Patent Grant
9752136
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 2, 2015, is named 44445002006_SL.txt and is 28,051 bytes in size.
The present invention relates to novel polypeptides derived from endolysins from a bacteriophage of Clostridium perfringens and to nucleic acid molecules encoding the same, as well as to compositions thereof. The invention also provides uses of such polypeptides and nucleic acid molecules in the diagnosis and treatment of conditions and diseases associated with microbial cells such as Clostridium perfringens. In particular, the invention provides a polypeptide having endolysin activity derived from bacteriophage φCP51 of Clostridium perfringens and to uses thereof.
Clostridium perfringens, one of the most pathogenic species in the Clostridium genus, is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. C. perfringens is a rod-shaped, Gram-positive, anaerobic spore-forming bacterium with the ability to produce potent toxins. According to the pattern of toxin expression, C. perfringens strains are classified into five toxinotypes: A, B, C, D and E. The interest in this microorganism is increasing because it is responsible for food poisoning, gas gangrene, necrotic enteritis (NE) both in humans and animals, and non-food borne gastrointestinal infections. In fact, C. perfringens is considered the third leading cause of human food poisoning-related death in the UK and USA.
A range of different alternatives to the conventional antimicrobial growth promoter approach are being investigated to reduce the numbers of C. perfringens pathogens in the food chain and to improve poultry health. In addition to vaccination and the use of natural antimicrobials, there are a number of examples of using probiotic microbes or lvtic enzymes to control NE in poultry.
Despite such control strategies, C. perfringens-associated food poisoning is classed among the most common gastrointestinal diseases in industrialized countries. One of the most frequently occurring infections caused by C. perfringens type A is NE in poultry; in countries where the use of antimicrobial growth promoters has been abolished, the incidence of NE has increased and the productivity of the poultry industry has been reduced.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.
There exists a growing need for new treatments and approaches for the control of C. perfringens.
Bacteriophages and their endolysins present an opportunity to control C. perfringens selectively without disturbing the balance of the microbiota in the gastrointestinal (GI) tract. Bacteriophage therapy is a growing area of research and tests in poultry have demonstrated the potential of bacteriophages to control levels of C. perfringens, Salmonella and Campylobacter. However, the narrow host range of bacteriophages and the rapid development of resistance to them can limit their application as control agents. At the end of their life cycle, lytic bacteriophages release their progeny by lysis of the host cell, through two proteins encoded by the lysis cassette. First, small hydrophobic proteins, termed holins, disrupt the cell membrane to produce holes through which endolysins can then pass. The endolysins are cell wall hydrolases, which directly target bonds in the peptidoglycan. The result of their activity is the destruction of the cell and the release of newly produced virions.
With few exceptions, endolysins consist of two domains: the N-terminal domain usually contains the catalytic activity of the enzyme (e.g., muramidase, glucosaminidase, endopeptidase or amidase) which specifically cleaves one of the major bonds found in the peptidoglycan, while the C-terminal domain binds to a specific substrate found in the cell wall of the host bacterium. Properties such as the specificity for their host pathogen, leading to minimal disturbance of the normal flora, the low risk of bacterial resistance and the ability to kill colonizing pathogens on mucosal surfaces make endolysins ideal anti-infective agents.
Recent studies have demonstrated the efficiency of endolysins in killing or controlling pathogenic bacteria in animal models; they have also shown efficacy against biofilms, as disinfectants and as the basis of detection systems for bacteria. Several bacteriophage endolysins which produce lytic activity against C. perfringens cells have been identified. A number have also been identified by sequencing and genome mining, producing a rich resource of information for such enzymes.
To find new control strategies to treat C. perfringens infection, the inventors investigated the activity and delivery of a bacteriophage endolysin. They succeeded in identifying a new endolysin, designated CP51L, which shows similarity to an N-acetylmuramoyl-L-alanine amidase domain but not to other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp51l gene was cloned and expressed in Escherichia coli and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested (see Examples below). A probiotic strain of Lactobacillus johnsonii FI9785 was engineered to produce a system for delivery of the endolysin to the gastrointestinal tract. The integration of the nisRK two component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (PnisA), while the use of a signal peptide (SLPmod) successfully secreted the active endolysin to the surrounding media. The high specificity and activity of the endolysin demonstrates its utility as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.
Lactobacillus johnsonii FI9785 is a poultry-isolated strain which has been shown to act as a competitive exclusion agent against C. perfringens in chickens. As well as acting as probiotics in their own right, lactic acid bacteria have also shown promise as delivery systems for the secretion of biologically active interleukins or peptidoglycan hydrolases. The promoter of the lantibiotic nisin A (PnisA) is induced by nisin A via signal transduction using the two component regulatory system NisRK. This system has been exploited to develop gene expression systems in Lactococcus lactis, Leuconostoc lactis and Lactobacillus helveticus. Here, the inventors have expressed the endolysin CP51L, originating from a prophage of C. perfringens (see Examples, below), both in Escherichia coli for enzymatic analysis and in a probiotic strain of L. johnsonii which was engineered for delivery to the GI tract.
Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. Nothing herein is to be construed as a promise.
It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.
The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.
A first aspect of the invention provides an isolated polypeptide which may comprise the amino acid sequence of SEQ ID NO:1, or a fragment, variant, derivative or fusion thereof which is capable of binding specifically to and/or lysing cells of Clostridium perfringens.
The amino acid sequence depicted below is that of the wildtype (i.e., naturally occurring) endolysin (CP51L) of bacteriophage φCP51 of Clostridium perfringens.
In one embodiment, the polypeptide is not a naturally occurring lysin of a bacteriophage of Clostridium perfringens (other than CP51L). Thus, the first aspect of the invention encompasses isolated polypeptides which may comprise or consist of the amino acid sequence of SEQ ID NO: 1 and non-naturally occurring fragments, variants, derivatives or fusions thereof.
The term ‘amino acid’ as used herein includes the standard twenty genetically-encoded amino acids and their corresponding stereoisomers in the ‘D’ form (as compared to the natural 1′ form), omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (e.g., a,a-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatised amino acids (see below).
Thus, when an amino acid is being specifically enumerated, such as ‘alanine’ or ‘Ala’ or ‘A’, the term refers to both L-alanine and D-alanine unless explicitly stated otherwise. Other unconventional amino acids may also be suitable components for polypeptides of the present invention, as long as the desired functional property is retained by the polypeptide. For the peptides shown, each encoded amino acid residue, where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.
Preferably, the polypeptide, or fragment, variant, fusion or derivative thereof, may comprise or consist of L-amino acids.
By “isolated” Applicants mean that the polypeptide of the invention, specifically the wildtype endolysin of bacteriophage CD51, is provided in a form other than that in which it may be found naturally. Preferably, the polypeptide is provided free from intact bacteriophage.
In one embodiment, the polypeptide of the invention is the naturally occurring endolysin of bacteriophage CP51 [SEQ ID NO: 1], provided in an isolated form.
Other naturally occurring lysins of a bacteriophage of Clostridium perfringens known in the prior art are specifically not encompassed by the first aspect of the invention. In particular, the following lysins of bacteriophage of Clostridium perfringens are explicitly excluded from the scope of the first aspect of the invention:
(a) the lysin ply φ3626 (see Zimmer M, Vukov N, Scherer S, Loessner M J. 2002. The murein hydrolase of the bacteriophage (I)3626 dual lysis system is active against all tested Clostridium perfringens strains. Appl Environ Microbiol. 68:5311-7);
(b) the lysin plyCP26F (see Simmons M, Donovan D M, Siragusa G R, Seal B S. 2010. Recombinant expression of two bacteriophage proteins that lyse Clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal active domains. J Agric Food Chem. 58:10330-7);
(c) the lysin plyCP39O (see Simmons M, Donovan D M, Siragusa G R, Seal B S. 2010. Recombinant expression of two bacteriophage proteins that lyse Clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal active domains. J Agric Food Chem. 58:10330-7);
(d) the lysin Pms (see Nariya H, Miyata S, Tamai E, Sekiya H, Maki J, Okabe A. 2011. Identification and characterization of a putative endolysin encoded by episomal phage φSM101 of Clostridium perfringens. Appl Microbiol Biotechnol. 90:1973-9); and
(e) the lysin PlyCM (see Schmitz J E, Ossiprandi M C, Rumah K R, Fischetti V A. 2011. Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens. Appl Microbiol Biotechnol. 89:1783-95).
In one embodiment, the polypeptide of the first aspect of the invention may comprise the amino acid sequence of SEQ ID NO: 1. For example, the polypeptide may consist of the amino acid sequence of SEQ ID NO: 1.
However, the first aspect of the invention also extends to fragments, variants, derivatives and fusions of the amino acid sequence of SEQ ID NO:1 which are capable of binding specifically to and/or lysing cells of Clostridium perfringens.
By “capable of binding specifically to cells of Clostridium perfringens” Applicants mean that the polypeptide is capable of binding preferentially to cells of Clostridium perfringens. However, it will be appreciated that such polypeptides may also bind preferentially to one or more additional types of cell.
By “capable of lysing cells of Clostridium perfringens” Applicants mean that the polypeptide, or fragment, variant, derivative or fusion, retains (at least in part) the ability of the wildtype endolysin of bacteriophage CP51 to lyse bacterial cells (e.g., cells of Clostridium perfringens). However, it will be appreciated that such polypeptides may also preferentially lyse one or more additional types of cell. It will be appreciated that such lytic activity should be cell-specific rather than a non-specific cytotoxic activity on all cell types. Such cell lysis activity may be determined using methods well known in the art, such as those described in detail in the Examples below (see also Loessner M J. 2005. Bacteriophage endolysins—current state of research and applications. Curr Opin Microbiol. 8:480-7, the disclosures of which are incorporated herein by reference). Preferably, the ability of polypeptides to lyse cells of Clostridium perfringens is determined using fresh cells.
Other types of cell to which the polypeptides of the invention may also bind and/or lyse may be selected from the group consisting of cells of Bacillus sp. and other Clostridium sp. For example, where the other type of cell is a Bacillus sp., the Bacillus sp. may be selected from the group consisting of Bacillus cereus (e.g., B. cereus NCIMB 11796) and B. subtilis (e.g., B. subtilis ATCC 6633). Alternatively, where the other type of cell is another Clostridium sp., the Clostridium sp. may be selected from the group consisting of C. acetobutylicum (e.g., C. acetobutylicum BL75141), C. bifermentans (e.g., C. bifermentans NCTC 13019) and C. beijerinckii (e.g., C. beijerinckii NCIMB 8052).
However, in one embodiment the polypeptide exclusively binds to and/or lyses cells of Clostridium sp. (e.g., C. perfringens). Preferably, such cell binding activity may be determined using methods well known in the art, in particular, those described in the Examples, below.
In a further embodiment, the polypeptide is substantially incapable of binding to and/or lysing cells selected from of the group consisting of Anaerococcus hydrogenalis DSMZ 7454, Bacillus amyloliquefaciens 0880, Bifidobacterium adolescentis DSMZ 20083, Bifidobacterium angulatum DSMZ 20098, Bifidobacterium bifidum DSMZ 20082, Bifidobacterium longum DSMZ 20219, Bifidobacterium pseudocatenulatum DSMZ 20438, Clostridium cellobioparum DSMZ 1351, Clostridium coccoides NCTC 11035, Clostridium colinum DSMZ 6011, Clostridium difficile NCTC 11204, Clostridium innocuum DSMZ 1286, Clostridium leptum DSMZ 753, Clostridium nexile DSMZ 1787, Clostridium ramosum DSMZ 1402, Clostridium sordellii NCTC 13356, Clostridium sporogenes ATCC 7886, Clostridium tyrobutyricum NCIMB 9582, Enterococcus faecalis FI10734, Enterococcus hirae FI10477, Eubacterium barkeri DSMZ 1223, Lactobacillus casei FI10736, Lactobacillus johnsonii FI9785, Lactobacillus plantarum FI08595, Lactobacillus rhamnosus FI10737, Lactococcus lactis MG1363, Leuconostoc mesenteroides subsp. Mesenteroides ATCC 8293, Listeria innocua NCTC 11288, Listeria ivanovii NCTC 11007, Micrococcus luteus FI10640, Pediococcus pentosaceus FI10642, Pediococcus acidilactici FI10738, Salmonella enterica serovar Typhimurium FI10739, Salmonella enterica serovar Enteritidis FI10113 and Staphylococcus aureus FI10139. Preferably, the polypeptide is incapable of lysing any of the cells in the preceding list.
In a preferred embodiment, the ability of polypeptides to lyse cells of Clostridium perfringens is determined using cells of strain NCTC 3110 and/or NCTC 8238.
It will be appreciated by persons skilled in the art that the polypeptide, or fragment, variant, derivative or fusion, need not retain all of the ability of the wildtype endolysin of bacteriophage CP51 to lyse bacterial cells. Rather, it is simply necessary for said polypeptide, fragment, variant, derivative or fusion to retain at least 10% of the ability of the wildtype endolysin of bacteriophage CP51 to lyse bacterial cells. Preferably, however, the polypeptide, fragment, variant, derivative or fusion exhibits at least 20%, for example at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more, of the ability of the wildtype endolysin of bacteriophage CP51 to lyse bacterial cells.
In one embodiment of the first aspect of the invention, the polypeptide may comprise or consist of a fragment of the amino acid sequence of SEQ ID NO: 1, which is capable of lysing cells of Clostridium perfringens.
The fragment may comprise or consist of at least 50 contiguous amino acids of SEQ ID NO: 1, for example at least 60, 70, 80, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265, 275, 285, 295, 300, 305, 310, 315, 320, 325, 335, 340, 345, 355, 360, 365, 370, 375 or 376 contiguous amino acids of SEQ ID NO: 1.
As noted above, it is well established that many bacteriophage endolysins consist of two distinct domains (for example, see Sheehan et al., 1996, FEMS Microbiology Letters 140:23-28, the disclosures of which are incorporated herein by reference). One is a catalytic domain that is responsible for cell wall degradation and these are known to exist in several different forms. The other domain is a cell wall binding domain that recognises a cell surface motif and permits attachment of the endolysin to that target cell. The precise pattern recognition involved in the latter is what provides the specificity.
The enzymatic domain can be identified by its amino acid homology to other similar regions of lytic enzymes that share the same type of lytic activity. In the case of the endolysin of bacteriophage CP51, Blastp analysis of CP51L identified 2 regions with similarity to conserved domains separated by a central section (see
The N-terminal region of the endolysin is most similar to domains associated with lysis, including the peptidoglycan recognition protein (PGRP, cd06583), lysozyme (PHA00447) and amidase_2 (N-acetylmuramoyl-L-alanine amidase, pfam01510). This is confirmed by alignment analysis of SEQ ID NO: 1 with known enzymatic domains, for example using the NCBI CDD search tool; see Marchler-Bauer & Bryant, 2004, Nuc. Acids Res. 32[W]:327-331, the disclosures of which are incorporated herein by reference).
The C-terminal region has similarity to a bacterial SH3 domain (SH3_3, pfam08239) towards the end of the sequence. SH3 (src Homology-3) domains are small protein modules containing approximately 50 amino acid residues (see Pawson T & Schlessingert J, 1993, SH2 and SH3 domains. Cum Biol. 3(7):434-42; Mayer B J, 2001, SH3 domains: complexity in moderation. J. Cell Sci. 114(7):1253-63; Gu J et al., 2011, LysGH15B, the SH3b domain of staphylococcal phage endolysin LysGH15, retains high affinity to staphylococci, Curr Microbiol. 63(6):538-42; Xu Q et al., 2009, Structural basis of murein peptide specificity of a gamma-D-glutamyl-I-diamino acid endopeptidase, Structure 17(2):303-13;). They are found in a great variety of intracellular or membrane-associated proteins (see Musacchio A, Gibson T, Lehto V P, & Saraste M, 1992, SH3—an abundant protein domain in search of a function. FEBS Lett. 307(1):55-61 Mayer B J & Baltimore D, 1993, Signalling through SH2 and SH3 domains. Trends Cell. Biol. 3(1):8-13; Pawson T, 1995, Protein modules and signalling networks. Nature 373(6515):573-80) for example, in a variety of proteins with enzymatic activity, in adaptor proteins that lack catalytic sequences and in cytoskeletal proteins, such as fodrin and yeast actin binding protein ABP-1.
The SH3 domain has a characteristic fold which consists of five or six beta-strands arranged as two tightly packed anti-parallel beta sheets. The linker regions may contain short helices (see Kuriyan J. & Cowburn D, 1997, Modular peptide recognition domains in eukaryotic signalling. Annu Rev Biophys Biomol Struct. 26:259-88). The surface of the SH3-domain bears a flat, hydrophobic ligand-binding pocket which consists of three shallow grooves defined by conservative aromatic residues in which the ligand adopts an extended left-handed helical arrangement. The ligand binds with low affinity but this may be enhanced by multiple interactions. The region bound by the SH3 domain is in all cases proline-rich and contains MOO as a core-conserved binding motif. The function of the SH3 domain is not well understood but they may mediate many diverse processes such as increasing local concentration of proteins, altering their subcellular location and mediating the assembly of large multiprotein complexes (Morton & Campbell, 1994, Curr. Biol. 4(7):615-7).
A homologue of the SH3 domain has been found in a number of different bacterial proteins including glycyl-glycine endopeptidase, bacteriocin and some hypothetical proteins.
In one embodiment, the polypeptide of the invention may comprise an enzymatic domain contained within amino acids 18 to 134 of SEQ ID NO:1. Thus, the fragment which may comprise the enzymatic domain may consist of the sequence of SEQ ID NO: 1 starting from any of amino acids 1, 5, 10, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 and ending at any of amino acids, 145, 140, 135, 134, 133, 132, 131, 130, 125, 120, 115, 110 or 105. For example, the fragment which may comprise the enzymatic domain may consist of amino acids 15 to 140 of SEQ ID NO: 1, or amino acids 18 to 134 of SEQ ID NO: 1, or any of the other possible permutations of the above start and end points.
In one embodiment, the polypeptide of the invention may comprise an SH3_3 domain contained within amino acids 322 to 375 of SEQ ID NO: 1. Thus, the fragment which may comprise the cell wall binding domain may consist of the sequence of SEQ ID NO: 1 starting from any of amino acids 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335 or 340 and ending at any of amino acids 350, 355, 360, 365, 370, 375 or 377. For example, the fragment which may comprise the cell wall binding domain may consist of amino acids 315 to 377 of SEQ ID NO: 1, or amino acids 320 to 376 of SEQ ID NO: 1, or any of the other possible permutations of the above start and end points.
Optionally, the polypeptide of the invention may comprise an amino acid sequence corresponding to a sequence within the central region of SEQ ID NO: 1. Thus, the polypeptide may comprise at least 20 contiguous amino acids from amino acids 135 to 321 of SEQ ID NO: 1, for example starting from any of amino acids 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 of SEQ ID NO: 1 and ending at any of amino acids 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210 or 200 of SEQ ID NO: 1. In one embodiment, this amino acid sequence may enhance the cell wall binding and/or lytic activity of the polypeptide of the invention.
The polypeptide of the first aspect of the invention preferably may comprise or consist of one or more fragments of the amino acid sequence of SEQ ID NO:1 corresponding to:
a. an enzymatic domain from within the N-terminal region of SEQ ID NO: 1 (i.e., from within amino acids 18 to 134 of SEQ ID NO:1);
b. a cell wall binding domain from within the C-terminal region of SEQ ID NO: 1 (i.e., from within amino acids 322 to 375 of SEQ ID NO:1); and/or
c. a further domain from within the central region of SEQ ID NO: 1 (i.e., from within amino acids 135 to 321 of SEQ ID NO:1).
Thus, in one embodiment, the polypeptide may comprise or consist of an enzymatic domain from within the N-terminal region of SEQ ID NO: 1 and a cell wall binding domain from within the C-terminal region of SEQ ID NO: 1.
However, it will be appreciated by persons skilled in the art that the cell wall binding domain of SEQ ID NO:1 may alternatively be fused or otherwise coupled to an enzymatic (lytic) domain from another source capable of lysing cells of Clostridium perfringens. The production of such chimeric lysins is described in Sheehan et al., 1996, FEMS Microbiology Letters 140:23-28, the disclosures of which are incorporated herein by reference). Thus, in an alternative embodiment, the polypeptide of the first aspect of the invention may comprise one or more fragments of the amino acid sequence of SEQ ID NO:1 corresponding to the cell wall binding domain but without an enzymatic domain of SEQ ID NO:1.
In an alternative embodiment, the polypeptide of the first aspect of the invention may comprise or consist of a variant of the amino acid sequence of SEQ ID NO:1, or of a fragment thereof, which is capable of lysing cells of Clostridium perfringens.
By ‘variant’ of the polypeptide Applicants include insertions, deletions and/or substitutions, either conservative or non-conservative, relative to the amino acid sequence of SEQ ID NO:1. In particular, the variant polypeptide may be a non-naturally occurring variant.
For example, the polypeptide may comprise an amino acid sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1, more preferably at least 70% or 80% or 85% or 90% identity to said sequence, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to said amino acid sequence.
It will be appreciated that the above sequence identity may be over the full length of the amino acid sequence of SEQ ID NO: 1 or over a portion thereof. Preferably, however, the sequence identity is over at least 50 amino acids of the amino acid sequence of SEQ ID NO: 1, for example at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265, 275, 285, 295, 300, 305, 310, 315, 320, 325, 335, 340, 345, 355, 360, 365, 370, 375 or 376 or more amino acids therein.
Percent identity can be determined by methods well known in the art, for example using the LALIGN program (Huang and Miller, Adv. Appl. Math. (1991) 12:337-357, the disclosures of which are incorporated herein by reference) at the ExPASy facility website using as parameters the global alignment option, scoring matrix BLOSUM62, opening gap penalty −14, extending gap penalty −4.
Alternatively, the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example AlignX, Vector NTI Advance 10 (from Invitrogen Corporation) or the GAP program (from the University of Wisconsin Genetic Computing Group).
It will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
Fragments and variants of the amino acid sequence of SEQ ID NO: 1 may be made using any of the methods of protein engineering, directed evolution and/or site-directed mutagenesis well known in the art (for example, see Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press, the disclosures of which are incorporated herein by reference).
It will be appreciated by skilled persons that the polypeptide of the invention, or fragment, variant or fusion thereof, may comprise one or more amino acids that are modified or derivatised. Thus, the polypeptide may comprise or consist of a derivative of the amino acid sequence of SEQ ID NO:1, or of a fragment or variant thereof.
Chemical derivatives of one or more amino acids may be achieved by reaction with a functional side group. Such derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine hydrochlorides, p-toluene sulphonyl groups, carboxybenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatised to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine and ornithine for lysine. Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained. Other included modifications are amidation, amino terminal acylation (e.g., acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g., with ammonia or methylamine), and the like terminal modifications.
It will be further appreciated by persons skilled in the art that peptidomimetic compounds may also be useful. Thus, by ‘polypeptide’ Applicants include peptidomimetic compounds which exhibit endolysin activity. The term ‘peptidomimetic’ refers to a compound that mimics the conformation and desirable features of a particular polypeptide as a therapeutic agent.
For example, the polypeptides described herein include not only molecules in which amino acid residues are joined by peptide (—CO—NH—) linkages but also molecules in which the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al. (1997) J. Immunol. 159, 3230-3237, the disclosures of which are incorporated herein by reference. Such retro-inverse peptides, which contain NH—CO bonds instead of CO—NH peptide bonds, are much more resistant to proteolysis. Alternatively, the polypeptide of the invention may be a peptidomimetic compound wherein one or more of the amino acid residues are linked by a -y(CH2NH)— bond in place of the conventional amide linkage.
It will be appreciated that the polypeptide may conveniently be blocked at its N- or C-terminus so as to help reduce susceptibility to exoproteolytic digestion, e.g., by amidation.
As discussed above, a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids may be used to modify polypeptides of the invention. In addition, a presumed bioactive conformation may be stabilised by a covalent modification, such as cyclisation or by incorporation of lactam or other types of bridges. Methods of synthesis of cyclic homodetic peptides and cyclic heterodetic peptides, including disulphide, sulphide and alkylene bridges, are disclosed in U.S. Pat. No. 5,643,872. Other examples of cyclisation methods are discussed and disclosed in U.S. Pat. No. 6,008,058, the relevant disclosures in which documents are hereby incorporated by reference. A further approach to the synthesis of cyclic stabilised peptidomimetic compounds is ring-closing metathesis (RCM).
In summary, terminal modifications are useful, as is well known, to reduce susceptibility by proteinase digestion and therefore to prolong the half-life of the peptides in solutions, particularly in biological fluids where proteases may be present. Polypeptide cyclisation is also a useful modification and is preferred because of the stable structures formed by cyclisation and in view of the biological activities observed for cyclic peptides.
Thus, in one embodiment the polypeptide, or fragment, variant, fusion or derivative thereof, is cycliC. However, in a preferred embodiment, the polypeptide, or fragment, variant, fusion or derivative thereof, is linear.
In a further embodiment of the first aspect of the invention, the polypeptide may comprise or consist of a fusion of the amino acid sequence of SEQ ID NO:1, or of a fragment, variant or derivative thereof.
By ‘fusion’ of a polypeptide Applicants include a polypeptide which is fused to any other polypeptide. For example, the polypeptide may comprise one or more additional amino acids, inserted internally and/or at the N- and/or C-termini of the amino acid sequence of SEQ ID NO:1, or of a fragment, variant or derivative thereof.
Thus, as described above, in one embodiment the polypeptide of the first aspect of the invention may comprise a fragment of SEQ ID NO: 1 consisting of the cell wall binding domain (or a variant of such a domain sequence which retains the cell wall binding activity thereof), to which is fused an enzymatic domain from a different source.
Examples of other suitable enzymatic domains include:
L-alanoyl-D-glutamate endopeptidase; D-glutamyl-m-DAP endopeptidase; interpeptide bridge-specific endopeptidase; N-acetyl-β-D-glucosaminidase (=muramoylhydrolase); N-acetyL-β-D-muramidase (=lysozyme); lytic transglycosylase.
Also N-acetylmuramoyl-L-alanine amidase from other sources could be utilised (see Loessner, 2005, Current Opinion in Microbiology 8:480-487, the disclosures of which are incorporated herein by reference).
In one embodiment, the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art. Similarly, the said polypeptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well-known Myc tag epitope. Fusions to any fragment, variant or derivative of said polypeptide are also included in the scope of the invention. It will be appreciated that fusions (or variants or derivatives thereof) which retain desirable properties, namely endolysin activity are preferred. It is also particularly preferred if the fusions are ones which are suitable for use in the methods described herein.
For example, the fusion may comprise a further portion which confers a desirable feature on the said polypeptide of the invention; for example, the portion may be useful in detecting or isolating the polypeptide, promoting cellular uptake of the polypeptide, or directing secretion of the protein from a cell. The portion may be, for example, a biotin moiety, a radioactive moiety, a fluorescent moiety, for example a small fluorophore or a green fluorescent protein (GFP) fluorophore, as well known to those skilled in the art. The moiety may be an immunogenic tag, for example a Myc tag, as known to those skilled in the art or may be a lipophilic molecule or polypeptide domain that is capable of promoting cellular uptake of the polypeptide, as known to those skilled in the art.
It will be appreciated by persons skilled in the art that the polypeptides of the invention also include pharmaceutically acceptable acid or base addition salts of the above described polypeptides. The acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful in this invention are those which form non-toxic acid addition salts, i.e. salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate [i.e. 1,1′-methylene-bis-(2-hydroxy-3 naphthoate)] salts, among others.
Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the polypeptides. The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of the present compounds that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.
The polypeptide, or fragment, variant, fusion or derivative thereof, may also be lyophilised for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilisation method (e.g., spray drying, cake drying) and/or reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that use levels may have to be adjusted upward to compensate. Preferably, the lyophilised (freeze dried) polypeptide loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (prior to lyophilisation) when rehydrated.
An essential feature of the polypeptides of the invention is the ability to lyse cells of Clostridium perfringens. Preferably, the polypeptide is capable of lysing cells of multiple strains of Clostridium perfringens. For example, the polypeptide may be capable of lysing one or more of the strains of Clostridium perfringens lysed by the CP51 lysin of SEQ ID NO: 1 (see Table 1 below).
In one embodiment, the polypeptides of the invention are substantially incapable of lysing bacteria which are commensal members of the microbiota of a healthy gut (and not known to cause adverse effects on the host). For example, it is advantageous if the polypeptide does not lyse cells of Clostridium clusters IV (e.g., Clostridium leptum), cluster XIVa (e.g., Clostridium nexile, Clostridium coccoides) or cluster XVI (e.g., Clostridium innocuum).
Most preferably, the polypeptide of the invention is capable of lysing cells of Clostridium perfringens strain NCTC 3110 and/or NCTC 8238. For example, the polypeptide may exhibit at least 10% of the lysis activity of the polypeptide of SEQ ID NO: 1 on cells of Clostridium perfringens strain NCTC 3110 and/or NCTC 8238, for example at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more. The polypeptide may even exhibit a greater lysis activity than the polypeptide of SEQ ID NO: 1 on cells of Clostridium perfringens strain NCTC 3110 and/or NCTC 8238, for example at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 500% or more.
Advantageously, the polypeptide is capable of lysing cells of pathogenic bacteria selectively, i.e. to a greater extent than cells of non-pathogenic bacteria.
Methods for the production of polypeptides, or a fragment, variant, fusion or derivative thereof, for use in the first aspect of the invention are well known in the art. Conveniently, the polypeptide, or fragment, variant, fusion or derivative thereof, is or may comprise a recombinant polypeptide.
Thus, a nucleic acid molecule (or polynucleotide) encoding the polypeptide, or fragment, variant, fusion or derivative thereof, may be expressed in a suitable host and the polypeptide obtained therefrom. Suitable methods for the production of such recombinant polypeptides are well known in the art (for example, see Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y., the relevant disclosures in which document are hereby incorporated by reference).
In brief, expression vectors may be constructed which may comprise a nucleic acid molecule which is capable, in an appropriate host, of expressing the polypeptide encoded by the nucleic acid molecule.
A variety of methods have been developed to operably link nucleic acid molecules, especially DNA, to vectors, for example, via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, e.g., generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3′-single-stranded termini with their 3′-5′-exonucleolytic activities, and fill in recessed 3′-ends with their polymerising activities.
The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a larger molar excess of linker molecules in the presence of an enzyme that is able to catalyse the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
The DNA (or in the case of retroviral vectors, RNA) is then expressed in a suitable host to produce a polypeptide. Thus, the DNA encoding the polypeptide may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the compound of the invention or binding moiety thereof. Such techniques are well known in the art.
The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector.
Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
Host cells that have been transformed by the expression vector are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
Many expression systems are known, including bacteria (for example, E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
The vectors typically include a prokaryotic replicon, such as the ColE1 ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, N.J., USA.
A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, N.J., USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
Other vectors and expression systems are well known in the art for use with a variety of host cells.
The host cell may be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, Md., USA (No. ATCC 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic and kidney cell lines. Yeast host cells include YPH499, YPH5200 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CRL 1658 and 293 cells which are human embryonic kidney cells. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.
Methods of cultivating host cells and isolating recombinant proteins are well known in the art. It will be appreciated that, depending on the host cell, the polypeptides of the invention produced may differ. For example, certain host cells, such as yeast or bacterial cells, either do not have, or have different, post-translational modification systems which may result in the production of forms of compounds of the invention which may be post-translationally modified in a different way.
Polypeptides of the invention may also be produced in vitro using a commercially available in vitro translation system, such as rabbit reticulocyte lysate or wheatgerm lysate (available from Promega). Preferably, the translation system is rabbit reticulocyte lysate. Conveniently, the translation system may be coupled to a transcription system, such as the TNT transcription-translation system (Promega). This system has the advantage of producing suitable mRNA transcript from an encoding DNA polynucleotide in the same reaction as the translation.
Automated polypeptide synthesisers may also be used, such as those available from CS Bio Company Inc, Menlo Park, USA.
Thus, a second aspect of the present invention provides an isolated nucleic acid molecule encoding a polypeptide according to the first aspect of the invention.
The nucleic acid molecule may be DNA (e.g., cDNA) or RNA.
In a preferred embodiment, the nucleic acid molecule may comprise or consist of the nucleotide sequence of SEQ ID NO 2:
A third aspect of the invention provides a vector which may comprise a nucleic acid molecule according to the second aspect of the invention. In one embodiment, the vector is an expression vector. Any suitable vector known in the art may be used. Preferably, the vector is selected from the group consisting of pET15b and pACYC184.
It will be appreciated by persons skilled in the art that the choice of expression vector may be determined by the choice of host cell. Thus, for expression of the polypeptides of the invention in Lactococcus lactis or Lactobacillus johnsonii, the nisin expression system could be used in which the polypeptide of the invention is expressed under the control of the promoter of the nisA operon using a background strain of Lactococcus lactis or Lactobacillus johnsonii which also expresses the nisR and nisK genes encoding a two component regulatory system. Under this system expression is positively regulated and induced by the provision of exogenous nisin (see de Ruyter at el., 1996, Applied and Environmental Microbiology 62:3662-3667, the disclosures of which are incorporated herein by reference).
In an alternative embodiment, the entire nisin biosynthesis gene cluster is provided within the same host cell, in which case the inducer is synthesised by that cell.
In a further alternative embodiment, the polypeptides of the invention may be expressed in Lactococcus lactis or Lactobacillus johnsonii under the control of the lactose catabolic operon, using either a plasmid-based or chromasomally integrated system (for example, see Payne et al., 1996, FEMS Microbiology Letters 136: 19-24 and van Rooijen et al., 1992, Journal of Bacteriology 174: 2273-2280, the disclosures of which are incorporated herein by reference).
As noted above, Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry.
Lactobacillus johnsonii FI9785 is a poultry-isolated strain which has been shown to act as a competitive exclusion agent against C. perfringens in chickens. As well as acting as probiotics in their own right, lactic acid bacteria have also shown promise as delivery systems for the secretion of biologically active interleukins or peptidoglycan hydrolases. The promoter of the lantibiotic nisin A (PnisA) is induced by nisin A via signal transduction using the two component regulatory system NisRK. This system has been exploited to develop gene expression systems in Lactococcus lactis, Leuconostoc lactis and Lactobacillus helveticus.
Hence, a fourth aspect of the invention provides a host cell which may comprise a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention. In one embodiment, the host cell is a microbial cell, for example a bacterial cell. Preferably, the host cell is non-pathogenic.
For example, the host cell may be selected from the group consisting of cells of Escherichia coli, Lactococcus sp., Bacteroides sp., Lactobacillus sp., Enterococcus sp. and Bacillus sp.
In a preferred embodiment, the host cell is a cell of Lactococcus lactis (e.g., L. lactis FI10676, L. lactis FI15876, L. lactis FI17847 and L. lactis UKLc10).
In an equally preferred embodiment, the host cell is a cell of Lactobacillus johnsonii (e.g., Lactobacillus johnsonii FI9785 or its derivative strains, for example FI10744, FI10386 or FI10844).
Alternatively, the host cell may be a yeast cell, for example Saccharomyces sp.
A fifth aspect of the invention provides a method for producing a polypeptide of the invention which may comprise culturing a population of host cells which may comprise a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention under conditions in which the polypeptide is expressed, and isolating the polypeptide therefrom.
A sixth aspect of the invention provides a pharmacological composition which may comprise:
(a) a polypeptide according to the first aspect of the invention;
(b) a nucleic acid molecule according to the second aspect of the invention;
(c) a vector according to the third aspect of the invention;
(d) a host according to the fourth aspect of the invention; and/or
(e) a bacteriophage capable of expressing a polypeptide according to the first aspect of the invention
and a pharmaceutically acceptable carrier, diluent or excipient.
As used herein, ‘pharmaceutical composition’ means a therapeutically effective formulation for use in the methods of the invention.
A ‘therapeutically effective amount’, or ‘effective amount’, or ‘therapeutically effective’, as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
In one embodiment of the invention, the pharmacological composition may comprise a polypeptide according to the first aspect of the invention.
The polypeptides can be formulated at various concentrations, depending on the efficacy/toxicity of the polypeptide being used. Preferably, the formulation may comprise the Polypeptide at a concentration of between 0.1 μM and 1 mM, more preferably between 1 μM and 100 μM, between 5 μM and 50 μM, between 10 μM and 50 μM, between 20 μM and 40 μM and most preferably about 30 μM. For in vitro applications, formulations may comprise similar concentrations of a polypeptide (however, it will be appreciated that higher concentrations may also be used).
Thus, the pharmaceutical formulation may comprise an amount of a polypeptide, or fragment, variant, fusion or derivative thereof, sufficient to inhibit at least in part the growth of cells of Clostridium perfringens in a patient who is infected or susceptible to infection with such cells. Preferably, the pharmaceutical formulation may comprise an amount of a polypeptide, or fragment, variant, fusion or derivative thereof, sufficient to kill cells of Clostridium perfringens in the patient.
It will be appreciated by persons skilled in the art that the polypeptides of the invention are generally administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA, the relevant disclosures in which document are hereby incorporated by reference).
For example, the polypeptides can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The polypeptides may also be administered via direct injection (for example, into the GI tract).
Preferably, however, the polypeptides and pharmaceutical compositions thereof are for oral administration.
Suitable tablet formulations may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxyl-propylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the polypeptides may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
The polypeptides can also be administered parenterally, for example, intravenously, intra-articularly, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
For oral and parenteral administration to human patients, the daily dosage level of the polypeptides will usually be from 1 to 1000 mg per adult (i.e. from about 0.015 to 15 mg/kg), administered in single or divided doses. For example, a dose of 1 to 10 mg/kg may be used, such as 3 mg/kg.
In an alternative embodiment of the invention, the pharmaceutical compositions do not comprise the polypeptide itself but instead may comprise a nucleic acid molecule capable of expressing said polypeptide. Suitable nucleic acid molecules, expression vectors, and host cells are described in detail above.
For example, a recombinant probiotic may be used (LAB strain, e.g., Lactococcus lactis or a Lactobacillus sp.).
In a further embodiment of the invention, the pharmaceutical compositions may comprise a bacteriophage capable of expressing a polypeptide according to the first aspect of the invention. For example, the wildtype bacteriophage φCP51 may be used to deliver a polypeptide according to the first aspect of the invention. Methods for performing such bacteriophage-based therapies are well known in the art (for example, see Watanabe et al., 2007, Antimicrobial Agents & Chemotherapy 51:446-452).
Thus, for treatment of bacterial infections described herein, the polypeptide of the invention may be administered as the cognate protein, as a nucleic acid construct, vector or host cell which expresses the cognate protein, as part of a living organism which expresses the cognate protein (including bacteriophages), or by any other convenient method known in the art so as to achieve contact of the lysin with its bacterial target, whether that be a pathogenic bacterium, such as C. perfringens, or another pathogen or potential pathogen, as further described herein.
Ideally, the protein is delivered to the GI tract in a protected form. This may be achieved by a wide variety of methods known in the art. For example, an appropriate dose of the lysin is microencapsulated in a form that survives the acidic conditions of the stomach, but which releases the protein as it enters the intestine. Delivery by a non-pathogenic microbe which survives GI tract transit, including but not limited to by Lactococcus lactis, Lactobacillus johnsonii, Lactobacillus sp., Bifidobacterium sp., Bacillus sp. or Bacteroides. Those skilled in the art are well aware of the options available for use of such means for GI tract delivery of active compounds such as the lysin disclosed herein. These means include intracellular production, secA secretion or secretion by means of another secretion pathway, and delivery by controlled lysis. Preferably the protein is not all released at one time, but is released increasingly as an administered bolus traverses through the GI tract. Alternatively, the lysin is introduced as part of a benign bacterium which expresses the lysin at the appropriate location or upon receipt of an appropriate signal in the GI tract. In a preferred embodiment disclosed herein, a non-pathogenic Lactococcus is engineered to express the CP51 lysin upon reaching a particular location in the GI tract. The expression signal may be defined by a pH sensitive promoter, or another means known in the art for this purpose.
Other means of delivery include the following:
(a) WO 2006/111553 (polyurea and other multilayer encapsulants);
(b) WO 2006/111570 and EP 1 715 739 (cyclodextrin encapsulation);
(c) WO 2006/100308 and EP 1 742 728 (for yeast and other microbial cell encapsulation technologies);
(d) U.S. Pat. No. 5,153,182, EP 1 499 183 and WO 03/092378; U.S. Pat. No. 6,831,070 (therapeutic gene product delivery by intestinal cell expression);
(e) U.S. Pat. No. 7,202,236 (pharmaceutical formulation for modified release);
(f) U.S. Pat. No. 5,762,904 (oral delivery of vaccines using polymerized liposomes, which may be modified to deliver the lysin of this invention),
(g) U.S. Pat. No. 7,195,906 (Bifidobacterium which may be modified to express the lysin according to this invention); and
(h) other means of delivery disclosed in the references cited therein,
all of which are herein incorporated by reference for purposes of enabling those skilled in the art to utilize the present disclosure to achieve the novel methods of delivery and compositions according to the present invention.
Thus, in a preferred embodiment of the pharmacological compositions of the invention, the polypeptide, nucleic acid molecule encoding the same, etc. is microencapsulated (e.g., within a stable chemical envelope, such as cyclodextrin or a lipid bilayer, or within a living or non-living microbial cell, such as an engineered Lactococcus or Lactobacillus cell). In this way, the polypeptide, nucleic acid molecule, etc. may be protected against acidic conditions of stomach en route to its site of action in the GI tract.
A seventh aspect of the invention provides a polypeptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or pharmacological composition according to the sixth aspect of the invention for use in medicine. Hence, the seventh aspect may be for use in a method for treatment of the human or animal body by surgery or therapy and/or diagnostic methods practised on the human or animal body.
An eighth aspect of the invention provides the use of a polypeptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or pharmacological composition according to the sixth aspect of the invention, in the preparation of a medicament for killing and/or inhibiting/preventing the growth of microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
A ninth aspect of the invention provides a polypeptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or pharmacological composition according to the sixth aspect of the invention for use in killing and/or inhibiting/preventing the growth of microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
A tenth aspect of the invention provides a method for killing and/or inhibiting/preventing the growth of microbial cells in a patient the method which may comprise administering to the patient a polypeptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or pharmacological composition according to the sixth aspect of the invention, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
An eleventh aspect of the invention provides the use of a polypeptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or pharmacological composition according to the sixth aspect of the invention in the preparation of a medicament for the treatment or prevention of a disease or condition associated with microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
A twelfth aspect of the invention provides a polypeptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or pharmacological composition according to the sixth aspect of the invention for use in the treatment or prevention of a disease or condition associated with microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
A thirteen aspect of the invention provides a method for the treatment or prevention of a disease or condition associated with microbial cells in a patient the method which may comprise administering to the patient a polypeptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or pharmacological composition according to the sixth aspect of the invention, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
By “a disease or condition associated with microbial cells in a patient” Applicants include diseases and conditions arising from or antagonised by infection of a patient with Clostridium perfringens. Such diseases and conditions include food poisoning, gas gangrene (Myonecrosis) and necrotic endocarditis (NE).
In one embodiment of the above method/use aspects of the invention, the polypeptide having the cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens is a polypeptide according to the first aspect of the invention, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis upon contact with a polypeptide of SEQ ID NO: 1 (see Table 1, below).
Preferably, the microbial cells may comprise or consist of Clostridium perfringens cells. Thus, the polypeptides having the cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens may be used to treat or prevent diseases and conditions associated with infection with Clostridium perfringens cells (such as food poisoning, gas gangrene (Myonecrosis) and necrotic endocarditis).
Persons skilled in the art will further appreciate that the uses and methods of the present invention have utility in both the medical and veterinary fields. Thus, the medicaments may be used in the treatment of both human and non-human animals (such as horses, cows, dogs and cats). Hence, the methods and uses may be for use in an organism belonging to a taxonomic superclass or class selected from the group consisting of Chondrichthyes (cartilaginous fish), Osteichthyes (bony fish), Actinopterygii (ray-finned bony fish), Sarcopterygii (lobe-finned fish), Tetrapoda (four-limbed vertebrates), Amphibia (amphibians), Reptilia (reptiles), Ayes (birds) and Mammalia (mammals).
In one preferred embodiment, the taxonomic class is Ayes (birds). Thus, the polypeptides of the invention may be for use in poultry (for example, selected from the group consisting of chicken, duck, goose, ostrich, pigeon, turkey, pheasant, guinea fowl, partridge and quail).
In another preferred embodiment, the taxonomic class is Mammalia (mammals), for example, the mammal may be selected from the group consisting of alpaca, banteng, bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit, reindeer, sheep, water buffalo, yak and human.
Most preferably, however, the polypeptides of the invention are for use in poultry.
By ‘treatment’ Applicants include both therapeutic and prophylactic treatment of a subject (or patient). In one embodiment, the uses and methods of the invention are for the treatment of an existing disease or condition. Alternatively or additionally, the uses and methods of the invention may be for prophylaxis. The term ‘prophylactic’ or ‘prophylaxis’ is used to encompass the use of a polypeptide or formulation described herein which either prevents or reduces the likelihood of infection with Clostridium perfringens in a patient or subject. The prophylaxis may be primary prophylaxis (i.e., to prevent the development of a disease) or secondary prophylaxis (where the disease has already developed and the patient is protected against worsening of this process). Preferably, the prophylaxis is primary prophylaxis.
As discussed above, the term ‘effective amount’ is used herein to describe concentrations or amounts of polypeptides according to the present invention which may be used to produce a favourable change in a disease or condition treated, whether that change is a remission, a favourable physiological result, a reversal or attenuation of a disease state or condition treated, the prevention or the reduction in the likelihood of a condition or disease state occurring, depending upon the disease or condition treated.
In one embodiment, the polypeptide according to the first aspect of the invention, nucleic acid according to the second aspect of the invention, vector according to the third aspect of the invention, host cell according to the fourth aspect of the invention or pharmacological composition according to the sixth aspect of the invention is administered in a single dose. Alternatively, the polypeptide, nucleic acid, vector, host cell, bacteriophage or pharmacological composition is administered as a plurality of doses (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more doses).
The polypeptide, nucleic acid, vector, host cell, bacteriophage or pharmacological composition is preferably administered at a frequency sufficient to maintain a continuous presence of the polypeptide according to the first aspect of the invention in the gastrointestinal (GI) tract of the subject. Preferably, the dose and dosage frequency is sufficient to prevent occurrence or recurrence of a disease or condition associated with microbial cells in a subject. Preferably, the dose and dosage frequency is sufficient to prevent occurrence or recurrence of growth impedance associated with microbial cells in a subject (e.g., C. perfringens).
In one embodiment, the uses and methods of the invention a host cell or pharmacological composition which may comprise a host cell is used to deliver the polypeptide of the first aspect of the invention (preferably a host cell).
It will be appreciated that the medicaments described herein may be administered to a subject in combination with one or more additional therapeutic agents.
For example, the medicaments described herein may be administered to a subject in combination with:
(a) one or more conventional antibiotic treatments (such as beta-lactams, aminoglycosides and/or quinolones);
(b) one or more additional lysins, or nucleic acid molecules, vectors, host cell or bacteriophage capable of expressing the same;
(c) one or more holins (such as the holing of φCP51: MENIFDYLKMGIVAIGTLFTWLLGAWDTPLVILIVLMALDYITGITKGYVNKDLSSNI GLKGIARKGVIFTILIVAVMLDRLLNTGNWIFRTLVCYFYIANEGISIIENASELGVPVP SKLKNALIQLKEDKEDHKKL [SEQ ID NO: 3]), or nucleic acid molecules, vectors, host cell or bacteriophage capable of expressing the same;
(d) one or more [antibiotics, or nucleic acid molecules, vectors, host cell or bacteria capable of expressing the same; and/or
(e) a therapy to neutralise the toxins released upon bacterial lysis of Clostridium perfringens cells within the gut. Suitable neutralising therapies may include antibodies (see Babcock et al., 2006, Infect. Immun. 74:6339-6347) and toxin-absorbing agents such as tolevamer (see Barker et al., 2006, Aliment. Pharmacol. Ther. 24:1525-1534).
A further aspect of the invention provides the use of a polypeptide having the cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens, or a nucleic acid molecule, vector, host cell or bacteriophage capable of expressing the same, for killing and/or inhibiting/preventing the growth of microbial cells in vitro and/or ex vivo, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin. For example, said polypeptides having endolysin activity may be used to clean surfaces, such as those in hospitals, kitchens, etc., which may be susceptible to contamination with such bacterial cells.
Preferably, the polypeptide having the cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens is a polypeptide according to the first aspect of the invention, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis upon contact with a polypeptide of SEQ ID NO: 1 (see Table 1, below). For example, the microbial cells may comprise or consist of Clostridium perfringens cells. Most preferably, the microbial cells may comprise or consist of cells of Clostridium perfringens NCTC 3110 and/or NCTC 8238.
A further aspect of the present invention provides a kit for detecting the presence of microbial cells in a sample, the kit which may comprise a polypeptide having the cell lysing activity and/or cell binding specificity of an endolysin from a bacteriophage of Clostridium perfringens, or a nucleic acid molecule, vector, host cell or bacteriophage capable of expressing the same, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
In a preferred embodiment, the polypeptide having the cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens is a polypeptide according to the first aspect of the invention, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis upon contact with a polypeptide of SEQ ID NO: 1 (see Table 1, below). For example, the microbial cells may comprise or consist of Clostridium perfringens cells. Most preferably, the microbial cells may comprise or consist of cells of Clostridium perfringens NCTC 3110 and/or NCTC 8238.
In a further embodiment of the kits of the invention, the polypeptide having the cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens is immobilised on a suitable surface, such as the surface of a multi-well plate.
The kits may be used in conjunction with any suitable sample of cells, such as tissue samples, cell culture samples and samples of cells derived from swabs (e.g., taken from a surface to be tested for contamination with microbial cells).
Optionally, the kit further may comprise a negative control sample (which does not contain cells of the type to be tested for, e.g., Clostridium perfringens cells) and/or a positive control sample (which contains cells of the type to be tested for).
Related aspects of the invention provide:
(a) the use of a polypeptide having the cell wall binding activity and/or cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens, or a nucleic acid molecule, vector, host cell or bacteriophage capable of expressing the same, in the preparation of a diagnostic agent for a disease or condition associated with microbial cells selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin;
(b) the use of a polypeptide having the cell wall binding activity and/or cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens, or a nucleic acid molecule, vector, host cell or bacteriophage capable of expressing the same, for the diagnosis of a disease or condition associated with microbial cells selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin;
(c) the use of a polypeptide having the cell wall binding activity and/or cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens, or a nucleic acid molecule, vector, host cell or bacteriophage capable of expressing the same, for detecting the presence of microbial cells in a sample in vitro and/or ex vivo, wherein the microbial cells selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin; and
(d) a method for the diagnosis of a disease or condition associated with microbial cells in a patient, the method which may comprise contacting a cell sample from a patient to be tested with a polypeptide having the cell wall binding activity and/or cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens, or a nucleic acid molecule, vector, host cell or bacteriophage capable of expressing the same, and determining whether the cells in the sample have been lysed thereby, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
In one embodiment of the above defined uses and methods of the invention, the polypeptide having the cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens is a polypeptide according to the first aspect of the invention, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis upon contact with a polypeptide of SEQ ID NO: 1 (see Table 1, below). Preferably, the microbial cells may comprise or consist of Clostridium perfringens cells. Thus, the polypeptides having the cell lysing activity of an endolysin from a bacteriophage of Clostridium perfringens may be used to diagnose diseases and conditions associated with infection with Clostridium perfringens cells (such as food poisoning, gas gangrene (Myonecrosis) and necrotic endocarditis (NE)). Most preferably, the microbial cells may comprise or consist of cells of Clostridium perfringens NCTC 3110 and/or NCTC 8238.
In such diagnostic uses and methods, lysis of cells may be detected using methods well known in the art. For example, levels of ATP may be measured as an indicator of cell lysis.
In an alternative embodiment of the above defined uses and methods of the invention, the polypeptide may comprise or consist of the cell wall binding domain of an endolysin from a bacteriophage of Clostridium perfringens. To permit detection, such a polypeptide may be fused to magnetic beads or used as a fusion protein which may comprise a suitable reporter (for example, green fluorescent protein).
Such diagnostic approaches are well established for endolysins from other systems, such as Listeria endolysins (for example, see Loessner et al., 2002, Mol Microbiol 44, 335-49; Kretzer et al., 2007, Applied Environ. Microbiol. 73:1992-2000, the disclosures of which are incorporated herein by reference; suitable assays are also available commercially, for example from Profos, Germany.
Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.
The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.
During sequencing of the genome of Clostridium perfringens strain 5147-97, a putative prophage was identified located within a gene for a proposed flavodoxin oxidoreductase. Mitomycin C induction of this strain released a bacteriophage whose morphological features examined by electron microscopy indicated it belonged to the family Siphoviridae. To determine the ends of the prophage, PCR was performed using primers facing outwards from the proposed end genes. This confirmed the presence of a circularised genome in PEG-precipitated bacteriophage particles. The 39108 bp genome includes 50 predicted open reading frames (ORFs), including two which may affect sporulation, and two predicted tRNAs.
C. perfringens is a Gram-positive, anaerobic, spore-forming bacterium whose production of toxins leads to debilitating infections in both humans and animals. Conditions include food poisoning, gas gangrene, non-foodborne gastrointestinal infections and necrotic enteritis (NE) [7]. In poultry, NE has a significant impact on health, productivity and the carriage of pathogenic bacteria in the food chain [17]. A range of novel approaches are being explored to control C. perfringens and the use of bacteriophages or their cell lytic endolysins may provide alternatives to conventional antimicrobials [9]. The increasing collection of C. perfringens phages presents opportunities both for control and for a better understanding of the relationship between the phage and the host [6, 8, 13, 18-20].
A single prophage sequence φCP51 was identified within the genome of C. perfringens 5146-97, a type A strain isolated from turkey (kindly provided by M. Brett, HPA, UK). Library construction and sequencing on the Illumina GAIIX was performed by TGAC (Norwich, UK). Reads were assembled with ABySS [15] and contigs were extended using CAP3 [5] then annotated by RAST [2]. Bacteriophage particles were induced with mitomycin C, harvested and assayed as described previously [14]. Filtered mitomycin C-induced supernatants were tested against 25 C. perfringens strains but no plaque formation was observed. However, phage particles were observed by electron microscopy of PEG-precipitated particles [11] which had been negatively stained in saturated uranyl acetate (
The φCP51 prophage genome was located between 2 host genome coding sequences which were annotated as fragments of a flavodoxin reductase. Two homologous 11 bp core regions (CTGGACATGCT [SEQ ID NO: 4]) were identified at the end of the first fragment and the beginning of the second, representing potential attachment sites. In silico removal of the prophage sequence and one of the core regions reconstituted a sequence which when translated showed 100% homology to complete putative FAD dependent oxidoreductases from C. perfringens genomes SM101 and D str. JGS1721. Similarly, circularization of the prophage at the core region joined the first and last ORFs in the prophage to generate a single ORF with homology to a recombinase, giving a final bacteriophage genome size of 39108 bp.
To confirm the extent of the phage genome, outward-facing primers were designed from the first (CP51_R1, 5-ATAGATCTTATAACGTCTCTCTTG [SEQ ID NO: 5]) and last (CP51_F2, 5-TCACCTATAGTTTTATTTGGAA [SEQ ID NO: 6]) prophage ORFs to cover the predicted circularized product of 326 bp. Using GoTaq (Promega), a product was successfully amplified from PEG-precipitated mitomycin C-induced supernatant and sequencing confirmed the expected recircularisation point.
Phage ORF locations and domain identifications were confirmed using Artemis [10] with BlastP searches [1] using the NCBI database. Start sites were selected as the best match to the consensus ribosome binding site sequence AGGAGG [SEQ ID NO: 7] 5 to 13 nt from the start codon. ATG was the most common start codon, but there were also incidences of alternative start codons TTG (ORFs 22, 30, 47), GTG (ORFs 17, 32, 49) and ATA (ORF 19). The G+C content of 28% matches that of the host bacterial genome.
BlastP analyses indicate that most of the ORFs show similarity to proteins from bacterial genomes rather than from phages, possibly matching proteins from other prophages. The organisation of modules was similar to that seen in φ3626 [20]. The first 25 ORFs, covering the proposed structural and lysis proteins, almost exclusively match C. perfringens sequences (
Two tRNAs (Tyr-ATA and Ser-GCT) were identified by RAST analysis; tRNAs in phage genomes have been associated with codon usage, corresponding to the codons that are highly used by phage genes and rare in the host genome [3]. However, analysis of the codon usage in φCP51 and the host genome showed no difference.
As more bacterial genomes are sequenced, there will be a continued opportunity for the identification of prophages, which can be used to add knowledge to phage biology or for isolation of phage encoded enzymes [12]. Although Applicants failed to demonstrate plaque formation, the φCP51 lysin is active (see Examples, below) indicating that the phage might be lytic on a suitable host. However there is a possibility that the phage is not able to progress through the life cycle due to unidentified defects within the genome.
Nucleotide Sequence Accession Number
The complete genome sequence of φCP51 was deposited in the GenBank database under accession number KC237729.
Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. To find new control strategies to treat C. perfringens infection, Applicants investigated the activity and delivery of a bacteriophage endolysin. Applicants identified a new endolysin, designated CP51L, which shows similarity to an N-acetylmuramoyl-L-alanine amidase domain but not to other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp51 l gene was cloned and expressed in Escherichia coli and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested. A probiotic strain of Lactobacillus johnsonii FI9785 was engineered to produce a system for delivery of the endolysin to the gastrointestinal tract. The integration of the nisRK two component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (PnisA), while the use of a signal peptide (SLPmod) successfully secreted the active endolysin to the surrounding media. The high specificity and activity of the endolysin suggest it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.
Materials and Methods
Bacterial Strains and Growth Conditions
C. perfringens strains listed in Table 1 were obtained from the NCTC (HPA, London, UK), or from in-house culture collections (IFR, Norwich, UK). C. perfringens 5416-97 is a Type A strain (11). Strains were maintained in Robertson's cooked-meat medium (SGL) at room temperature and were grown anaerobically at 37° C. in brain heart infusion broth (BHI, Oxoid) supplemented with vitamin K [50 mg/l], hemin [5 mg/l], resazurin [1 mg/l], and L-cysteine[0.5 g/l]), termed BHI+C. E. coli strains were grown in L broth with shaking at 37° C. and L. johnsonii strain FI9785 (20) and derivatives were grown in MRS broth (Oxoid) at 37° C. Commensal, environmental and clostridial strains were obtained from IFR culture collections, the DSMZ (Braunschweig, Germany) or the NCIMB (Aberdeen, UK), and were grown as recommended by DSMZ or in BHI+C. The following strains were used for lysin specificity tests: Anaerococcus hydrogenalis DSMZ 7454, Bacillus amyloliquefaciens 0880, Bacillus cereus NCIMB 11796, Bacillus subtilis ATCC 6633, Bifidobacterium adolescentis DSMZ 20083, Bifidobacterium angulatum DSMZ 20098, Bifidobacterium bifidum DSMZ 20082, Bifidobacterium longum DSMZ 20219, Bifidobacterium pseudocatenulatum DSMZ 20438, Clostridium acetobutylicum BL75141, Clostridium bifermentans NCTC 13019, Clostridium beijerinckii NCIMB 8052, Clostridium cellobioparum DSMZ 1351, Clostridium coccoides NCTC 11035, Clostridium colinum DSMZ 6011, Clostridium difficile NCTC 11204, Clostridium innocuum DSMZ 1286, Clostridium leptum DSMZ 753, Clostridium nexile DSMZ 1787, Clostridium ramosum DSMZ 1402, Clostridium sordellii NCTC 13356, Clostridium sporogenes ATCC 17886, Clostridium tyrobutyricum NCIMB 9582, Enterococcus faecalis FI10734, Enterococcus hirae FI10477, Eubacterium barkeri DSMZ 1223, Lactobacillus casei FI10736, L. johnsonii FI9785, Lactobacillus plantarum FI08595, Lactobacillus rhamnosus FI10737, Lactococcus lactis MG1363, Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293, Listeria innocua NCTC 11288, Listeria ivanovii NCTC 11007, Micrococcus luteus F110640, Pediococcus pentosaceus FI10642, Pediococcus acidilactici FI10738, Salmonella enterica serovar Typhimurium FI10739, Salmonella enterica serovar Enteritidis FI10113, Staphylococcus aureus FI10139.
Endolysin CP51L Analysis and Subcloning
The endolysin sequence is available in the φCP51 genome nucleotide sequence, accession number KC237729. Endolysin amino acid similarities and conserved domains were determined using Blastp (1) and the NCBI non-redundant protein sequences database. Amino acid alignments were performed using the ClustalW algorithm in Vector NTI (Invitrogen).
Genomic DNA was extracted from cells of C. perfringens 5416-97 (grown to mid-exponential phase) using the Genomic DNA extraction kit with a Genomic Tip 20/G column (Qiagen) as described by the manufacturer, with the addition of 50 U mutanolysin (Sigma) to aid cell lysis. The putative endolysin gene cp51l was amplified from genomic DNA using Phusion DNA polymerase (Finnzymes). To facilitate cloning into the Ndel and Xhol sites of expression vector pET15b (Novagen), primers were designed to incorporate an Ndel site at the initiating methionine codon CP51L_NDE (5′—GAATGTCATATGTATATAAATCAATCA-3′ [SEQ ID NO: 8], altered nucleotides underlined throughout) and a Xhol site downstream of the stop codon CP51L_XHO (5′-ACTCGAGGTGGGATAATTCCTACC-3′ [SEQ ID NO: 9]). Due to the presence of an internal Ndel site, internal primers were designed to create a 1 bp substitution (T771C) without changing the resulting amino acid sequence. Primer CP51L_GTG (5′-TATTGCATGTGGTAGTCAAAAGAT-3′ [SEQ ID NO: 10]) was used in conjunction with CP51L_NDE and primer CP51L_CAC (5′-TTGACTACCACATGCAATATAAAAATG-3′ [SEQ ID NO: 11]) was paired with CP51L_XHO, then the two products were spliced and amplified using overlap extension PCR with primers CP51L_NDE and CP51L_XHO. The splice product was restricted with Ndel and Xhol (New England Biolabs) and cloned into restricted pET15b that had been dephosphorylated with Antarctic Phosphatase (New England Biolabs) using Fastlink ligase (Epicentre). Ligation products were transformed into chemically competent E. coli TOP10 cells (Invitrogen) and transformants were selected with ampicillin (100 μg/ml). After confirmation by sequencing, construct pET15b-cp51l and the empty control vector pET15b were transformed for expression into chemically competent E. coli BL21(DE3) cells (Invitrogen).
An endolysin expression and secretion construct was produced in pUK200 (47) placing the endolysin coding sequence downstream of the signal peptide SLPmod (7) and a 6×His tag, all under the control of the promoter Pnisa (pUK200-slpmod-6×His-cp51l). Ligation products were transformed into electrocompetent E. coli MC 1022, selected with chloramphenicol (15 μg/ml) and confirmed by sequencing. To provide the two-component regulatory system required for PnisA controlled expression, the nisRK genes from Lactococcus lactis FI5876 (5) were inserted into the chromosome of L. johnsonii FI9785 using the thermosensitive pG+host9 vector (27). An integration region was amplified from L. johnsonii FI9785 genomic DNA and the resulting 1033 bp product was cloned into the blunt-ended Spel site of pG+host9 (creating plasmid pFI2657). A 2.4 kb region encoding nisRK was amplified from the genome of L. lactis FI5876 using the primer pair nisRK_F (5′-CCCGGGAGAATCTTAAAGAGTCTAGGG-3′ [SEQ ID NO: 12]) and nisRK R (5′-AAAAAGTAATCCTTAGAGATTAC-3′ [SEQ ID NO: 13]) and cloned into a blunt-ended BstEll site located within the integration region of pF12657, creating pFI2652. Subsequently, L. johnsonii FI9785 was transformed with pFI2652 and the nisRK sequences were integrated into the chromosome by gene replacement (27) using 30° C. as the permissive temperature and 42° C. as the non-permissive temperature, to create the nisRK-carrying strain FI10744. The lysin expression construct and the control vector pUK200 were transformed into electrocompetent L. johnsonii FI10744 as described (14) and positive transformants were selected using chloramphenicol (7.5 μg/ml) to give the endolysin delivery strain FI10744-L and the vector control FI10744-V.
Protein Expression, Analysis, and Partial Purification
Crude protein extracts were produced from IPTG-induced E. coli BL21(DE3) cells containing pET15b-cp51l or pET15b in either NP buffer (20 mM sodium phosphate buffer pH 6.5), TN buffer (20 mM Tris-HCl, 50 mM NaCl pH 7.5), or elution buffer (EB, 50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole pH 8) by bead beating as previously described (29). His-CP51L was partially purified using the nickel-nitrilotriacetic acid (Ni-NTA) Fast Start kit (Qiagen). L. johnsonii cells grown to mid-exponential phase were incubated for 2 h with or without nisin (10 ng/ml). Cells were harvested by centrifugation for 10 min at 2,500×g and 4° C. and the cell pellet was frozen while the supernatant was concentrated 20-fold using Amicon columns (Ultra-4 ultracell-30 k, Millipore) or 166-fold by shaking for 5 min after the addition of an equal volume of 100% ethanol pre-cooled to −80° C., followed by centrifugation for 30 min at 10,000×g and 4° C. The resulting pellet was freeze-dried in liquid nitrogen prior to resuspension in EB. Crude cell extracts were produced by bead beating in EB as with E. coli. Protein quantification, electrophoresis and Western blot analysis with a His Tag® monoclonal antibody (Novagen) were all performed as described previously (29).
Lysis Assays
The lysis of C. perfringens NCTC 3110 cells resuspended in phosphate buffered saline (PBS) was assessed by turbidity reduction assay as previously described (29) using the appropriate buffer controls. Cells for lysis assays were either used immediately after harvest (fresh′) or were flash frozen in liquid nitrogen and stored at −20° C. until use (frozen). Hen egg white lysozyme (Sigma) was used as a positive control at 500 U per 300 μl assay. The activity profile of crude protein extracted in NP buffer under various pH conditions was examined by adjusting the PBS to pH values between 4.5 and 8.5, while activity in different media was assessed by replacing the PBS with BHI+C or chemostat medium, a complex medium used for in vitro fermentations (CM: peptone water 2 g/l (Oxoid), yeast extract 2 g/l (Oxoid), NaCI 0.1 g/l, K2HPO4 0.04 g/l, KH2PO4 0.04 g/I, MgSO4.7H2O 0.01 g/l, CaCl2.6H2O 0.01 g/l, NaHCO3 2 g/l, Tween 80 2 ml, hemin 0.02 g/l dissolved in 400 μL, 1 M NaOH, vitamin K1 10 μl of 5% v/v aqueous solution, cysteine HCl 0.5 g/l, bile salts 0.5 g/l, 1% glucose all from Sigma, (28)). Activity against different species was assessed using 10 μg of Ni-NTA partially purified protein during a 1 h incubation. The endolysin activity of crude protein extracts and concentrated culture supernatants from the engineered L. johnsonii strains was measured by plate assay as described previously (41). Briefly, C. perfringens cells were grown to stationary phase in 100 ml BHI broth, autoclaved, pelleted by centrifugation then resuspended in 1 ml of PBS and added to 100 ml of PBS with 1% agar at 55° C. After pouring into plates, small holes were punched into the seeded agar and 10 μl of protein extract (equivalent to 30 μg) or 20 μl of concentrated supernatants were loaded into the wells and the plates were then incubated for 24 h at 37° C. To demonstrate lytic activity from growing colonies, C. perfringens cells were processed in the same way then incorporated into plates containing MRS agar with 7.5 μg/ml chloramphenicol. Overnight cultures of FI10744-L and FI10744-V were subsequently streaked onto these plates and the plates were incubated for 1-3 d at 37° C.
C. perfringens NCTC 3110 viability assays were performed by adding 166 μg of Ni-NTA partially purified CP51L protein to 5 ml of pre-reduced BHI+C under anaerobic conditions. The protein was added to the media either immediately before C. perfringens inoculation (20 μl of a fresh overnight culture) or during the early exponential phase of growth (optical density [OD600] ca. 0.3). To determine C. perfringens viability, samples were taken every 2 h for 8 h and serially diluted in PBS before being plated onto BHI agar and incubated for 24 to 48 h at 37° C. Results were calculated as the total number of bacteria from triplicate counts.
Results
Characterisation of the CP51L Endolysin
The cp51l gene was identified previously within the genome of a mitomycin C-inducible prophage of C. perfringens 5416-97 (φCP51, see Examples, below). Blastp analysis of CP51L identified 2 regions with similarity to conserved domains separated by a central section (
Expression and Activity of CP51L
CP51L was expressed in E. coli as a His-tagged protein, giving a product of C. 45 kDa which was visible in crude extracts and bound a His-tag antibody (
Ni-NTA columns were used to partially purify His-tagged CP51L from E. coli cells to assess lytic activity and host range of the endolysin. Strong lytic activity was shown by an effective drop in the OD600 reading within 6 min of addition of 1, 10 or 30 μg of partially-purified protein to frozen cells of C. perfringens (
The partially purified CP51L was stable at 4° C. for several months and its activity was not affected by a 30 min incubation at 37° C. or 45° C. However, treatment at 65° C. for 30 min, or 42° C. overnight, or a pasteurization treatment at 72° C. for 20 s all abolished lytic activity. Stability at room temperature was monitored over 3 weeks, the activity remaining unchanged after 8 days compared to a sample stored at 4° C. After 15 d and 22d storage at room temperature the endolysin activity had reduced to C. 83% and C. 62% respectively, compared to the protein samples stored at 4° C.
Lysis assays were performed to examine the activity of CP51L when cells were suspended in broth media such as BHI+C or CM at pH 9 (
The host range of CP51L was tested using frozen cells of C. perfringens strains and a variety of clostridial and non-clostridial species by incubating the cells with 1 μg of partially-purified lysin. All of the 25 C. perfringens strains tested were sensitive to the endolysin, but the speed of lysis varied greatly (Table 1). In some strains the drop in turbidity was immediate, while with others there was a lag of up to C. 20 mins before lysis was detected. Sensitivity to the positive control lysozyme was also variable, with some strains being insensitive while others exhibited an effective lysis. There was no apparent relationship between sensitivity to the CP51L lysin and to lysozyme. In addition, CP51L also showed activity against C. bifermentans, C. acetobutylicum, C. beijerinckii, B. subtilis and B. cereus cells (Table 1) but failed to cause lysis of a range of other bacteria (listed in Materials and Methods).
L. johnsonii as an Endolysin Delivery Vehicle
The cp51L gene was cloned into an expression and delivery system which combined expression from the PnisA in conjunction with the chromosomally-located signal transduction nisRK genes with a signal peptide to secrete the lysin. The His-tagged CP51L endolysin was not detectable by Western blotting in 10 μg of crude protein extracts from L. johnsonii carrying the lysin expression construct (FI10744-L). However, the FI10744-L extracts demonstrated a lytic activity that was absent from the extracts of the empty vector control (FI10744-V) (
C. perfringens is widely distributed in the intestine of animals, especially poultry, and can be pathogenic to the host. It is responsible both for severe infections and important economic losses (22, 38); targeting the pathogen in poultry will have impacts not just on poultry welfare and production but also on carriage in the food chain and the incidence of human infections. In this study Applicants have examined the ability of a novel endolysin to kill C. perfringens cells and to control populations in co-culture when secreted by a probiotic strain of L. johnsonii.
The gene for CP51L is located within a prophage which is inducible by mitomycin C (see Examples, below). In common with 3 other endolysins which have been demonstrated to show activity against C. perfringens, it is predicted to be an amidase (40, 49). This enzymatic type is encoded commonly in prophage lysin-like enzymes found in the genomes of C. perfringens, and the presence of an SH3 domain is also very prevalent in these proteins (39). In CP51L the predicted SH3_3 domain is present in the C-terminal region commonly associated with endolysin specificity (8, 25). The C-terminal and the central region between the amidase domain and the SH3_3 domain showed high similarity to other putative amidases from C. perfringens, suggesting that these regions may represent common binding domains for C. perfringens cell wall recognition.
When expressed from E. coli, CP51L showed a potent lytic activity against C. perfringens NCTC 3110, a Type B strain, and was also active against all 25 other C. perfringens strains tested. However, activity varied widely between strains, even between 2 strains of the same Type A (NCTC 8238 and 8239). Although sensitivity to the lysozyme positive control also varied to a lesser extent between strains, there was no relationship between the speed of lysis with lysozyme and that with CP51L, suggesting that the variation in CP51L activity may be associated with efficacy of recognition by the cell wall binding domains rather than with access of lytic enzymes to the peptidoglycan or intrinsic resistance of cells to lysis. The sensitivity of other selected clostridial and Bacillus spp. might indicate shared binding targets in the cell walls of these species. This large difference in activity between strains was also found with the amidase endolysins Ply3626, PIyCP39O and PlyCP26F and the glucosyl hydrolase endolysins SM101 and PlyCM (34, 39-40, 49), although CP51L shows a slightly less stringent host specificity than most of these lysins. The high stability, activity at physiologically relevant pH values and temperatures and the ability to lyse cells in complex media suggests that the endolysin is able to function in GI tract conditions, without causing collateral damage to the normal microflora.
L. johnsonii FI9785 was previously shown to reduce colonization and shedding of C. perfringens in chickens after a single oral dose (20), making it an ideal tool for prophylaxis. The use of a delivery system incorporating the signal peptide SLPmod, which has previously been demonstrated to be effective in the delivery of interleukin-12 to the murine gut by Lactococcus lactis (7), gave successful production and secretion of a functional enzyme, as demonstrated by the lysis assays. Further, the combination of the nisin A promoter and the chromosomally located signal transduction genes gave constitutive production without the need for nisin induction, which is of great potential benefit for in vivo applications. Production in L. johnsonii was lower than that in E. coli and this is an area which may benefit from improvement. Another possibility is co-delivery or co-application with other antimicrobials—the combination of a phage endolysin with nisin achieved a strong synergistic effect against Staphylococcus aureus (9) and the ability of lactobacilli to produce a range of bacteriocins, in addition to their other probiotic properties (21), make them valuable candidates as vehicles to deliver specific antimicrobials to gut communities.
The probiotic L. johnsonii has the capability to exclude C. perfringens from the GI tract of poultry birds. In this study Applicants have demonstrated its ability to express a biologically active anti-C. perfringens endolysin, indicating that the engineered strain has the potential to be an even more effective control agent for this pathogen.
The problem of food-borne pathogens associated with meat consumption has a significant impact on both health and the economy. Clostridium perfringens is frequently found in food and the environment and produces potent toxins that have a negative impact on both human and animal health and particularly on the poultry industry. The probiotic Lactobacillus johnsonii FI9785 has been demonstrated to competitively exclude Clostridium perfringens in poultry. Applicants have investigated the interaction between wild type L. johnsonii FI9785 or an engineered strain expressing a cell wall hydrolysing endolysin and C. perfringens in vitro, using a batch culture designed to simulate human gastrointestinal tract conditions. Applicants' aim was to understand the mechanism of competitive exclusion and to assess the efficacy of the endolysin as a novel antimicrobial for the control of Clostridium perfringens in the gut environment. Co-cultures of L. johnsonii and C. perfringens indicated that acid production by the probiotic is important in pathogen control. The co-culture of the endolysin-secreting L. johnsonii with C. perfringens showed that the engineered strain had the potential to control the pathogen.
1.1 Clostridium perfringens—Pathogenesis and Control Strategies
Clostridium perfringens is a Gram-positive, spore forming, anaerobic bacterium that is widely distributed in the intestines of people and animals and is also common in the environment and easily found in a variety of foods. C. perfringens is classified into 5 types (A, B, C, D, E) based on toxin production (Miyamoto et al., 2012; Petit et al., 1999). It is the causative agent of several disease in humans and animals such as gas gangrene or necrotic enteritis (NE), and is also a common source of food poisoning (Keyburn et al., 2008; Lindstrom et al., 2011). It is the cause of both severe infections and important economic losses, especially in poultry (Lee et al., 2011; Scallan et al., 2011).
NE is one of the most important enteric diseases in poultry and causes high costs to the poultry industry. Antibiotics can be used for the control of NE in poultry, but in some cases the development of resistance by C. perfringens strains has been described (Johansson et al., 2004). The ban, in some countries, of the prophylactic use of antibiotics for the control of NE in poultry feed was initiated in response to the spread of antibiotic resistance in human and animal bacterial pathogens (Chapin et al., 2005). However the ban has increased the emergence of diseases that were previously controlled, such as NE in the poultry industry (Lee et al., 2011; Van Immerseel et al., 2009). For this reason a range of novel strategies to combat C. perfringens are being exploited such as vaccination (Lee et al., 2011), natural antimicrobials and enzymes (Liu et al., 2010; Timbermont et al., 2010; Zhang et al., 2010), probiotic microrganisms (Gil de los Santos et al., 2012; Kizerwetter-Swida and Binek, 2009; La Ragione et al., 2004) or bacteriophages (Miller et al., 2010).
1.2 Potential of Probiotics and Endolysins as Control Agents
Probiotics are defined by the World Health Organisation as ‘Live microorganisms, which when administered in adequate amounts confer a health benefit on the host’. Many of the known probiotics are lactic acid bacteria; they are normally derived from healthy intestinal microflora and can exert several positive effects (Lebeer et al., 2008). Lactobacilli have been showed to exert health benefits in both humans and animals, preventing or treating several diseases caused by pathogens. Meta-analysis showed that certain lactobacilli can prevent necrotizing enterocolitis in preterm neonates (Alfaleh et al., 2011), reduce the occurrence of C. difficile associated diarrhoea (McFarland, 2006) or reduce the Listeria monocytogenes count in acquired listeriosis (Archambaud et al., 2012). In animal models probiotics have been explored as alternatives to conventional antimicrobials to control problems such as pathogenic avian influenza (Seo et al., 2012) or disease caused by Salmonella enterica (Chen et al., 2012).
Different proprieties contribute to the positive effects of probiotics on health, including both interactions between microbes (competition for nutrients, the production of antimicrobial compounds, competitive exclusion) and effects on the host (Lebeer et al., 2008). In recent years, the development of probiotics engineered for the secretion of heterologous proteins has increased, showing potential for the treatment or prevention of disease. Probiotic microorganisms have been demonstrated as potential tools for the delivery of biologically active molecules such as interleukins (Bract et al., 2006; Fernandez et al., 2009) or antivirals (Moeini et al., 2011). They have also successfully expressed cell wall hydrolases such as endolysins (Turner et al., 2007). Endolysins are enzymes produced by bacteriophages in order to lyse host cells and release new virions.
Their lytic ability and specificity has been exploited to develop novel antimicrobial activities against a range of Gram positive pathogens (Fischetti, 2010). Although potent, endolysins are susceptible to proteases and delivery to the gastrointestinal (GI) tract is likely to be problematic. However, expression and secretion from lactic acid bacteria represents a method for continuous production and delivery in the gut environment. Endolysin CP51L has been shown to be active against C. perfringens in vitro and constitutive expression and export of this endolysin from the lactic acid bacterium L. johnsonii FI9785 has recently been demonstrated (see Examples, below).
1.3 Development of a Novel Control Strategy
In Applicants' study the activity of both an established probiotic and an engineered derivative was investigated for the control of C. perfringens. L. johnsonii FI9785 is a poultry-isolated strain which has been confirmed in vivo as a competitive exclusion agent against C. perfringens in chickens (La Ragione et al., 2004). In this work, Applicants have performed co-culture experiments to explore the ability of L. johnsonii to control C. perfringens. In addition, to improve the performance of L. johnsonii Applicants have investigated the ability of a strain engineered for the expression and delivery of endolysin CP51L to control C. perfringens in vitro in conditions designed to simulate the GI tract.
2.1 Bacterial Strains and Growth Conditions
C. perfringens strain NCTC 3110 was obtained from the National Collection of Type Cultures (Public Health Laboratory, London, UK). The strain was grown anaerobically overnight at 37° C. in BHI (Oxoid) supplemented with vitamin K [10 μl; 50 mg/l], hemin [5 mg/l], resazurin [1 mg/l], and L-cysteine[0.5 g/l]. Stocks were maintained at 4° C. in Robertson's cooked-meat medium (SGL, Corby, UK). Lactobacillus johnsonii strain FI9785 (La Ragione et al., 2004) and derivatives were grown in MRS broth or agar (Oxoid) at 37° C. Co-cultures and in vitro batch cultures were performed in chemostat media (CM: peptone water 2 g/l (Oxoid), yeast extract 2 g/l (Oxoid), NaCI 0.1 g/l, K2HPO4 0.04 g/l, KH2PO4. 0.04 g/l, MgSO4.7H2O 0.01 g/l, CaCl2.6H2O 0.01 g/l, NaHCO3 2 g/l, Tween 80 2 ml, hemin 0.02 g/l dissolved in 400 μl 1 M NaOH, vitamin K1 10 μl of 5% v/v aqueous solution, cysteine HCl 0.5 g/l, bile salts 0.5 g/I, 1% glucose, all from Sigma (Mandalari et al., 2007)).
The endolysin delivery system involved Lactobacillus johnsonii strain FI10744, constructed by the insertion of the nisRK genes into the chromosome, expressing plasmid pUK200-PnisA-slpmod-6×His-cp51l (FI10744-L) which allowed constitutive expression and signal peptide-mediated secretion of histidine-tagged endolysin (see Examples, below). Strain FI10744-V contained the empty pUK200 vector as a control; both strains were grown with chloramphenicol (7.5 μg/ml) to select for the plasmid except during co-culture assays, where no antibiotics were present.
2.2 In Vitro Batch Culture and Viability Assessment
Batch cultures were carried out over 48 h according to the procedure described previously (Mandalari et al., 2007). Briefly, each reactor consisted of a 300 ml water jacketed fermentation vessel (Shoam Scientific, UK), containing 250 ml chemostat medium, magnetically stirred and maintained at 37° C. by a circulating water bath. Culture pH was automatically controlled and maintained by addition of 1M HCl or 1M NaOH. Anaerobic conditions were maintained by sparging the vessels with oxygen-free nitrogen. Vessels with media were autoclaved at 121° C. for 15 min then equilibrated overnight to create anaerobic conditions prior to the inoculation of the microorganisms. Initial mini-batch culture tests were performed in 25 ml culture of the same medium under anaerobic conditions at 37° C. but without stirring or pH control. Bacteria were inoculated from overnight cultures in MRS or BHI with supplements (L. johnsonii and C. perfringens respectively) with antibiotic selection for plasmids where appropriate.
At specific time points samples were withdrawn and serial dilutions were performed anaerobically in PBS. Both diluted and undiluted samples were plated in triplicate using a spot-plate method on BHI agar with gentamycin (20 μg/ml) to enumerate C. perfringens, or MRS agar grown aerobically and containing colistin sulphate (50 μg/ml) to select for L. johnsonii. Strains L. johnsonii FI10744-L and FI10744-V were plated on MRS with or without chloramphenicol (7.5 μg/ml) to assess plasmid loss in the absence of selection and were incubated aerobically. Plates were incubated for 24 to 48 h and results were calculated as the total colony forming units (cfu) from triplicate spots.
2.3 Lysis Assay
Endolysin-mediated lysis of C. perfringens NCTC 3110 cells resuspended in phosphate buffered saline (PBS) was assessed by turbidity reduction assay as previously described (Mayer et al., 2008). At the end of the batch culture experiments, spent medium was tested to assess endolysin activity. To assess the stability of the endolysin in spent media, endolysin protein was produced from cells of Escherichia coli expressing construct pET15b-cp 51l (see Examples, below) and partially purified using a nickel-nitrilotriacetic acid (Ni-NTA) Fast Start kit (Qiagen) as described previously (Mayer et al., 2008). 1 ml of spent medium was harvested, filtered and endolysin was added to a final concentration of 0.33 μg/pl. 30 μl samples (equivalent to 10 μg) were taken at intervals for 96 h and used in turbidity reduction assays, with elution buffer EB (50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole pH 8) as a negative control. Assays were performed in duplicate.
3.1 Co-Culture of C. perfringens and L. johnsonii
The ability of the probiotic L. johnsonii FI9785 to inhibit the growth of C. perfringens in vitro was evaluated in co-culture experiments carried out in a complex growth medium formulated to simulate the conditions of the GI tract, where both the microorganisms could survive but where growth was limited.
Preliminary tests in 25 ml without pH control revealed a significant decrease in the viability of C. perfringens when co-inoculated with L. johnsonii compared to pure culture (
3.2 In Vitro Batch Fermentation
To study the interaction of the microorganisms, the co-culture of C. perfringens with L. johnsonii FI9785 was repeated in larger volumes in an in vitro batch fermentation model with a controlled pH. At time 0, cells were added from overnight cultures to give an estimated final concentration of c. 2×106 cells/ml of each bacterium in vessel 1 or 2×106 cells/ml of pure cultures in control vessels 2 (C. perfringens) and 3 (L. johnsonii). The first test was conducted for 24 h at a pH range between 6.8 and 7.2 (
3.3 Effect of Endolysin Delivery on C. perfringens
Batch culture experiments were repeated using a strain of L. johnsonii (FI10744-L) which had been engineered for antimicrobial production. This strain expressed a histidine-tagged endolysin CP51L from the vector pUK200-PnisA-sIpmod-6×His-cp51l which produced constitutive expression via an interaction between the nisin A promoter and chromosomally located nisRK genes and export from the cell via the signal peptide SLPmod (see Examples, below). The effect of this strain in co-culture with C. perfringens was compared to that of strain FI10744-V carrying an empty vector control. As the previous experiments had indicated a lower ability of L. johnsonii to survive and replicate in batch culture, L. johnsonii strains were inoculated at a c. 10-fold excess to C. perfringens and a pH range between 5.8 and 6.2 was used to improve their viability over the 48 h test period. L. johnsonii FI10744-L did show some ability to reduce the numbers of C. perfringens compared to a co-culture containing L. johnsonii FI10744-V, but results varied with replicate experiments (
3.4 Endolysin Stability
No lytic activity was observed when spent medium was tested to assess endolysin activity, indicating that the lysin concentration was not sufficient for effective lysis in the turbidity reduction assays. Lysis assays conducted in spent media showed no drop in activity for the first 6 h, followed by a slight decrease at 8 h; after 24 h the activity was reduced by 50% and at 72 h and 96 h lysis was similar to that in the buffer control (
In this work Applicants have investigated the potential of the probiotic competitive exclusion agent L. johnsonii and an engineered derivative to control the pathogen C. perfringens in vitro. To simulate the conditions of the GI tract, a batch fermentation system was used together with a complex medium. Preliminary work showed that although pathogen numbers were clearly reduced in co-culture, the addition of pH controls in batch culture removed this effect, indicating that the production of acids may be an important feature of the ability of L. johnsonii to exclude C. perfringens. The production of lactic acid by lactobacilli is established as an important facet of their antimicrobial nature (Lebeer et al., 2008), and the pH of the medium also clearly had an effect on L. johnsonii viability.
To attempt to improve control of the pathogen, a strain which produced and secreted biologically active endolysin CP51L was tested. This system exported the endolysin via a signal peptide which has been proven to effectively deliver murine interleukin-12 from Lactococcus lactis to the murine gut (Fernandez et al., 2009). In 2 out of 3 co-culture experiments, expression of the endolysin led to a reduction in the pathogen counts compared with co-cultures of C. perfringens with the L. johnsonii vector control. Lytic activity of the endolysin in the chemostat medium has previously been demonstrated (see Example B) and although the concentration of the endolysin in the medium from the co-culture experiments was not concentrated enough to detect using turbidity reduction assays, these experiments demonstrate the potential of the endolysin to improve control of C. perfringens. Although batch cultures are stirred continuously one can expect to obtain areas where the clostridial cells are not inevitably next to L. johnsonii cells and so are able to survive and multiply. In all batch co-cultures L. johnsonii strains failed to maintain their starting inoculum levels and this may also have had an effect on their ability to control C. perfringens. Activity in the GI tract, where L. johnsonii survives well, would be expected to function better.
This work confirms that L. johnsonii has the potential to control C. perfringens and that endolysin expression and delivery can increase the efficacy of the probiotic.
C. perfringens NCTC 3110
C. perfringens NCTC 8238
C. perfringens NCTC 8239
C. perfringens 5146-97b
C. perfringens 5810-97b
C. perfringens 2144-98b
C. perfringens 2118-98b
C. perfringens 2536-01b
C. perfringens 2551-01b
C. perfringens 4519-98b
C. perfringens 2151-88b
C. perfringens 6081-97b
C. perfringens 3011-98b
C. perfringens 2540598b
C. perfringens F3278
C. perfringens N151/151
C. perfringens N 147/155A
C. perfringens DP2
C. perfringens DP3
C. perfringens DP5
C. perfringens CH1
C. perfringens CH2
C. perfringens FD00389c
C. perfringens FD00413c
C. perfringens FD00412c
B. cereus NCIMB 11796
B. subtilis ATCC 6633
C. acetobutylicum BL75141
C. bifermentans NCTC 13019
C. beijerinckii NCIMB 8052
aPercent drop in OD600 in 4 min linear lysis (mean ± standard deviation),
bDr M. Brett, (HPA, UK)
cProf. R La Ragione (VLA, UK).
perfringens E str. JGS1987,
perfringens F262, 0.0)
perfringens B str. ATCC 3626,
Bacillus subtilis and
acidilactici DSM 20284, 2e−04)
pneumoniae SPNA45, 6e−35)
perfringens WAL-14572, 0.0)
perfringens C str. JGS1495,
perfringens F262, 0.0)
youngiae DSM 17734, 3e−12);
difficile ATCC 43255 1e−06]
bacterium9_1_43BFAA, 4e−53)
bacterium 9_1_43BFAA, 2e−39)
botulinum H04402 065, 4e−05)
plantarum subsp. plantarum
sphaeroides 2.4.4, 2e−19)
perfringens E str. JGS1987,
botulinum A2 str. Kyoto, 2e−35)
perfringens E str. JGS1987,
australicus RC3, 1e−54)
perfringens E str. JGS1987,
perfringens WAL-14572,
perfringens E str. JGS1987,
hafniense Y51, 2e−25)
ac denotes reverse strand,
bamino acids,
cHP, hypothetical protein
The invention is further described by the following numbered paragraphs:
1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1, or a fragment, variant, derivative or fusion thereof which is capable of binding specifically to and/or lysing cells of Clostridium perfringens.
2. A polypeptide according to Paragraph 1 wherein the fragment, variant, derivative or fusion thereof exhibits at least 60% identity to the amino acid sequence of SEQ ID NO: 1.
3. A polypeptide according to any one of the preceding paragraphs wherein the fragment, variant, derivative or fusion thereof is not a naturally occurring lysin of a bacteriophage of Clostridium perfringens.
4. An isolated polypeptide according to any one of the preceding paragraphs capable of binding specifically to cells of Clostridium perfringens.
5. An isolated polypeptide according to any one of the preceding paragraphs capable of lysing cells of Clostridium perfringens.
6. An isolated polypeptide according to any one of the preceding paragraphs capable of binding specifically to and lysing cells of Clostridium perfringens.
7. An isolated polypeptide according to any one of the preceding paragraphs comprising the amino acid sequence of SEQ ID NO: 1.
8. An isolated polypeptide according to Paragraph 7 consisting of the amino acid sequence of SEQ ID NO: 1.
9. An isolated polypeptide according to any one of the preceding paragraphs comprising or consisting of a fragment of the amino acid sequence of SEQ ID NO: 1.
10. An isolated polypeptide according to Paragraph 9 wherein the fragment comprises at least 50 contiguous amino acids of SEQ ID NO: 1, for example at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375 or 376 contiguous amino acids of SEQ ID NO: 1.
11. An isolated polypeptide according to Paragraph 9 or 10 wherein the fragment comprises or consists of the enzymatic (lytic) domain of SEQ ID NO: 1.
12. An isolated polypeptide according to any one of Paragraphs 9 to 11 wherein the fragment comprises or consists of the cell wall binding domain of SEQ ID NO: 1.
13. An isolated polypeptide according to any one of the preceding paragraphs comprising or consisting of a variant of the amino acid sequence of SEQ ID NO:1, or of a fragment thereof.
14. An isolated polypeptide according to Paragraph 13 wherein the variant comprises or consists of an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 1, or to a fragment thereof, more preferably at least 80% or 85% or 90% identity to said sequence, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to said amino acid sequence.
15. An isolated polypeptide according to any one of the preceding paragraphs comprising or consisting of a derivative of the amino acid sequence of SEQ ID NO:1, or of a fragment or variant thereof.
16. An isolated polypeptide according to any one of the preceding paragraphs comprising or consisting of a fusion of the amino acid sequence of SEQ ID NO: 1, or of a fragment, variant or derivative thereof.
17. An isolated polypeptide according to Paragraph 16 comprising or consisting of one or more additional amino acids inserted at the N- and/or C-termini of the amino acid sequence of SEQ ID NO: 1, or of a fragment, variant or derivative thereof.
18. An isolated polypeptide according to Paragraph 16 or 17 comprising or consisting of the cell wall binding domain of SEQ ID NO:1 and an enzymatic (lytic) domain different to that in SEQ ID NO: 1.
19. An isolated polypeptide according to any one of the preceding paragraphs wherein the polypeptide is capable of lysing cells of multiple strains of Clostridium perfringens.
20. An isolated polypeptide according to any one of the preceding paragraphs wherein the polypeptide is capable of lysing one or more cell types selected from the group consisting of cells of Bacillus sp. and other Clostridium sp.
21. An isolated peptide according to Paragraph 20 wherein the polypeptide is capable of lysing cells of Bacillus sp. selected from the group consisting of Bacillus cereus (e.g., B. cereus NCIMB 11796) and B. subtilis (e.g., B. subtilis ATCC 6633).
22. An isolated peptide according to Paragraph 20 wherein the polypeptide is capable of lysing cells of other Clostridium sp. selected from the group consisting of C. acetobutylicum (e.g., C. acetobutylicum BL75141), C. bifermentans (e.g., C. ifermentans NCTC 13019) and C. beijerinckii (e.g., C. beijerinckii NCIMB 8052).
23. An isolated polypeptide according to any one of the preceding paragraphs wherein the polypeptide is substantially incapable of lysing cells selected from the group consisting of Anaerococcus hydrogenalis DSMZ 7454, Bacillus amyloliquefaciens 0880, Bifidobacterium adolescentis DSMZ 20083, Bifidobacterium angulatum DSMZ 20098, Bifidobacterium bifidum DSMZ 20082, Bifidobacterium longum DSMZ 20219, Bifidobacterium pseudocatenulatum DSMZ 20438, Clostridium cellobioparum DSMZ 1351, Clostridium coccoides NCTC 11035, Clostridium colinum DSMZ 6011, Clostridium difficile NCTC 11204, Clostridium innocuum DSMZ 1286, Clostridium leptum DSMZ 753, Clostridium nexile DSMZ 1787, Clostridium ramosum DSMZ 1402, Clostridium sordellii NCTC 13356, Clostridium sporogenes ATCC 7886, Clostridium tyrobutyricum NCIMB 9582, Enterococcus faecalis FI10734, Enterococcus hirae FI10477, Eubacterium barkeri DSMZ 1223, Lactobacillus casei FI10736, Lactobacillus johnsonii FI9785, Lactobacillus plantarum FI08595, Lactobacillus rhamnosus FI10737, Lactococcus lactis MG1363, Leuconostoc mesenteroides subsp. Mesenteroides ATCC 8293, Listeria innocua NCTC 11288, Listeria ivanovii NCTC 11007, Micrococcus luteus FI10640, Pediococcus pentosaceus FI10642, Pediococcus acidilactici FI10738, Salmonella enterica serovar Typhimurium FI10739, Salmonella enterica serovar Enteritidis FI10113 and Staphylococcus aureus FI10139.
24. An isolated polypeptide according to any one of the preceding paragraphs wherein the polypeptide is capable of lysing cells of Clostridium perfringens NCTC 3110 and/or NCTC 8238.
25. An isolated polypeptide according to Paragraph 24 wherein the polypeptide exhibits at least 10% of the lysis activity of the polypeptide of SEQ ID NO: 1 on cells of Clostridium perfringens NCTC 3110 and/or NCTC 8238, for example at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more.
26. An isolated polypeptide according to Paragraph 25 wherein the polypeptide exhibits at least 100% of the lysis activity of the polypeptide of SEQ ID NO: 1 on cells of Clostridium perfringens NCTC 3110 and/or NCTC 8238, for example at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 500% or more.
27. An isolated polypeptide according to any one of the preceding paragraphs wherein the polypeptide is capable of lysing cells of pathogenic bacteria selectively.
28. An isolated polypeptide according to any one of the preceding paragraphs wherein the polypeptide is a recombinant polypeptide.
29. An isolated nucleic acid molecule encoding a polypeptide according to any one of Paragraphs 1 to 28.
30. A nucleic acid molecule according to Paragraph 29 wherein the nucleic acid molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 2.
31. A vector comprising a nucleic acid molecule according to Paragraph 29 or 30.
32. A vector according to Paragraph 31 wherein the vector is an expression vector.
33. A vector according to Paragraph 31 or 32 wherein the vector is selected from the group consisting of pET15b and pACYC 184.
34. A host cell comprising a nucleic acid molecule according to Paragraph 29 or 30 or a vector according to any one of Paragraphs 31 to 33.
35. A host cell according to Paragraph 34 wherein the host cell is capable of expressing a polypeptide according to any one of Paragraphs 1 to 28.
36. A host cell according to Paragraph 34 or 35 wherein the host cell is a microbial cell.
37. A host cell according to any one of Paragraphs 34 to 36 wherein the host cell is a bacterial cell.
38. A host cell according to Paragraph 36 or 37 wherein the host cell is non-pathogenic.
39. A host cell according to any one of Paragraph 34 to 38 wherein the host cell is selected from the group consisting of cells of Escherichia coli, Lactococcus sp., Bacteroides sp, Lactobacillus sp., Enterococcus sp. and Bacillus sp.
40. A host cell according to Paragraph 38 wherein the host cell is a Lactococcus lactis or Lactobacillus johnsonii cell.
41. A host cell according to Paragraph 40 wherein the host cell is a Lactococcus lactis selected from the group consisting of L. lactis FI10676, L. lactis FI15876, L. lactis FI17847 and L. lactis UKLc10.
42. A host cell according to Paragraph 40 wherein the host cell is a Lactobacillus johnsonii selected from the group consisting of L. johnsonii FI9785, L. johnsonii FI10744, L. johnsonii FI10836 and L. johnsonii FI10844.
43. A method for producing a polypeptide according to any one of Paragraphs 1 to 28 comprising culturing a population of host cells comprising a nucleic acid molecule according to Paragraph 29 or 30 or a vector according to any one of Paragraphs 31 to 33 under conditions in which the polypeptide is expressed, and isolating the polypeptide therefrom.
44. A pharmacological composition comprising:
45. A pharmacological composition according to Paragraph 44 comprising a polypeptide according to any one of Paragraphs 1 to 28.
46. A pharmacological composition according to Paragraph 44 or 45 for oral administration.
47. A pharmacological composition according to Paragraph 44 to 46 wherein the polypeptide is microencapsulated.
48. A pharmacological composition according to any one of Paragraphs 44 to 47 capable of delivering the polypeptide to the GI tract.
49. A pharmacological composition according to any one of Paragraphs 44 to 48 comprising a nucleic acid molecule according to Paragraph 29 or 30 and/or a vector according any one of Paragraphs 31 to 33.
50. A pharmacological composition according to any one of Paragraphs 44 to 48 comprising a host cell according to any one of Paragraphs 34 to 42.
51. A pharmacological composition according to Paragraph 50 comprising a non-pathogenic bacterial host cell which is genetically engineered to express a polypeptide according to any one of Paragraphs 1 to 28 and to release said polypeptide upon reaching a predetermined location within the GI tract.
52. A pharmacological composition according to any one of Paragraphs 44 to 51 comprising a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28.
53. A pharmacological composition according to any one of Paragraphs 44 to 52 wherein the composition allows sustained or slow-release of the polypeptide within the GI tract.
54. A polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28 for use in medicine.
55. Use of a polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28 in the preparation of a medicament for killing and/or inhibiting/preventing the growth of microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
56. A polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28 for use in killing and/or inhibiting/preventing the growth of microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
57. A method for killing and/or inhibiting/preventing the growth of microbial cells in a patient the method comprising administering to the patient a polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
58. Use of a polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28 in the preparation of a medicament for the treatment or prevention of a disease or condition associated with microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
59. A polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28 for use in the treatment or prevention of a disease or condition associated with microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
60. A method for the treatment or prevention of a disease or condition associated with microbial cells in a patient the method comprising administering to the patient polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
61. Use of a polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28 for killing and/or inhibiting/preventing the growth of microbial cells in vitro and/or ex vivo, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
62. The use according to Paragraph 55 or 58, the polypeptide, nucleic acid, vector, host or bacteriophage for use according to Paragraph 56 or 59 the method according to Paragraph 57 or 60 or the use according to Paragraph 61 wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis upon contact with a polypeptide of SEQ I D NO: 1.
63. The use according to Paragraph 55, 58 or 62, the polypeptide, nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59 or 62, or the method according to Paragraph 57, 60 or 62 or the use according to Paragraph 61 or 62 wherein the microbial cells are Clostridium perfringens cells.
64. The use according to Paragraph 55, 58, 62 or 63; the polypeptide, nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59, 62 or 63; or the method according to Paragraph 57, 60, 62 or 63, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with the endolysin of SEQ ID NO: 1.
65. The use according to Paragraph 55, 58 or 62 to 64; the polypeptide, nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59 or 62 to 64; or the method according to Paragraph 57, 60 or 62 to 64, wherein the polypeptide, nucleic acid, vector, host cell, bacteriophage or pharmacological composition is for use in an organism belonging to a taxonomic superclass or class selected from the group consisting of Chondrichthyes (cartilaginous fish), Osteichthyes (bony fish), Actinopterygii (ray-finned bony fish), Sarcopterygii (lobe-finned fish), Tetrapoda (four-limbed vertebrates), Amphibia (amphibians), Reptilia (reptiles), Ayes (birds) and Mammalia (mammals).
66. The use according to Paragraph 55, 58 or 62 to 65; the polypeptide nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59 or 62 to 65; or the method according to Paragraph 57, 60 or 62 to 65, wherein the taxonomic class is Aves (birds).
67. The use according to Paragraph 66; the polypeptide, nucleic acid, vector, host or bacteriophage for use according to Paragraph 66; or the method according to Paragraph 66, for use in poultry.
68. The use according to Paragraph 67; the polypeptide, nucleic acid, vector, host or bacteriophage for use according to Paragraph 67; or the method according to Paragraph 67, wherein the poultry is selected from the group consisting of chicken, duck, goose, ostrich, pigeon, turkey, pheasant, guinea fowl, partridge and quail.
69. The use according to Paragraph 55, 58 or 62 to 65; the polypeptide the nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59 or 62 to 65; or the method according to Paragraph 57, 60 or 62 to 65 wherein the taxonomic class is Mammalia (mammals).
70. The use according to Paragraph 69; the polypeptide, nucleic acid, vector, host or bacteriophage for use according to Paragraph 69; or the method according to Paragraph 69, wherein the mammal is selected from the group consisting of alpaca, banteng, bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit, reindeer, sheep, water buffalo, yak and human.
71. The use according to Paragraph 70; the polypeptide, nucleic acid, vector, host or bacteriophage for use according to Paragraph 70; or the method according to Paragraph 70, wherein the mammal is human.
72. The use according to Paragraph 55, 58 or 62 to 71; the polypeptide nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59 or 62 to 71; or the method according to Paragraph 57, 60 or 62 to 71, for the treatment of an existing disease or condition.
73. The use according to Paragraph 55, 58 or 62 to 71; the polypeptide nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59 or 62 to 71; or the method according to Paragraph 57, 60 or 62 to 71 for prophylaxis.
74. The use according to Paragraph 73; the polypeptide, nucleic acid, vector, host or bacteriophage for use according to Paragraph 73; or the method according to Paragraph 73, wherein the prophylaxis is primary or secondary prophylaxis.
75. The use according to Paragraph 74; the polypeptide, nucleic acid, vector, host or bacteriophage for use according to Paragraph 74; or the method according to Paragraph 74, wherein the prophylactic use is primary prophylaxis.
76. The use according to Paragraph 55, 58 or 62 to 75; the polypeptide nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59 or 62 to 75; or the method according to Paragraph 57, 60 or 62 to 75, wherein the polypeptide, nucleic acid, vector, host cell, bacteriophage or pharmacological composition is administered as a single dose.
77. The use according to Paragraph 55, 58 or 62 to 75; the polypeptide nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59 or 62 to 75; or the method according to Paragraph 57, 60 or 62 to 75, wherein the polypeptide, nucleic acid, vector, host cell, bacteriophage or pharmacological composition is administered as a plurality of doses (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more doses).
78. The use according to Paragraph 77; the polypeptide, nucleic acid, vector, host or bacteriophage for use according to Paragraph 77; or the method according to Paragraph 77, wherein the polypeptide, nucleic acid, vector, host cell, bacteriophage or pharmacological composition is administered in a frequency such that the polypeptide according to any one of Paragraphs 1-27 is continuously present in the GI tract of the patient.
79. The use according to Paragraph 55, 58 or 62 to 78; the polypeptide nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59 or 62 to 78; or the method according to Paragraph 57, 60 or 62 to 78, wherein the polypeptide, nucleic acid, vector, host cell, bacteriophage or pharmacological composition is administered in a frequency sufficient to prevent occurrence or recurrence of a disease or condition associated with microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with endolysin of SEQ I D NO: 1.
80. The polypeptide, nucleic acid, vector, host cell, bacteriophage or pharmacological composition for use according to Paragraph 15 wherein the polypeptide, nucleic acid, vector, host cell, bacteriophage or pharmacological composition is administered in a frequency sufficient to prevent occurrence or recurrence of a disease or condition associated with microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells.
81. The use according to Paragraph 55, 58 or 62 to 80; the polypeptide nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59 or 62 to 80; or the method according to Paragraph 57, 60 or 62 to 80, wherein the polypeptide, nucleic acid, vector, host cell, bacteriophage or pharmacological composition is a host cell or pharmacological composition comprising the same.
82. The use according to Paragraph 55, 58 or 62 to 80; the polypeptide nucleic acid, vector, host or bacteriophage for use according to Paragraph 56, 59 or 62 to 80; or the method according to Paragraph 57, 60 or 62 to 80, wherein the polypeptide, nucleic acid, vector, host cell, bacteriophage or pharmacological composition is a host cell.
83. A kit for detecting the presence of microbial cells in a sample, the kit comprising a polypeptide according to any one of Paragraphs 1 to 27, a nucleic acid molecule according to Paragraph 27 or 28, a vector according to any one of Paragraphs 29 to 31 or a host according to any one of Paragraphs 32 to 39, a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 27, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
84. The kit according to Paragraph 83 wherein the microbial cells are Clostridium perfringens cells.
85. The kit according to Paragraph 83 or 84 wherein the microbial cells are Clostridium perfringens NCTC 3110 and/or NCTC 8238 cells.
86. The kit according to any one of Paragraphs 83 to 85 wherein the polypeptide is immobilised on a surface.
87. The kit according to any one of Paragraphs 83 to 86 wherein the sample is a cell sample.
88. The kit according to any one of Paragraphs 83 to 87 wherein the sample is derived from a swab taken from a surface to be tested for contamination with microbial cells.
89. The kit according to any one of Paragraphs 83 to 88 further comprising a negative control sample.
90. The kit according to any one of Paragraphs 83 to 89 further comprising a positive control sample.
91. Use of a polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28, in the preparation of a diagnostic agent for a disease or condition associated with microbial cells selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
92. A polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28 for use in the diagnosis of a disease or condition associated with microbial cells selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
93. Use of a polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28, for detecting the presence of microbial cells in a sample in vitro and/or ex vivo, wherein the microbial cells selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
94. A method for the diagnosis of a disease or condition associated with microbial cells in a patient, the method comprising contacting a cell sample from a patient to be tested with a polypeptide according to any one of Paragraphs 1 to 28, a nucleic acid molecule according to Paragraph 29 or 30, a vector according to any one of Paragraphs 31 to 33 or a host according to any one of Paragraphs 34 to 42, or a bacteriophage capable of expressing a polypeptide according to any one of Paragraphs 1 to 28, and determining whether the cells in the sample have been lysed thereby, wherein the microbial cells are selected from the group consisting of Clostridium perfringens cells and other bacterial cells susceptible to lysis with said endolysin.
95. The use according to Paragraph 91, polypeptide for use according to Paragraph 92, use according to Paragraph 93 or method according to Paragraph 94 wherein the microbial cells are Clostridium perfringens cells.
96. The use according to Paragraph 91, polypeptide for use according to Paragraph 92, use according to Paragraph 93 or method according to Paragraph 94 wherein the microbial cells are Clostridium perfringens NCTC 3110 and/or NCTC 8238 cells.
97. An isolated polypeptide substantially as herein described with reference to the examples.
98. An isolated nucleic acid molecule substantially as herein described with reference to the examples.
99. A vector substantially as herein described with reference to the examples.
100. A host cell substantially as herein described with reference to the examples.
101. A method for producing a polypeptide substantially as herein described with reference to the examples.
Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1302042.5 | Feb 2013 | GB | national |
This application is a continuation-in-part application of international patent application Serial No. PCT/GB2014/050275 filed 31 Jan. 2014, which published as PCT Publication No. WO 2014/122435 A1 on 14 Aug. 2014, which claims benefit of GB patent application Serial No. 1302042.5 filed 5 Feb. 2013. The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
| Number | Name | Date | Kind |
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| 7371375 | Zimmer et al. | May 2008 | B2 |
| 8962297 | Seal et al. | Feb 2015 | B2 |
| 20050153415 | Zimmer et al. | Jul 2005 | A1 |
| 20120052546 | Seal | Mar 2012 | A1 |
| 20120134972 | Mayer | May 2012 | A1 |
| 20160040148 | Mayer et al. | Feb 2016 | A1 |
| Number | Date | Country |
|---|---|---|
| 03066845 | Aug 2003 | WO |
| 2010003943 | Jan 2010 | WO |
| 2010136754 | Dec 2010 | WO |
| 2012030535 | Mar 2012 | WO |
| Entry |
|---|
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| Zimmer et al., Appl. Environ. Microbiol. 68:5311-5317, 2002. |
| Gervasi et al., Appl. Microbiol. Biotechnol. 98:2495-2505, Aug. 2013. |
| GenBank Accession No. KC237729, Apr. 2013, 17 pages. |
| Sang, H., Mechanisms of Development 121:1179-1186, 2004. |
| International Search Report dated Jun. 2, 2014, which issued during prosecution of International Application No. PCT/GB2014/050275. |
| Database UniProt “Subname: Full=Putative N-acetylmuramoyl-L-alanine amidase” retrieved from EBI Accession No. UNIPROT: H7CV24, May 16, 2012. |
| Gervasi, et al. “Expression and delivery of an endolysin to combat Clostridium perfringens” Applied Microbiology and Biotechnology, Aug. 2013, 98(6):2495-2505. |
| Nariya, et al. “Identification and characterization of a putative endolysin encoded by episomal phage phiSM101 of Clostridium perfringens” Applied Microbiology and Biotechnology, Apr. 2011, 90(6):1973-1979. |
| Nowell, et al. “Genome Sequencing and Analysis of a Type A Clostridium perfringens Isolate from a Case of Bovine Clostridial Abomasitis” PLOS One, Mar. 2012, 7(3):e32271. |
| Schmitz, et al. “Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens” Applied Microbiology and Biotechnology, Mar. 2011, 89(6):1783-1795. |
| Simmons, et al. “Recombinant Expression of Two Bacteriophage Proteins That Lyse Clostridium perfringens and Share Identical Sequences in the C-Terminal Cell Wall Binding Domain of the Molecules but Are Dissimilar in Their N-Terminal Active Domains” Journal of Agricultural and Food Chemistry, Oct. 2010, 58(19)10330-10337. |
| Annex A: Comparison between Seq Id No. 1 (i.e. CP51L) and D1 (i.e. p1y3626), Apr. 20, 2017. |
| Annex B: Comparison between Seq Id No. 1 (i.e. CP51L) and D2 (i.e. plyS9) Apr. 20, 2017. |
| Annex C: Comparison between Seq Id No. 1 (i.e. CP51L) and plyCP39O of D3 and D4, Apr. 20, 2017. |
| Annex D: Comparison between Seq Id No. 1 (i.e. CP51L) and plyCP26F of D3 and D4, Apr. 20, 2017. |
| Annex E: Comparison between Seq Id No. 1 (i.e. CP51L) and D6 (i.e. plyCM), Apr. 20, 2017. |
| Number | Date | Country | |
|---|---|---|---|
| 20160017307 A1 | Jan 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/GB2014/050275 | Jan 2014 | US |
| Child | 14818927 | US |
