Antimicrobial peptides

Abstract

The invention relates to bacteriophage encoded enzymes and enzyme variants having antimicrobial activity, to methods of making such enzymes and enzyme variants, and to bacteriostatic and bactericidal methods using such enzyme and enzyme variants.

Description

[0001] ANTIMICROBIAL PEPTIDES

[0002] FIELD OF THE INVENTION

[0003] This invention relates generally to at least one bacteriophage encoded enzyme, particularly an endolysin, having antimicrobial activity. The invention also relates generally to methods of making such an enzyme, and to bacteriostatic and bactericidal methods using such an enzyme.

[0004] BACKGROUND TO THE INVENTION

[0005] Antibacterial resistance is a potent threat to human health. It is estimated that Antimicrobial Resistance (AMR) will cause about 10 million deaths per annum, accompanied by a cumulative loss of 60 to 100 trillion USD from the global economy during the period of 2014-2050. The World Health Organization (WHO) has issued a list of 12 bacterial groups for which the new treatments are urgently needed. Among those, the top three (critical) are Gram-negative bacteria: carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacterales, as well as extended spectrum 0-lactamases- (ESBL-)producing Enterobacteriaceae.

[0006] Gram-negative (or double-membrane or diderm) bacteria pose a particular problem due to possession of a highly impermeable outer membrane (absent from Gram-positive or monoderm bacteria such as Staphylococcus or Streptococcus}, and an array of multi-drug efflux pumps. Gram-negative pathogens are therefore intrinsically resistant to many existing antibiotics and are associated with a low success rate of antimicrobial development.

[0007] There are three major approaches that are used currently to combat multidrug-resistant bacterial pathogens: discovering novel antimicrobials; e.g., new antibiotics, repurposing drugs already approved for other diseases and employing drug combination therapy. One problem with the use of antibiotics against Gram-negative bacteria (e.g. Escherichia co / / and Salmonella species) lies in the nature of the cell envelope of these organisms. Gram-negative bacteria are highly resistant to large antimicrobial compounds (i.e., molecules above a certain molecular weight). In particular, antibiotics whose molecular weight is over 600 Da cannot cross the Gram-negative outer membrane to access targets inside the cell. This is due to the specific structure of the outer layer of the outer membrane (lipopolysaccharide or LPS). Therefore, many antibiotics now used against bacteria that are resistant to carbapenems, 0-lactams or quinolones are ineffective against the Gram-negative bacteria due to being larger than 600 Da (e.g., vancomycin, M.W. 1,449 Da). Furthermore, many Gram-negative bacteria (including Enterobacterales) have a wide range of active efflux pumps that remove xenobiotics, including antibiotics and bile salts, from the cells, rendering them recalcitrant to many xenobiotic agents. Accordingly, there is a need in the art for new antimicrobial compounds (e.g., antibiotics) that are effective at inhibiting and / or preventing the growth of Gram-negative bacteria associated with a wide range of diseases and conditions.

[0008] It is an object of the invention to provide at least one antimicrobial compound (e.g., an antibiotic compound) that inhibits the growth and / or proliferation of at least one Gram-negative bacterial species and / or that is effective in treating and / or preventing at least one bacterial infection, disease or condition in a subject in need thereof that is caused by, or associated with at least one Gram-negative bacterial species, and / or to at least provide the public with a useful choice.

[0009] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0010] SUMMARY OF THE INVENTION

[0011] In one aspect the invention relates to an isolated polypeptide comprising at least 95% amino acid sequence identity to SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 or a functional variant or derivative thereof.

[0012] In another aspect the invention relates to an isolated polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 or functional variant or derivative thereof.

[0013] In another aspect the invention relates to an isolated polynucleotide or functional fragment or derivative thereof comprising at least 70% nucleic acid sequence identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 15.

[0014] In another aspect the invention relates to a polynucleotide construct comprising a polynucleotide or functional variant or derivative thereof as described herein.

[0015] In another aspect the invention relates to a vector comprising a polynucleotide or functional variant or derivative thereof as described herein.

[0016] In another aspect the invention relates to an isolated host cell comprising a polypeptide or functional variant or derivative thereof, a polynucleotide or functional variant or derivative thereof, a polynucleotide construct or a vector as described herein. In another aspect the invention relates to a composition comprising, consisting or consisting essentially of an isolated polypeptide, an isolated polynucleotide, a polynucleotide construct, a vector and / or an isolated host cell as described herein and a carrier, diluent or excipient.

[0017] In another aspect the invention relates to a method of making an antibacterial polypeptide (AP), the method comprising expressing a polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 in an isolated host cell.

[0018] In another aspect the invention relates to a method of making an antibacterial polypeptide (AP), the method comprising chemically synthesizing a polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13.

[0019] In another aspect the invention relates to a method of inhibiting the growth and / or proliferation of at least one Gram-negative bacterial species and / or of killing at least one Gram-negative bacterial species comprising contacting the Gram-negative bacterial species with an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13.

[0020] In another aspect the invention relates to a method of inhibiting the growth and / or proliferation of at least one Gram-negative bacterial species and / or of killing at least one Gram-negative bacterial species comprising contacting the Gram-negative bacterial species with a composition comprising an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 and a carrier, diluent or excipient.

[0021] In another aspect, the invention relates to a method of treating a Gram-negative bacterial infection, disease or condition comprising administering an isolated an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 to a subject in need thereof.

[0022] In another aspect, the invention relates to a method of treating a Gram-negative bacterial infection, disease or condition comprising administering a composition comprising an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 to a subject in need thereof.

[0023] Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE FIGURES

[0024] The invention will now be described by way of example only and with reference to the drawings in which:

[0025] Figure 1 - Comparison of Gram positive and Gram-negative cell wall components

[0026] Figure 2 - Activity of Ecol45 against membrane permeabilized bacteria. (A) Ecol45 showed activity at a range of protein concentrations on membrane permeabilized E. co / / TOP10 cells. (B) Ecol45 showed activity against membrane permeabilized Gram-negative bacteria Klebsiella pneumoniae and Sa / / 77cwe / / aTyphimurium but did not display activity against the Gram-positive bacteria Staphylococcus aureus.

[0027] Figure 3 - Bactericidal activity of Ecol45 (20 pM) against antimicrobial resistant clinical

[0028] E. coH, K. pneumoniae and P. aeruginosa isolates

[0029] Figure 4 - Bactericidal activity of Ecol45 derived polypeptides

[0030] (A) Ecol45 derived polypeptides A (ECOalpha_5_6_7), B (ECOalpha 6_7), C (ECOalpha_5_6), and D (ECOalpha_8) with predicted antimicrobial activity.

[0031] (B) Bactericidal activity of Ecol45 derived polypeptides ECO145alpha5_6_7 (13 pM), ECO145alpha7 (27 pM), ECO145alpha5_6 (27 pM) and ECO145alpha8 (35 pM) against clinical drug-resistant E. coH A0077. Also, synergistic bactericidal activity of same polypeptides with another endolysin, LysFl, (9 pM) against E. colikQQ77.

[0032] (C) Bactericidal activity of ECOalpha_5_6_7 against clinical antimicrobial-resistant Gram-negative bacterial isolates including E. coiian P. aeruginosa.

[0033] (D) Bactericidal activity of ECOalpha_5_6_7 (13 pM) against clinical antimicrobial-resistant Gramnegative bacterial isolates including Klebsiella aerogenes, Enterobacter cloacae, K. pneumoniae and E. coii.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] Definitions

[0036] The following definitions are presented to better define the present invention and as a guide for those of ordinary skill in the art in the practice of the present invention.

[0037] Unless otherwise specified, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains. Examples of definitions of common terms in microbiology, molecular biology and biochemistry can be found in Methods for General and Molecular Microbiology, 3rd Edition, C. A. Reddy, et al. (eds.), ASM Press, (2008); Encyclopedia of Microbiology, 2nd ed., Joshua Lederburg, (ed.), Academic Press, (2000); Microbiology By Cliffs Notes, I. Edward Alcamo, Wiley, (1996); Dictionary of Microbiology and Molecular Biology, Singleton et al. (2d ed.) (1994); Biology of Microorganisms 11thed., Brock et al., Pearson Prentice Hall, (2006); Genes IX, Benjamin Lewin, Jones & Bartlett Publishing, (2007); The Encyclopedia of Molecular Biology, Kendrew et al. (eds.), Blackwell Science Ltd., (1994) and Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Robert A. Meyers (ed.), VCH Publishers, Inc., (1995).

[0038] The term "polynucleotide(s)," as used herein, refers in its broadest sense to a single or doublestranded deoxyribonucleotide or ribonucleotide polymer of any length, and includes as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers, fragments, polynucleotide constructs, vectors and modified polynucleotides. Reference to nucleic acids, nucleic acid molecules, nucleotide sequences and polynucleotide sequences is to be similarly understood.

[0039] Nucleic acids as contemplated herein may be, or include (but not limited thereto), deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a 0-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization), threose nucleic acids (TNAs), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA), glycol nucleic acids (GNAs), or chimeras or combinations thereof.

[0040] The term "polynucleotide construct" refers to a polynucleotide molecule, usually double-stranded DNA, which has been conjugated to another polynucleotide molecule. Non-limiting examples include a polynucleotide construct made by inserting a first polynucleotide molecule into a second polynucleotide molecule or made by conjugating a first polynucleotide molecule to a second polynucleotide molecule. Polynucleotide constructs can be readily made by the skilled person, for example, by restriction / ligation as known in the art.

[0041] A polynucleotide construct may contain the necessary elements that permit transcription of a polynucleotide molecule, and, optionally, for translating the transcript into a polypeptide. A polynucleotide molecule comprised in and / or by the polynucleotide construct may be derived from a host cell or may be derived from a different cell or organism and / or may be a recombinant polynucleotide. Once inside a host cell a polynucleotide construct may become integrated in the host chromosomal DNA or remain non-integrated, such as in a plasmid. The polynucleotide construct may be comprised within or linked to a vector. The term "vector" as used herein refers to any type of polynucleotide molecule that may be used to manipulate genetic material so that it can be amplified, replicated, manipulated, partially replicated, modified and / or expressed, but not limited thereto. In some embodiments a vector may be used to transport a polynucleotide or polynucleotide construct comprised in that vector into a cell or organism. In some embodiments the vector comprises the necessary regulatory elements to allow expression of a polypeptide from a polynucleotide or polynucleotide construct comprised in the vector. In some embodiments a vector is selected from the group consisting of plasmids, bacterial artificial chromosomes (BACs), Pl- derived artificial chromosomes (PACs), yeast artificial chromosomes (YACs), bacteriophage, phagemids, and cosmids. In a preferred embodiment, a vector is a plasmid.

[0042] The term "coding region" or "open reading frame" (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and / or a polypeptide under the control of appropriate regulatory sequences. The coding sequence is identified by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a polynucleotide construct, a "coding sequence" is capable of being expressed when it is operably linked to promoter and terminator sequences and / or other regulatory elements.

[0043] Additionally, the term "open reading frame" refers to a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA). An open reading frame encodes a polypeptide.

[0044] The term "noncoding region" refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5' UTR and the 3' UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency.

[0045] The term "3' untranslated region" (3'UTR) is used herein as understood by the skilled person and refers to a region of an mRNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation). The 3'UTR does not comprise an open reading frame and / or is not translated into a polypeptide.

[0046] The term "5' untranslated region" (5'UTR) is used herein as understood by the skilled person and refers to a region of an mRNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome). The 5'UTR does not comprise an open reading frame and / or is not translated into a polypeptide.

[0047] As used herein, the term "polyA tail" means a region of mRNA that is downstream (i.e., 3') from the 3' UTR and that contains multiple, consecutive adenosine monophosphates (A residues). As is appreciated in the art, the function of the poly(A) tail is to protect an mRNA from enzymatic degradation as well as to facilitate both transcription termination and mRNA export from the nucleus. The number of consecutive A residues in a "poly A tail" may vary; e.g., from 10 to 300. By way of example only, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 A residues.

[0048] "Operably-linked" means that the sequence to be expressed is placed under the control of regulatory elements.

[0049] "Regulatory elements" as used herein refers to any nucleic acid sequence element that controls or influences the expression of a polynucleotide insert from a vector or polynucleotide construct and includes promoters, transcription control sequences, translation control sequences, origins of replication, tissue-specific regulatory elements, temporal regulatory elements, enhancers, polyadenylation signals, repressors, and terminators. Regulatory elements can be "homologous" or "heterologous" to the polynucleotide insert to be expressed from a polynucleotide construct or vector as described herein. When a polynucleotide construct or vector as described herein is present in a cell, a regulatory element can be "endogenous", "exogenous", "naturally occurring" and / or "non- naturally occurring" with respect to cell.

[0050] Terminators are sequences, which terminate transcription, and are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.

[0051] The term "promoter" refers to non-transcribed cis-regulatory elements upstream of the coding region that regulate the transcription of a polynucleotide sequence. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes. In one non-limiting example, bacterial promoters may comprise a "Pribnow box" (also known as the -10 region), and other motifs that are bound by transcription factors and promote transcription. Promoters can be homologous or heterologous with respect to polynucleotide sequence to be expressed. When the polynucleotide sequence is to be expressed in a cell, a promoter may be an endogenous or exogenous promoter. Promoters can be constitutive promoters, inducible promoters or regulatable promoters as known in the art.

[0052] The term "polypeptide(s)," as used herein, is used in a broad sense to include naturally occurring polypeptides, artificial polypeptides, synthetic polypeptides, gene products, homologs, orthologs, para logs, variants, fragments, and other equivalents, as well as analogs of such as would be appreciated by a skilled person in the art. A polypeptide may be a single molecule or may part of a molecular complex. Such complexes include, but are not limited to, dimers, trimers, tetramers, hexamers, and the like. A polypeptide can comprise a single chain of amino acids (i.e., a single polypeptide), or, in the case of a molecular complex, multiple chains of amino acids (multiple polypeptides). Frequently, molecular complexes comprising multiple polypeptides comprise disulfide bridges or linkages between certain amino acid residues. As used herein, the term "polypeptide" also refers to polymers of amino acid residues comprising at least one modified amino acid residue, including as a non-limiting example, an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0053] "Naturally occurring" as used herein with reference to a polypeptide or polynucleotide refers to a polynucleotide or polypeptide sequence having a primary nucleic acid or amino acid sequence that is found in nature. A synthetic polynucleotide or polypeptide sequence that is identical to a wild-type polynucleotide sequence is, for the purposes of this disclosure, considered a naturally occurring sequence. What is important for a naturally occurring polynucleotide or polypeptide sequence is that the actual sequence of nucleotide bases or amino acid residues that make up the polynucleotide or polypeptide respectively, is as found or as known from nature.

[0054] The term "wild-type" is used here as generally understood in the art. For example, a wild-type polynucleotide sequence is a naturally occurring polynucleotide sequence, but not limited thereto. A naturally occurring polynucleotide sequence also refers to variant polynucleotide sequences as found in nature that differ from wild-type. For example, allelic variants and naturally occurring recombinant polynucleotide sequences due to hybridization or horizontal gene transfer, but not limited thereto.

[0055] "Non-naturally occurring" as used herein with reference to a polypeptide or polynucleotide refers to a polynucleotide or polypeptide consisting of a primary nucleic acid or amino acid sequence that is not found in nature. Such peptides are also called "artificial polypeptides" (and grammatical variations thereof) herein.

[0056] Examples of non-naturally occurring polynucleotide and polypeptide sequences include artificially produced mutant and variant polynucleotide and polypeptide sequences, made for example by point mutation, insertion, or deletion, domain rearrangement, but not limited thereto. Non-naturally occurring polynucleotide and polypeptide sequences also include chemically evolved sequences. What is important for a non-naturally occurring polynucleotide or polypeptide sequence as described herein is that a polynucleotide or polypeptide consisting of the actual sequence of nucleotide bases or amino acid residues that make up that polynucleotide or polypeptide respectively, is not found in or known from nature.

[0057] The term "functional variant or derivative thereof" of a polypeptide refers to a variant amino acid sequence or subsequence of the polypeptide that performs a function that is required for the function of the polypeptide, typically the biological activity and / or binding of that polypeptide and / or that provides the three-dimensional structure of the polypeptide. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or functional polypeptide derivative thereof performs the polypeptide activity.

[0058] A "functional variant or derivative thereof" of a polypeptide, including a fusion polypeptide, is one that comprises additions, substitutions and / or deletions of non-essential amino acid residues, and / or of non-essential amino acid sequences where "non-essential" means amino acid residues or sequences that do not affect the functionality of the expressed polypeptide.

[0059] For the absence of doubt, a "functional variant or derivative" of a polypeptide as described herein has hydrolase activity, preferably a murein hydrolase or peptidoglycan hydrolase activity.

[0060] "Isolated" as used herein with reference to polynucleotide or polypeptide sequences describes a sequence that has been removed from its natural cellular environment or from a cellular environment in which it was synthesized or expressed. An isolated molecule may be obtained by any method or combination of methods as known and used in the art, including biochemical, recombinant, and synthetic techniques. The polynucleotide or polypeptide sequences may be prepared by at least one purification step.

[0061] The term "recombinant" refers to a polynucleotide sequence that is removed from polynucleotide sequences that surround it in its natural context and is recombined with other polynucleotide sequences that are not present in its natural context. In one non-limiting example, a recombinant polynucleotide is an isolated endogenous polynucleotide conjugated to an isolated exogenous polynucleotide. A further non-limiting example of a recombinant polynucleotide is an isolated polynucleotide operatively linked to a heterologous promoter. A "recombinant" polypeptide sequence is produced by translation from a "recombinant" polynucleotide sequence.

[0062] As used herein, the term "variant" refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polypeptides useful in the invention have biological activities that are the same or similar to those of a corresponding wild type molecule; i.e., the parent polypeptides or polynucleotides. In these embodiments the variants are termed functional variants.

[0063] The term "variant" with reference to polypeptides also encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 35%, preferably at least 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, preferably at least 99% identity to a sequence of the present invention. Identity is found over a comparison window of at least 2 amino acid positions, preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, at least 300 amino acid positions, preferably over the entire length of an isolated polypeptide as described herein. In certain embodiments, variants of the polypeptides described herein have biological activities that are similar, or that are substantially similar to their corresponding wild type molecules. In certain embodiments the similarities are similar activity and / or binding specificity.

[0064] In certain embodiments, variants of polypeptides described herein have biological activities that differ from their corresponding wild type molecules. In certain embodiments the differences are altered activity and / or binding specificity.

[0065] Polypeptide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences, and which could not reasonably be expected to have occurred by random chance.

[0066] Polypeptide sequence identity and similarity can be determined readily by those of skill in the art.

[0067] A variant polypeptide includes a polypeptide wherein the amino acid sequence differs from a polypeptide herein by one or more conservative amino acid or non-conservative substitutions, deletions, additions or insertions which do not affect the biological activity of the peptide.

[0068] Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0069] Analysis of evolved biological sequences has shown that not all sequence changes are equally likely, reflecting at least in part the differences in conservative versus non-conservative substitutions at a biological level. For example, certain amino acid substitutions may occur frequently, whereas others are very rare. Evolutionary changes or substitutions in amino acid residues can be modelled by a scoring matrix also referred to as a substitution matrix. Such matrices are used in bioinformatics analysis to identify relationships between sequences and are known to the skilled worker.

[0070] Other variants include peptides with modifications which influence peptide stability. Such variants may also be referred to as artificial peptides and may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are such variants that include non-naturally occurring amino acid residues e.g. D-amino acids or non-naturally occurring synthetic amino acids. Non-naturally occurring amino acids are amino acids that are not one of the canonical 20 amino acids of the genetic code. Non-naturally occurring amino acids are organic compounds having similar structures to naturally occurring amino acids but with structural modifications that mimic the structure and bioactivity of the natural molecule. Non-naturally occurring amino acids can share side chain and / or backbone structure with their naturally occurring counterparts and include, for example, amino acids or analogs of amino acids other than the 20 naturally-occurring amino acids, e.g. beta or gamma amino acids and cyclic analogs. Certain non- naturally occurring amino acids are described in US20230332199, the entirety of which is hereby incorporated by reference.

[0071] Substitutions, deletions, additions or insertions may be made by mutagenesis methods known in the art. A skilled worker will be aware of methods for making phenotypically silent amino acid substitutions. See for example Bowie etal, 1990, Science 247, 1306.

[0072] A polypeptide as used herein can also refer to a polypeptide that has been modified during or after synthesis, for example, by biotinylation, benzylation, glycosylation, phosphorylation, amidation, by derivatization using blocking / protecting groups and the like. Such modifications may increase stability or activity of the polypeptide.

[0073] Variant polynucleotide sequences preferably exhibit at least 50%, at least 60%, preferably at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, preferably at least 99% identity to a sequence of the present invention. Identity is found over a comparison window of at least 8 nucleotide positions, preferably at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, at least 5000 nucleotide positions, preferably over the entire length of a polynucleotide as described herein.

[0074] Polynucleotide variants encompass those which exhibit sufficient nucleic acid sequence identity to one or more of the specifically identified sequences described herein so as to likely preserve the functional equivalence of those sequences, and which could not reasonably be expected to have occurred by random chance. Such variants are also known as functional variants. Polynucleotide variants and / or functional variants may be derivatized as known in the art to include non-naturally occurring nucleic acid residues. Additionally, polynucleotide variants include polynucleotides that have been codon- optimized for expression of an encoded polypeptide.

[0075] Polynucleotide sequence identity and similarity can be determined readily by those of skill in the art.

[0076] Variant polynucleotides also encompass polynucleotides that differ from the polynucleotide sequences described herein but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.

[0077] Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie etal., 1990, Science 247, 1306). The terms "modulate(s) expression", "modulated expression" and "modulating expression" of a polynucleotide or polypeptide, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide to be expressed is modified thus leading to modulated expression of an isolated polypeptide or polynucleotide as described herein. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations. The "modulated expression" can be related to an increase or decrease in the amount of messenger RNA and / or polypeptide produced and may also result in an increase or decrease in the activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.

[0078] The terms "modulate(s) activity", "modulated activity" and "modulating activity" of a polynucleotide or polypeptide, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide to be expressed is modified thus leading to modulated expression of a polynucleotide or modulated expression or activity of polypeptide as described herein. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations. The "modulated activity" can be related to an increase or decrease in the amount of messenger RNA and / or polypeptide produced and may also result in an increase or decrease in the functional activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.

[0079] The terms "antibiotic" and "antibiotic compound" and grammatical variations thereof are used interchangeably in the context of the present disclosure and should be taken to mean the same thing. A non-limiting example of an antibiotic compound is an antimicrobial peptide as described herein.

[0080] In the context of the present disclosure, "inhibiting the growth and / or proliferation" of at least one Gram-negative bacterial species refers to no detectable increase in the number of bacteria present, and / or in the duration of the bacterial presence or infection under the conditions that otherwise stimulate bacterial multiplication (in the absence of an antimicrobial compound; e.g., an antibiotic as described herein).

[0081] In some embodiments, "inhibiting the growth and / or proliferation" of at least one Gram-negative bacterial species is determined by comparative assay of the optical density at 600nm over time, of a Gram-negative bacterial control culture vs. a Gram-negative bacterial culture treated with an antibacterial combination or composition as described herein. In some embodiments, inhibition is observed when the optical density of the treated culture is less than 10% of the optical density relative to the control culture.

[0082] In the context of the present disclosure, "killing" of bacteria refers to decrease in the number of viable Gram-negative bacterial cells remaining in a population of Gram-negative bacterial cells exposed to an antimicrobial compound as described herein as compared to the number of viable Gram-negative bacterial cells in an untreated population. In some embodiments, "killing" of Gram-negative bacteria is determined by measuring decrease in the number of viable bacterial cells at set time points during culturing in the presence of an antimicrobial compound ("time-kill curve").

[0083] The phrase "a Gram-negative bacterial infection, disease or condition" as used herein refers to any bacterial infection, disease or condition that is caused by or associated with a particular species of Gram-negative bacteria.

[0084] As used herein, the terms "treat", "treating" and "treatment" refer to therapeutic measures which reduce, alleviate, ameliorate, manage, prevent, restrain, stop or reverse bacterial infection caused by or associated with Gram-negative bacterial species, including the symptoms associated with or related to such a bacterial infection. The subject may show observable or measurable (statistically significant) decrease in one or more of the symptoms associated with or related bacterial infection as known to those skilled in the art, as indicating improvement.

[0085] The term "effective amount" as used herein means an amount effective to protect against, delay, reduce, stabilize, improve or treat a bacterial infection, disease and / or condition as known in the art, and / or as described herein. In particular, an "therapeutically effective amount" of an antimicrobial compound as described is an amount that is sufficient to achieve at least a lessening of the symptoms associated with a bacterial infection that is being or is to be treated or that is sufficient to achieve a reduction in bacterial growth, or that is sufficient to increase in bacterial susceptibility to other therapeutic agents or natural immune clearance.

[0086] In some embodiments, an effective amount is an amount sufficient to achieve a statistically different result as compared to an untreated control.

[0087] As used herein the term "artificial peptides" refers to a synthesized or engineered peptide that is an isolated peptide that consists of an amino acid sequence that is not found isolated in nature. While the primary sequence of an artificial peptide as described herein may be found embedded in a larger polypeptide molecule that may exist in nature, the artificial peptides described herein are molecules that themselves do not exist as isolated peptides in nature and that exhibit markedly different properties than any larger polypeptides that they may be a subsequence(s) of. The term "artificial peptide" is used synonymously with artificial polypeptide herein and specifically contemplates all of the polypeptide modifications described herein.

[0088] The term "about" when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, "about 100" means from 90 to 110 and "about six" means from 5.4 to 6.6.

[0089] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting statements in this specification that include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

[0090] The term "consisting essentially of as used herein means the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0091] The term "consisting of" as used herein means the specified materials or steps of the claimed invention, excluding any element, step, or ingredient not specified in the claim.

[0092] The term "at least one" as used herein with reference to described features, including but not limited to "at least one inducible promoter", "at least one selective marker", "at least one auxotrophic marker" and other such usages, means that at least one of the stated features is present. However, this term as used herein also specifically contemplates as an embodiment, the singular "the", "a", "an", and / or "one" (including other such grammatical variations).

[0093] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0094] Description

[0095] In the work disclosed herein the inventors have identified several powerful endolysins, prime candidates for use as novel antimicrobial compounds, particularly against Gram-negative bacteria. The functional characterization disclosed herein provides the first demonstration of the novel cell killing properties of antimicrobial peptides, Ecol45, ECO145alpha5_6, ECO145alpha5_6_7, ECO145alpha7 and ECO145alpha8.

[0096] Endolysins

[0097] Endolysins are enzymes encoded by virulent phages, produced at the end of the phage lytic cycle. Endolysins specifically cleave bonds of the bacterial cell wall peptidoglycan. Degrading the cell wall causes bacterial lysis to occur, releasing progeny phages (Young, 1992). Exogenous application of recombinant endolysins can also cause cell lysis, killing the bacteria (Loessner, 2005). This phenomenon has led to a growing number of studies focused on the implementation of various endolysins as antibacterial agents (Love et al., 2018). While the peptidoglycan of Gram-positive bacteria is exposed and easily accessed by exogenously applied endolysins, the peptidoglycan of Gram-negative bacteria, such as Escherichia coii, Salmonella enterica, Pseudomonas aeruginosa and Klebsiella pneumoniae, is protected by an outer membrane layer (Figure 1). To kill Gram-negative bacteria with endolysins, the enzyme must first penetrate the outer membrane layer to gain access to the peptidoglycan (Fischetti, 2010). This is a significant technical barrier to the development of endolysin-based therapeutics against Gram-negative bacteria.

[0098] Disclosed herein is the inventor's work identifying the antibacterial function of Ecol45 (Liao et al., 2019). This endolysin belongs to the T4 lysozyme-like family of endolysins (Wohlkonig et al., 2010a). Previous studies of other T4 lysozyme-like endolysins, such as the E. coH phage FAHEcl endolysin LysFl (Hudson et al., 2013, 2015), have not identified cell lysis when the outer membrane is intact (Love et al., 2021a). In contrast, the inventors have determined that Ecol45 penetrates the outer membrane of Gram-negative bacteria, providing access to the catalytic substrate of Ecol45 - the peptidoglycan layer.

[0099] The inventors have further identified that endolysin Ecol45 of Escherichia phage vB_EcoS-Rol45clw has intrinsic antibacterial activity against antimicrobial resistant Escherichia coii, Klebsiella pneumoniae, and Pseudomonas aeruginosa strains causing 1.25-log, 0.49-log, and 0.59-log killing, respectively (Figure 3). Without wishing to be bound by theory, the inventors believe that based on biophysical, microbiological and insiiico studies, that the antimicrobial activity of ECO145 has two phases: 1) Ecol45 first penetrates the outer membrane of Gram-negative bacteria, either by destabilising the membrane or by inducing pore formation. This penetration then provides 2) access to the peptidoglycan cell wall layer of the bacteria, enabling the catalytic domain of this enzyme to lyse the peptidoglycan.

[0100] As used herein, the terminology "Ecol45" means the GenBank amino acid sequence having Accession No: AUX83761.1. The nucleic acid sequence encoding the wild-type Escherichia phage vB_EcoS- Rol45clw Endolysin Ecol45-protein is the GenBank nucleic acid sequence having Accession No.: Genome: MG852086.2, Locus tag: vBEcoSRol45clw_00053.

[0101] The amino acid sequence of the Ecol45 wild type is shown in SEQ ID NO: 1 (Table 1). The nucleic acid sequence of the Ecol45 wild type is shown in SEQ ID NO: 3 (Table 1).

[0102] As disclosed herein, the inventors have synthesised a series of artificial peptides from specific helices of the wild-type Ecol45 protein. These artificial peptides are not themselves found naturally occurring and exhibit markedly different properties than the Ecol45 endolysin from which they are derived.

[0103] Disclosed herein are the following four artificial peptides: a) ECO145alpha5_6, which consists of alpha-helices 5 and 6 (SEQ ID NO: 4) b) ECO145alpha5_6_7 peptide, which consists of alpha-helices 5, 6 and 7 of Endolysin Ecol45 (SEQ ID NO: 7) c) ECO145alpha7, which consists of just alpha-helix 7 (SEQ ID NO: 10) d) ECO145alpha8, which consists of just alpha-helix 7; (SEQ ID NO: 13)

[0104] The amino acid sequences of these artificial peptides are shown in Table 1. Also shown in Table 1 are the nucleic acid sequences encoding these peptides. Also as disclosed herein, the inventors have determined that artificial peptide a) ECO145alpha5_6_7 kills drug resistant clinical E. coH , K. pneumoniae and P. aeruginosa strains (Figure 4). Accordingly, the inventors have shown by this work that artificial peptides as described herein, particularly ECO145alpha5_6_7 and ECO145alpha7, have potent bactericidal alone, and synergistic bactericidal activity against Gram-negative bacteria when combined with other antimicrobial suitable candidates for replacing or substituting for known antibiotics.

[0105] Specifically, artificial peptides ECO145alpha5_6_7 and ECO145alpha7 have potent antibacterial activity against Escherichia coii causing 4.46-log and 0.66-log killing in PBS (Figure 4B).

[0106] Additionally, artificial peptides ECO145alpha5_6_7 and ECO145alpha7 act synergistically with another endolysin, LysFl, demonstrating potent antibacterial activity against Escherichia coii'm causing >5-log (kills all bacterial cells) and 1.25-log killing in PBS. (Figure 4B)

[0107] Although previous work with endolysins has investigated the antibacterial properties of certain fused endolysins with peptides derived from other organisms to produce chimeric enzymes comprising antimicrobial peptides from different organisms (e.g., artilysin), antimicrobial peptides comprising shuffled endolysin domains termed chimeolysins, endolysins comprising bacteriocin domains termed lysocins and endolysins fused to phage receptor binding proteins termed innolysins, the inventors believe they are the first to use artificial peptides constructed from selected structural domains of endolysins to produce potent antibacterial endolysins. Moreover, the inventor's selection of specific sub-sequences from ECO145 could not have been predicted, based on what was known in the art, to provide the potent antimicrobial peptides described herein.

[0108] Accordingly, in one aspect the invention relates to an isolated polypeptide comprising at least 95% amino acid sequence identity to SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 or a functional variant or derivative thereof.

[0109] In one embodiment the isolated polypeptide or functional variant or derivative thereof comprises at least 96%, preferably at least 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13.

[0110] In one embodiment the isolated polypeptide or functional variant or derivative thereof is a hydrolase, preferably a murein hydrolase or peptidoglycan hydrolase.

[0111] In one embodiment the isolated polypeptide comprises, consists essentially or consists of an amino acid sequence having at least 96%, preferably at least 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 4 or a functional variant or derivative thereof. In one embodiment the isolated polypeptide comprises, consists essentially or consists of SEQ ID NO: 4 or a functional variant or derivative thereof. In one embodiment the isolated polypeptide comprises, consists essentially or consists of an amino acid sequence having at least 96%, preferably at least 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 7 or a functional variant or derivative thereof. In one embodiment the isolated polypeptide comprises, consists essentially or consists of SEQ ID NO: 7 or a functional variant or derivative thereof.

[0112] In one embodiment the isolated polypeptide comprises, consists essentially or consists of an amino acid sequence having at least 96%, preferably at least 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 10 or a functional variant or derivative thereof. In one embodiment the isolated polypeptide comprises, consists essentially or consists of SEQ ID NO: 10 or a functional variant or derivative thereof.

[0113] In one embodiment the isolated polypeptide comprises, consists essentially or consists of an amino acid sequence having at least 96%, preferably at least 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 13 or a functional variant or derivative thereof. In one embodiment the isolated polypeptide comprises, consists essentially or consists of SEQ ID NO: 13 or a functional variant or derivative thereof.

[0114] In another aspect the invention relates to an isolated polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 or functional variant or derivative thereof.

[0115] In one embodiment the isolated polypeptide or functional variant or derivative thereof comprises at least 96%, preferably at least 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13.

[0116] In one embodiment the isolated polypeptide or functional variant or derivative thereof is a hydrolase, preferably a murein hydrolase or peptidoglycan hydrolase.

[0117] In one embodiment the isolated polypeptide comprises, consists essentially or consists of an amino acid sequence having at least 96%, preferably at least 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 4 or a functional variant or derivative thereof. In one embodiment the isolated polypeptide comprises, consists essentially or consists of SEQ ID NO: 4 or a functional variant or derivative thereof.

[0118] In one embodiment the isolated polypeptide comprises, consists essentially or consists of an amino acid sequence having at least 96%, preferably at least 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 7 or a functional variant or derivative thereof. In one embodiment the isolated polypeptide comprises, consists essentially or consists of SEQ ID NO: 7 or a functional variant or derivative thereof. In one embodiment the isolated polypeptide comprises, consists essentially or consists of an amino acid sequence having at least 96%, preferably at least 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 10 or a functional variant or derivative thereof. In one embodiment the isolated polypeptide comprises, consists essentially or consists of SEQ ID NO: 10 or a functional variant or derivative thereof.

[0119] In one embodiment the isolated polypeptide comprises, consists essentially or consists of an amino acid sequence having at least 96%, preferably at least 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 13 or a functional variant or derivative thereof. In one embodiment the isolated polypeptide comprises, consists essentially or consists of SEQ ID NO: 13 or a functional variant or derivative thereof.

[0120] The polypeptides or functional variants or derivatives as described herein can be produced by the skilled worker as known in the art. In some embodiments the polypeptides or functional variants or derivatives thereof are expressed from a polynucleotide or functional variant or derivative thereof. In some embodiments the polynucleotide or functional variant or derivative thereof is comprised in and expressed from a polynucleotide construct or vector.

[0121] In other embodiments the polypeptides or functional variants or derivatives thereof are chemically synthesized. A chemically synthesized polypeptide or functional variant (including orthologues) or derivative thereof as described herein can comprise non-naturally occurring amino acid residues as described herein.

[0122] In one embodiment the polypeptide or functional variant or derivative thereof is chemically synthesised by solid phase peptide synthesis (SPPS). In one embodiment the chemically synthesised polypeptide or functional variant or derivative thereof is not phosphorylated, acetylated, methylated, succinylated, glutathionylated, carbonylated, oxidated or deaminated.

[0123] In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof has hydrolase activity, preferably murein hydrolase or peptidoglycan hydrolase activity, and comprises at least one AA alteration, preferably at least one conservative AA alteration. In one embodiment the alteration is a substitution, addition, insertion or deletion. In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof has hydrolase activity, preferably murein hydrolase or peptidoglycan hydrolase activity, and comprises 20, preferably 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, preferably 1 AA alterations, preferably conservative AA alterations. In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof has hydrolase activity, preferably murein hydrolase or peptidoglycan hydrolase activity, and comprises 5, 4, 3, 2 or 1 AA alterations, preferably conservative AA alterations.

[0124] In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof has hydrolase activity, preferably murein hydrolase or peptidoglycan hydrolase activity, and comprises at least one non-naturally occurring AA. In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof comprises at least one modified AA residue, preferably an artificial chemical analogue of a corresponding naturally occurring AA. In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof comprises at least one non-naturally occurring AA residue. In one embodiment the at least one non-naturally occurring AA residue is a D-amino acid residue. In one embodiment the at least one non-naturally occurring AA residue comprises a modified side chain.

[0125] In some embodiments, the chemically synthesized polypeptide or functional variant or derivative thereof is bacteriostatic or bactericidal, preferably bactericidal.

[0126] In another aspect the invention relates to an isolated polynucleotide or functional variant or derivative thereof comprising at least 70% SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 15.

[0127] In one embodiment the polynucleotide comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% nucleic acid sequence identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 15. In one embodiment the polynucleotide comprises, consists essentially of or consists of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 15.

[0128] In one embodiment the isolated polynucleotide encodes a polypeptide or functional variant or derivative thereof that is a hydrolase, preferably a murein hydrolase or peptidoglycan hydrolase.

[0129] In one embodiment the polynucleotide comprises or consists essentially of a polynucleotide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% nucleic acid sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6. In one embodiment the polynucleotide comprises, consists essentially of or consists of SEQ ID NO: 5 or SEQ ID NO: 6.

[0130] In one embodiment the polynucleotide comprises or consists essentially of a polynucleotide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% nucleic acid sequence identity to SEQ ID NO: 8 or SEQ ID NO: 9. In one embodiment the polynucleotide comprises, consists essentially of or consists of SEQ ID NO: 8 or SEQ ID NO: 9.

[0131] In one embodiment the polynucleotide comprises or consists essentially of a polynucleotide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% nucleic acid sequence identity to SEQ ID NO: 11 or SEQ ID NO: 12. In one embodiment the polynucleotide comprises, consists essentially of or consists of SEQ ID NO: 11 or SEQ ID NO: 12.

[0132] In one embodiment the polynucleotide comprises or consists essentially of a polynucleotide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% nucleic acid sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15. In one embodiment the polynucleotide comprises, consists essentially of or consists of SEQ ID NO: 14 or SEQ ID NO: 15.

[0133] In another aspect the invention relates to a polynucleotide construct comprising a polynucleotide or functional variant or derivative thereof as described herein.

[0134] In one embodiment the polynucleotide construct comprises at least one heterologous regulatory element. In one embodiment the polynucleotide construct is comprised in a vector. In one embodiment the vector is an expression vector. In one embodiment the vector is selected from the group consisting of plasmids, BACs, (PACs), YACs, bacteriophage, phagemids, and cosmids.

[0135] Preferably the vector is a plasmid.

[0136] In another aspect the invention relates to a vector comprising an isolated polynucleotide or polynucleotide construct according to the invention.

[0137] In one embodiment the vector is selected from the group consisting of plasmids, BACs, (PACs), YACs, bacteriophage, phagemids, and cosmids. Preferably the vector is a plasmid.

[0138] A polynucleotide or polynucleotide construct as described herein can be incorporated into any suitable vector capable of expressing that polynucleotide or, where applicable, of expressing an encoded polypeptide as described herein in vitro or in a host cell. In one embodiment the vector is an expression vector.

[0139] Examples of suitable expression vectors include, but not limited to, plasmid DNA vectors, viral DNA vectors (such as adenovirus and adeno-associated virus), or viral RNA vectors (such as a retroviral vectors). In some embodiments the plasmid and / or phage vectors may be selected from the following vectors or variants thereof including pUC18, pU19, Mpl8, Mpl9, ColEl, PCR1 and pKRC; lambda gtlO and M13 plasmids such as pBR322, pACYC184, pT127, RP4, plJlOl, SV40 and BPV. Also included are vectors such as, but not limited to, cosmids, YACS, BACs shuttle vectors such as pSA3, PAT28 transposons (such as described in US 5,792,294) and the like.

[0140] Suitable viral vectors include but are not limited to vectors derived from adenovirus (AV); adeno- associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. Viral vectors employed herein can be appropriately modified by pseudo typing with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins.

[0141] A polynucleotide construct or vector comprising a polynucleotide construct as described herein can be used to drive expression of a polypeptide as described herein. In one embodiment, the polynucleotide construct comprises a polynucleotide as described herein operatively linked to 5' or 3' untranslated regulatory element. The design of a particular polynucleotide construct will depend on various factors including the host cells in which the operatively linked polynucleotide is to be expressed and the desired level of polynucleotide expression. Likewise, the selection of various promoters, enhancers and / or other genetic elements for a polynucleotide construct will depend on various factors including the host cells and expression levels discussed above.

[0142] In one embodiment, the polynucleotide construct comprises a homologous promoter operatively linked to a polynucleotide as described herein. In another embodiment, the polynucleotide construct comprises a heterologous promoter operatively linked to a polynucleotide as described herein. In one embodiment, the homologous or heterologous promoter is an inducible, repressible or regulatable promoter. A suitable promoter may be chosen and used under the appropriate conditions to direct high-level expression of a polynucleotide of the invention. Many such elements are described in the literature and are available through commercial suppliers.

[0143] By way of example only, promoters useful in polynucleotide constructs as described herein can be any suitable eukaryotic or prokaryotic promoter. In one embodiment, the eukaryotic promoter can be a eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III).

[0144] Expression levels of an operably linked polynucleotide in a particular cell type will be determined by the nearby presence (or absence) of specific gene regulatory sequences (e.g., enhancers, silencers and the like). Any suitable promoter / enhancer combination (see: Eukaryotic Promoter Data Base EPDB) can be used to drive expression of a polynucleotide as described herein.

[0145] Additional promoters useful in polynucleotide constructs or vectors as described herein may include 0- lactamase, alkaline phosphatase, tryptophan, and tac promoter systems, and yeast promoters including 3-phosphoglycerate kinase, enolase, hexokinase, pyruvate decarboxylase, glucokinase, and glyceraldehydrate-3-phosphanate dehydrogenase but not limited thereto.

[0146] Prokaryotic promoters useful in polynucleotide constructs or vectors as described herein include constitutive promoters such as the int promoter of bacteriophage lamda and the bla promoter of the beta-lactamase gene sequence of pBR322 and regulatable promoters (such as lacZ, recA and gal), but are also not limited thereto. A ribosome binding site upstream of the CDS may also be required for expression.

[0147] Enhancers useful in a polynucleotide construct or vector as described herein may include SV40 enhancer, cytomegalovirus early promoter enhancer, globin, albumin, insulin and the like, but again are not necessarily limited to. In one embodiment, polypeptide expression from a polynucleotide construct or vector may be driven by a T3, T7 or SP6 cytoplasmic expression system.

[0148] In another aspect the invention relates to an isolated host cell comprising a polypeptide or functional variant or derivative thereof, a polynucleotide or functional variant or derivative thereof, a polynucleotide construct or a vector as described herein. In one embodiment the isolated host cell is a prokaryotic or eukaryotic cell. Prokaryotes most commonly employed as host cells are strains of Escherichia coii (E. coii). In one embodiment the isolated host cell is E. co / / strain BL21(DE3).

[0149] Other prokaryotic hosts may include Pseudomonas, Bacillus, Serratia, Klebsiella, Streptomyces, Listeria, Salmonella and Mycobacteria but are not limited thereto.

[0150] In one embodiment the eukaryotic cell is an animal cell, a plant cell, a fungal cell or a protist cell. In one embodiment the animal cell is an insect cell or a mammalian cell. In one embodiment the mammalian cell is a human cell in vitro or ex vivo. In one embodiment the mammalian cell is a human cell that is not in the human body.

[0151] In another aspect the invention relates to a composition comprising, consisting or consisting essentially of an isolated polypeptide, an isolated polynucleotide, a polynucleotide construct, a vector and / or an isolated host cell as described herein and a carrier, diluent or excipient.

[0152] In one embodiment the composition is a bactericidal or bacteriostatic composition.

[0153] In one embodiment the composition comprises or consists essentially of the isolated polypeptide and a carrier, diluent or excipient. In one embodiment the composition comprises or consists essentially of the isolated polynucleotide and a carrier, diluent or excipient. In one embodiment the composition comprises or consists essentially of the polynucleotide construct and a carrier, diluent or excipient. In one embodiment the composition comprises or consists essentially of the vector and a carrier, diluent or excipient. In one embodiment the composition comprises or consists essentially of the isolated host cell and a carrier, diluent or excipient.

[0154] In one embodiment the carrier, diluent or excipient is a buffer. In one embodiment the buffer is a zwitterionic buffer. In one embodiment the zwitterionic buffer is selected from the group consisting of MES, MOPS, HEPES and TRIS, preferably MES. In one embodiment the buffer is an inorganic buffer. In one embodiment the inorganic buffer is selected from the group consisting of citrate, acetate, phosphate and cacodylate. Buffers with low concentrations of chloride ions are preferred to prevent precipitation of AgCl. In one embodiment the buffer maintains the composition in a pH range of about 6 to about 8 or of about 6 to 8 or of 6 to about 8 or of 6 to 8, preferably about 6.5 to about 7.5 or about 6.5 to 7.5 or 6.5 to about 7.5 or 6.5 to 7.5, preferably about pH 6.5, 7 or 7.5, preferably at pH 6.5±.2, 7±.2 or 7.5±.2, preferably at pH 6.5, 7 or 7.5, preferably at pH 6.5.

[0155] In one embodiment the composition is a pharmaceutical composition, wherein the carrier, diluent or excipient is a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment the pharmaceutical composition consists or consists essentially of an isolated polypeptide as described herein and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment the pharmaceutical composition comprises or consists essentially of an effective amount of the isolated polypeptide, preferably a therapeutically effective amount of the isolated polypeptide.

[0156] In one embodiment the pharmaceutical composition consists of an effective amount of the isolated polypeptide and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment the pharmaceutical composition consists of a therapeutically effective amount of the isolated polypeptide and a pharmaceutically acceptable carrier, diluent or excipient.

[0157] In one embodiment an effective amount is an amount of the isolated polypeptide that, when contacted to a Gram-negative bacterial species as described herein, kills at least 50%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, at least 99.99%, at least 99.995%, or preferably at least 99.999% of the at least one Gram-negative bacterial species as described herein for any aspect of the invention. In this embodiment, the at least one Gram-negative bacterial species comprises a starting population of cells (i.e., the number of cells before treatment begins) of at least 1.0 x 105cells.

[0158] In one embodiment killing of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, at least 99.99%, at least 99.995%, or preferably at least 99.999% of the Gram-negative bacterial species occurs in less than about 48 hours, preferably less than about 24 hours, preferably less than about 12 hours, preferably less than about 6 hours, preferably less than about 4 hours after the Gram-negative bacterial species is contacted. Preferably killing occurs in less than 4 hours.

[0159] In one embodiment killing occurs in less than about 3 hours, preferably less than about 2 hours, less than about 1 hour, less than about 45 min, preferably less than about 30 min. In one embodiment killing occurs in less than 3 hours, preferably less than 2 hours, lh, 45m, preferably less than 30 min.

[0160] In one embodiment the composition is a cosmetic composition comprising an isolated polypeptide as described herein and a cosmetically acceptable carrier, diluent or excipient.

[0161] In one embodiment the cosmetic composition consists essentially of an isolated polypeptide as described herein and a cosmetically acceptable carrier, diluent or excipient. In one embodiment the cosmetic composition consists of an isolated polypeptide as described herein and a cosmetically acceptable carrier, diluent or excipient.

[0162] A composition as described herein may be formulated as a solid, liquid, paste, gel, emulsion, micro or nano emulsion, cream, ointment, lotion, liniment, solution, suspension, stick, block, pill, lozenge, powder, slurry, mist or vapour for use to inhibit the growth and / or proliferation of at least one Gramnegative bacterial species, or to treat a Gram-negative bacterial infection, disease and / or condition as described herein. A composition as described herein may also be immobilized on plastic or other types of films and / or encapsulated (including micro or nano encapsulated) for use to inhibit the growth and / or proliferation of at least one Gram-negative bacterial species, or to treat a Gramnegative bacterial infection, disease and / or condition as described herein.

[0163] In some embodiments, a composition as described herein may also contain other additives such as stabilising agents, preservatives, solubilizers, colouring agents, chelating agents, gel forming agents, ointment bases, pH-regulators, anti-oxidants, perfumes and skin protective agents, but not limited thereto. If the composition is in the form of a shampoo or soap, the composition may further comprise foaming agents, pearling agents and / or conditioners. Typical preservatives that may be used include the parabens, formaldehyde, Kathan CG, Bronidox, Bronopol, p-chlorom- cresol, chlorhexidine, benzalkonium chloride, etc.

[0164] In one embodiment the composition, pharmaceutical or cosmetic composition comprises acceptable carriers, particularly pharmaceutically acceptable or cosmetically acceptable carriers, proteins, small peptides, salts, excipients, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and / or other carriers. Such compositions and formulations can be used as described herein.

[0165] An acceptable carrier, particularly a pharmaceutically acceptable or cosmetically acceptable carrier may be liquid or solid and is selected in view of a planned manner of use, application and / or administration. In some embodiments, a pharmaceutically or cosmetically acceptable carrier may be selected to provide for the desired bulk, consistency, or other desirable pharmaceutical or cosmetic property that is to be used or delivered in a particular context as described herein.

[0166] In some embodiments a pharmaceutically or cosmetically acceptable carrier may include binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone (PVP) or hydroxypropyl methylcellulose, and the like, fillers such as lactose or other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate, etc.).

[0167] An isolated polypeptide as described herein may be formulated as a composition, particularly a pharmaceutical or cosmetic composition, by determining an appropriate mode of use, application and / or administration of the composition with reference to the literature and as described herein, and then formulating the composition for such mode. By way of non-limiting example, a formulation of the composition as a pharmaceutical composition for topical application would be preferred for inhibiting the growth and / or proliferation of certain Gram-negative bacteria, or for the treatment and prevention of certain Gram-negative bacterial infections, diseases and / or conditions of the skin or mucosa that are caused by and / or associated with at least one Gram-negative bacterial species. In another non-limiting embodiment, a formulation of the composition as a pharmaceutical composition for systemic application would be preferred for the treatment of systemic or localized internal bacterial infections, diseases and / or conditions of the skin or mucosa that are caused by and / or associated with at least one Gram-negative bacterial species.

[0168] In one embodiment an isolated polypeptide or pharmaceutical composition as described herein is formulated for administration, or is in a form for administration, to a subject in need thereof. In one embodiment administration is selected from the group consisting of is topical, intranasal, epidermal, transdermal, oral or parenteral. In one embodiment parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration. In one embodiment administration is by aerosol delivery.

[0169] In one embodiment the isolated polypeptide or pharmaceutical composition is formulated for, or is in a form for, parenteral administration in any appropriate solution, including sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

[0170] In one embodiment the isolated polypeptide or pharmaceutical composition is formulated for, or is in the form of, an injection. In one embodiment, injection is into or near the infected area. In one embodiment the infected area is the ear, eye, nose, throat or mouth.

[0171] In one embodiment the isolated polypeptide or pharmaceutical composition is formulated for, or is in a form for, oral administration in powders or granules, aqueous or non-aqueous suspensions or solutions, sprays, capsules, gels, pills, lozenges, emulsions, nano emulsions or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0172] In one embodiment the isolated polypeptide, pharmaceutical composition or cosmetic composition is formulated for, or is in a form for topical, aerosol, or direct administration in transdermal patches, subdermal implants, ointments, lotions, creams, gels, drops, pastes, suppositories, sprays, liquids, liposomes, emulsions, nano emulsions and powders. In such embodiments, conventional pharmaceutical and cosmetic carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0173] In one embodiment, the direct administration is direct application or local application. In one embodiment direct or local application comprises application of the antibacterial combination in combination with a delivery reagent or additional anti-microbial agent. In one embodiment the additional anti-microbial agent is an endolysin.

[0174] In one embodiment the isolated polypeptide or pharmaceutical composition is formulated for injection.

[0175] In one embodiment, injection is into or near the infected area. In one embodiment the infected area is the ear, eye, nose, throat or mouth. The appropriate mode of administration of an isolated polypeptide or composition containing such as described herein can be determined with reference to the literature and as described herein. By way of non-limiting example, a systemic application would be preferred for the treatment and prevention of certain microbial infections whereas a local application would be preferred for the treatment of others but not limited thereto.

[0176] In some embodiments, an isolated polypeptide or pharmaceutical composition as described herein may be formulated according to conventional pharmaceutical practice and may be: Semisolid formulations: Gels, pastes, mixtures. Liquid formulations: Solutions, suspensions, drenches, emulsions. As indicated, an isolated polypeptide or pharmaceutical composition as described herein may comprise a polypeptide comprising, consisting or consisting essentially of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 or a functional analogue or derivative thereof as described herein. In some embodiments, such a composition may also comprise an additional active agent. In some embodiments the additional active agent is an endolysin.

[0177] In some embodiments, a composition as described herein can be formulated as, or provided in the form of, a topical composition. A number of different types of topical compositions, including but not limited to topical compositions that are pharmaceutical compositions, can be prepared including peroral, parenteral, intravenous, vaginal, or rectal compositions (but not limited thereto) as described herein, and by following the guidelines for topical application, for example, according to conventional formulation practice, see, e.g., "Remington's Pharmaceutical Sciences" and "Encyclopaedia of Pharmaceutical Technology". Cosmetic, hair care and skin care compositions may also be prepared as topical compositions.

[0178] In one embodiment the composition is in the form of or is formulated as a topical composition. In one embodiment the topical composition is also a pharmaceutical or cosmetic composition as described herein. In one embodiment the antibacterial combination or composition is in the form for, or is formulated for, topical administration. In one embodiment topical administration is to an object or part thereof, preferably to a surface of the object, or a part thereof.

[0179] In one embodiment topical administration is to an animal or part of an animal, preferably a mammal, preferably a human. In one embodiment topical administration to an animal comprises administration to a wound, a burn, an ulcer, ulcus curis, acne, gonorrhoea (including urethritis, endocervicitis and proctitis), gas gangrene, scarlatina, erysipelas, sycosis barbae, folliculitis, impetigo contagiosa, or impetigo bullosa.

[0180] In some embodiments, topical administration is onto or close to an effected area of the body. In some embodiments, topical administration is onto an exterior part of the body. In one embodiment the exterior part of the body is the hair or skin or a part thereof. Topical administration may be by simple application of the composition such as by smearing a creme, ointment, lotion or gel comprising the antibacterial combination onto or around an area to be treated, or from which at least one Gram-negative bacterial species is to be inhibited or killed, or using a soap, detergent, disinfectant or shampoo for the same purpose. Alternatively, it may involve the use of an applicator or device suitable for enhancing the establishment of contact between the combination or composition and the substrate to which it is applied such as by the use of occlusive dressings or plasters comprising the composition or by way of a brush to apply a soap or shampoo. By way of non-limiting example, a composition of the invention may be impregnated or distributed onto pads, plasters, strips, gauze, sponge materials or cotton or wool pieces. In some embodiments, topical administration comprises spraying or misting an area to be treated or from which at least one Gramnegative bacterial species is to be inhibited or killed.

[0181] In some embodiments, the topical composition comprises or consists essentially of about 0.0001-20%, preferably 0.0001-20%, by weight (w / w) of an isolated polypeptide as described herein on the total weight of each component in the composition. By way of non-limiting example, the topical composition comprises a total of about 0.0001-15% w / w, preferably 0.0001-10% w / w, 0.0001-8% w / w, 0.0001-6% w / w, 0.0001-5% w / w, 0.0001-4% w / w, 0.0001-3% w / w, 0.0001-2% w / w, preferably 0.0001-1% w / w of the isolated polypeptide.

[0182] In some embodiments the topical composition is applied from once to 10 times daily. In some embodiments the topical composition is applied at least once, preferably at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times preferably at least ten times daily. The number of applications may be determined by the skilled person based on the disclosure provided herein and common general knowledge. The number of applications will include consideration of the extent to which the growth and / or proliferation of bacteria is to be inhibited in the target area and / or the type, severity and localisation of the bacterial infection, disease and / or condition being treated.

[0183] For topical pharmaceutical applications, a composition ad described may be formulated in accordance with conventional pharmaceutical practice with pharmaceutical excipients conventionally used for topical applications. The nature of the vehicle employed in the preparation of any particular composition will depend on the method intended for administration of that composition.

[0184] Vehicles other than water can be used in topical compositions and can include solids or liquids such as emollients, solvents, humectants, thickeners, and powders. A skilled worker will appreciate that such vehicles may be used as appropriately in other compositions described herein that comprise the isolated polypeptide as described herein including pharmaceutical and cosmetic compositions, as well as disinfectant compositions. Examples of each of these types of vehicles, which can be used singly or as mixtures of one or more vehicles, are as follows: Emollients, such as stearyl alcohol, glyceryl, monoricinoleate, glyceryl monostearate, propane-1, 2-diol, butane-l,3-diol, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di methyl polysiloxane, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate;

[0185] Solvents, such as water, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulfoxide, tetra hydrofuran, vegetable and animal oils, glycerol, ethanol, propanol, propylene glycol, and other glycols or alcohols, fixed oils; humectants or moistening agents, such as glycerin, sorbitol, sodium 2- pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin; powders, such as chalk, talc, kaolin, starch, and derivatives thereof, gums, colloidal silicon dioxide, sodium polyacrylate, chemically modified magnesium aluminium silicate, hydrated aluminium silicate, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate; gelling or swelling agents, such as pectin, gelatin and derivatives thereof, cellulose derivatives such as methyl cellulose, carboxymethyl cellulose or oxidised cellulose, cellulose gum, guar gum, acacia gum, karaya gum, tragacanth gum, bentonite, agar, alginates, carbomer, gelatine, bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum, hectorite, ispaghula husk, xanthan gum; polymers, such as polylactic acid or polyglycolic acid polymers or copolymers thereof, paraffin, polyethylene, polyethylene oxide, polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone; surfactants, such as non-ionic surfactants, e.g. glycol and glycerol esters, macrogol ethers and esters, sugar ethers and esters, such as sorbitan esters, ionic surfactants, such as amine soaps, metallic soaps, sulfated fatty alcohols, alkyl ether sulfates, sulfated oils, and ampholytic surfactants and lecitins; buffering agents, such as sodium, potassium, aluminium, magnesium or calcium salts (such as the chloride, carbonate, bicarbonate, citrate, gluconate, lactate, acetate, gluceptate or tartrate).

[0186] For topical applications, the pH of a composition as described herein may be about 3 to about 9, preferably about 4 to about 8, preferably about 5, about 6 or about 7. In some embodiments the pH of a composition as described herein is between 3 and 9, preferably between, 4 and 8, between 5 and 8, between 6 and 8, preferably 7. Depending on the intended use of the composition, buffering agents as described herein and as known in the art may be used obtain the pH desired.

[0187] In some embodiments, an isolated polypeptide or composition as described herein, preferably a cosmetic composition as described herein, is formulated for, or is in the form of a hair care product or skin care lotion. In some embodiments, an isolated polypeptide or composition as described herein is formulated as a soap, detergent, body wash, shampoo, lotion, ointment, toothpaste or foam spray. In one embodiment an isolated polypeptide or composition as described herein is formulated as or is in the form of a coating.

[0188] Many diseases arise from a primary infection with any bacterium but are caused by the secondary spread of the infectious agent from a primary infection site. Accordingly, the inventors believe that an isolated polypeptide or composition comprising such as described herein will be useful for inhibiting the growth and / or proliferation of bacteria, particularly Gram-negative bacteria, including prophylactically, and for treating bacterial infections, diseases and / or conditions in a subject in need thereof. The inventors also believe that an isolated polypeptide or composition as described herein is useful for the manufacture of a medicament for the prophylaxis and / or treatment of bacterial infections, diseases and / or conditions, particularly Gram-negative bacterial infections, diseases or conditions as described herein.

[0189] Specifically contemplated as embodiments of any of the above compositions is the addition of a further anti-bacterial agent.

[0190] In some embodiments the combination of the isolated polypeptide and the further antibacterial agent in the composition provides a synergistic combination. In some embodiments the composition comprising the isolated polypeptide and the further antibacterial agent is a synergistic composition.

[0191] In some embodiments the further antibacterial agent is an endolysin. In one embodiment the endolysin is selected from the group consisting of Stel, LysF8 and LysFl, preferably the further antibacterial agent is LysFl.

[0192] In another aspect the invention relates to a method of making an antibacterial polypeptide (AP), the method comprising expressing a polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 in an isolated host cell.

[0193] In some embodiments, the method further comprises culturing the host cell and optionally purifying the AP from the host cell and / or culture to obtain an isolated polypeptide.

[0194] In some embodiments the method further comprises a step, after obtaining the isolated polypeptide, of modifying the isolated polypeptide to remove any naturally occurring post translational modifications.

[0195] In some embodiments the method further comprises a step, after obtaining the isolated polypeptide, of modifying the isolated polypeptide to include at least one non-naturally occurring post translational modification. In one embodiment the polypeptide or functional variant or derivative thereof comprises at least 96%, 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13.

[0196] In one embodiment the polypeptide or functional variant or derivative thereof is a hydrolase, preferably a murein hydrolase or peptidoglycan hydrolase.

[0197] In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of an amino acid sequence having at least 96%, 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 1. In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of SEQ ID NO: 1.

[0198] In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of an amino acid sequence having at least 96%, 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 4. In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of SEQ ID NO: 4.

[0199] In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of an amino acid sequence having at least 96%, 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 7. In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of SEQ ID NO: 7.

[0200] In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of an amino acid sequence having at least 96%, 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 10. In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of SEQ ID NO: 10.

[0201] In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of an amino acid sequence having at least 96%, 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 13. In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of SEQ ID NO: 13.

[0202] In one embodiment the method comprises an initial step of transforming the isolated host cell with an isolated polynucleotide encoding the polypeptide or a functional variant or derivative thereof as described herein.

[0203] In one embodiment expressing the polypeptide or functional variant or derivative thereof comprises expressing a polynucleotide comprising, consisting essentially of or consisting of a nucleic acid sequence having at least 70% nucleic acid sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 15. In one embodiment the polynucleotide or functional variant or derivative thereof comprises, consists essentially of or consists of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% nucleic acid sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 15.

[0204] Specifically contemplated as further embodiments are all of the embodiments in the previous aspects of the invention that relate to each of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 15 separately, as well as to polynucleotide constructs or vectors comprising each of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 15 separately as described herein.

[0205] In another aspect the invention relates to a method of making an antibacterial polypeptide (AP), the method comprising chemically synthesizing a polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13.

[0206] In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of an amino acid sequence having at least 96%, 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 4. In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of SEQ ID NO: 4.

[0207] In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of an amino acid sequence having at least 96%, 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 7. In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of SEQ ID NO: 7.

[0208] In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of an amino acid sequence having at least 96%, 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 10. In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of SEQ ID NO: 10.

[0209] In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of an amino acid sequence having at least 96%, 97%, 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 13. In one embodiment the polypeptide or functional variant or derivative thereof comprises, consists essentially or consists of SEQ ID NO: 13.

[0210] In one embodiment the polypeptide or functional variant or derivative thereof is chemically synthesised by solid phase peptide synthesis (SPPS). In one embodiment the chemically synthesised polypeptide or functional variant or derivative thereof is not phosphorylated, acetylated, methylated, succinylated, glutathionylated, carbonylated, oxidated or deaminated. In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof has hydrolase activity, preferably murein hydrolase or peptidoglycan hydrolase activity, and comprises at least one AA alteration, preferably at least one conservative AA alteration. In one embodiment the alteration is a substitution, addition, insertion or deletion. In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof has hydrolase activity, preferably murein hydrolase or peptidoglycan hydrolase activity, and comprises 20, preferably 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, preferably 1 AA alterations, preferably conservative AA alterations. In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof has hydrolase activity, preferably murein hydrolase or peptidoglycan hydrolase activity, and comprises 5, 4, 3, 2 or 1 AA alterations, preferably conservative AA alterations.

[0211] In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof has hydrolase activity, preferably murein hydrolase or peptidoglycan hydrolase activity, and comprises at least one non-naturally occurring AA. In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof comprises at least one modified AA residue, preferably an artificial chemical analogue of a corresponding naturally occurring AA. In one embodiment the chemically synthesized polypeptide or functional variant or derivative thereof comprises at least one non-naturally occurring AA residue. In one embodiment the at least one non-naturally occurring AA residue is a D-amino acid residue. In one embodiment the at least one non-naturally occurring AA residue comprises a modified side chain.

[0212] In some embodiments, the chemically synthesized polypeptide or functional variant or derivative thereof is bacteriostatic or bactericidal, preferably bactericidal.

[0213] In one embodiment the polypeptide or functional variant or derivative thereof is chemically synthesized with at least one non-naturally occurring amino acid residue or other non-naturally occurring feature.

[0214] In another aspect the invention relates to a method of inhibiting the growth and / or proliferation of at least one Gram-negative bacterial species and / or of killing at least one Gram-negative bacterial species comprising contacting the Gram-negative bacterial species with an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13.

[0215] In another aspect the invention relates to a method of inhibiting the growth and / or proliferation of at least one Gram-negative bacterial species and / or of killing at least one Gram-negative bacterial species comprising contacting the Gram-negative bacterial species with a composition comprising an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 and a carrier, diluent or excipient. Specifically contemplated as embodiments of the methods of inhibiting aspects above are the following embodiments. References to polypeptides in the following embodiments are understood to also encompass functional variants and derivatives thereof.

[0216] In one embodiment the at least one Gram-negative bacterial species is a species or strain of Enterobacterales, Pasteurellales or Neisseriales.

[0217] In one embodiment the strain of Enterobacterales, Pasteurellales or Neisseriales is a drug-resistant strain.

[0218] In one embodiment the species or strain of Enterobacterales are chosen from the genera Escherichia, preferably E. coir, Salmonella, preferably S. enterica, S. enteritidis, S. infantis, S. dubiin, S. typhimurium, S. typhi, S. paratyphi, S. schottmulleri, or S. choieraesuiff, Citrobacter, preferably Citrobacter gillenii, C. amaionaticus, C. koseri, or C. freundir, Serratia, preferably S. marscences or S.iiquifaciens,- Shigella, preferably S sonnei, S fiexneri, S dysenteriae or S boydii, Klebsiella, preferably K pneumoniae, K oxytoca, K. ozaenae a \ K. rhinoscleromatiff, Enterobacter, preferably E. bugandensis, E. cloacae, E. huaxiensis, E. chuandaensiff, Cronobacter, preferably C. sakazakii and C. malonaticus ; Acinetobacter, preferably , baumannir, Yersinia, preferably Y. enteroiitica, Y. pseudotuberculosis or Y. pestis; Morganeiia, preferably M. morganii; Proteus, preferably P. vulgaris, P. mirabiiiff, and Pseudomonas, preferably P. aeruginosa,- or from the family Pasteurellales, genera Haemophilus, preferably Haemophilus influenzae, or from the family Neisseriales, genera

[0219] Neisseria, preferably Neisseria gonorrhoeae.

[0220] Preferably the species of Enterobacterales are species or strains of Escherichia, preferably E. coir, Salmonella, preferably S enterica, S enteritidis, S infantis, S dublin, S typhimurium, S typhi, S paratyphi, S schottmulleri or S choieraesuiff, Citrobacter, preferably C. gillenii, C. amaionaticus, C. koseri or C. freundir, Shigella, preferably S sonnei, S fiexneri, S dysenteriae or S boydii; Klebsiella, preferably K pneumoniae K oxytoca, K ozaenae a \ K rhinoscleromatiff, Enterobacter, preferably E. bugandensis, E. cloacae, E. huaxiensis, E. chuandaensiff, Cronobacter, preferably C sakazakii and C maionaticuff, Acinetobacter, preferably A. baumannir, and Pseudomonas, preferably P. aeruginosa.

[0221] Preferably the species of Enterobacterales are species or strains of Escherichia, preferably E. coir, Salmonella, preferably S enterica, S enteritidis, S typhimurium, S typhi, S paratyphi, S schottmulleri or S choieraesuiff, Klebsiella, preferably K. pneumoniae, and Pseudomonas, preferably P. aeruginosa.

[0222] In one embodiment contacting comprises contacting an object or part thereof that comprises the at least one Gram-negative bacterial species. In one embodiment contacting comprises contacting a surface in and / or on the object or part thereof. In one embodiment contacting is for a sufficient time to allow the isolated polypeptide or composition to inhibit the growth and / or proliferation of the at least one Gram-negative bacterial species on and / or in the object.

[0223] In one embodiment sufficient time is at least 30 seconds, preferably at least 1 minute, preferably at least 5 min, preferably at least 10 min, preferably at least 20 min, preferably at least 30 min, preferably at least 40 min, preferably at least 1 hour, preferably at least 2 hours, preferably at least 3 hours, preferably at least 5 hours, preferably at least 12 hours.

[0224] In one embodiment sufficient time is about 30 seconds, preferably about 1 minute, preferably about 5 min, preferably about 10 min, preferably about 20 min, preferably about 30 min, preferably about 40 min, preferably about 1 hour, preferably about 2 hours, preferably about 3 hours, preferably about 5 hours, preferably about 12 hours.

[0225] In one embodiment contacting comprises directly or indirectly applying the isolated polypeptide or composition to the object or part thereof. In one embodiment applying is directly applying. In one embodiment applying is indirectly applying.

[0226] In one embodiment applying comprises applying the isolated polypeptide or composition to the object or part thereof at least two times. In one embodiment applying is applying as a coating or partial coating.

[0227] In some embodiments, applying comprises applying the isolated polypeptide or composition at least lx, or 2x, or 3x, or 4x, or 5x, or 6x, or 7x, or 8x, or 9x, preferably lOx, or more. In some embodiments applying is least lx per day (lx / d), at least 2> d, at least 3x / d, at least 4x / day, at least 5x / day, at least 6x / day, at least 7> day, at least 8x / day, at least 9x / day, at least lOx / day.

[0228] In one embodiment the object is an animal or part thereof. In one embodiment the animal is a mammal. In one embodiment the mammal is selected from the group consisting of canines, felines, bovines, ovines, cervines, caprines, porcines, lagomorphs, rodents, camelids and hominids.

[0229] In one embodiment the mammal is a human.

[0230] In one embodiment the mammal is a non-human mammal selected from the group consisting of cats, dogs, rats, stoats, ferrets, possums, guinea pigs, mice, hamsters, zebra, elephants, lions, tigers, cheetah, monkeys, apes, macaques, tarsiers, lemurs, giraffes, prairie dogs, meerkats, bears, otters, tapiers, cows, horses, pigs, sheep, goats, deer, minks, and hippopotami.

[0231] In one embodiment the animal is a bird selected from the group consisting of chickens, ducks, pheasants, pigeons, ostriches, turkeys and geese.

[0232] In one embodiment the part of the animal is the hair, skin or hide, preferably human, cow, deer, sheep or horsehair, skin or hide. In one embodiment, the isolated polypeptide or composition is formulated as part of, or as a coating, or is in the form of, a coating or a partial coating.

[0233] In one embodiment the polypeptide or composition is formulated as, or as part of, or is in a form of, a disinfectant, a detergent, a wash, a soap or a shampoo.

[0234] In one embodiment the object is in inanimate article, material or substance, or part thereof. In one embodiment the object is an object on which Gram-negative bacterial species are known or suspected of being present and / or growing.

[0235] In one embodiment the object is used in food processing, hygiene, medicine, dentistry or any other industry where contamination by Gram-negative bacterial species poses a health risk and / or is desired to be prevented and / or reduced. In some embodiments the object is selected from the group consisting of medical devices, surgical devices, surgical instruments, surgical implants, stents, catheters, dental devices, dental instruments dental prostheses, dental implants, contact lenses, bandages, wound dressings, and food processing equipment.

[0236] Specifically contemplated as embodiments of the above method of inhibiting aspects are all of the embodiments set out herein relating to the aspects of the invention directed to an isolated polypeptide and compositions as described herein. Specifically encompassed in these embodiments are the technical features and specific steps recited in these previous aspects including as directed to isolated polypeptides and isolated polynucleotides (including % nucleic acid and amino acid sequence identity), polynucleotide constructs, vectors, host cells and compositions.

[0237] In another aspect the invention relates to the use of of an isolated polypeptide or functional variant or derivative thereof as described herein and / or a composition as described herein for inhibiting the growth and / or proliferation of at least one Gram-negative bacterial species, and / or for killing at least one Gram-negative bacterial species.

[0238] Specifically contemplated as embodiments of this use aspect of the invention are all of the embodiments set in the previous aspects of the invention relating to isolated polypeptides and polynucleotides, polynucleotide constructs, vectors, compositions and methods of making and use. Specifically encompassed in these embodiments are the technical features and specific steps recited in these previous aspects including as directed to isolated polypeptides and polynucleotides (including amino acid and nucleic acid sequence identity), polynucleotide constructs, vectors, host cells and compositions.

[0239] In another aspect, the invention relates to a method of treating a Gram-negative bacterial infection, disease or condition comprising administering an isolated an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 to a subject in need thereof. In another aspect, the invention relates to a method of treating a Gram-negative bacterial infection, disease or condition comprising administering a composition comprising an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 to a subject in need thereof.

[0240] Specifically contemplated as embodiments of the methods of treating aspects above are the following embodiments. References to polypeptides in the following embodiments are understood to also encompass functional variants and derivatives thereof.

[0241] In one embodiment the bacterial infection, disease and / or condition is caused by or is associated with at least one Gram-negative bacterial species or strain.

[0242] In one embodiment the at least one Gram-negative bacterial species is a species or strain of Enterobacterales, Pasteurellales or Neisseriales.

[0243] In one embodiment the strain of Enterobacterales, , Pasteurellales or Neisseriales is a drug-resistant strain.

[0244] In one embodiment the species or strain of Enterobacterales are chosen from the genera Escherichia, preferably E. coir, Salmonella, preferably S. enterica, S. enteritidis, S. infantis, S. dubiin, S. typhimurium, S. typhi, S. paratyphi, S. schottmulleri, or S. choieraesuiff, Citrobacter, preferably Citrobacter gillenii, C. amaionaticus, C. koseri, or C. freundir, Serratia, preferably S. marscences or S.iiquifaciens,- Shigella, preferably S sonnei, S fiexneri, S dysenteriae or S boydii, Klebsiella, preferably K pneumoniae, K oxytoca, K. ozaenae a \ K. rhinoscleromatiff, Enterobacter, preferably E. bugandensis, E. cloacae, E. huaxiensis, E. chuandaensiff, Cronobacter, preferably C. sakazakii and C. malonaticus ; Acinetobacter, preferably , baumannir, Yersinia, preferably Y. enteroiitica, Y. pseudotuberculosis or Y. pestis; Morganeiia, preferably M. morganii; Proteus, preferably P. vulgaris, P. mirabiiiff, and Pseudomonas, preferably P. aeruginosa,- or from the family Pasteurellales, genera Haemophilus, preferably Haemophilus influenzae, or from the family Neisseriales, genera

[0245] Neisseria, preferably Neisseria gonorrhoeae.

[0246] Preferably the species of Enterobacterales are species or strains of Escherichia, preferably E. coir, Salmonella, preferably S enterica, S enteritidis, S infantis, S dublin, S typhimurium, S typhi, S paratyphi, S schottmulleri or S choieraesuiff, Citrobacter, preferably C. gillenii, C. amaionaticus, C. koseri or C. freundir, Shigella, preferably S sonnei, S fiexneri, S dysenteriae or S boydii; Klebsiella, preferably K pneumoniae K oxytoca, K ozaenae a \ K rhinoscleromatiff, Enterobacter, preferably E. bugandensis, E. cloacae, E. huaxiensis, E. chuandaensiff, Cronobacter, preferably C sakazakii and C maionaticuff, Acinetobacter, preferably A. baumannir, ; and Pseudomonas, preferably P. aeruginosa. Preferably the species of Enterobacterales are species or strains of Escherichia, preferably E. coir, Salmonella, preferably S. enterica, S. enteritidis, S. typhimurium, S. typhi, S. paratyphi, S. schottmuHeri or S. choleraesuis, Klebsiella, preferably K. pneumoniae, and Pseudomonas, preferably P. aeruginosa.

[0247] In one embodiment the bacterial infection is selected from the group consisting of nosocomial infections, eye infections, nose infections, ear infections, mouth infections, throat infections, lung infections caused by and / or associated with the at least one Gram-negative bacterial species.

[0248] In one embodiment the infection, disease or condition is selected from the group consisting of halitosis, sore throat, orbital cellulitis, conjunctivitis, otitis media, sinusitis, pneumonia, diphtheria, pertussis, epiglottitis, nasopharyngitis, bronchitis, tonsillitis, gastritis, typhus, gastroenteritis, pseudomembranous colitis, dysentery, enterocolitis, peritonitis, abscess, pertussis, cholera, pestis, cystitis, pneumonia, meningitis, and Crohn's disease.

[0249] In one embodiment, administration is local or systemic administration. In one embodiment, administration is topical, intranasal, epidermal, and transdermal, oral or parenteral. In one embodiment oral administration comprises aerosol delivery. In one embodiment oral delivery comprises application of a liquid, gel, creme, ointment, or slurry. In one embodiment oral delivery comprises delivery of a solid, preferably a powder.

[0250] In one embodiment, parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration.

[0251] In one embodiment, administration is transient administration. In one embodiment transient administration comprises administration of an isolated polypeptide or composition as described herein for a sufficient period of time to provide a treatment or achieve a therapeutic result without the presence of the antibacterial combination or composition being harmful or causing significant deleterious effects to the subject. Administration can be rapid (e.g., by injection) or can occur over a period of time (e.g., by slow infusion or administration of slow-release formulations).

[0252] Specifically contemplated as embodiments of this treating aspect of the invention are all of the embodiments set in the previous aspects of the invention relating to isolated polypeptides and polynucleotides, polynucleotide constructs, vectors, compositions and methods of making and use. Specifically encompassed in these embodiments are the technical features and specific steps recited in these previous aspects including as directed to isolated polypeptides and polynucleotides (including amino acid and nucleic acid sequence identity), polynucleotide constructs, vectors, host cells and compositions.

[0253] A particular and effective dosage regime according to a method of treating a bacterial infection, disease or condition as described herein will be dependent on severity of the infection, disease and / or condition to be treated and on the responsiveness of the treated subject to the course of treatment. An effective treatment may last from several hours to several days to several months, or until an acceptable therapeutic outcome is affected or assured or until an acceptable reduction of the infection is observed.

[0254] An optimal dosing schedule (s) may be calculated from drug accumulation as measured in the body of a treated subject. It is believed to be within the skill of persons in the art to be able to easily determine optimum and / or suitable dosages, dosage formulations and dosage regimes. Of course, the optimum dosages may vary depending on the relative potency of the isolated polypeptide or composition as described herein but will be estimable from an EC50s found to be effective in suitable cells in vitro and in an appropriate in vivo animal model. In general, dosage is from 0.0001 g to 1 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, but not limited thereto.

[0255] In another aspect the invention relates an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 for use in treating a Gram-negative bacterial infection, disease and / or condition.

[0256] In another aspect the invention relates a composition comprising an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 for use in treating a Gram-negative bacterial infection, disease and / or condition.

[0257] Specifically contemplated as embodiments of these composition for use aspects of the invention are all of the embodiments set in the previous aspects of the invention relating to isolated polypeptides and polynucleotides, polynucleotide constructs, vectors, compositions and methods of making and use. Specifically encompassed in these embodiments are the technical features and specific steps recited in these previous aspects including as directed to isolated polypeptides and polynucleotides (including amino acid and nucleic acid sequence identity), polynucleotide constructs, vectors, host cells and compositions.

[0258] In another aspect the invention relates to the use of an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 in the manufacture of a medicament for treating a Gram-negative bacterial infection, disease, and / or condition.

[0259] In another aspect the invention relates to the use of an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13 in the manufacture of a medicament for treating a Gram-negative bacterial infection, disease, and / or condition. Specifically contemplated as embodiments of these "use in the manufacture of a medicament" aspects of the invention are all of the embodiments set in the previous aspects of the invention relating to isolated polypeptides and polynucleotides, polynucleotide constructs, vectors, compositions and methods of making and use. Specifically encompassed in these embodiments are the technical features and specific steps recited in these previous aspects including as directed to isolated polypeptides and polynucleotides (including amino acid and nucleic acid sequence identity), polynucleotide constructs, vectors, host cells and compositions.

[0260] Specifically contemplated for the composition for use and use in the manufacture of a medicament aspects of the invention are the Gram-negative bacterial species or strains as set out in the embodiments within the previous aspects of the invention.

[0261] Additionally, in one embodiment the use or the medicament comprises an effective amount of the isolated polypeptide or functional variant or derivative thereof or of the composition. In one embodiment the effective amount is a therapeutically effective amount.

[0262] In another aspect the invention relates to the use of an isolated polypeptide as described herein to make a cosmetic composition.

[0263] Specifically contemplated as embodiments of use aspect of the invention are all of the embodiments set in the previous aspects of the invention relating to isolated polypeptides and polynucleotides, polynucleotide constructs, vectors, compositions and methods of making and use. Specifically encompassed in these embodiments are the technical features and specific steps recited in these previous aspects including as directed to isolated polypeptides and polynucleotides (including amino acid and nucleic acid sequence identity), polynucleotide constructs, vectors, host cells and compositions.

[0264] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents; or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0265] The invention will now be illustrated in a non-limiting way by reference to the following examples. EXAMPLES

[0266] Example 1 - Materials and Methods

[0267] Expression and purification of Ecol45 and derivative polypeptides

[0268] The genes for Ecol45 and its derivative polypeptides (ECOalpha5_6, ECOalpha5_6_7, ECOalpha7, ECOalpha8) used in this study were each synthesized and cloned into the expression vector pET28(a)- TEV between Ndel and Xhol restriction sites by Genscript Biotech Corporation (Piscataway, NJ, USA). The genes of interest were expressed with an N-terminal polyhistidine expression tag and a TEV protease recognition sequence. Constructs were transformed into BL21(DE3) cells and cultured in LB medium, supplemented with kanamycin (30 pg mL-1), at 37 °C with shaking at 180 rpm. Protein expression was induced mid-exponential phase (ODeoonm ~0.6) with isopropyl 0-D-1- thioga lactopyranoside (final concentration of 1 mM) at 37 °C for 3.5 h. Cells were harvested at 8,000 x g for 15 min at 4 °C in a Sorvall™ Lynx 6000 Centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). The pellet was resuspended in lysis buffer (20 mM sodium phosphate, 150 mM sodium chloride, 20 mM imidazole, pH 6.5) and cells were lysed for 10 min by sonication at 70%, with a 0.5 s on / off cycle using an ultrasonic processer UP200S (Hielscher, Teltow, Germany) on ice. Cell debris was pelleted at 18,000 x g at 4 °C for 15 min.

[0269] Ecol45 and the derivative polypeptides were each purified by using a two-step procedure of immobilized metal affinity chromatography followed by size exclusion chromatography. The cell-free supernatant was loaded onto a 5 mL HisTrap FF column (GE Healthcare, Chicago, IL, USA) equilibrated with lysis buffer. The column was then washed with lysis buffer until a steady baseline UV (AbS280nm) reading was reached. Ecol45 or the derivative polypeptides were then eluted in the elution buffer (20 mM sodium phosphate, 150 mM sodium chloride, 500 mM imidazole, pH 6.5). Fractions containing Ecol45 or the derivative polypeptides were identified with SDS-PAGE analysis and then pooled. Pooled protein was spin-concentrated to 1 mL with a Vivaspin 6, 10,000 MWCO PES spin concentrator (Sartorius, Gottingen, Germany). The concentrated protein was centrifuged at 18,000 x g at 4 °C for 15 min and the supernatant was loaded onto a HiLoad 16 / 60 Superdex 200 column (GE Healthcare, Chicago, IL, USA) equilibrated with a size exclusion buffer (phosphate-buffered saline, pH 7.4).

[0270] Ecol45 is a phage derived protein which displays endolysin activity

[0271] We verified that the annotated endolysin gene from vB_EcoS-Rol45clw (Liao et al., 2019) displays the predicted endolysin activity by incubating purified protein with membrane permeabilized E. co / / cells. Sequence analysis identified Ecol45 as a member of the Lyz endolysin autolysin (cd00737), phage lysozyme (pfam00959) and glycoside hydrolase 24 (GH24) families, and therefore is expected to cleave the 0(1-4) glycosidic bond between / V-acetylmuramic acid and / V-acetylglucosamine in peptidoglycan. Following recombinant expression and purification, endolysin activity was determined using a turbidity reduction assay (Briers et al., 2007) The endolysin, denoted Ecol45, was recombinantly expressed and purified. For the functional assay, E. a? / / cells were treated with chloroform to permeabilize the outer membrane exposing the peptidoglycan, the substrate of endolysins (Schmelcher et al., 2012). The permeabilized cells were incubated with varying concentrations of Ecol45 and the optical density of the suspension was measured at 600 nm (ODeoonm). A decrease in absorbance was observed for all enzyme concentrations tested indicating that the peptidoglycan was degraded by Ecol45, causing cell lysis. There was a linear enzyme dose-activity relationship from 50 ng mL-1to 400 ng mL-1Ecol45 (Figure 2 A). These data confirm that Ecol45 is catalytically active and displays endolysin activity.

[0272] Turbidity reduction assay

[0273] The peptidoglycan degrading activity of Ecol45 was evaluated against Gram-positive Staphylococcus aureus ATCC 25923 and outer membrane-permeabilized Gram-negative bacteria E. co / / TOP10, S. typhimurium \ 2 and K pneumoniae ATCC 13883 by measuring reductions of ODeoonm. Permeabilizing was performed using the chloroform-Tris-HCI technique (Lavigne et al., 2004). Briefly, cells were grown at 37 °C until they reached an ODeoonm of 0.6. Cells were centrifugated at 4,000 x g at 4 °C for 15 min and resuspended in 50 mM Tris-HCI (pH 7.7) saturated with chloroform. Cells were incubated for 45 minutes then washed and resuspended in 10 mM sodium phosphate buffer to an ODeoonm of 1.0. Protein (30 pL), at various concentrations, was combined with bacterial cells (270 pL) and the change in ODeoonm was measured over time. Muralytic activity of Ecol45 was described in units, where 1 unit corresponds to the amount of protein resulting in an ODeoonm decrease in culture turbidity of 0.001 min-1(Briers et al., 2007a).

[0274] When we incubated Ecol45 with membrane permeabilized K. pneumoniae and S. typhimurium a reduction in the turbidity of the cell suspension was observed (Figure 2B). No endolysin activity was observed when Ecol45 was incubated with the Gram-positive bacteria S. aureus which has a different peptidoglycan chemotype (Figure 2B).

[0275] Antibacterial assay of Ecol45

[0276] Unexpectedly we found that endolysin Ecol45 is uniquely able to kill E. co / / cells without the addition of outer membrane permeabilizers. For endolysins to overcome the outer membrane of Gram-negative bacteria and subsequently cause cell lysis, often they must first be modified or used in combination with outer membrane permeabilizing agents (Briers & Lavigne, 2015a; Loessner, 2005). Previous studies on gram-negative outer membrane permeabilization have indicated that fusion of positive and amphipathic peptides that are not structured may be key in the destabilization of LPS (Briers & Lavigne, 2015). However, Ecol45 does not have a positive and amphipathic unstructured peptide connected to the enzyme and further that the C-terminal region (where in Arti lysins the peptide is attached) is well folded (unlike the work of Artilysi ns) and critical for the function of the enzyme. Therefore, to test the ability of Ecol45 to permeabilize the outer membrane of E. coii e measured the bactericidal activity of Ecol45 against E. coii cells. E. co / ZTOPlO, E. coh'P CC 25922, S. typhimurium T and K. pneumoniae ATCC 13883 were grown up in LB media to mid-exponential phase (~0.6 ODeoonm). Cells were then washed and resuspended in 10 mM HEPES-NaOH (pH 7.4) and then diluted 100-fold to a final density of ±106CFU mL-1. Cell culture was incubated with protein (14 pM final final), or PBS buffer for 30 minutes at 37 °C. Appropriate cell dilutions were then performed and plated on LB agar plates in triplicate. Plates were incubated at 37 °C overnight and then colonies were counted. Antibacterial activity was reported as the log reduction of cells (Logic (No / Ni), with No = initial number of cells counted when incubated with buffer and Ni = number of cells after treatment with endolysins Three biological repeats with technical triplicates were produced for all endolysin assays.

[0277] A reduction of live E. co / / cells was observed when incubated with Ecol45 (0.5 mg mL-1), with a 1.2 ± 0.1 log reduction in 30 minutes. In contrast, Ecol45 was not as effective against either K. pneumoniae or S. typhimurium cells giving less than 1 logic reduction. We reasoned that the higher antibacterial activity against E. co / / cells was not due to the substrate specificity of Ecol45 because Gram-negative bacteria such as E. coii, K. pneumoniae and S. typhimurium shave, the peptidoglycan chemotype Aly (Schleifer & Kandler, 1972).

[0278] Further testing of Ecol45 against carbapenamase resistant clinical E. co / / isolates (each with different sequence types and resistance genes), showed a significant cell killing ability, with log reductions of - 1.3 ± 0.4 logic, (95% cells killed) Figure 3. Furthermore, testing of carbapenamase resistant clinical isolates of K. pneumoniae (each with different sequence types and resistance genes) showed a significant cell killing ability, with log reductions of 0.5 ± 0.2 logic, (70% cells killed) Figure 3. In addition, testing of carbapenamase resistant clinical isolates of P. aeruginosa (each with different sequence types and resistance genes) showed a significant cell killing ability, with log reductions of 0.6 ± 0.3 logic, (80% cells killed) Figure 3.

[0279] Antibacterial assay of polypeptides derived from Ecol45

[0280] Despite the protein being well folded and without an unstructured peptide region (as in the Artilysins), we investigated whether any sequence within Ecol45 might possess antimicrobial function separate to the peptidoglycan degrading catalytic domain. Therefore, we created several polypeptides from the core of Ecol45 (Figure 4 A). The selected peptides were synthesized to study their AMP properties.

[0281] Bacterial isolates were grown up in LB media to mid-exponential phase (~0.6 ODeoonm). Cells were then washed and resuspended in 10 mM HEPES-NaOH (pH 7.4) and then diluted 100-fold to a final density of ±106CFU mL-1. Cell culture was incubated with the following peptides and proteins: ECO145alpha5_6_7 (13 pM), ECO145alpha7 (27 pM), ECO145alpha5_6 (27 pM), ECO145alpha8 (35 pM), endolysin, and LysFl (9 pM), or with PBS buffer for 30 minutes at 37 °C. Appropriate cell dilutions were then performed and plated on LB agar plates in triplicate. Plates were incubated at 37 °C overnight and then colonies were counted. Antibacterial activity was reported as the log reduction of cells (Logic (No / Ni), with No = initial number of cells counted when incubated with buffer and Ni = number of cells after treatment with endolysins Three biological repeats with technical triplicates were produced for all peptides assayed.

[0282] Unexpectedly, bactericidal assays conducted with peptide ECOalpha_5_6_7 showed significantly enhanced (>100 fold increased) killing with log reductions of 4.46 logic, (99.997% cells killed) of E. coH compared to the parental Ecol45 endolysin (2.1 logic reduction) (Figure 4 B). Peptide ECOalpha_7 also showed killing of E. coH with 0.66 logic reductions (78% cells killed) (Figure 4B).

[0283] We tested if there was any synergy in antibacterial activity between the polypeptides from Ecol45 and an intact endolysin (LysFl). Unexpectedly, there was a synergy between ECOalpha_5_6_7 and LysFl with >5 logic reductions (no live cells detected, >99.999% cell killed) in E. co / i. This compares to a 4.46 logic reduction with ECOalpha_5_6_7 alone (>50% increase). There was also a notable increase in the activity of ECOalpha_7 against E. co / i ihen introduced with LysFl, with 1.26 logic reductions (94% cells killed) compared to 0.66 logic reductions with ECOalpha_7 alone.

[0284] We next tested the activity of ECOalpha_5_6_7 on carbapenamase resistant clinical isolates of E. coH and P. aeruginosa. Again, ECOalpha_5_6_7 showed significantly increased activity on these isolates compared to Ecol45, with 3.8 ± 1.1 logic reductions for E. coH, compared to the parental Ecol45 endolysin (2.1 logic reduction) (Figure 4 C). For P. aeruginosa .4 logic ± 0.5 reductions were achieved, a significant increase compared to the parental Ecol45 endolysin (0.6 logic) (Figure 4 C).

[0285] Testing of the antimicrobial activity of the peptide ECO145alpha5_6_7 was also carried out using two additional species, Klebsiella aerogenes and Enterobacter cloacae, and to another class of antimicrobial resistance, extended-spectrum beta-lactamase producing clinical isolates of Klebsiella pneumoniae and Enterobacter cloacae. These experiments were performed identically to the bacteriocidal assay method described above, except that incubation time was increased to 150 minutes. Results are discussed below.

[0286] ECO145alpha5_6_7 has bactericidal activity agianst carbapenemase resistant clinical isolates of Klebsiella aerogenes (Figure 4D). We observed 1.9 logic ± 0.2 reductions for ECO145alpha5_6_7 treated cells when compared to the PBS control.

[0287] ECO145alpha5_6_7 has bactericidal activity against extended-spectrum beta-lactamase resistant clincial isolates of Klebsiella pneumoniae (Figure 4D). Reductions of 2.9 logic ± 0.5 were found for cells treated with ECO145alpha5_6_7 compared to PBS controls.

[0288] ECO145alpha5_6_7 has bactericidal activity against extended-spectrum beta-lactamase resistant clinical isolates of Enterobacter cloacae (Figure 4D). Compared to the PBS control, ECO145alpha5_6_7 was able to achieve reductions of 3.0 logic ± 0.3 of these bacteria. ECO145alpha5_6_7 has bactericidal activity against extended-spectrum beta-lactamase resistant clincial isolates of E. co / / (Figure 4D). We observed 3.5 logic ± 0.3 reductions for ECO145alpha5_6_7 treated cells compared to the PBS control.

[0289] Table 1 - Nucleic acid and Amino acid sequences

[0290] NB: plasmid nucleotide sequences are slightly different to the original bacteriophage sequences from the NCBI database due to the need for codon optimisation for protein expression.

Claims

What we claim is:

1. An isolated polypeptide comprising at least 95% amino acid sequence identity to SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 4, or SEQ ID NO: 13 or a functional variant or derivative thereof.

2. The isolated polypeptide of claim 1 that comprises SEQ ID NO: 7 or a functional variant or derivative thereof.

3. The isolated polypeptide of claim 1 that consists of SEQ ID NO: 7 or a functional variant thereof.

4. The isolated polypeptide of claim 1 that comprises SEQ ID NO: 10 or a functional variant or derivative thereof.

5. The isolated polypeptide of claim 1 that consists of SEQ ID NO: 10 or a functional variant or derivative thereof6. An isolated polynucleotide encoding an isolated polypeptide of any one of claims 1 to 5.

7. A composition comprising an isolated polypeptide as defined in any one of claims 1 to 6 or a polynucleotide as defined in claim 6 and a carrier, diluent or excipient, preferably wherein the composition is a bactericidal or bacteriostatic composition.

8. The composition of claim 7 that is a pharmaceutical composition.

9. A method of inhibiting the growth and / or proliferation of at least one Gram-negative bacterial species and / or of killing at least one Gram-negative bacterial species comprising contacting the Gram-negative bacterial species with an isolated polypeptide or functional variant or derivative thereof comprising at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 4, or SEQ ID NO: 13.

10. The method of claim 9 wherein the isolated polypeptide or functional variant or derivative thereof comprises SEQ ID NO: 1 or a functional variant or derivative thereof.

11. The method of claim 9 or 10 wherein the isolated polypeptide or functional variant or derivative thereof consists of SEQ ID NO: 1 or a functional variant thereof.

12. The method of claim 9 wherein the isolated polypeptide or functional variant or derivative thereof comprises SEQ ID NO: 7 or a functional variant or derivative thereof.

13. The method of claim 9 or 10 wherein the isolated polypeptide or functional variant or derivative thereof consists of SEQ ID NO: 7 or a functional variant thereof.

14. The method of claim 9 wherein the isolated polypeptide or functional variant or derivative thereof comprises SEQ ID NO: 10 or a functional variant or derivative thereof.

15. The method of claim 9 or 10 wherein the isolated polypeptide or functional variant or derivative thereof consists of SEQ ID NO: 10 or a functional variant thereof.

16. The method of any one of claims 9 to 15 wherein the at least one Gram-negative bacterial species is a species or strain of Enterobacterales.

17. The method of claim 16 wherein the species or strain of Enterobacterales are chosen from the genera Escherichia, preferably E. coir, Klebsiella, preferably K. pneumoniae, K. oxytoca, K ozaenae v Pseudomonas, preferably P. aeruginosa.

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