Patent Application
20140286904
This invention relates to an antimicrobial peptide having potent antibacterial activity against a range of bacteria, a nucleic acid encoding the antimicrobial peptide, a strain of Lactobacillus salivarius bacteria capable of expressing the antimicrobial peptide, an immunity protein conferring resistance to the antimicrobial effects of the antimicrobial peptide and a nucleic acid encoding the cognate immunity protein, and recombinant bacteria transformed with a nucleic acid encoding the antimicrobial peptide and/or a nucleic acid encoding the cognate immunity protein.
The genus Lactobacillus contains in excess of 145 species whose habitats range from the extremities of soil and plants to the mammalian gastrointestinal tract (GIT). These bacteria are renowned for the rich diversity of antimicrobial peptides (bacteriocins) they produce, each of which is assigned a name specific to the individual producing member species. Additionally, these antimicrobial peptides show great diversity with respect to structure and mode of action varying from extensively post-translationally modified lantibiotics such as plantaricin C to large unmodified heat labile proteins such as helveticin.
Lactobacillus salivarius is a species particularly associated with the mammalian GIT, and has a number of associated probiotic attributes. Several studies have revealed favourable immunomodulatory properties of certain L. salivarius strains. The human ileal isolate L. salivarius UCC118 as well as L. salivarius CECT 5713, isolated from a mother and child pair, were both found to reduce colonic inflammation in animal models of colitis reducing the levels of pro-inflammatory cytokines such as TNF-α and IL-12. Likewise, L. salivarius-mediated stimulation of the anti-inflammatory cytokine IL-10 reduced lipopolysaccharide (LPS)-induced inflammatory responses in murine bone-marrow-derived macrophages in vitro. Interestingly, oral administration of L. salivarius UCC118 was also reported to reduce tumour development and mortality in IL-10 knockout mice. Pre-treatment of intestinal epithelial cells with L. salivarius UCC118 was found to significantly reduce Salmonella typhimurim-induced pro-inflammatory responses (IL-8 production) in vitro. In addition, a strain of L. salivarius was among six select strains chosen to formulate a multi-species probiotic for combating disease in critically ill patients due to its positive immunomodulatory and potent antimicrobial activity. The ability of a L. salivarius component of another five-strain probiotic combination, L. salivarius DPC6005, to dominate over four co-administered strains within the porcine ileal digesta and mucosa. This strain produced a potent antimicrobial activity of due to production of the antimicrobial peptide salivaricin P.
L. salivarius produces a range of antimicrobial compounds which have exhibited inhibitory activity toward several important gastrointestinal pathogens. Indeed, acid production mediated by L. salivarius induced reductions in the prevalence of peridontopathic pathogens in the oral cavity. Indeed, the corresponding strains were purported to be effective for the probiotic treatment of periodontal diseases. Strain-specific inhibition of Helicobacter pylori was also partly ascribed to acid production by inhibitory L. salivarius strains. The potent activity of L. salivarius CECT 5713 was ascribed to a combination of factors including acid and hydrogen peroxide production. This strain was also capable of inducing mucin production in HT29 cells in vitro. Adherence to hog mucin and co-aggregation with Salmonella (thereby reducing adhesion of the pathogen to mucin), was also demonstrated by strain CECT 5713 and likely contributed to the protective effect of this strain against Salmonella infection in a murine model. Furthermore, anti-staphylococcal activity associated with strain CECT 5713 may suggest its use as a preferential alternative to conventional antibiotics in the treatment of infectious mastitis in women during lactation. This effect was also a purported consequence of acid and hydrogen peroxide production. However, the presence of the genetic determinants for production of the abp118 antimicrobial peptide were recently revealed in the genome of L. salivarius CECT 5713, the expression of which may also be a contributing factor to the potent antimicrobial activity of this strain. Recent safety assessments of strain CECT 5713 in an animal model, six-month old infants and healthy adults confirmed the beneficial immune modulatory properties and the safety of this strain for human consumption.
The range of characterized antimicrobial peptides produced by L. salivarius extends from the class IIa pediocin-like antimicrobial peptide OR-7 to two-component class IIb antimicrobial peptides, abp118, salivaricin P and variants thereof, as well as the recently described salivaricin T and class IId linear non-pediocin-like antimicrobial peptides such as salivaricin B. Each of these antimicrobial peptides has demonstrated inhibitory activity towards gastrointestinal or urogenital pathogens. Significantly, L. salivarius strains NRRL B-30514 and UCC118 have demonstrated OR-7- and abp118-mediated in vivo protection against Campylobacter jejuni and Listeria monocytogenes infection, respectively.
O'Shea et al. (Characterization of enterocin- and salivaricin-producing lactic acid bacteria from the mammalian gastrointestinal tract; FEMS Microbiol Lett, vol. 291(1), pp. 24-34 (2009)) describes the isolation of a number of antimicrobial producing LAB from the mammalian gut, including a strain L. salivarius DPC6502 that is characterised as expressing a two-component class II salivaricin P-like bacteriocin and of having a spectrum of inhibition that is restricted to closely relates strains of lactic acid bacteria (LAB). International Patent Publication No. WO 98/35014 describes human isolates L. salivarius UCC1, now known as L. salivarius AH4331, and L. salivarius UCC118 and variants thereof having the same antimicrobial and adhesive properties as said L. salivarius UCC1 and L. salivarius UCC118. Arihara et al. (Salivacin 140, a novel bacteriocin from lactobacillus salivarius subsp. Salicinius T140 active against pathogenic bacteria; Letters in Applied Microbiology, vol. 22, pp. 420-424 (1996)) describe the isolation of strain L. salivarius T140 which produces salivaricin 140.
It is an object of the present invention to provide alternative antimicrobial peptides which have a broad host range and which can function as a probiotic property.
Broadly, this invention provides a novel broad spectrum antimicrobial peptide, designated Bactofensin LS1, with potent anti-L. monocytogenes and anti-S. aureus activity which has been identified in L. salivarius. Genomic sequence analysis of the producing strain revealed the genetic determinants responsible for antimicrobial peptide production. The antimicrobial peptide locus encodes a highly basic antimicrobial peptide and an extremely unusual immunity system, and in this respect Bactofensin LS1 bears a closer resemblance to certain eukaryotic cationic antimicrobial peptides than to bacteriocins produced by bacteria. Indeed, the amino acid sequence of the cationic antimicrobial peptide did not display significant homology with previously characterized bacteriocins. In addition, Bactofensin LS1 displayed antimicrobial activity at micromolar concentrations, as do other eukaryotic antimicrobial peptides.
The invention relates to an antimicrobial peptide of SEQ ID NO: 2 (Bactofensin LS1), or a variant thereof having at least 90% sequence identity with SEQ ID NO: 2, and wherein the antimicrobial peptide of SEQ ID NO: 2, or the variant thereof, have potent antibacterial activity against Listeria monocytogenes and Staphylococcus aureus. The antimicrobial peptide of SEQ ID NO: 2, and variants thereof as defined above, are hereafter referred to as “antimicrobial peptide of the invention”.
Ideally, the antimicrobial peptide of the invention has an antibacterial activity against Listeria monocytogenes and Staphylococcus aureus of at least 40 μM, 30 μM, 20 μM, 10 μM, 9 μM, 8 μM, 7 μM, 6 μM or 5 μM.
The invention also provides an isolated antimicrobial peptide having potent antibacterial activity against Listeria monocytogenes and Staphylococcus aureus, produced by an isolated strain of the invention, wherein the variant retains the phenotypic characteristics of the isolated bacteria, and expresses an antimicrobial peptide having potent antibacterial activity against Listeria monocytogenes and Staphylococcus aureus.
The invention also relates to a nucleic acid encoding the antimicrobial peptide of the invention.
The invention also relates to a nucleic acid having a sequence of SEQ ID NO: 1, or a variant thereof having at least 90% sequence identity with SEQ ID NO: 1, and wherein the nucleic acid of SEQ ID NO: 1, or the variant thereof, encode an antimicrobial peptide having potent antibacterial activity against Listeria monocytogenes and Staphylococcus aureus. The nucleic acid of SEQ ID NO: 1, and variants thereof as defined above, are hereafter referred to as “nucleic acid of the invention”.
The Applicants have also surprisingly discovered a gene in Lactobacillus salivarius DPC6502 strain which encodes a protein that confers immunity on the host strain to the antibacterial effects of the antimicrobial peptide of the invention (termed “cognate immunity protein”). Thus, the invention also relates to an isolated protein having an amino acid sequence of SEQ ID NO: 8, or an isolated variant thereof comprising a sequence having at least 90% sequence identity with SEQ ID NO: 8, wherein the isolated protein or variant thereof is capable of conferring immunity on a host strain, typically a Lactobacillus host strain, ideally a Lactobacillus salivarius host strain, to the antibacterial effects of the antimicrobial peptide of the invention. The isolated protein of SEQ ID NO: 8, and isolated variants thereof, are hereafter referred to as “cognate immunity proteins of the invention”.
The invention also relates to a nucleic acid encoding a cognate immunity protein of the invention, for example a nucleic acid comprising a nucleic acid of SEQ ID NO: 7.
The invention also provides a recombinant vector comprising one or more of: a nucleic acid of the invention; a nucleic acid encoding an antimicrobial peptide of the invention; and a nucleic acid encoding a cognate immunity protein of the invention. In a particularly preferred embodiment of the invention, the recombinant vector comprises a nucleic acid encoding an antimicrobial peptide of the invention (for example a nucleic acid of SEQ ID NO: 1 or a nucleic acid encoding an antimicrobial peptide of SEQ ID NO: 2); and a nucleic acid of SEQ ID NO 7 or a nucleic acid encoding a cognate immunity protein of the invention (for example a nucleic acid encoding a protein of SEQ ID NO: 8).
The invention also relates to a host cell transformed by a recombinant vector of the invention (hereafter “host cell of the invention”).
The invention also relates to an antimicrobial peptide of the invention for use as a medicament.
The invention also relates to an antimicrobial peptide of the invention for use as an antibacterial agent or an antibiotic.
The invention also relates to an antimicrobial peptide of the invention for use in treating or preventing a disease or condition characterised by growth of Listeria or Staphylococcus aureus.
The invention also relates to a pharmaceutical composition comprising an antimicrobial peptide of the invention in combination with a suitable pharmaceutical excipient.
The invention also relates to an antibacterial or antibiotic formulation comprising the antimicrobial peptide of the invention.
The Applicants have surprisingly discovered that the isolate described in O'Shea et al (2009) was incorrectly characterised, and that the strain in fact produces a different bacteriocin to that described previously. Additionally, the antibacterial activity was incorrectly characterised as being restricted to closely related bacterial strains, whereas that Applicants have now surprisingly discovered that the antibacterial activity is broader than initially described, and includes activity against Listeria and Staphylococcus. The invention thus also relates to an isolated Lactobacillus salivarius DPC6502 strain as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 41840 on 9 May 2011 (deposited in the name of Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland), and variants thereof, wherein the variants typically retain the phenotypic characteristics of the isolated bacteria as described below, and wherein the isolated bacteria and variants thereof express an antimicrobial peptide of SEQ ID NO: 2 (designated Bactofensin LS1), or a variant thereof having at least 90% sequence identity to SEQ ID NO: 2, and wherein the antimicrobial peptide of SEQ ID NO: 2, or the variant thereof, have potent antibacterial activity against Listeria monocytogenes and Staphylococcus aureus (and typically also L. delbrueckii subsp. bulgaricus). The isolated strain of the invention and variants thereof are hereafter referred to as “isolated strain of the invention”.
The invention also relates to an isolated strain of the invention, or a transformed host cell, of the invention, for use as a probiotic culture.
The invention also relates to an isolated strain of the invention, or an antimicrobial peptide of the invention, for use as a biopreservative, for use in treating microbial infections and clinical infections, for use as a disinfectant, for use as an antibiotic in animal husbandry applications, for use in reducing the incidence of sensitive methicillin-resistant S. aureus (MRSA) in animals, especially pigs, and for use as an antimicrobial against L. delbrueckii subsp. bulgaricus, Listeria and Staphylococcus aureus.
The invention also relates to a formulation comprising an isolated strain of the invention, or an antimicrobial peptide of the invention, or a variant strain of the invention or a variant antimicrobial peptide of the invention. Suitably, the formulation is a pharmaceutical formulation and additionally comprises a pharmaceutically acceptable carrier. Alternatively, the formulation may be a comestible product, for example a food product. Ideally, the food product is a fermented food, for example a fermented dairy product such as a yoghurt. The formulation may also be a hygiene product, for example an antibacterial formulation, or a fermentation product such as a fermentation broth. For formulations that comprise the antimicrobial peptide of the invention, it will be appreciated that the peptide may be directly added to the formulation, or it may be produced in-situ in the formulation by a bacteria, for example the isolated strain of the invention or a variant thereof.
The invention also relates to a formulation of the invention for use in treating or preventing methicillin-resistant Staphalococcus aureus growth or infection in animals.
The antimicrobial produced by L. salivarius DPC6502 described herein differs considerably from that of L. salivarius UCC118. L. salivarius UCC118 produces a two-component class IIb bacteriocin abp118, which requires two peptides for activity and is hydrophobic in nature (Flynn et al., 2002). Li et al., (2007) reported the anti-Listeria activity of L. salivarius AH4331 and confirmed the presence of the abp118 structural genes in this strain, indicating that strain AH4331 has the same antimicrobial properties as L. salivarius UCC118. According to WO 98/35014, L. salivarius UCC118 exhibits a broad spectrum of antimicrobial activity but does not inhibit closely related lactobacilli. Bactofensin LS1 produced by L. salivarius DPC6502 is a one peptide class IId bacteriocin which is hydrophilic in nature which also has a broad antimicrobial spectrum but also inhibits strains closely related to the producer including L. salivarius UCC118. Therefore, L. salivarius DPC6502 does not have the same antimicrobial properties of L. salivarius UCC118 and thus cannot be classified as a variant thereof as described in WO 98/35014. Salivaricin 140 has not been described in detail. However, unlike L. salivarius DPC6502, Arihara et al., (1996) reported that the cell free supernatant of strain L. salivarius T140 does not exhibit antimicrobial activity in agar well diffusion assays. As strain L. salivarius T140 of Ahira et al. (1996) does not possess the same antimicrobial phenotype as L. salivarius DPC6502, it should not be considered a variant thereof.
The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:—
Broadly, the invention is based on the discovery of an antimicrobial peptide designated Bactofensin LS1 which has potent antibacterial activity against a broad spectrum of bacteria, including the pathogenic Listeria monocytogenes and Staphylococcus aureus strains. The amino acid sequence of Bactofensin LS1 is provided in SEQ ID NO: 2. A source of Bactofensin LS1 is a strain of Lactobacillus salivarius that was isolated from porcine jejunum bacterial isolates, designated Lactobacillus salivarius DPC6502, which has been deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 41840 on 9 May 2011 (in the name of Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland). The applicants have also discovered a protein expressed by Lactobacillus salivarius DPC6502 that confers immunity on its host against the antibacterial effects of Bactofensin LS1. This protein, designed an “immunity protein” has an amino acid sequence provided in SEQ ID NO: 8.
The term “variant” as applied to Lactobacillus salivarius DPC6502 should be understood to mean strains of Lactobacillus salivarius that express an antimicrobial peptide of SEQ ID NO: 2, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 2 having potent antibacterial activity against Listeria monocytogenes and Staphylococcus aureus. Preferably, the term variant should be understood to mean progeny (unmodified descendents), modified descendents, or derivatives of Lactobacillus salivarius DPC6502, for example strains which are genetically modified to alter the genotype of the bacteria, or strains which are altered by natural processes such as selection or serial passage.
The term “variants” as applied to the antimicrobial peptide of SEQ ID NO: 2 should be understood to mean peptides comprising a sequence having at least 90% sequence identity with SEQ ID NO: 2, and having potent antibacterial activity against Listeria monocytogenes and Staphylococcus aureus. The peptides will generally have less than 100 residues, 60 residues, 50 residues, 40 residues, 30 residues or 25 residues, and can include immature forms of the peptide of SEQ ID NO: 2, for example a precursor peptide such as in SEQ ID NO. 11. Suitably, the variants consist of, or comprise, a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 2. Variants of the antimicrobial peptide of SEQ ID NO: 2 shall preferably be taken to mean peptides having amino acid sequences which are substantially identical to SEQ ID NO: 2. Thus, for example, the term should be taken to include proteins or polypeptides that are altered in respect of one or more amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Preferably such alterations involve the insertion, addition, deletion and/or substitution of 5 or fewer amino acids, more preferably of 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only. Insertion, addition and substitution with natural and modified amino acids is envisaged. The variant may have conservative amino acid changes, wherein the amino acid being introduced is similar structurally, chemically, or functionally to that being substituted. Typically, Bactofensin LS1 proteins of the invention which have been altered by substitution or deletion of residues that are critical to its antibacterial activity will be excluded from the term “variant”. The term sequence identity comprises both sequence identity and similarity, i.e. a polypeptide sequence that shares 90% amino acid identity with SEQ ID NO: 2 is one in which any 90% of aligned residues are either identical to, or conservative substitutions of, the corresponding residues in SEQ ID NO: 2. The term “variant” is also intended to include chemical derivatives of Bactofensin LS1 protein, i.e. where one or more residues of Bactofensin LS1 is chemically derivatized by reaction of a functional side group. Also included within the term variant are Bactofensin LS1 molecules in which naturally occurring amino acid residues are replaced with amino acid analogues. Details of amino acid analogues will be well known to those skilled in the art.
Proteins and polypeptides (including variants and fragments thereof) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid. The proteins and peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984)).
The term “variant” as applied to a nucleic acid of SEQ ID NO: 1 (the nucleic acid encoding the antimicrobial peptide of SEQ ID NO: 2) should be understood to mean a nucleic acid that includes a sequence having at least 90% sequence identity with SEQ ID NO: 1, and which encode an antimicrobial peptide having potent antibacterial activity against Listeria monocytogenes and Staphylococcus aureus. The term should also be understood to mean nucleic acids that encode a variant antimicrobial peptide of the invention. Suitably, the variants have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1. The term also includes nucleic acids encoding the precursor peptide of SEQ ID NO. 11, for example, the nucleic acid of SEQ ID NO. 13.
The invention also relates to an isolated cognate immunity protein of the invention. This term should be understood to mean a protein of SEQ ID NO: 8, or an isolated variant thereof having 90% sequence identity with SEQ ID NO: 8, wherein the isolated protein or variant thereof is capable of conferring immunity on a host strain, typically a Lactobacillus host strain, ideally a Lactobacillus salivarius host strain, to the antibacterial effects of the antimicrobial peptide of the invention. Thus, a bacteria which expresses an immunity protein of the invention will be immune to the antibacterial effects of the antimicrobial peptide of the invention, or will have increased immunity compared to a bacteria which does not express the immunity protein of the invention. Suitably, the variants comprise a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 8.
The invention also relates to a recombinant vector comprising a nucleic acid encoding an antimicrobial peptide of the invention, a nucleic acid encoding a cognate immunity protein of the invention, or one or more nucleic acids encoding an antimicrobial peptide and a cognate immunity protein of the invention. The nucleic acids may be cloned as separate entities (i.e. distinct nucleic acid constructs), or in a same construct, under distinct promoter regions or in a single operon. Typically, the nucleic acids are cloned into a recombinant vector (for example a plasmid) which is capable of replicating in the host bacteria. Typical plasmids contain, in addition to the cloned insert, a selection gene (i.e. antibiotic resistance, a dye etc) and an origin of replication effective in the host bacterium. The plasmid may also comprise regulatory sequences, for example promoters, terminators and/or enhancers. Examples of such vectors are pNZ44 (McGrath S, Fitzgerald G F, van Sinderen D (2001) Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis. Appl. Environ. Microbiol. 67 (2): 608-616)) and pCI372 (Hayes F, Daly C, Fitzgerald G F (1990) Identification of the Minimal Replicon of Lactococcus lactis subsp. lactis UC317 Plasmid pCI305. Appl. Environ. Microbiol. 56: 202-209)). However, any recombinant vector suitable for replicating in a host bacteria known to the person skilled in the art may be used.
The nucleic acid may also be cloned into an integrative cassette suitable for integration into the genome of suitable host bacteria. Such an integrative cassette typically comprises a nucleic acid encoding an antimicrobial peptide of the invention or a cognate immunity protein of the invention, or both, linked to (or flanked by) one or several sequences allowing integration, preferably site-specific integration. Such sequences may be for instance nucleic acid sequences homologous to a targeted region of the genome, allowing integration through crossing over. Various techniques can be used to insert a nucleic acid into a host bacteria, for example through natural transformation or electroporation.
The host bacteria suitable for cloning the antimicrobial peptide and/or the cognate immunity protein may be selected from any host bacteria known to a person skilled in the art such as, for example, Lactococcus, Lactobacillus and Enterococcus.
In this regard, the term “animal husbandry-related applications” should be understood to mean that the antimicrobial peptide can be used for reducing the incidence of S. aureus or for the treatment of Staphylococcus aureus-related infections in animals such as for example mastitis, or as a probiotic trait for producing strains in animal feed applications (the antimicrobial peptide should contribute to the dominance of producing strains in the intestine by killing competing flora).
In the specification, the term “potent antibacterial activity against Listeria monocytogenes and Staphylococcus aureus” as applied to the antimicrobial peptides (AMPs) of the invention should be understood to mean that a concentration of AMP in the range of 0.1 μM to 50 μM is sufficient to inhibit the growth of both S. aureus DPC5246 and L. Monocytogenes NCTC11994 by 50% using the broth-based MIC50 assay described below. In this specification, the term “antimicrobial activity of at least XμM against Listeria monocytogenes and Staphylococcus aureus” as applied to the antimicrobial peptides (AMPs) of the invention should be understood to mean that a concentration of AMP of at most XμM is sufficient to inhibit the growth of both S. aureus DPC5246 and L. Monocytogenes NCTC11994 by 50% using the broth-based MIC50 assay described below.
In one embodiment, the term “cognate immunity protein” should be understood to mean a protein, which, when expressed, increases strain resistance to the antimicrobial peptide by increasing the content of esterified D-alanyl groups of techoic acids on the bacterial cell surface thereby resulting in a decrease in the net negative charge of the bacterial cell wall and reducing the potential of the initial electrostatic interaction of the cell with the cationic antimicrobial peptide.
In the specification, the term “isolated” should be considered to mean material removed from its original environment in which it naturally occurs, for example, in this instance a bacterial strain of the mammalian gut and/or an antimicrobial peptide designated Bactofensin LS1 and/or a cognate immunity protein. The removed material is typically cultivated, purified and cultured separately from the environment in which it was located. Thus, the purified isolated bacterial strain in this instance ideally does not contain any significant amounts of other bacterial strains. The isolated strain or variant of the invention may be provided in a viable or non-viable form, and in a culturable or non-culturable form. The invention also relates to an isolated strain of the invention, or variant thereof, of an antimicrobial peptide designated Bactofensin LS1 and a cognate immunity protein, in any format, for example a freeze-dried form, a suspension, a powder, or a broth, for example a fermentation broth or an extract from a fermentation broth that is enriched in the antimicrobial peptide of the invention.
The term “freeze-dried form” should be understood to mean that the strain, the antimicrobial peptide designated Bactofensin LS1 or the cognate immunity protein of the invention, optionally together with other ingredients including, for example, preservatives, is frozen and then the ice crystals in the frozen strain are sublimated under vacuum.
In the specification, the term “mammal” or “individual” as employed herein should be taken to mean a human; however it should also include higher mammals for which the prophylaxis, therapy or use of the invention is practicable, for example, pigs. The term “animal” should be understood to include any animal including humans.
In this specification, the term “administering” should be taken to include any form of delivery that is capable of delivering the bacterial strain to a site of infection, including local delivery, intravenous delivery, oral delivery, intranasal delivery, intramuscular delivery, intrathecal delivery, transdermal delivery, inhaled delivery and topical delivery. Methods for achieving these means of delivery will be well known to those skilled in the art of drug delivery. For treatment or prophylaxis of MRSA or Staphylococcus or Lysteria infections, especially chronic MRSA infections in hospital patients, intranasal delivery is ideal as it delivers the bacterial strain expressing the antimicrobial peptide designated Bactofensin LS1 or isolated antimicrobial peptide designated Bactofensin LS1 of the invention directly to the nasal mucous membrane where the target bacteria mucoid infections exist and are ordinarily difficult to remove.
In this specification, the term “pharmaceutical composition” should be taken to mean compositions comprising a therapeutically effective amount of the antimicrobial peptide designated Bactofensin LS1, or variants thereof, that in one embodiment are produced in-situ in the composition by a bacterial strain, and a pharmaceutically acceptable carrier or diluent. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the bacterial strain and/or antimicrobial peptide is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
“Effective amount” refers to the amount or dose of the antimicrobial peptide designated Bactofensin LS1, or variants thereof, upon single or multiple dose administration to the patient, which provides the desired effect in the patient under treatment. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of bacterial strain expressing the antimicrobial peptide or the isolated antimicrobial peptide administered, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual patient; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
The term “comestible product” should be understood to include products that are intended to be consumed by ingestion by humans or animals, such as foods and drinks. In particular, the comestible product is a food or drink product intended for consumption by humans, for example a fermented product or a diary product, especially a fermented dairy product such as a yoghurt.
Indicator strains employed for antimicrobial characterization included Escherichia coli DH5α, nontoxigenic E. coli O157:H7 strain P1432, Enterobacter sakazakii NCTC08155, Listeria innocua DPC3572, Listeria monocytogenes NCTC11994, Staphylococcus aureus DPC5246, and methicillin-resistant S. aureus (MRSA) DPC5646, all of which were grown aerobically at 37° C. in BHI (Merck, Darmstadt, Germany). L. salivarius strains used in this study are listed in Table 1 below. All lactobacilli were routinely cultured under anaerobic conditions at 37° C. in MRS medium (Difco Laboratories, Detroit, Mich.), unless otherwise stated. Anaerobic conditions were maintained with the use of anaerobic jars and anaerocult A gas packs (Merck).
Lactobacillus salivarius strains used in this study.
Lactobacillus salivarius UCC118
Lactobacillus salivarius DPC6502
Lactobacillus salivarius DPC6488
Lactobacillus salivarius DPC6005
Lactobacillus salivarius 7.3
Lactobacillus salivarius DPC6189
Lactobacillus salivarius DPC6027
Lactobacillus salivarius DPC6196
Purification of the Hydrophilic Antimicrobial Peptide Produced by L. salivarius DPC6502.
The antimicrobial peptide was purified from a 0.5 L overnight culture of L. salivarius DPC6502 grown in MRS-IM-G media. The cells were removed by centrifugation at 8,000×g for 15 min, and the supernatant applied to a column containing 10 ml SP sepharose Fast flow cation-exchange resin (GE Healthcare, UK) previously equilibrated with 50 mM sodium acetate buffer, pH 4.5. The column was washed with 50 mM sodium acetate buffer, pH 4.5 containing 0.3 M NaCl, the bioactive peptide was subsequently eluted from the column using 50 mM sodium acetate buffer, pH 4.5 containing 1.0 M NaCl. The eluate was applied to a 2.0 g (12 ml volume) C18 Bond Elute column (Phenomenex, Cheshire, UK) pre-equilibrated with methanol and water. The column was then washed with 0.1% (vol/vol) trifluoroacetic acid (TFA) and the bioactive peptide eluted with 70% (vol/vol) propan-2-ol containing 0.1% (vol/vol) TFA. The propan-2-ol was removed by rotary evaporation and 250 μl aliquots of the resultant preparation were applied to a Jupiter proteo reversed-phase high-performance liquid chromatography (RP-HPLC) column (250.0×4.6 mm, 4 μm particle size, 90 {acute over (Å)} pore size; Phenomenex). The column was pre-equilibrated with 10% acetonitrile containing 0.1% (vol/vol) TFA, followed by separation and elution of the bioactive peptide by gradient RP-HPLC using 0.1% (vol/vol) TFA and acetonitrile concentrations that ranged from 10% to 30% (vol/vol), from 5 to 45 min at a flow rate of 1.0 ml min−1 Absorbance was monitored at a wavelength of 214 nm. Antimicrobial peptide activity was monitored throughout the purification procedure by well diffusion assay using the sensitive indicator strain L. delbrueckii subsp. bulgaricus LMG6901. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (AXIMA-TOF2; ShimadzuBiotech, Manchester, UK) analysis was performed on bioactive fractions as described previously (Cotter, P. D., Deegan, L. H., Lawton, E. M., Draper, L. A., O'Connor, P. M., Hill, C. and Ross, R. P. (2006) Complete alanine scanning of the two-component lantibiotic lacticin 3147: generating a blueprint for rational drug design. Mol Microbiol, 62, 735-747). Fractions of interest were further purified by reapplying them to the RP-HPLC column under the conditions described above. N-terminal sequence analysis of the purified antimicrobial peptide by Edman degradation was performed by Aberdeen Proteomics (University of Aberdeen, Scotland). Reduction and alkylation of the cysteine residues was performed using dithiolthreitol (DTT) and iodoacetamide.
The sequence of the genomic DNA extracted from L. salivarius DPC6502 was determined through random shotgun pyrosequencing (Beckman Coulter Genomics, USA). The draft genome assembly was processed using the annotation software package GAMOLA. The gene model was determined with Gene Locator and Interpolated Markov ModelER 3.02 (GLIMMER). Sequence similarity analyses were performed using gapped BLASTp algorithm and the non-redundant database provided by the NCBI (ftp://ftp.ncbi.nih.gov/blast/db).
The Bactofensin LS1 peptide was synthesized according to the deduced amino acid sequence of the chromosomally located antimicrobial peptide structural gene (DLSL—0050) using microwave-assisted solid-phase peptide synthesis (MW-SPPS) performed on a CEM Liberty™ microwave peptide synthesiser using a H-Cys-HMPB-ChemMatrix® resin (PCAS Biomatrix Inc., Quebec, Canada). The synthetic peptide was purified by RP-HPLC as described above. Fractions containing a peptide with the desired molecular mass, as identified by MALDI-TOF MS, were pooled and lyophilised using a Genevac HT 4× evaporator (Genevac Ltd. Ipswitch, United Kingdom). The peptide was dissolved in 70% (vol/vol) propan-2-ol at a concentration of 5 mg ml−1 and stored at −20° C. Appropriate dilutions of the peptide in 50 mM sodium phosphate buffer, pH 6.8, were used to determine the specific activity of the synthetic analogue.
A microtiter plate assay system was used to determine the minimum concentration of the synthetic Bactofensin LS1 analogue required to inhibit the growth of a range of pathogenic indicator strains (Table 1), by 50% (MIC50). The microtitre plate was first treated with bovine serum albumin (BSA) to prevent adherence of the peptide to the sides of the wells. Each plate included triplicate assays at each concentration of synthetic Bactofensin LS1 examined. Each well contained a total volume of 200 μl, comprised of purified Bactofensin LS1, and 150 μl of a 1-in-10 dilution of the indicator culture (A590 of 0.1) in BHI broth. Control wells contained media only (blanks), and untreated indicator culture. The microtiter plate cultures were incubated at 37° C. for 24 h, and the optical densities at 590 nm (OD590) recorded at 30 mM intervals (GENios plus; TECAN, Switzerland). Triplicate readings were averaged and blanks were subtracted from these readings. The amount of antimicrobial peptide that inhibited the indicator strain by 50% was defined as 50% of the final OD590±0.05 of the untreated control culture.
Detection of salS.
The presence of salS was determined by PCR using template DNA from six additional genetically distinct intestinal L. salivarius isolates (Table 1) and the primer pair: SalSF 5′ CAGTCGACAATGATCATGATGGAGTAGCG 3′ (SEQ ID NO. 3) and SalSR 5′ GGAAGTAAGTAGGGTTTTGATAAGGTGTC 3′ (SEQ ID NO. 4) to amplify a product of 430 nucleotides. This was performed using Expand High Fidelity PCR system (Roche) according to the manufacturers' instructions. The products derived from PCR were purified and sequenced (Beckman coulter genomics), and analysis of DNA sequence data was performed using LASERGENE 6 software (DNAStar Inc., Madison, Wis.). Template DNA of L. salivarius DPC6502 and L. salivarius UCC118 were used as positive and negative controls, respectively.
L. salivarius DPC6502 from porcine jejunal digesta was described previously (O'Shea et al., 2009) as an abp118-variant producer. Recent array comparative genomic hybridization (aCGH) analyses revealed the absence of abp118-related homologues in strain DPC6502. Indeed, this strain was the most divergent of seven L. salivarius test strains compared with the genome of the reference strain L. salivarius UCC118 (Claesson, M. J., Li, Y., Leahy, S., Canchaya, C., van Pijkeren, J. P., Cerdeno-Tarraga, A. M., Parkhill, J., Flynn, S., O'Sullivan, G. C., Collins, J. K., Higgins, D., Shanahan, F., Fitzgerald, G. F., van Sinderen, D. and O'Toole, P. W. (2006) Multireplicon genome architecture of Lactobacillus salivarius. Proc Natl Acad Sci USA, 103, 6718-6723), exhibiting just 78% conservation of strain UCC118-specific gene content.
Characterization of the Antimicrobial Phenotype of L. salivarius DPC6502.
The peptide responsible for the antimicrobial activity of L. salivarius DPC6502 was purified from an overnight culture of the strain using cation-exchange chromatography and subsequent MALDI-TOF MS analysis revealed an associated mass of 2,785 Da in the active fractions (
Comparative genomic analyses have previously revealed considerable intraspecies diversity in L. salivarius. A study of the relatedness between seven intestinal L. salivarius isolates, with an antimicrobial peptide-positive genotype, and the genome sequenced human probiotic strain L. salivarius UCC118 revealed that the Bactofensin LS1-producing porcine intestinal isolate DPC6502 differed most extensively from UCC118. As a consequence of this divergence, the fact that no porcine-derived L. salivarius isolates have been sequenced to date and the production of an apparently novel antimicrobial peptide, the genome of DPC6502 was targeted for genome sequencing.
Characterization of the Bactofensin LS1 Locus in the Genome of L. salivarius DPC6502.
Scanning of the entire draft genome sequence for a gene corresponding to the amino acid sequence of Bactofensin LS1, as determined by Edman degradation, revealed a chromosomally located open reading frame (ORF), DLSL—0050, present on DPC6502-specific gene cluster 1 which was designated bfls1. The unidentified amino acids (aa) at positions 4 and 7 in the sequence determined by Edman degradation were, on the basis of the corresponding codons, found to be lysine and cysteine residues respectively, as shown in
A database search did not identify any homologues for this ORF, indicating that DPC6502 produces a novel antimicrobial peptide, designated Bactofensin LS1. The mature Bactofensin LS1 peptide (SEQ ID No. 2) is highly basic containing eight positively charged residues, largely concentrated at the N-terminus (
Typically, class II antimicrobial peptide structural genes are co-transcribed with an ORF encoding a cognate immunity protein located downstream of the structural gene to provide self-protection for the producing strain. The deduced 396 aa product (SEQ ID NO: 8 (see
The availability of the structural gene and deduced peptide sequences facilitated the production of synthetic Bactofensin LS1. MS analysis revealed a mass of 2,784 Da for the synthetic form of the peptide, which displayed anti-Listeria and anti-S. aureus activity that was similar to that of the purified natural Bactofensin LS1 peptide. This suggests that the disulfide bond of the natural form of Bactofensin LS1 is not crucial for activity. As a consequence of this, and the fact that the synthetic approach provided access to larger quantities of peptide, the synthetic Bactofensin LS1 peptide was employed for further antimicrobial activity assays, which on this occasion took the form of more sensitive broth-based minimum inhibitory concentration 50 (MIC50) assays. These investigations revealed that although concentrations of up to 50 μM of synthetic Bactofensin LS1 did not inhibit E. coli and E. sakazakii strains, a concentration of 5 μM was sufficient to inhibit the growth of both S. aureus DPC5246 and L. monocytogenes NCTC 11994 by 50% (
the Bactofensin LS1 Locus is a Novel Hyper-Variable Gene Cluster Characteristic of L. salivarius of Porcine Origin.
The ability of genetically distinct strains, and in several cases distinct species, to produce similar or identical antimicrobial peptides is a common phenomenon of LAB. It is evident from this study and others that there is a relatively high content of hyper-variable gene clusters in L. salivarius. It is now also apparent that DPC6502 contains many features not previously associated with L. salivarius, despite the current availability of two complete L. salivarius genome sequences and two draft genome assemblies (accession no. ACGT00000000 and AEBA00000000), all of which correspond to strains of human origin. To investigate if the Bactofensin LS1 locus described herein is among the hyper-variable loci of the flexible gene pool of L. salivarius, six further L. salivarius isolates of human and porcine intestinal origin were investigated for the presence of the Bactofensin LS 1 structural gene. A PCR product corresponding to this gene could not be generated from the genomic DNA of the two strains of human origin, DPC6488 and DPC6196. However, all four porcine strains, (L. salivarius DPC6005, DPC6027, DPC6189, and 7.3) were positive for bfls1, and sequencing of the PCR products generated confirmed 100% identity with the Bactofensin LS1 structural gene of DPC6502 in each case. These strains also produce a two-component class IIb antimicrobial peptide, salivaricin P.
In the specification the terms “comprise, comprises, comprised and comprising” or any variation thereof and the terms “include, includes, included and including” or any variation thereof are considered to be totally interchangeable and they should all be afforded the widest possible interpretation and vice versa. The term comprising may be substituted with the term “consisting of” or “consisting essentially of”.
The invention is not limited to the embodiments hereinbefore described but may be varied in both construction and detail.
| Number | Date | Country | Kind |
|---|---|---|---|
| 11175589.8 | Jul 2011 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/064841 | 7/27/2012 | WO | 00 | 6/10/2014 |
