCCAACTCCTGAACAATGGCTTGAGAGGGCTACAAGATTGGTTACTCAAGCTATTGCTGATAAGAAACAACTTCAAACTACAAATAACACATTGATTAAGAACTCTCCAACTCCTCTTGAAAGCAAAAAGCTATCTATAATGGTGAACTTTTGGGTTGATGAGATTGCTTCATTGCAAGCTAGGCTTGTTAAATTGAATGCTGAGACTACAAGAAGGAGAACAGAA GCTGAGGAACAGGCTAAGGCTGAGGAAGAGATTAAGGGCGCTATCAAGTTCACCGCCGACTTCTACAAAGAAATCGGCGAGAAGTACGGCGCTCAGATGACTGCTTCTGCTACTGATCTTGCTGAGACTGCTAAGGGTAAGACCCTTAGGTCAGCAGAAGAGGCTCTGAAGGCTTTCGATCAGTACAAGGATCACCTGGACAAGAAGTTCAGCGCTGCTGATAGGGCTGCTATTGTGAACGCTCTGGATTCTCTTGACAGGGCTGAGCTTGCTAAGAACCTGAACCTGTTCGCTAAAGGCTTCGGCTACACCTCTAAGGCATTCGATGTGTACGATCTGGTGGAAGAGGTGAAGAAAAGCTACGCTAGCGGCGATTGGAACAACACCGCTCTTAAGGTTGAGACTCTGTTCGCTGGTTCTGCTGCTACCGGTTTGATTGCTTTCGCTTTCGGTGTGACTGTGTCTACCCCTGTTGGTATTGTGGCTTTCGCTCTGATTATGGCTCTGGTGAGCGCTTACATCGATGACGCTCATGTGAAGCAGTTCAACGATGCTCTGGATGCTATCCTGCCTTAA

[0097] SEQ ID NO: 15: The second segment of Pflu794 KASDQANNVDIEEAILFTTTFYENLTEKYGEKVSAVAKELAESAKGKTMRSSKEALQTFEKYKDTYNGRFRSRDRREVDRALKSLDKELLSKNLAKFSKAFGSVSKIGDLTEVFIELENSIRTGDWKPLLLTLEGIGLGMAGTYLVAAVFGISATTPLGIVVFAVLMAATSAYIDDDLVKKVNKDLFGF SEQ ID NO: 16: Coding sequence of the second segment of Pflu794 AAGGCTAGCGACCAGGCTAACAACGTTGACATCGAAGAGGCTATCCTGTTCACCACCACCTTCTACGAGAACCTCACTGAGAAGTACGGCGAGAAGGTTAGCGCTGTGGCTAAAGAACTTGCTGAGAGCGCTAAGGGCAAGA CCATGAGGTCATCTAAAGAGGCTCTGCAGACCTTCGAGAAGTACAAGGATACCTACAACGGCCGGTTCAGGTCTAGGGATAGAAGGGAAGTTGATCGGGCTCTGAAGTCCCTTGACAAAGAGCTGCTGTCTAAGAACCTCGCC AAGTTCTCTAAGGCTTTCGGCTCCGTGTCTAAGATCGGTGATCTTACCGAGGTGTTCATCGAGTTGGAGAACTCTATCAGGACCGGCGATTGGAAGCCTCTTCTGCTTACCCTTGAAGGTATCGGTCTTGGTATGGCTGGAA CCTACCTTGTTGCTGCTGTGTTCGGTATCTCTGCTACTACCCCTCTTGGCATTGTGGTGTTCGCTGTTCTTATGGCTGCTACCAGCGCTTACATCGATGATGACCTGGTGAAGAAGGTGAACAAGGACCTGTTCGGCTTCTAA

[0098] SEQ ID NO: 17: S5-Pflu794, pyocin S5 portion is underlined MSNDNEVPGSMVIVAQGPDDQYAYEVPPIDSAAVAGNMFGDLIQREIYLQKNIYYPVRSIFEQGTKEKKEKINKKVSDQVDGLLKQITQGKREATRQERVDVMSAVLHKMESDLEGYKKTFTKGPFIDYEKQSSLSIYEAWVKIWEKNSWEERKKYPFQQLVRDELERAVAYYKQDSLSEAVKVLRQELNKQKALKEKEDLSQLERDYRTRKANLEMKVQSELDQAGSALPPLVSPTPEQWLERATRLVTQAIADKKQLQTTNNTLIKNSPTPLEKQKAIYNGELLVDEIASLQARLVKLNAETTRRRTEKASDQANNVDIEEAILFTTTFYENLTEKYGEKVSAVAKELAESAKGKTMRSSKEALQTFEKYKDTYNGRFRSRDRREVDRALKSLDKELLSKNLAKFSKAFGSVSKIGDLTEVFIELENSIRTGDWKPLLLTLEGIGLGMAGTYLVAAVFGISATTPLGIVVFAVLMAATSAYIDDDLVKKVNKDLFGF Sequence number 18: S5-Pflu794 coding sequence, the piosin S5 part is underlined ATGTCCAATGATAACGAAGTGCCAGGTTCAATGGTTATAGTTGCTCAAGGTCCAGATGATCAATACGCTTACGAAGTTCCTCCAATCGATTCTGCTGCTGTTGCTGGAAATATGTTCGGAGATCTTATCCAAAGGGAAATATATCTTCAAAAGAATATCTATTACCCAGTTAGATCTATCTTCGAGCAAGGAACAAAGGAAAAGAAAGAGATTAATAAGAAAGTTTCAGATCAAGTTGATGGACTTTTGAAGCAAAATTACACAAGGAAAGAGAGAAGCTACTAGGCAAGAGAGAGTTGATGTTATGTCAGCTGTTCTTCATAAGATGGAATCTGATTTGGAGGGATATAAGAAAACTTTTACAAAGGGTCCTTTTATTGATTACGAAAAGCAATCTTCACTTTCAATCTATGAAGCTTGGGTTAAGATTTGGGAGAAAAATTCTTGGGAGGAGAGAAAGAAAT ACCCTTTTCAACAACTTGTTAGGGATGAATTGGAGAGAGCTGTTGCTTATTACAAGCAAGATTCTCTTTCAGAAGCTGTTAAAGTTCTTAGGCAAGAGTTGAATAAGCAAAAGGCTTTGAAGGAAAAGGAGGATCTTTCACAATTGGAAAGAGATTACAGGACAAGAAAGGCTAATCTTGAGATGAAGGTTCAATCTGAGTTGGATCAAGCTGGATCAGCTCTTCCACCTTTGGTTTCTCCAACTCCTGAACAATGGCTTGAGAGGGCTACAAGATTGGTTACTCAAGCTATTGCTGATAAGAAACAACTTCAAACTACAAATAACACATTGATTAAGAACTCTCCAACTCCTCTTGAAAGCAAAAAGCTATCTATAATGGTGAACTTTTGGGTTGATGAGATTGCTTCATTGCAAGCTAGGCTTGTTAAATTGAATGCTGAGACTACAAGAAGGAGAACAGAA AAGGCTAGCGACCAGGCTAACAACGTTGACATCGAAGAGGCTATCCTGTTCACCACCACCTTCTACGAGAACCTCACTGAGAAGTACGGCGAGAAGGTTAGCGCTGTGGCTAAAGAACTTGCTGAGAGCGCTAAGGGCAAGACCATGAGGTCATCTAAAGAGGCTCTGCAGACCTTCGAGAAGTACAAGGATACCTACAACGGCCGGTTCAGGTCTAGGGATAGAAGGGAAGTTGATCGGGCTCTGAAGTCCCTTGACAAAGAGCTGCTGTCTAAGAACCTCGCCAAGTTCTCTAAGGCTTTCGGCTCCGTGTCTAAGATCGGTGATCTTACCGAGGTGTTCATCGAGTTGGAGAACTCTATCAGGACCGGCGATTGGAAGCCTCTTCTGCTTACCCTTGAAGGTATCGGTCTTGGTATGGCTGGAACCTACCTTGTTGCTGCTGTGTTCGGTATCTCTGCTACTACCCCTCTTGGCATTGTGGTGTTCGCTGTTCTTATGGCTGCTACCAGCGCTTACATCGATGATGACCTGGTGAAGAAGGTGAACAAGGACCTGTTCGGCTTCTAA

[0099] Sequence number 19: The second segment of Pflu618 AEEAAKRIDDYKRAVAFVADANKYILEKYGANLHQVVMDLQKDISGKKIRSYAEAMQTFEAVRTNPNARLSPQDTRAVVDALNALDKATYMDSVNKLAKGFGVTGKIVQAHSVIEKTVIGFRDGNWKPLILELESIALGAGAGAAVATLLAVFSPGFAASAIGIVAVGVAIATIASLLNADNVEKINVFISDHLETALKEQR SEQ ID NO:20: Coding sequence of the second segment of Pflu618 GCTGAAGCTGAGGCTAAGAGAATTGATGATTACAAAAGGGCTGTTGCTTTCGTTGCTGATGCTAATAAGTACATCTTGGAAAAGTACGGTGCTAATCTTCATCAAGTTGTTATGGATTTGCAAAAGGATATCTCTGGAAAGAAAATTAGATC ATACGCTGAAGCTATGCAAACTTTTGAGGCTGTTAGAACAAATCCAAATGCTAGGCTTTCTCCTCAAGATACTAGGGCTGTTGTTGATGCTTTGAATGCTCTTGATAAGGCTACATACATGGATTCTGTTAATAAGTTGGCTAAAGGATTTG GTGTTACTGGAAAGATTGTTCAAGCTCATTCAGTTATTGAAAGACAGTTATTGGTTTTAGAGATGGAAATTGGAAACCACTTATTTTGGAACTTGAGTCAATTGCTTTGGGAGCTGGTGCTGGAGCTGCTGTTGCTACACTTTTGGCTGTT TTCTCTCCTGGATTTGCTGCTTCAGCTATTGGTATTGTTGCTGTTGGAGTTGCTATTGCTACTATTGCTTCTCTTTTGAACGCTGATAACGTTGAGAAGATTAATGTTTTTATTTCAGATCATTTGGAAACAGCTCTTAAAGAGCAAAGGTAA

[0100] SEQ ID NO:21: S5-Pflu618, pyocin S5 portion is underlined MSNDNEVPGSMVIVAQGPDDQYAYEVPPIDSAAVAGNMFGDLIQREIYLQKNIYYPVRSIFEQGTKEKKEKINKKVSDQVDGLLKQITQGKREATRQERVDVMSAVLHKMESDLEGYKKTFTKGPFIDYEKQSSLSIYEAWVKIWEKNSWEERKKYPFQQLVRDELERAVAYYKQDSLSEAVKVLRQELNKQKALKEKEDLSQLERDYRTRKANLEMKVQSELDQAGSALPPLVSPTPEQWLERATRLVTQAIADKKQLQTTNNTLIKNSPTPLEKQKAIYNGELLVDEIASLQARLVKLNAETTRRRTEAEAEAKRIDDYKRAVAFVADANKYILEKYGANLHQVVMDLQKDISGKKIRSYAEAMQTFEAVRTNPNARLSPQDTRAVVDALNALDKATYMDSVNKLAKGFGVTGKIVQAHSVIEKTVIGFRDGNWKPLILELESIALGAGAGAAVATLLAVFSPGFAASAIGIVAVGVAIATIASLLNADNVEKINVFISDHLETALKEQR

[0101] SEQ ID NO: 22: S5-Pflu618 coding sequence, the pyocin S5 part is underlined ATGTCCAATGATAACGAAGTGCCAGGTTCAATGGTTATAGTTGCTCAAGGTCCAGATGATCAATACGCTTACGAAGTTCCTCCAATCGATTCTGCTGCTGTTGCTGGAAATATGTTCGGAGATCTTATCCAAAGGGAAATATATCTTCAAAAGAATATCTATTACCCAGTTAGATCTATCTTCGAGCAAGGAACAAAGGAAAAGAAAGAGATTAATAAGAAAGTTTCAGATCAAGTTGATGGACTTTTGAAGCAAAATTACACAAGGAAAGAGAGAAGCTACTAGGCAAGAGAGAGTTGATGTTATGTCAGCTGTTCTTCATAAGATGGAATCTGATTTGGAGGGATATAAGAAAACTTTTACAAAGGGTCCTTTTATTGATTACGAAAAGCAATCTTCACTTTCAATCTATGAAGCTTGGGTTAAGATTTGGGAGAAAAATTCTTGGGAGGAGAGAAAGAAAT ACCCTTTTCAACAACTTGTTAGGGATGAATTGGAGAGAGCTGTTGCTTATTACAAGCAAGATTCTCTTTCAGAAGCTGTTAAAGTTCTTAGGCAAGAGTTGAATAAGCAAAAGGCTTTGAAGGAAAAGGAGGATCTTTCACAATTGGAAAGAGATTACAGGACAAGAAAGGCTAATCTTGAGATGAAGGTTCAATCTGAGTTGGATCAAGCTGGATCAGCTCTTCCACCTTTGGTTTCTCCAACTCCTGAACAATGGCTTGAGAGGGCTACAAGATTGGTTACTCAAGCTATTGCTGATAAGAAACAACTTCAAACTACAAATAACACATTGATTAAGAACTCTCCAACTCCTCTTGAAAGCAAAAAGCTATCTATAATGGTGAACTTTTGGGTTGATGAGATTGCTTCATTGCAAGCTAGGCTTGTTAAATTGAATGCTGAGACTACAAGAAGGAGAACAGAA GCTGAAGCTGAGGCTAAGAGAATTGATGATTACAAAAGGGCTGTTGCTTTCGTTGCTGATGCTAATAAGTACATCTTGGAAAAGTACGGTGCTAATCTTCATCAAGTTGTTATGGATTTGCAAAAGGATATCTCTGGAAAGAAAATTAGATCATACGCTGAAGCTATGCAAACTTTTGAGGCTGTTAGAACAAATCCAAATGCTAGGCTTTCTCCTCAAGATACTAGGGCTGTTGTTGATGCTTTGAATGCTCTTGATAAGGCTACATACATGGATTCTGTTAATAAGTTGGCTAAAGGATTTGGTGTTACTGGAAAGATTGTTCAAGCTCATTCAGTTATTGAAAAGACAGTTATTGGTTTTAGAGATGGAAATTGGAAACCACTTATTTTGGAACTTGAGTCAATTGCTTTGGGAGCTGGTGCTGGAGCTGCTGTTGCTACACTTTTGGCTGTTTTCTCTCCTGGATTTGCTGCTTCAGCTATTGGTATTGTTGCTGTTGGAGTTGCTATTGCTACTATTGCTTCTCTTTTGAACGCTGATAACGTTGAGAAGATTAATGTTTTTATTTCAGATCATTTGGAAACAGCTCTTAAAGAGCAAAGGTAA

[0102] SEQ ID NO: 23: The second segment of Ppu259 AEAEAKRIDDYKRAVAYVADANKFILEKYGAKLHQVVMDLQKDVSGKKIRSYNEALRTFEQVRTNPNARLSPQDTRAVVDALNALDKATYMDSVNRLAKGFGVTGKIVQAHSVVEKAIIGFQDGNWKPLLLEFESIAAGAGAGLLVALIAPPVLAAFSFPPVIAVVATGLLVAGVAALLDAKTVEKINDTIFTLVETTPAH Sequence number 24: Coding sequence of the second segment of Ppu259 GCAGAGGCTGAGGCTAAGAGGATCGATGATTACAAGCGGGCTGTTGCTTACGTGGCCGATGCTAACAAGTTCATCCTCGAGAAGTACGGTGCCAAGCTTCATCAGGTGGTGATGGATCTGCAGAAGGACGTTAGCGGCAAGAAAATCCGGTCTTACAACGAGGCTCTTAGGACCTTTGAGCAGGTCAGGACTAATCCTAACGCTAGGTTGTCTCCTCAGGACACTAGGGCTGTTGTGGATGCTCTTAACGCTCTGGACAAGGCCACCTACATGGATTCTGTGAACAGGCTGGCTAAAGGTTTCGGTGTGACCGGTAAGATTGTGCAGGCTCATTCTGTGGTCGAGAAGGCTATTATCGGTTTCCAGGATGGCAACTGGAAGCCTCTTTTGCTCGAGTTCGAGTCTATTGCTGCTGGTGCTGGTGCAGGTTTGCTTGTGGCTCTTATTGCTCCTCCTGTGCTGGCTGCTTTTTCTTTCCCACCTGTGATTGCTGTGGTGGCTACTGGTCTTTTGGTTGCTGGTGTTGCTGCTCTGCTTGACGCTAAGACTGTCGAGAAGATCAACGACACCATCTTCACCCTGGTTGAGACTACTCCTGCTCACTAA

[0103] Sequence number 25: S5-Ppu259, the pyocin S5 part is underlined MSNDNEVPGSMVIVAQGPDDQYAYEVPPIDSAAVAGNMFGDLIQREIYLQKNIYYPVRSIFEQGTKEKKEKINKKVSDQVDGLLKQITQGKREATRQERVDVMSAVLHKMESDLEGYKKTFTKGPFIDYEKQSSLSIYEAWVKIWEKNSWEERKKYPFQQLVRDELERAVAYYKQDSLSEAVKVLRQELNKQKALKEKEDLSQLERDYRTRKANLEMKVQSELDQAGSALPPLVSPTPEQWLERATRLVTQAIADKKQLQTTNNTLIKNSPTPLEKQKAIYNGELLVDEIASLQARLVKLNAETTRRRTEAEAEAKRIDDYKRAVAYVADANKFILEKYGAKLHQVVMDLQKDVSGKKIRSYNEALRTFEQVRTNPNARLSPQDTRAVVDALNALDKATYMDSVNRLAKGFGVTGKIVQAHSVVEKAIIGFQDGNWKPLLLEFESIAAGAGAGLLVALIAPPVLAAFSFPPVIAVVATGLLVAGVAALLDAKTVEKINDTIFTLVETTPAH

[0104] SEQ ID NO: 26: S5-Ppu259 coding sequence, the piosin S5 part is underlined ATGTCCAATGATAACGAAGTGCCAGGTTCAATGGTTATAGTTGCTCAAGGTCCAGATGATCAATACGCTTACGAAGTTCCTCCAATCGATTCTGCTGCTGTTGCTGGAAATATGTTCGGAGATCTTATCCAAAGGGAAATATATCTTCAAAAGAATATCTATTACCCAGTTAGATCTATCTTCGAGCAAGGAACAAAGGAAAAGAAAGAGATTAATAAGAAAGTTTCAGATCAAGTTGATGGACTTTTGAAGCAAAATTACACAAGGAAAGAGAGAAGCTACTAGGCAAGAGAGAGTTGATGTTATGTCAGCTGTTCTTCATAAGATGGAATCTGATTTGGAGGGATATAAGAAAACTTTTACAAAGGGTCCTTTTATTGATTACGAAAAGCAATCTTCACTTTCAATCTATGAAGCTTGGGTTAAGATTTGGGAGAAAAATTCTTGGGAGGAGAGAAAGAAAT ACCCTTTTCAACAACTTGTTAGGGATGAATTGGAGAGAGCTGTTGCTTATTACAAGCAAGATTCTCTTTCAGAAGCTGTTAAAGTTCTTAGGCAAGAGTTGAATAAGCAAAAGGCTTTGAAGGAAAAGGAGGATCTTTCACAATTGGAAAGAGATTACAGGACAAGAAAGGCTAATCTTGAGATGAAGGTTCAATCTGAGTTGGATCAAGCTGGATCAGCTCTTCCACCTTTGGTTTCTCCAACTCCTGAACAATGGCTTGAGAGGGCTACAAGATTGGTTACTCAAGCTATTGCTGATAAGAAACAACTTCAAACTACAAATAACACATTGATTAAGAACTCTCCAACTCCTCTTGAAAGCAAAAAGCTATCTATAATGGTGAACTTTTGGGTTGATGAGATTGCTTCATTGCAAGCTAGGCTTGTTAAATTGAATGCTGAGACTACAAGAAGGAGAACAGAA GCAGAGGCTGAGGCTAAGAGGATCGATGATTACAAGCGGGCTGTTGCTTACGTGGCCGATGCTAACAAGTTCATCCTCGAGAAGTACGGTGCCAAGCTTCATCAGGTGGTGATGGATCTGCAGAAGGACGTTAGCGGCAAGAAAATCCGGTCTTACAACGAGGCTCTTAGGACCTTTGAGCAGGTCAGGACTAATCCTAACGCTAGGTTGTCTCCTCAGGACACTAGGGCTGTTGTGGATGCTCTTAACGCTCTGGACAAGGCCACCTACATGGATTCTGTGAACAGGCTGGCTAAAGGTTTCGGTGTGACCGGTAAGATTGTGCAGGCTCATTCTGTGGTCGAGAAGGCTATTATCGGTTTCCAGGATGGCAACTGGAAGCCTCTTTTGCTCGAGTTCGAGTCTATTGCTGCTGGTGCTGGTGCAGGTTTGCTTGTGGCTCTTATTGCTCCTCCTGTGCTGGCTGCTTTTTCTTTCCCACCTGTGATTGCTGTGGTGGCTACTGGTCTTTTGGTTGCTGGTGTTGCTGCTCTGCTTGACGCTAAGACTGTCGAGAAGATCAACGACACCATCTTCACCCTGGTTGAGACTACTCCTGCTCACTAA

[0105] SEQ ID NOS: 27 - 38: Primer sequences shown in Supplementary Table 2 SEQ ID NO: 39: Second segment of PyoS5 (killing domain) aerkaaeeqal qdaikftadf ykevtekfga rtsemarqla egargknirs saeaiksfek hkdalnkkls lkdrqaiaka fdsldkqmma kslekfskgf gvvgkaidaa slyqefkist etgdwkpffv kietlaagaa aswlvgiafa tatatpigil gfalvmavtg amidedllek annlvisi References

[0106] TIFF2025512803000005.tif112155 TIFF2025512803000006.tif231154 TIFF2025512803000007.tif234156 TIFF2025512803000008.tif81154

Claims

1. An antimicrobial protein comprising a polypeptide including a first polypeptide segment and preferably a second polypeptide segment adjacent thereto, The first polypeptide segment is one of the following groups, (a-i) to (d-i): (a-i) A segment of the amino acid sequence of SEQ ID NO: 1, or (b-i) A segment having sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95% with respect to the amino acid sequence of SEQ ID NO: 1, or (c-i) A segment having sequence similarity of at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% to the amino acid sequence of SEQ ID NO: 1, or (d-i) A segment having 1 to 60, preferably 1 to 45, more preferably 1 to 30, and most preferably 1 to 15 amino acid residue substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of SEQ ID NO:

1. Selected from, The second polypeptide segment is the following group, (a-i)' to (d-iii)': (a-i)' A segment of the amino acid sequence of SEQ ID NO: 3, or (a-ii)' A segment of the amino acid sequence of SEQ ID NO: 7, or (a-iii)' A segment of the amino acid sequence of SEQ ID NO: 11, or (b-i)' A segment having sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% with respect to the amino acid sequence of SEQ ID NO: 3, or (b-ii) A segment having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 7, or (b-iii)' A segment having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 11, or (c-i)' A segment having sequence similarity of at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably up to 98% to the amino acid sequence of SEQ ID NO: 3, or (c-ii) A segment having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably up to 98% sequence similarity to the amino acid sequence of SEQ ID NO: 7, or (c-iii)' A segment having at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably up to 98% sequence similarity to the amino acid sequence of SEQ ID NO: 11, or (d-i)' A segment having substitutions, additions, insertions, and / or deletions of 1 to 38, preferably 1 to 28, more preferably 1 to 19, and most preferably 1 to 10 amino acid residues compared to the amino acid sequence of SEQ ID NO: 3, or (d-ii)' A segment having substitutions, additions, insertions, and / or deletions of 1 to 38, preferably 1 to 28, more preferably 1 to 19, and most preferably 1 to 10 amino acid residues compared to the amino acid sequence of SEQ ID NO: 7, or (d-iii)' A segment having 1 to 38, preferably 1 to 28, more preferably 1 to 19, and most preferably 1 to 10 amino acid residue substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of SEQ ID NO:

11. The antimicrobial protein selected from the above.

2. (A-i) The amino acid sequence of Sequence ID No. 5, or (A-ii) The amino acid sequence of sequence number 9, or (A-iii) The amino acid sequence of Sequence ID No. 13, or (B-i) An amino acid sequence having at least 80% sequence identity with the amino acid sequence of Sequence ID No. 5, or (B-ii) An amino acid sequence having at least 80% sequence identity with the amino acid sequence of Sequence ID No. 9, or (B-iii) An amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 13, or (C-i) An amino acid sequence having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 5, or (C-ii) An amino acid sequence having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 9, or (C-iii) An amino acid sequence having at least 85% sequence similarity to the amino acid sequence of Sequence ID No. 13, or (D-i) An amino acid sequence having 1 to 100 amino acid residues substituted, added, inserted, and / or deleted from the amino acid sequence of SEQ ID NO: 5, or (D-ii) An amino acid sequence having 1 to 100 amino acid residues substituted, added, inserted, and / or deleted from the amino acid sequence of SEQ ID NO: 9, or (D-iii) Amino acid sequence having 1 to 100 amino acid residue substitutions, additions, insertions, and / or deletions to the amino acid sequence of SEQ ID NO: 13 An antimicrobial protein comprising a polypeptide containing or consisting of.

3. (A-i) The amino acid sequence of Sequence ID No. 5, or (A-ii) The amino acid sequence of sequence number 9, or (A-iii) The amino acid sequence of Sequence ID No. 13, or (B-i) An amino acid sequence having at least 85%, preferably at least 90%, and more preferably at least 95% sequence identity with the amino acid sequence of Sequence ID No. 5, or (B-ii) An amino acid sequence having at least 85%, preferably at least 90%, and more preferably at least 95% sequence identity with the amino acid sequence of Sequence ID No. 9, or (B-iii) An amino acid sequence having at least 85%, preferably at least 90%, and more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 13, or (C-i) An amino acid sequence having at least 90%, preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO: 5, or (C-ii) An amino acid sequence having at least 90%, preferably at least 95%, and most preferably at least 98% sequence similarity to the amino acid sequence of SEQ ID NO: 9, or (C-iii) An amino acid sequence having at least 90%, preferably at least 95%, most preferably at least 98% sequence similarity to the amino acid sequence of Sequence ID No. 13, or (D-i) An amino acid sequence having 1 to 75, preferably 1 to 50, more preferably 1 to 25, and most preferably 1 to 12 amino acid residues substituted, added, inserted, and / or deleted from the amino acid sequence of SEQ ID NO: 5, or (D-ii) An amino acid sequence having 1 to 75, preferably 1 to 50, more preferably 1 to 25, and most preferably 1 to 12 amino acid residues substituted, added, inserted, and / or deleted from the amino acid sequence of SEQ ID NO: 9, or (D-iii) An amino acid sequence having 1 to 75, preferably 1 to 50, more preferably 1 to 25, and most preferably 1 to 12 amino acid residues substituted, added, inserted, and / or deleted from the amino acid sequence of SEQ ID NO:

13. An antimicrobial protein comprising a polypeptide containing or consisting of.

4. The antimicrobial protein according to claim 1, wherein the first segment is located at the N-terminus of the second segment in the amino acid sequence of the antimicrobial protein.

5. A composition comprising the protein according to any one of claims 1 to 4.

6. The composition according to claim 5, further comprising the second bacteriocin according to any one of claims 1 to 4.

7. A pharmaceutical composition comprising an antimicrobial protein as defined in any one of claims 1 to 4 and one or more pharmaceutically acceptable carriers or excipients.

8. A pharmaceutical composition comprising a sterile aqueous solution containing the antimicrobial protein or a solid preparation, wherein the solid preparation is preferably a powder suitable for reconstitution in an aqueous liquid medium or a solid preparation suitable for administration as an aerosol, according to claim 7.

9. The composition according to claim 5 for use in therapeutic purposes.

10. The composition according to claim 5 for use in the treatment or prevention of bacterial infections, preferably bacterial infections caused by the genus Pseudomonas, and more preferably bacterial infections caused by Pseudomonas aeruginosa.

11. The composition according to claim 5 for the treatment of lung infection or keratitis.

12. The composition according to claim 5, for use in the treatment of pulmonary infections by administration to the lungs of mammals as a solid or liquid aerosol; or for use in the treatment of keratitis by administration to the affected eye of a mammal in aqueous solution.

13. Use of an antimicrobial protein as defined in any one of claims 1 to 4 in the manufacture of a pharmaceutical product for treating or preventing bacterial infections in mammals.

14. The use according to claim 13, wherein the pharmaceutical is a pharmaceutical for treating a bacterial lung infection by being administered to the lungs of a mammal as a solid or liquid aerosol; or a pharmaceutical for treating bacterial keratitis by being administered to the affected eye of a mammal in aqueous solution.

15. A nucleic acid molecule comprising a nucleic acid sequence encoding a protein or polypeptide according to any one of claims 1 to 4.

16. The nucleic acid molecule according to claim 15, wherein the nucleic acid sequence comprises a polynucleotide described in any one of sequence numbers 6, 10, or 14, or a polynucleotide encoding a polypeptide as defined in claim 2 or 3.

17. A bacterial cell or eukaryotic cell comprising the nucleic acid molecule described in claim 15.

18. A bacterial cell or eukaryotic cell comprising the antimicrobial protein described in any one of claims 1 to 4.

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