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WO2011065854A1 - Peptides de phage entérococcique et procédés d'utilisation de ceux-ci - Google Patents

Peptides de phage entérococcique et procédés d'utilisation de ceux-ci Download PDF

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Publication number
WO2011065854A1
WO2011065854A1 PCT/PT2010/000050 PT2010000050W WO2011065854A1 WO 2011065854 A1 WO2011065854 A1 WO 2011065854A1 PT 2010000050 W PT2010000050 W PT 2010000050W WO 2011065854 A1 WO2011065854 A1 WO 2011065854A1
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Prior art keywords
peptide
polypeptide
chimeric polypeptide
faecalis
lysin
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Ceased
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PCT/PT2010/000050
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English (en)
Inventor
Miguel Ângelo DA COSTA GARCIA
Madalena Maria Vilela Pimentel
Carlos Jorge SOUSA DE SÃO JOSÉ
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Bluepharma - Industria Farmaceutica Sa
Technophage Investigacao e Desenvolvimento em Biotecnologia SA
Original Assignee
Bluepharma - Industria Farmaceutica Sa
Technophage Investigacao e Desenvolvimento em Biotecnologia SA
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Publication of WO2011065854A1 publication Critical patent/WO2011065854A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed to isolated and chimeric polypeptides of
  • Enterococcal bacteriophage origin having antibiotic activity and use thereof in the treatment and control of bacterial infections.
  • the present invention is directed to the use of a novel antibacterial peptide derived from bacteriophage 168 and chimeric constructs thereof, including chimeric constructs with an antibacterial peptides derived from bacteriophage 170, and their use for the treatment and control of infections caused by gram-positive bacteria, including Enterococcus faecalis.
  • Bacteriophage are viruses that specifically infect and lyse bacteria. Phage therapy, a method of using whole phage viruses for the treatment of bacterial infectious diseases, was introduce by Felix d'Herelle, who discovered phage around 1920. In the beginning of the 20 th century, there were various studies of the application of phage for therapy in humans as well as in animals. In 1940 Eli Lilly Company produced 7 phage products for human use, including phage preparations for treating different sicknesses caused by Staphylococcus sp., E. coli, and other pathogenic bacteria. These preparations were utilized to treat infections that cause abscesses, purulent wounds, vaginitis, acute chronic upper-respiratory tract infections, and mastoid infections.
  • phage therapy preparations including those originating from well-established companies in the United States and other countries, consisted of raw lysates of the host bacteria treated with the phage of interest. Thus, the preparations had bacterial components, including endotoxins, that could have adverse effects in patients treated with these preparations, particularly those receiving intravenous administration.
  • bacteriophage for therapeutic ends continued jointly with, or in place of antibiotics, in Eastern Europe and in the former Soviet Union where access to antibiotics was limited.
  • phage lysins are most promising of the strategies currently in development.
  • Preparations of purified endolysins can be used as therapeutic agents, per se, or combined with classic antibiotics.
  • the addition of exogenous lysins to susceptible gram-positive bacteria can cause complete lysis in the absence of bacteriophage (Loeffler et al.. 2001 , Science 294:2170- 2172; Shuch et al., 2002, Nature 418:884-889).
  • Microscopic images of bacteria treated with a lysin indicate that these en2ymes exercise their lethal effect by digesting peptidoglycan, leading to the formation of holes in the cell wall.
  • the inside of a bacterium is hypertonic, and when the bacterial wall loses its structural integrity the result is the extrusion of the cytoplasmic membrane and hypertonic lysis.
  • phage lysins destroy the peptidoglycan directly, exercising their lytic effect seconds after being administered.
  • the lysins can also destroy the cell wall of bacteria that are not growing and are insensitive to many antibiotics.
  • two lysins, or two lysin catalytic domains, that have differing targets may attack the peptidoglycan in multiple regions, presenting a synergistic effect.
  • Increased antibiotic resistance has turned attention to phage lysins as antibacterial agents, as well as to the development of chimeric constructs of such lysins.
  • Chimeric lysins have been constructed by re-combining catalytic domains of different lysins, in attempts for example to produce lysins with different bacterial and catalytic specificities (Fischetti VA, 2010, '''Bacteriophage endolysins: A novel anti-infective to control Gram-positive pathogens '" Intl J. Med. Microbiol. 300(6): 357-62).
  • Prior work with such constructs generally has focused on lysins directed against Pneamococcus and Staphylococcus spp.
  • catalytic domains of lytic enzymes for S pneumoniae phage were swapped to create a lysin having the same binding domain for pneumococci, but able to cleave a different peptidoglycan bond (Garcia et al., 1990, "Modular organization of the lytic enzymes of S pneumoniae phage
  • Enterococcal phage lysins having been described by other than the instant inventors. (See, e.g., WO 2010/090542 and WO 2010/041970, each hereby incorporated by reference in its entirety). Moreover, most lysins investigated to date are specific to species (or subspecies) of bacteria from which they are derived. For example, it has been shown that lysins isolated from streptococcal phage only kill certain streptococci and that lysins produced by pneumococcal phage only kill pneumococci (Fishcetti, 2005, Trends in Microbio 13:491-496).
  • VRE vancomycin-resistant enterococci
  • Enterococci can cause a variety of infections, including endocarditis, bacteremia, meningitis, and surgical would infections, and also are capable of colonizing environmental surfaces, including medical equipment, for prolonged periods (Arias, CA, et al., 2008,
  • E. faecalis E. faecium, both gram-positive bacteria that colonize the mouth, vagina, and lower intestines. While they normally cause no adverse effects in humans, high-level antibiotic resistance can lead to these organisms becoming a significant source of nosocomial infections, particularly in
  • the present invention is directed to isolated and chimeric polypeptides of
  • Enterococcal bacteriophage origin having antibiotic activity and use thereof in the treatment, prevention, and control of bacterial infections, particularly Enterococcal infections.
  • the present invention is directed to chimeric polypeptides comprising the catalytic domains of two or more Enterococcal bacteriophage endolysins, where the chimeric
  • polypeptides show increased lytic performance towards Enterococcus bacteria compared to the native bacterophage endolysins.
  • Increased lytic performance includes an increased spectrum of activity against Enterococcus bacteria species and/or strains; as well as an increased ability to kill and/or inhibit the growth and reproduction of Enterococcus bacteria.
  • the catalytic domains of the chimeric polypeptides may be from the same or different bacteriophages, which may have the same or different bacterial hosts, e.g., bacterial hosts of the same or different species, or of the same or different bacterial strain.
  • the native endolysins are from bacteriophages that natively infect E. faecalis, in particular strains E. faecalis 1518/05 and E. faecalis 926/095.
  • the chimeric polypeptides of the invention include a
  • CHAP domain of an endolysin from bacteriophage F 168/08 in particular a CHAP domain from Lysl 68 (SEQ ID NO:7).
  • the CHAP domain is combined with an amidase domain of an endolysin from bacteriophage F 170/08, in particular an amidase-2 domain from Lysl 70 (SEQ ID NO:5).
  • the CHAP domain comprises or consists of the amino acid sequence SEQ ID NO:7, or a fragment thereof, having antimicrobial or antibiotic activity against Enterococcus sp., particularly, E. faecalis and/or E. faecium.
  • a peptide is used that corresponds to the CHAP domain and comprises a fragment, variant or derivative of SEQ ID NO:7, wherein the fragment, variant or derivative has antibiotic activity or antimicrobial activity ⁇ e.g., lytic killing activity) against a Gram-positive bacteria, e.g., Enterococcus sp.. particularly, E. faecalis and/or E. faecium.
  • the invention provides for peptides having an amino acid sequence with at least 60%, 65%, 70%, 75%, 85%, 95%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to an amino acid sequence of the same length (i.e., consisting of the same number of residues) and having amino acid sequence SEQ ID NO:7, or a fragment thereof.
  • the amidase domain comprises or consists of the amino acid sequence SEQ ID NO:5, or a fragment thereof, having antimicrobial or antibiotic activity against E. faecalis.
  • a peptide is used that corresponds to the amidase domain and comprises a fragment, variant or derivative of SEQ ID NO:5, wherein the fragment, variant or derivative has antibiotic activity or antimicrobial activity (e.g., lytic killing activity) against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E. faecalis and/or E. faecium.
  • antibiotic activity or antimicrobial activity e.g., lytic killing activity
  • the invention provides for peptides having an amino acid sequence with at least 60%, 65%, 70%, 75%, 85%», 95%, 90%, 95%. 96%, 97%, 98%, 99%, or greater sequence identity to an amino acid sequence of the same length (i.e., consisting of the same number of residues) and having amino acid sequence SEQ ID NO:5, or a fragment thereof.
  • the chimeric polypeptide comprises or consists of the amino acid sequence SEQ ID NO: 9, or a fragment thereof, having antimicrobial or antibiotic activity against Enterococcus sp., particularly, E. faecalis and/or E. faecium.
  • the chimeric polypeptide comprises a fragment, variant or derivative of SEQ ID NO:9, wherein the fragment, variant or derivative has antibiotic activity or antimicrobial activity (e.g., lytic killing activity) against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E. faecalis and/or E. faecium.
  • the invention provides for chimeric polypeptides having an amino acid sequence with at least 60%, 65%, 70%, 75%, 85%, 95%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to an amino acid sequence of the same length (i.e. , consisting of the same number of residues) and having amino acid sequence SEQ ID NO:9, or a fragment thereof.
  • the present invention is directed to peptides obtained from bacteriophage F 168/08, which peptides exhibit antibiotic activity against a Gram-positive bacterium, e.g., Enterococcus sp.. particularly, E. faecalis and/or E. faecium, as well as to chimeric constructs thereof, including chimeric constructs with antibacterial peptides obtained from bacteriophage F 170/08.
  • the peptide of the invention comprises or consists of the amino acid sequence SEQ ID NO:7.
  • the peptide of the invention comprises a fragment, variant, or derivative of SEQ ID NO: 7, wherein said fragment, variant or derivative has antibiotic (e.g., lytic killing activity) activity against a Gram-positive bacteria, e.g., Enterococcus sp.. particularly, E. faecalis and/or E. faecium.
  • antibiotic e.g., lytic killing activity
  • the invention provides for peptides having an amino acid sequence with at least 60%.
  • the invention also encompasses polynucleotides that encode the polypeptides of the invention.
  • the invention also relates to a vector comprising said nucleic acid.
  • said vector is an expression vector.
  • the invention further provides host cells containing a vector comprising polynucleotides encoding the polypeptides of the invention.
  • the invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding a peptide of phage 168, or active fragment thereof, which polypeptide or fragment exhibits antibiotic activity (e.g., lytic killing activity) against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E. faecalis and/or E. faecium.
  • the invention provides for a nucleic acid comprising or consisting of the nucleic acid sequence SEQ ID NO:8, or a fragment thereof.
  • the invention also relates to a vector comprising said nucleic acid.
  • said vector is an expression vector.
  • the invention further provides host cells containing a vector comprising polynucleotides encoding the polypeptides of the invention.
  • the invention provides a chimeric nucleic acid comprising a nucleic acid sequence encoding a peptide of phage 168 corresponding to a catalytic domain, or active fragment thereof, combined with a catalytic domain of a heterologous lysin protein.
  • the invention provides for a nucleic acid comprising or consisting of the nucleic acid sequence SEQ ID NO: 10, or a fragment thereof.
  • the invention also relates to a vector comprising said chimeric nucleic acid.
  • said vector is an expression vector.
  • the invention further provides host cells containing a vector comprising polynucleotides encoding the chimeric polypeptides of the invention.
  • the invention encompasses methods for the evaluation of antibiotic activity of isolated peptides and chimeric polypeptides (e.g., killing based on the antimicrobial activity and/or lytic activity of the peptides and polypeptides of the invention).
  • Antibiotic activity may be assessed by any method known in the art and/or described herein.
  • antibiotic activity is assessed by culturing Gram-positive bacteria according to standard techniques (e.g., in liquid culture or on agar plates), contacting the culture with peptides and/or polypeptides of the invention and monitoring cell growth after said contacting.
  • the bacteria e.g., E. faecalis.
  • OD optical density
  • bacterial colonies can be allowed to form on an agar plate, the plate exposed to a peptide and/or polypeptide of the invention, and the subsequent growth of the colonies evaluated compared to control plates. Decreased size of colonies, or decreased total numbers of colonies indicate a peptide and/or polypeptide with antibiotic activity.
  • the present invention encompasses methods for the production of peptides and polypeptides of the invention or active fragments thereof, particularly for use in pharmaceutical compositions, e.g., antibiotic or antimicrobial compositions.
  • the peptides and polypeptides of the invention are isolated directly from cell cultures (e.g. bacterial cell cultures) infected with bacteriophage 168 or bacteriophage 170, using standard techniques known in the art and/or described herein.
  • the peptides and polypeptides of the present invention are produced by recombinant means using an expression vector comprising a nucleic acid sequence encoding a peptide or polypeptide of the invention, e.g., SEQ ID NO: 6, 8 or 10, or an active fragment, derivative, or variant thereof (i.e., which active fragment has antibiotic activity).
  • an expression vector comprising a nucleic acid sequence encoding a peptide or polypeptide of the invention, e.g., SEQ ID NO: 6, 8 or 10, or an active fragment, derivative, or variant thereof (i.e., which active fragment has antibiotic activity).
  • the peptides and polypeptides of the invention or fragments thereof can be produced by any method known in the art for the production of a polypeptide, in particular, by chemical synthesis or by recombinant expression techniques.
  • the invention relates to a method for recombinants producing a lysin peptide or chimeric polypeptide of the invention, or active fragment thereof, said method comprising: (i) constructing a nucleic acid encoding said peptide or polypeptide; (ii) culturing in a medium a host cell comprising said nucleic acid, under conditions suitable for the expression of said peptide or polypeptide; and (iii) recovering said peptide or polypeptide from said medium.
  • the nucleic acid sequence encoding the lysin peptide or chimeric polypeptide of the invention is operably linked to a heterologous promoter, meaning combination with a promoter not naturally found with the sequence.
  • the present invention encompasses pharmaceutical compositions comprising isolated peptides and/or chimeric polypeptides derived from bacteriophage 168, in particular isolated peptides or chimeric polypeptides having antimicrobial and/or antibiotic activity.
  • the pharmaceutical compositions of the invention may additionally comprise a pharmaceutically acceptable carrier, excipient, or stabilizer.
  • the pharmaceutical compositions comprise a polypeptide having the amino acid sequence of SEQ ID NO: 7.
  • the pharmaceutical compositions comprise a polypeptide that is a variant, derivative or fragment of SEQ ID NO: 7, wherein the variant, derivative or fragment retains antimicrobial activity against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E.
  • the pharmaceutical compositions comprise a chimeric polypeptide having the amino acid sequence of SEQ ID NO:9.
  • the pharmaceutical compositions comprise a chimeric polypeptide that is a variant, derivative or fragment of SEQ ID NO:9, wherein the variant, derivative or fragment retains antimicrobial and/or antibiotic activity against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E. faecalis and/or E. faecium.
  • the pharmaceutical compositions of the invention are antibiotic compositions for the treatment, prevention, and/or amelioration of symptoms of a disease or disorder associated with infection by a Gram-positive bacteria in a subject in need thereof.
  • another aspect of the invention relates to a method of treating a bacterial infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition.
  • the subject receiving a pharmaceutical composition of the invention may be a mammal (e.g., bovine, ovine, caprine, equid, primate (e.g., human), rodent, lagomorph) or avain (e.g., chicken, duck, goose).
  • "treatment" refers to both therapeutic treatment and
  • compositions of the present invention may be used in the treatment or management of infections associated with, but not limited to. Gram-positive bacteria, such as. Enterococcus, in particular, Enterococcus faecalis and/or Enterococcus faecium, as well as Staphylococcus aureus (including MRSA), Staphylococcus haemolyticus. Staphylococcus epidermidis. Bacilus subtilis, Bacilus licheniformis. Streptococcus o, Micrococcus luteus, Escherichia coli, and combinations thereof.
  • the pharmaceutical compositions of the invention are of use in the treatment of conditions associated with infection by vancomycin-resistant strains of
  • the pharmaceutical compositions may also be used to treat conditions or disorders associated with bacterial infections including, but not limited to, endocarditis, bacteremia, diverticulitis, meningitis, urinary tract infection, and surgical wound infections.
  • the invention provides for the use of lysin peptides or chimeric polypeptides as a single agent therapy.
  • the lysin peptides and chimeric polypeptides of the present invention may be combined with one or more lysins from a bacteriophage other than bacteriophage 168, and/or other than with lysins from bacteriophage 170.
  • the invention provides for the use of a lysin peptide or chimeric polypeptide, or active fragment, variant, derivative thereof, in combination with a standard or experimental treatment for Gram-positive bacterial infection.
  • the invention provides for the use of a lysin peptide, chimeric polypeptide, or active fragment of either, that has been chemically conjugated to still another therapeutic molecule (e.g., peptide or non-peptide cytotoxin).
  • a lysin peptide, chimeric polypeptide, or active fragment of either that has been chemically conjugated to still another therapeutic molecule (e.g., peptide or non-peptide cytotoxin).
  • a lysin peptide, chimeric polypeptide, or active fragment of either that has been chemically conjugated to still another therapeutic molecule (e.g., peptide or non-peptide cytotoxin).
  • peptide or non-peptide cytotoxin e.g., peptide or non-peptide cytotoxin
  • anti-inflammatory agents e.g., penicillin, synthetic penicillins, bacitracin, methicillin, cephalosporin, polymyxin, cefaclor, cefadroxil, cefamandole nafate, cefazolin, cefixime, cefmetazole, cefonioid, cefoperazone, ceforanide, cefotanme, cefotaxime, cefotetan, cefoxitin, cefpodoxime, proxetil, ceftazidime, ceftizoxime, ceftriaxone, cefriaxone moxalactam, cefuroxime, cephalexin, cephalosporin C, cephalosporin C sodium salt, cephalothin, cephalothin sodium salt, cephapirin, cephradine, cefuroximeaxetil, dihydratecepha
  • standard chemotherapeutic antibiotic agents e.g., penicillin, synthetic penicillins
  • compositions of the invention may be administered by any method known in the art suitable for administration of an antibiotic compound, e.g., via oral or parenteral (e.g., inhalation, intramuscular, intravenous, or epidermal) delivery.
  • oral or parenteral e.g., inhalation, intramuscular, intravenous, or epidermal
  • compositions of the present invention may also be used for traditionally non-therapeutic uses such as antibacterial agents in cosmetics, or in sprays or solutions for use on solid surfaces to prevent the colonization of Gram-positive bacteria (e.g., as a disinfectant or anti-infectant).
  • non-therapeutic uses such as antibacterial agents in cosmetics, or in sprays or solutions for use on solid surfaces to prevent the colonization of Gram-positive bacteria (e.g., as a disinfectant or anti-infectant).
  • the present invention is also directed to methods for screening peptides for antibiotic activity.
  • the method comprises screening contiguous amino acid sequences of at least 6, 10, 15, 20 or 25 residues in length from SEQ ID NO:5, 7 or 9 for antimicrobial activity, e.g., antimicrobial activity against Enterococcus sp., particularly, E.
  • said antibiotic and/or targeting activity measured by the peptide's or polypeptide's ability to inhibit bacterial growth in agar or liquid culture.
  • FIG. 1 shows the lytic activity of lysins Lysl68, Lysl 70, and Lysl 70-168, tested for four different amounts, in 98 clinic strains of Enterococcus.
  • FIG. 2 shows Lysl 68 and Lysl 70 activity as the percent reduction in turbidity for each of three E. faecalis strains, E. faecalis 926/05, E. faecalis 1518/05, and E. faecalis 1915/05, after addition of 5 ⁇ g/mL of each lysin.
  • FIG. 3 shows a cell viability assay for each of three E. faecalis strains, E. faecalis
  • FIG. 4 shows a therapeutic evaluation for Lysl 68 and Lysl 70 in the hearts of 3 female Wistar rats, where buffer was used as negative control and the treatment was carried out after 24 hours of heart infection.
  • FIG. 5 shows a therapeutic evaluation of Lys 168 and Lysl 70 in the blood of 3 female Wistar rats, where buffer was used as negative control and the treatment was carried out after 24 hours of heart infection.
  • FIG. 6 shows a therapeutic evaluation of Lys 168 and Lys 170 in the hears of 1 male and 3 female Wistar rats, where the male heart and buffer were used as negative controls and the treatment was carried out after 19-24 hours of heart infection.
  • polypeptide As used herein, the terms “polypeptide”, “peptide,” and “protein” are used interchangeably to refer to an amino acid sequence of any length. In general, however, “peptide” refers to shorter sequences, e.g., a fragment of a full-length polypeptide or protein, including a functional fragment corresponding to an enzymatic domain that retains its functionality separate from the rest of the polypeptide or protein from which it is derived.
  • Protein generally refers to an amino acid sequence expressed and found naturally by an organism in nature; while
  • polypeptide generally refers to a recombinant and/or chimeric product, but also can include the general meanings of "protein” and "peptide.”
  • fragment refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 1 75 amino acid residues, or at least contiguous 200 amino acid residues of the amino acid sequence of a second polypeptide.
  • the fragment is a functional fragment in that it retains at least one function of the second polypeptide (e.
  • the term "isolated" in the context of a peptide, polypeptide, or fusion protein refers to a peptide, polypeptide, or fusion protein that is substantially free of cellular material or contaminating proteins from the cell or tissue source from which it is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a peptide, polypeptide, or fusion protein in which the peptide, polypeptide, or fusion protein is separated from cellular components of the cells from which it is isolated or recombmantly produced.
  • a peptide, polypeptide, or fusion protein that is substantially free of cellular material includes preparations of a peptide, polypeptide, or fusion protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as "contaminating protein").
  • heterologous protein also referred to herein as "contaminating protein”
  • the peptide, polypeptide, or fusion protein is recombinanfly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • the peptide, polypeptide, or fusion protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.
  • a peptide, polypeptide, or fusion protein e.g., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the peptide, polypeptide, or fusion protein. Accordingly such preparations of a peptide, polypeptide, or fusion protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the peptide, polypeptide, or fusion protein of interest.
  • nucleic acid molecules refers to a first nucleic acid molecule which is separated from other nucleic acid molecules which are present in the natural source of the first nucleic acid molecule.
  • an "isolated"" nucleic acid molecule such as a cDNA molecule, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized and may be free of other cDNA or other genomic DNA molecules, e.g., where it has been isolated from other clones in a nucleic acid library.
  • purified in the context of a lysin or chimeric lysin in accordance with the instant invention, means that the lysin or chimeric lysin construct has been measurably increased in concentration by any purification process, including but not limited to, column chromatography, HPLC, precipitation, electrophoresis, etc., thereby partially, substantially, or completely removing impurities such as precursors or other chemicals involved in preparing the lysin or chimeric lysin.
  • any purification process including but not limited to, column chromatography, HPLC, precipitation, electrophoresis, etc.
  • compositions intended for administration to humans ordinarily must be of high purity in accordance with regulatory standards (e.g., of higher purity than isolated proteins for laboratory use).
  • derivative in the context of polypeptides refers to a polypeptide that comprises an amino acid sequence which has been altered by the introduction of amino acid residue substitutions, deletions, or additions.
  • derivative as used herein also refers to a polypeptide that has been modified, i.e., by the covalent attachment of any type of molecule to the polypeptide.
  • polypeptides may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation,
  • a derivative polypeptide may be produced by chemical
  • a derivative polypeptide may contain one or more non-classical amino acids.
  • a polypeptide derivative may possess a similar or identical function as the polypeptide from which it was derived, or it may possess an improved function.
  • the term "derived" as used in reference to a polypeptide "derived” from an organism may also refer to isolation of a polypeptide directly from said organism (e.g. bacterial cells or phage).
  • chimeric refers to a construct derived from two or more heterologous sources.
  • a chimeric gene or chimeric nucleic acid can comprise sequences derived from a first nucleic acid combined with sequences derived from a second nucleic acid, where the first and second nucleic acids are native to different types of bacteriophage or naturally occur in different polypeptides of a single type of bacteriophage.
  • the sequences from each nucleic acid typically correspond to coding sequences for a functional domain of the respective encoded polypeptides, e.g., a catalytic domain of a lysin.
  • the heterologous nucleic acid sequences may be combined in frame, e.g., by recombinant means, so as to encode a fusion protein or chimeric polypeptide, which can be expressed thereform under appropriate conditions.
  • a chimeric polypeptide can be engineered to include the full sequence of two or more native proteins, or only a portion of either. Chimeric polypeptides generally are created to impart functionality from each of the original proteins to the resulting chimeric polypeptides.
  • the dual (or higher order) functionality of fusion proteins is made possible by the fact that protein functional domains are generally modular, such that a linear portion of a polypeptide constituting a given domain, such as catalytic domain, may be removed from the rest of the protein without destroying its enzymatic capability.
  • a chimeric nucleic acid or chimeric polypeptide comprising sequences derived from two or more different lysin genes or
  • polypeptides can be referred to as a "chimeric lysin” or “chimeric lysin construct”.
  • endolysin is used interchangeably with the term
  • Endolysins are double-stranded DNA bacteriophage-encoded proteins, produced towards the end of a lytic life cycle of the bacteriophage, and designed to attack the
  • Endolysins are also capable of degrading peptidoglycan when applied exogenously to a cell wall, e.g., as isolated and/or recombinant polypeptides, usually resulting in rapid lysis of the bacterial cell wall.
  • Gram-positive phage lysins usually have a modular domain structure, with the N-terminal domain containing the catalytic domain and the C-terminal domain containing a binding or targeting domain that binds to a specific substrate of the host bacterium cell wall.
  • Enzymatic activities of the catalytic domains include, e.g., an endo- ⁇ - ⁇ - acetylglucosaminidase or N-acetylmuramidase activity (lysozyme activities), which act on the carbohydrate moiety of a bacterial cell wall; an endopeptidase activity, which acts on the peptide cOrss-bridge; or an N-acetylmuramoyl-L-alanine amidase activity (amidase activity), which attacks the amide bond connecting the glycan strand and peptide moieties.
  • endo- ⁇ - ⁇ - acetylglucosaminidase or N-acetylmuramidase activity lysozyme activities
  • endozyme activities which act on the carbohydrate moiety of a bacterial cell wall
  • an endopeptidase activity which acts on the peptide cOrss-bridge
  • CHAP domain refers to a conserved amidase domain found in several phage-encoded peptidoglycan hydrolases and stands for “cysteine, histidine-dependent amidohydrolases/peptidases.” See, e.g., Rigden D, et. al., Trends Biochem Sci. 2003 May 28(5): 230-4. It is found in a superfamily of amidases, including GSP amidase and peptidoglycan hydrolases.
  • the family includes at least two different types of peptidoglycan cleavage activities: L-muramoyl-L-alanine amidase and D-alanyl-glycyl endopeptidase activity.
  • CHAP domains generally contain conserved cysteine and histidine residues and hydrolyze ⁇ -glutamyl-containing substrates. These cysteine residues are believed to be essential for the activity of several of these amidases, and their thiol groups appear to function as the nucleophiles in the catalytic
  • CHAP domains are often found in association with other domains that cleave peptidoglycan, e.g., acting in a cooperative manner to cleave specialized substrates. See also, Bateman A, et al.. Trends Biochem Sci. 2003 May 28(5): 234-7.
  • the term "host cell” refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. It can also refer to a cell infected, particularly naturally infected, with a particular bacteriophage. Progeny of a host cell may not be identical to the parent cell transfected with the nucleic acid molecule, or infected with the phage, due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule or phage genetic material into the host cell genome.
  • the term “in combination” refers to the use of more than one prophylactic and/or therapeutic agent.
  • the use of the term “in combination” does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject with a disease or disorder.
  • a first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent (different from the first prophylactic or therapeutic agent) to a subject with a disease or disorder.
  • a second prophylactic or therapeutic agent different from the first prophylactic or therapeutic agent
  • nucleic acids and “nucleotide sequences” include
  • DNA molecules e.g., cDNA or genomic DNA
  • RNA molecules e.g., mRNA
  • Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases.
  • Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes.
  • the nucleic acids or nucleotide sequences can be single- stranded, double-stranded, may contain both single-stranded and double-stranded portions, and may contain triple-stranded portions, but preferably are double-stranded DNA.
  • prophylactic agent and “prophylactic agents” refer to peptides, polypeptides, and/or proteins of the invention, which can be used in the prevention, delay of the onset of, slowing the progression of, amelioration, or management of one or more symptoms of a disease or disorder, or of the underlying cause of the disease or disorder, associated with infection by a Gram-positive bacteria and, in particular, associated with infection by an Enterococcus.
  • therapeutic agent refers to peptides, polypeptides, and/or proteins of the invention that can be used in the treatment, management, or amelioration of one or more symptoms of a disease or disorder, or of the underlying cause of the disease or disorder, associated with infection by a Gram-positive bacteria and. in particular, associated with infection by an Enterococcus.
  • the term "therapeutically effective amount” refers to that amount of a therapeutic agent sufficient to result in amelioration of one or more symptoms of a disease or disorder (e.g., a disease or disorder associated with infection by Gram-positive bacteria and, in particular, associated with infection by an Enterococcus) in a subject; or to result in a reduction in total bacterial burden in said subject, in particular a reduction in total Enterococcal bacterial burden.
  • a disease or disorder e.g., a disease or disorder associated with infection by Gram-positive bacteria and, in particular, associated with infection by an Enterococcus
  • the terms “treat”, “treatment” and “treating” refer to the amelioration of one or more symptoms associated with an infection by Gram-positive bacteria, in particular, associated with an infection by Encterococcus or to the reduction in total bacterial burden, in particular, a reduction in total Enterococcal bacterial burden, resulting from the administration of one or more peptides, polypeptides, and/or proteins of the invention.
  • antibiotic activity refers to the ability to kill and/or inhibit the growth or reproduction of a microorganism and can be used interchangeably with “antimicrobial activity”.
  • antibiotic or antimicrobial activity is assessed by culturing Gram-positive bacteria according to standard techniques (e.g., in liquid culture or on agar plates), contacting the culture with peptides, polypeptides, and/or proteins of the invention and monitoring cell growth after said contacting.
  • standard techniques e.g., in liquid culture or on agar plates
  • the bacteria e.g., E. faecalils or E.
  • faecium may be grown to a optical density ("OD") representative of a midpoint in exponential growth of the culture; the culture exposed to one or more concentrations of one or more polypeptides of the invention; and the OD monitored relative to a control culture.
  • OD optical density
  • Decreased OD relative to a control culture is representative of a polypeptide exhibiting antibiotic activity (e.g., exhibiting lytic killing activity).
  • bacterial colonies can be allowed to form on an agar plate, the plate exposed to a polypeptide of the invention, and subsequent growth of the colonies evaluated related to control plates. Decreased size of colonies, or decreased total numbers of colonies, indicate a polypeptide with antibiotic activity.
  • a fragment, variant, or derivative of a lysin polypeptide having antibiotic or antimicrobial activity refers to the fragment having the catalytic ability to bring about host bacterial cell death and/or to bring about inhibition of growth or reproduction thereof, or to the fragment having such catalytic ability as well as targeting activity towards the host, as defined below.
  • targeting activity refers to the ability of a lysin polypeptide to direct catalytic activity, such as antibiotic or antimicrobial activity, to a given bacterial host cell.
  • Targeting activity may be associated with a particular region or domain of the polypeptide, such that, e.g., a chimeric construct comprising a targeting domain of a first lysin polypeptide, native to a first host species, can direct the catalytic activity, such as the antibiotic activity, of the chimeric construct to bacterial cells of first host species.
  • a chimeric construct comprising a targeting domain of a first lysin polypeptide, native to a first host species
  • the catalytic activity such as the antibiotic activity
  • Targeting domain refers to a functional domain of a lysin polypeptide capable of directing the lysin polypeptide to a host cell, e.g., E. faecalis, thereby facilitating lytic action upon the host cell.
  • a "targeting domain”, for example, can correspond to the cell wall binding domain of a lysin polypeptide.
  • antimicrobial activity or “antibiotic activity” refers to both or either functionality, that is. to the catalytic and/or targeting activities to bring about cell death of gram-positive bacteria, e.g., E. faecalis, the native host for phage 168.
  • antibiotic activity refers to both or either functionality, that is. to the catalytic and/or targeting activities to bring about cell death of gram-positive bacteria, e.g., E. faecalis, the native host for phage 168.
  • the invention is directed to polypeptides isolated from a phage that infects gram-positive bacteria.
  • the polypeptides have antimicrobial (e.g., lytic) and/or targeting activity against one or more strains of E. faecalis.
  • polypeptides are provided that exhibit antimicrobial and/or targeting activity against vancomycin-resistant strains of Enterococcus (VRE).
  • VRE vancomycin-resistant strains of Enterococcus
  • polypeptides having antimicrobial and/or targeting activity against one or more bacterial pathogens such as Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, S. haemolyticus, S. epidemidis, Bacilus subtilis, Bacilus licheniformis, Streptococcus o. Micrococcus luteus. and Escherichia coli are provided herein.
  • the polypeptide of the invention is isolated from bacteriophage 168, which infects the host E. faecalis.
  • bacteriophage 168 is used interchangeably with the terms “bacteriophage F 168/08" or "phage 168.”
  • the polypeptide is an isolated lysin peptide from phage 168, the lysin peptide comprising the amino acid sequence of SEQ ID NO:7 or a fragment thereof havng antimicrobial activity against E. aecalis.
  • the peptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 7, or a fragment thereof, which peptide exhibits antibiotic and/or targeting activity against E. faecalis.
  • Sequence identity with respect to the peptide or polypeptide sequences disclosed herein is defined as the percentage of amino acid residues that are identical in a candidate sequence of the same length ⁇ i.e., consists of the same number of residues) as the amino acid sequences of the present invention.
  • the present invention also encompasses variants, derivatives, and/or fragments of SEQ ID NO: 7, retaining antimicrobial activity and/or targeting activity to at least one Gram-positive bacterial strain or species.
  • the invention is directed to isolated peptides, polypeptides, and/or proteins of the present invention recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to therapeutic agents, e.g., small molecules, heterologous polypeptides, or catalytic domains of heterologous polypeptides, to generate fusion proteins or chimeric polypeptides.
  • therapeutic agents e.g., small molecules, heterologous polypeptides, or catalytic domains of heterologous polypeptides.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences or through chemical conjugation.
  • Non-limiting examples of therapeutic agents to which the peptide, polypeptides, or protein of the invention may be conjugated are peptide or non-peptide cytotoxins (including antimicrobials and/or antibiotics), tracer/marker molecules (e.g., radionuclides and fluorophores), and other antibiotic compounds as known in the art.
  • the present invention is directed to chimeric polypeptides comprising the catalytic domains of two or more Enterococcal bacteriophage endolysins, where the chimeric polypeptides show increased lytic performance towards Enterococcus bacteria compared to the native bacterophage endolysins.
  • the catalytic domains of the chimeric polypeptides may be from the same or different bacteriophages, which may have the same or different bacterial hosts, e.g., bacterial hosts of the same or different species, or of the same or different bacterial strain.
  • the native endolysins are from bacteriophages that natively infect E.
  • the invention in directed to chimeric polypeptides where at least one domain of a polypeptide isolated from phage 168, or a fragment thereof, is combined with at least one domain of a heterologous protein.
  • Preferable chimeric constructs include the fusion of a catalytic domain of a lysin isolated from phage 168 ⁇ e.g., Lysl 68) with a catalytic domain of a lysin isolated from phage 170 ⁇ e.g., Lysl 70), which infect hosts of the Enterococcus species.
  • Lysl 70-168 comprises an amidase domain of Lysl 70 and a CHAP domain of Lys 168.
  • the chimeric polypeptide Lys 170- 168 comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 9, which chimeric polypeptide exhibits antibiotic or antimicrobial activity against E. faecalis.
  • Sequence identity with respect to the chimereic polypeptide sequences disclosed herein also is defined as the percentage of amino acid residues that are identical in a candidate sequence of the same length ⁇ i.e., consists of the same number of residues) as the amino acid sequences of the present invention.
  • the present invention also encompasses variants, derivatives and/or fragments of SEQ ID NO: 9 retaining
  • the chimeric polypeptides and variants, derivatives, and/or fragments thereof improve the properties of Lysl 68 and/or Lysl 70, e.g., in terms of increased solubility, yield, stability, and/or lytic performance, such as including an increased lytic spectrum of activity towards Enterococcus species and/or other Gram-positive bacteria.
  • the isolated and chimeric polypeptides of the present invention may be administered alone or incorporated into a pharmaceutical composition for the use in treatment or prophylaxis of bacterial infections caused by gram-positive bacteria, including Enterococcus faecalis.
  • the pharmaceutical composition may be an antibiotic composition.
  • the polypeptides may be combined with a pharmaceutically acceptable carrier, excipient, or stabilizer.
  • Examples of pharmaceutically acceptable carriers, excipients and stabilizers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysin; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM as known in the art.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • low molecular weight polypeptides proteins, such as serum album
  • the pharmaceutical composition of the present invention can also include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients.
  • a polypeptide of the present invention may also be combined with one or more therapeutic and/or prophylactic agents useful for the treatment of infection with gram-positive bacteria ⁇ e.g. one or more antibiotics and/or lysins as are known in the art).
  • therapeutic agents that may be used in combination with the polypeptide of the invention include standard antibiotics agents, anti-inflammatory agents, and antiviral agents.
  • Standard antibiotics that maybe used with pharmaceutical compositions comprising polypeptides of the invention include, but are not limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, rifamycin, naphthomycin, geldanamycin, ansamitocin, carbacephems. imipenem, meropenem, ertapenem, faropenem, doripenem, panipenem/betamipron, biapenem, PZ-601 , cephalosporins, cefacetrile, cefadroxil.
  • cefalexin cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cefonicid, cefprozil, cefuroxime, cefuzonam, cefrnetazole, cefotetan, cefoxitin, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, ceftnenoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, latamoxef, cefclidine, cefepime, cefluprenam, cefosel
  • roxithromycin, aztreonam, pencillin and penicillin derivatives actinomycin, bacitracin, colistin, polymyxin B, cinoxacin, flumequine, nalidixic acid, oxolinic acid, piromidic acide, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin.
  • rufloxacin balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, garenoxacin, gemifloxacin, stifloxacin, trovalfloxacin, prulifloxacin, acetazolamide, benzolamide,
  • ethoxyzolamide furosemide, hydrochlorothiazide, indapamide, mafendide, mefruside, metolazone, probenecid, sulfacetamide, sulfadimethoxine, sulfadoxine, sulfanilamides, sulfamethoxazole, sulfasalazine, sultiame, sumatriptan, xipamide, tetracycline, chlortetracycline, oxytetracycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline,
  • antibiotics for use in the management, prevention, and/or treatment of enterococcal infections include ⁇ -lactams, aminoglycosides, glycopeptides, lipoglycopeptides, lipopeptides such as daptomycin, oxazolidinones such as linezolid, glycylcyclines such as tigecycline, and pristinamycins such as quinupristin-dalfopristin.
  • the combination of one or more polypeptides of the invention and one or more antibiotics as known in the art may enhance ⁇ e.g., additively or synergistically) the therapeutic effect of the
  • polypeptide of the invention for a given infection, such as an Enterococcal infection.
  • the chimeric polypeptides of the present invention comprise a combination where heterologous lysins are recombinantly fused, preferably where a catalytic domain of a lysin isolated from phage 168 is joined to a catalytic domain of a heterologous lysin, such as a lysin from phage 170.
  • the lysin construct comprises an amidase-2 domain of the lysin from phage 170 and a CHAP domain from the lysin from phage 168, which both natively infect Enlerococcus species.
  • the chimeric construct in accordance with the instant invention targets gram-positive bacteria, including
  • the present invention provides chimeric lysin constructs that permit strain and species cross- reactivity in accordance with a goal of the invention. In some particularly preferred
  • this cross-reactivity serves to improve the lytic performance of the chimeirc polypeptides on certain gram-positive bacteria, including E. faecalis, compared to the lytic activity of Lysl 70 and/or Lysl 68.
  • polypeptides of the present invention also may be combined with one or more lysins isolated from a bacteriophage other than bacteriophage 170 and 168, in particular, another Enterococcus phage lysin.
  • Lysins in general, either have amidase, endopeptidase, muramidase, or glucosaminidase activity. Therefore, the combination of catalytic domains of lysins, especially those of different enzymatic activities, is contemplated by the presented invention, which in preferred embodiments produces lysins with increased lytic performance towards
  • compositions can be administered using various modes of administration.
  • they may be administered by inhalation, in the form of a suppository or pessary, topically (e.g., as a lotion, solution, cream, ointment, or dusting powder), epidermally (e.g., by use of a skin patch), orally (e.g., as a tablet, (e.g., a tablet containing excipients such as starch or lactose), a capsule, ovule, elixir, solution, or suspension, optionally containing flavoring or coloring agents and/or excipients), or they can be injected parenterally, for example intravenously, intramuscularly, or subcutaneously.
  • a suppository or pessary topically (e.g., as a lotion, solution, cream, ointment, or dusting powder), epidermally (e.g., by use of a skin patch), orally (e.g., as a tablet
  • compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • the polypeptides of the present invention may be combined with one, or a combination of carriers, which include but are not limited to, an aqueous liquid, an alcohol base liquid, a water soluble gel, a lotion, an ointment, a nonaqueous liquid base, a mineral oil base, a blend of mineral oil and petrolatum, lanolin, liposomes, proteins carriers such as serum albumin or gelatin, powdered cellulose carmel, and combinations thereof.
  • a topical mode of delivery may include a smear, a spray, a time-release patch, a liquid-absorbed wipe, and combinations thereof.
  • the polypeptide of the invention may be applied to a patch either directly or in one of the carriers.
  • the patches may be damp or dry, wherein the lysin or chimeric lysin is in a lyophilized form on the patch.
  • the carriers of topical compositions may comprise semi-solid and gel-like vehicles that include a polymer thickener, water, preservatives, active surfactants, emulsifiers, antioxidants, sun screens, and/or a solvent or mixed solvent system.
  • U.S. Patent No. 5,863,560 discloses a number of different carrier combinations that can aid in the exposure of skin to a medicament.
  • the therapeutic agent of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray, e.g., presentated from a pressurized container, pump, spray, or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2- tetrafluoroethane (HFA)
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2- tetrafluoroethane (HFA
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurized container, pump, spray, or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the agent and a suitable powder base such as lactose or starch.
  • the therapeutic compositions may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment, or dusting powder.
  • the therapeutic agent of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route.
  • the compounds can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH-adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
  • the tablets 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, certain complex silicates, and/or granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be included.
  • 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
  • Dosages and desired drug concentrations of the pharmaceutical compositions of the present invention may vary depending on the particular use. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. In vitro and in vivo animal experiments can provide reliable guidance for the determination of effective doses in human therapy, such as those provided in the Examples below. Interspecies scaling of effective doses can be performed by one of ordinary skill in the art following the principles described by Mordenti, J. and Chappell, W., "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Ding Development, Yacobi et al., Eds., Pergamon Press, New York 1989. pp42-96 (hereby incorporated by reference in its entirety).
  • the polypeptides of the present invention have antibiotic activity against a number of gram-positive bacteria, including Enterococcus faecalis, and including several vancomycin-resistant strains of Enterococcus bacteria. Therefore, the polypeptides of the present invention may be used in methods of treating infections associated with bacteria against which it has lytic activity (e.g., antibiotic or antimicrobial activity) in both humans and animals.
  • compositions of the present invention may be used to treat an infection caused by one or more of the following Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus (including MRSA), S. haemolyticus, S. epidermidis, Bacilus subtilis, Bacilus
  • the polypeptides of the invention may also exhibit antibiotic or antimicrobial activity (e.g., lytic killing activity) again Gram-negative bacteria or bacteria that are not classified as either Gram-positive or Gram-negative.
  • the polypeptides of the invention may be used to treat, prevent, and/or manage infections associated with non-Gram- positive bacteria.
  • diseases that are caused by infection of gram-positive bacteria include, but are not limited to, endocarditis, bacteremia, diverticulitis, meningitis, urinary tract infection, and surgical wound infections, as well as post-operative endophtalmitis, other infections of the central nervous system, other wound infections (e.g., diabetic foot ulcers), pneumonia, osteomylelitis, sepsis, and mastitis.
  • polypeptides of the present invention may be used in anti-infective
  • compositions for controlling gram-positive bacteria including E. faecalis, in order to prevent or reduce the development of serious infections.
  • the lysins or lysin constructs of the present invention may also be incorporated into formulations such as sprays or ointments for controlling colonization of Gram- positive bacteria on the skin and other solid surfaces, including medical devices like catheters.
  • the present invention also encompasses diagnostic methods for determining the causative agent in a bacterial infection.
  • the method comprises culturing bacteria isolated from a bacterial infection and measuring the susceptibility to the antimicrobial polypeptides of the present invention, wherein susceptibility to the polypeptide indicates the presence of certain Gram-positive bacteria and the lack of susceptibility indicates the presence of non-responsive bacteria (e.g., non-responsive Gram-negative or non-responsive Gram-positive bacteria).
  • the bacteria may be collected from, e.g., pus, urine, exudate from a wound, vaginal secretions, or any other bodily fluid infected with the bacteria.
  • the invention also encompasses variants of the lysin polypeptides, or active fragments, or derivatives thereof, isolated from bacteriophage 168.
  • the invention encompasses an amino acid sequence variant of SEQ ID NO:7, or active fragment or derivative thereof.
  • Amino acid sequence variants of the polypeptides of the invention can be created such that they are substitutional, insertional, or deletion variants.
  • Deletion variants lack one or more residues of the native protein which are not essential for function (e.g., antimicrobial and/or targeting activity). Insertional mutants typically involve the addition of material at a nonterminal point in the polypeptide.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative
  • substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • point mutagenesis may be employed to identify with particularity which amino acid residues are important in the antibiotic activities.
  • one of skill in the art will be able to generate single base changes in the DNA strand to result in an altered codon and a missense mutation.
  • mutation of the amino acids of a protein creates an equivalent, or even an improved, second-generation molecule.
  • certain amino acids may be substituted for other amino acids in a protein structure without detectable loss of function (e.g., antibiotic and/or targeting activity).
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, interaction with a peptidoglycan within the outer coat of a Gram-positive bacteria.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics; for example: isoleucine (+4.5); valine (+4.2); leucine (+3. 8) ; phenylalanine (+2.8); cysteine/cystine (+2.5): methionine (+1.9): alanine (+1 .8); glycine (-0.4); threonine(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (- 1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • hydrophilicity indices are within ⁇ 2, preferably ⁇ 1 , or most preferably ⁇ 0.5 of each other.
  • the invention also encompasses chimeric polypeptides comprising a sequence corresponding to a catalytic domain of a first Enterococcal bacteriophge endolysin and a sequence corresponding to a catalytic domain of a second Enterococcal bacteriophage endolysin.
  • the catalytic domains may be an amidase domain, preferably an amidase-2 domain, and/or a CHAP domain, for example an amidase-2 domain of one lysin fused to the CHAP domain of a heterologous lysin.
  • the chimeric polypeptide shows increased lytic performance towards Enterococcus bacteria compared to said first and/or said second phage lysin.
  • Increased lytic performance may refer to an increased spectrum of activity, e.g., against a larger number or a more diverse group of Enteroccocus species, of E. faecalis strains, E.
  • Increase lytic performance may also refer to an increased ability to kill and/or inhibit the growth or reproduction of a microorganism, in particular Enterococcus bacteria.
  • the chimeric polypeptide may be constructed using lysins from the same or different bacteriophages. Where the lysins come from different bacteriophages, the different bacteriophages may have bacterial hosts that are of the same or different bacterial species, or the different bacteriophages may have bacterial host that are of the same or different bacterial strain. In some embodiments, the chimeric polypeptides are derived from bacteriophage F 168/08, which chimeric polypeptides exhibit antibiotic activity against a Gram-positive bacterium, e.g., against an Enterococcus species, such as E. faecalis.
  • the chimeric polypeptide may be derived from a lysin isolated from phage 168, or fragment or variant thereof, which is recombinantly fused to a heterologouos lysin.
  • the invention in directed to chimeric polypeptides where at least one domain of a lysin isolated from phage 168, or fragment or variant thereof, is combined with at least one domain of a heterologous lysin, or a fragment or variant thereof.
  • Preferable chimeric constructs include combination of a catalytic domain of a lysin isolated from phage 168, such as a catalytic domain of Lysl68, with a catatlytic domain of a lysin isolated from phage 170, which has antimicrobial or antibiotic activity against E. faecalis, such as a catalytic domain of Lysl 70.
  • the chimeric lysin comprises a CHAP domain of Lysl 68 recombinantly fused to an amidase or amidase-2 domain of Lysl 70.
  • the chimeric polypeptides of the invention comprise catalytic domains of lysins from bacteriophages having hosts from different strains of E. faecalis, such as from E. faecalis 926/05 and E. faecalis 1518/05.
  • one endolysin catalytic domain originates from bacteriophage F 170/08 and the other from bacteriophage F 168/08.
  • one endolysin is Lysl 70, corresponding to SEQ ID NO: 1 ; and/or one endolysin is Lysl68 corresponding to SEQ ID NO: 3.
  • the chimeric polypeptide comprises a catalytic domain of an endolysin having an amino acid sequence with 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 1 , which polypeptide exhibits antibiotic and/or targeting activity against Enterococcus sp..
  • E. faecalis and/or E. faeciuM and/or a catalytic domain of an endolysin having an amino acid sequence with 60%. 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%. or greater sequence identity to SEQ ID NO: 3, which polypeptide exhibits antibiotic and/or targeting activity against Enterococcus sp., particularly, E. faecalis and/or E. faecium.
  • the chimeric polypeptide comprises a catalytic domain comprising SEQ ID NO:5, or a fragment thereof having antimicrobial activity against Enterococcus sp., particularly, E. faecalis and/or E. faecium.
  • the catalytic domain has an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 5 or a fragment thereof, and exhibits antibiotic and/or targeting activity against Enterococcus sp.. particularly, E. faecalis and/or E. faecium.
  • the chimeric polypeptide comprises a catalytic domain comprising SEQ ID NO: 7, or a fragment thereof having antimicrobial activity against Enterococcus sp., particularly, E. faecalis and/or E. faecium.
  • the catalytic domain has an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO: 7 or a fragment thereof, and exhibits antibiotic and/or targeting activity against Enterococcus sp., particularly, E.faecalis and/or E. faecium.
  • the chimeric polypeptide comprises the amino acid sequence SEQ ID NO:9, or a fragment thereof having antimicrobial or antibiotic activity against at least one Enterococcus species, e.g., E. faecalis.
  • the chimeric polypeptide comprises a fragment, variant, or derivative of SEQ ID NO:9, wherein the fragment, variant or derivative has antibiotic activity or antimicrobial activity against a Gram-positive bacteria, e.g., Enterococcus sp., particularly, E. faecalis and/or E. faecium.
  • Amino acid sequence variants of the chimeric polypeptides of the present invention can be created as described above with respect to isolated polypeptides of the invention, for example, by substitutions, insertions, deletions, and the like, preferably to generate further improved second- (or third- or more) generation molecules.
  • the chimeric polypeptide comprises an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to SEQ ID NO:9 or a fragment thereof, and exhibits antibiotic and/or targeting activity against Enterococcus sp., particularly, E. faecalis and/ 'or E. faecium
  • Enterococcus sp. particularly, E. faecalis and,' 'or E. faecium.
  • E. faecalis Enterococcus sp.
  • E. faecalis Enterococcus sp.
  • E. faecalis Enterococcus sp.
  • E. faecalis Enterococcus sp.
  • E. faecalis Enterococcus sp.
  • ' 'or E. faecium particularly preferred
  • the chimeric polypeptides and variants, derivatives, and/or fragments thereof show improved properties, e.g., with respect to increased solubility, yield, stability, and/or lytic performance, such as including an increased lytic spectrum towards Enterococcus species and/or other Gram-positive bacteria, compared to the native isolated polypeptides.
  • the present invention further provides compositions comprising one or more polypeptides of the invention and one or more differing prophylactic or therapeutic agents, and methods for treatment of bacterial infection in a subject in need thereof, (e.g., preventing, treating, delaying the onset of, slowing the progression of, or ameliorating one or more symptoms associated with an infection by gram-positive bacteria) comprising administering to said subject one or more of said compositions.
  • Therapeutic or prophylactic agents include, but are not limited to, peptides, polypeptides, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.
  • Any agent which is known to be useful, or which has been used, or is currently being used for prevention or treatment of infection by a gram-positive bacteria, or for the prevention, treatment, or amelioration of one or more symptoms associated with an infection by a gram-positive bacteria, can be used in combination with the antibiotic or antimicrobial polypeptide in accordance with the invention described herein.
  • fusion protein refers to the use of a fusion protein or chimeric polypeptide, wherein an isolated polypeptide of the invention is covalently or non- covalently joined to another polypeptide, as described above.
  • Preferable fusion proteins include chimeric polypeptides of Lysl68 with one or more heterologous lysins. such as Lysl 70 or another lysin from Enterococcus phages.
  • Lysl 70 a heterologous lysin from Enterococcus phages.
  • the invention provides polynucleotides comprising a nucleotide sequence encoding a polypeptide of the invention.
  • the invention also encompasses polynucleotides that hybridize under high stringency, intermediate, or lower stringency hybridization conditions, to polynucleotides that encode a polypeptide of the invention.
  • High stringency conditions can include, but are not limited to, those that (1 ) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.001 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ, during hybridization, a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1 % bovine serum albumin/0.1 % Ficoll/0.1 %
  • 5XSSC 0.75 M NaCl, 0.075 M sodium citrate
  • 50 mM sodium phosphate pH 6.8
  • Modely stringent conditions are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2.sup.nd Ed.. New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent than those described above.
  • moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5XSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 XSSC at about 37-50°C.
  • 5XSSC 150 mM NaCl, 15 mM trisodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5X Denhardt's solution 10% dextran sulfate
  • 20 mg/mL denatured sheared salmon sperm DNA followed by washing the filters in 1 XSSC at about 37-50°C.
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • a polynucleotide encoding a polypeptide of the invention may be generated from nucleic acid from a suitable source, e.g., bacteriophage 168, as described in the Examples below. If a source containing a nucleic acid encoding a particular polypeptide is not available, but the amino acid sequence of the polypeptide of the invention is known, a nucleic acid encoding the polypeptide may be chemically synthesized and cloned into replicable cloning vectors using methods well known in the art.
  • nucleotide sequence of the polynucleotide of the invention may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.
  • Chimeric polynucleotides of the invention encompass nucleotide sequences encoding a chimeric polypeptide of the invention, such as chimeric polypeptides comprising a catalytic domain of a lysin isolated from phage 168 fused to a catalytic domain of a lysin isolated from a heterologous lysin, preferably a heterologous phage lysine, such as a catalytic domain of a lysin from phage 170.
  • the invention also encompasses polynucleotides that hybridize under high stringency, intermediate, or lower stringency hybridization conditions, e.g., as defined supra, to chimereic polynucleotides that encode a chimeric polypeptide of the invention
  • Chimeric polynucleotides may be obtained by recombinant techniques, as are well known and routinely practiced in the art.
  • Recombinant chimeric polynucleotides typically are created by joining two or more genes, or portions thereof, which originally coded for separate proteins.
  • the individual sequences typically correspond to coding sequences for a functional domain of each of the respective proteins, such that the chimeric polypeptide encodes a fusion protein having dual functionality.
  • functional domains may correspond to modular catalytic domains, such as domains having lytic acitivity, including, e.g., amidase activity, and the coding sequences for the different catalytic domains fused together.
  • a first coding sequence, or portion thereof may be joined in frame to a second coding sequence, or portion thereof, which typically is achieved through ligation or overlap extension PCR.
  • Ligation is used with the conventional method of creating chimeric genes, called the "cassette mutagenesis method.”
  • DNA can be cut into specific fragments by restriction endonucleases acting at restriction endonuclease recognition sites, and the specific fragments can be then ligated.
  • a particular fragment can be substituted with a
  • One such approach involves modified overlap extension PCR to create chimeric genes in the absence of restriction enzymes in three steps: (i) a conventional PCR step, using primers partially complementary at their 5' ends to the adjacent fragments that are to be fused to create the chimeric molecule; (ii) a second PCR step where the PCR fragments generated in the first step are fused using the complementary extremities of the primers; and (iii) a third step involving PCR amplification of the fusion product.
  • the final PCR product is a chimeric gene built up with the different amplified PCR fragments. See, e.g.,
  • nucleic acid encoding the chimeric polypeptide may be chemically synthesized.
  • the corresponding nucleotide sequence may be devised, chemically synthesized, and cloned into replicable cloning vectors using, e.g., well known methods in the art.
  • the Examples below provide additional details for creating the chimeric polynucleotide, SEQ ID NO: 10, encoding a chimeric polypeptide of the invention.
  • the vector for the production of the molecules may be produced by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequences for the molecules of the invention and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • An expression vector comprising the nucleotide sequence of a molecule identified by the methods of the invention can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells then can be cultured by conventional techniques to produce the molecules of the invention.
  • conventional techniques e.g., electroporation, liposomal transfection, and calcium phosphate precipitation
  • the Examples below provide additional details for producing chimeric polypeptides according to SEQ ID NO:9 from chimeric polynucleotides encoding same, after transfection of vectors comprising SEQ ID NO: 10 into competent cells for expression.
  • the host cells used to express the molecules identified by the methods of the invention may be either bacterial cells (nonsusceptible to the lysin protein, lysin construct, or fragment thereof of the invention) such as Escherichia coli in certain embodiments.
  • a variety of host-expression vector systems may be utilized to express molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of the molecules of the invention may be expressed and subsequently purified; and also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the molecules of the invention in situ.
  • microorganisms such as bacteria that are not susceptible to the lysin protein, lysin construct, or fragment of the invention ⁇ e.g., E. coli and B. subtilis in some embodiments) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing coding sequences for molecules encompassed by the invention; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing sequences encoding molecules encompassed by the invention; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing sequences encoding molecules encompassed by the invention; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid)
  • microorganisms such
  • Per C.6 cells human retinal cells developed by Crucell harboring recombinant expression constructs containing sequences encoding molecules encompassed by the invention operatively linked to promoters, e.g., promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; or the vaccinia virus 7.5K promoter).
  • promoters e.g., promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; or the vaccinia virus 7.5K promoter).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the molecule being expressed. For example, when a large quantity of such a protein is to be produced, e.g., for the generation of pharmaceutical compositions of a polypeptide, vectors which direct the expression of high levels of fusion protein products and that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2: 1791.
  • telomere sequence may be ligated individually into a vector in frame with a lac Z coding region so that a fusion protein is produced
  • pIN vectors Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509; each of which is hereby incorporated by reference in its entirety
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix of glutathione- agarose beads, followed by elution in the presence of free gluta-thione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites, so that the expressed product can be released from the GST moiety.
  • the Examples below provide additional details for producing chimeric polypeptides according to SEQ ID NO:9 from chimeric polynucleotides encoding same, using E. coli BL21 as the expression system.
  • Autographa californica nuclear polyhedrosis virus can be used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the polypeptide coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).
  • the polypeptide coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene e.g., then may be inserted in the adenovirus genome by in vitro or in vivo recombination.
  • Insertion into a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing the inserted polypeptide molecule in infected hosts (e.g., see Logan & Shenk. 1984, Proe. Natl. Acad. Sci. USA 81 :355-359, hereby incorporated by reference in its entirety).
  • Specific initiation signals may also be required for efficient translation of inserted coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • translational control signals and/or initiation codons can be of a variety of origins, both natural and synthetic, including exogenous sources.
  • the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, Bittner et al., 1987, Methods in Enzymol. 153:51 -544, hereby incoporated by reference in its entirety).
  • cell lines which stably express a polypeptide of the invention may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1 -2 days in an enriched media, and then switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may be advantageously used to engineer cell lines which express the polypeptides of the invention.
  • engineered cell lines may be particularly useful in screening and evaluation of bacterial species susceptible to the polypeptides of the invention.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977. Cell 1 1 : 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48: 202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes, which can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981 , Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981 , Proc. Natl. Acad. Sci.
  • the expression levels of a polypeptide of the invention can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning. Vol. 3, Academic Press, New York, 1987). Briefly, when a marker in the vector system expressing a polypeptide of interest is amplifiable, increasing the level of inhibitor present in the host cell culture can increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence encoding the polypeptide of interest, production of the polypeptide also will increase (Crouse et al., 1983, Mol. Cell. Biol. 3 :257).
  • polypeptide of the invention may be purified by any method known in the art for purification of polypeptides, for example, by chromatography (e.g., ion exchange, affinity, and/or sizing column chromatography),
  • Bacteriophage 168 was isolated from E. faecalis 926/05; bacteriophage 170 was isolated from E. faecalis 1518/05.
  • Genomic DNA can be isolated from a stock of phage 168 and a stock of phage 170, each obtained from a clinical isolate of E. faecalis. . and the genomic DNA of the phages can be extracted, cloned, and sequenced according to protocols as follows.
  • Preparation of stock phage 168 and stock phage 170 can be carried out using the protocols described in Carlson ., 2005, "Working with bacteriophages: common techniques and methodological approaches," in utter, E. Sulakvelidze, A. (eds.) Bacteriophages: Biology and Applications. 5 th ed. CRC press ("Carlson;” hereby incorporated by reference in its entirety).
  • the stock phage 168 and 170 each can be concentrated by precipitation with PEG according to the protocol described in Yamamato et al., 2004, PNAS 101 :6415-6420 (hereby incorporated by reference in its entirety) and Carlson.
  • the phage 168 and phage 170 stocks can be incubated in 1 M NaCl for one hour at 4°C with agitation. Next, PEG 8000 (AppliChem, Cheshire, MA) can be added gradually until a final concentration of 10% (p/v) is reached. The compositions then can be each incubated overnight at 4°C. After the incubation period, each composition can be centrifuged at 10000 x g for 30 minutes at 4°C.
  • the sediments then can be re-suspended in SM (0.05 M Tris-HCl at pH 7.4, 0.1 M NaCl, 10 mM MgS0 4 and gelatin at 1% p/v) and centrifuged again at 1000 rpm at 4°C for 10 minutes. The supernatant containing each suspended phage can saved for further purification.
  • Purification of phages 168 and 170 can be achieved using a CsCl gradient as described by Carlson. Removal of CsCl from each of the phage stocks can be achieved through dialysis.
  • a dialysis membrane Cellu.Sep H I High Grade Regenerated Cellulose Tubular Membrane (Cellu. Sep, River Street, USA) can be prepared according to the
  • the dialysis may consist of a first incubation of 30 minutes in 100 mM Tris-HCl and 3 M NaCl (pH 7.4) at 4°C. This can be followed by a second incubation of 30 minutes in 100 mM Tns-HCl and 0.3 M NaCl (pH 7.4) at 4°C. After dialysis, each of the suspended phages can be removed from the interior of the dialysis bag and stored at 4°C.
  • Phage 168 and phage 170 DNA can be obtained from each stock phage, respectively, purified on CsCl. To 5 ml of each purified phage can be added 20 mM EDTA at pH 8.0. SDS at 0.5% (p/v) and Proteinase at a final concentration of 40 ⁇ g/ml. Each mixture then can be incubated at 56°C for one hour. This may be followed by successive extractions in phenol:chloroform:alcohol at a proportions of 25:24: 1, until the interface between the aqueous and organic phases becomes clear. Each aqueous phase then can be treated with an equal volume of chloroform and centrifuged at 13,0000 x g for 10 minutes at 4°C.
  • Each aqueous phase can be once again removed and the DNA precipitated by adding two volumes of absolute ethanol and incubating for thirty minutes at 20°C.
  • the samples then can be centrifuged at 1 1 ,000 x g for 30 minutes at 4°C.
  • the pellets then can be washed with 70% ethanol at room temperature and re- suspended in 50 ⁇ of ultra-pure water (Gibco, California).
  • the DNA concentration for each phage then can be determined by measuring the absorbance at 260 nm in a ND-1000
  • Spectrophotometer The integrity of each isolated phage DNA then can be analyzed by electrophoresis on a 1 % agarose gel.
  • the phage 168 and 170 DNA each can be sequenced, and the open reading frames (ORFs) coding for amino acid sequences identified, using the tools described under the
  • Target DN A and amino acid sequences can be carried out by using ExPASy (Expert Protein Analysis System) of the Swiss Institute of Bioinformatics. Additional analysis also can be carried out using the programs Translate Tool, Prosite, and ProtPram. The homology of the target amino acid sequences with sequences in the UniProt Knowledgebase database can be performed using FASTA3. Sequence alignments can be performed using ClustalW. Both programs can be accessed through the European Molecular Biology Laboratory - European Bioinformatics Institute (EMBL-EBI) website. The determination of the secondary structure of the target sequence can be carried out using the program Foldlndex
  • Sequencing of the bacteriophage genomes can allow identification of potential open reading frames (ORFs) within the genome.
  • ORFs open reading frames
  • the putative ORFs of bacteriophages can be translated into their corresponding amino acid sequences and the amino acids sequences can be used to search the UniProt Knowledgebase using the program FASTA3. Alignment with other known lysin proteins from other bacteriophages can allow identification of the phage 170 and phage 168 lysin proteins (Lysl 70, SEQ ID NO: 1 and Lys l 68, SEQ ID NO: 3, respectively) and their corresponding gene sequences (SEQ ID NO:2 and SEQ ID NO:4, respectively).
  • Lys 168 was isolated from bacteriophage 168 and Lys 170 was isolated from bacteriophage 170.
  • the lysins also can be isolated from recombinant cells expressing the phage proteins from plasmids encoding same.
  • lysins Lys 168 (237aa) and Lys 170 (289aa) were amplified using specific primers respectively from bacteriophages F168 and F170; and were cloned in vectors pIVEX 2.3d (Roche) and pTrCHisA (Invitrogen) using E. coli MRF' XLl-blue (Stratagene). E. coli BL21 was used for expression.
  • the plasmids can be constructed by inserting the sequence of cDNA
  • the PCR reaction can be set up using the following conditions: puReTaq Ready-to-Go PCR Beads (Amersham Biosciences, U.K.), 200 ng of genomic DNA from phage 168 or phage 170, the primers at a final concentration of 0.4 pmol/ ⁇ and ultra-pure water to a final volume of 25 ⁇ .
  • the following thermocycler conditions can be used: 1 minute at 95°C for 1 cycle, 1 minute at 95°C +1 minute at 57°C + l minute at 72°C for 30 cycles, and 5 minutes at 72°C for one cycle.
  • the vectors can be digested with restriction enzymes, such as Bam HI, Hindlll and
  • the restriction digest mixture can be prepared according to the manufacturer's instructions.
  • the fragments of DNA resulting from the digestion of the vectors as well the amplified DNA of Lys l 68 and Lysl 70 can be run on a 1 % agarose gel.
  • the DNA then can be purified from the gel using the High Pure PCR Product Purification Kit (Roche, Germany) according to the manufacturer's instructions.
  • the purified vector DNA and the cDNA encoding Lysl 68 and Lysl 70 can be combined in a ratio of 1 :5 moles along with 1 of T4 DNA ligase (New England Biolabs, Frankfort, Germany) 10 x ligation buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, pH 7.5 at 25°C) and ultra-pure water at a final volume of 20 ⁇ .
  • the ligation mixture can be incubated overnight at 22°C followed by transformation of E. coli strain BL21. The transformation can be done according to the protocol described previously.
  • Transformants can be selected by plating in a Petri dish containing LB agar and the respective selection marker. Colonies of transformants then can be used to inoculate LB broth containing the appropriate selection marker and incubated overnight. The cultures then can be centrifuged and the DNA extracted as described previously.
  • Correct insertion of the cloned fragment can be determined by digestion of the recombinant plasmid using the same restriction enzymes used to construct the plasmids. All methods and procedures for cloning the Lysl 68 and Lysl 70 fragment can be done according to standard protocols. Recombinant plasmids containing the DNA of interest corresponding to the DNA of Lysl 68 or Lysl 70 can be sequenced by Macrogen (Coreia do Sul).
  • OE-PCR Overlap-Extension by Polymerase Chain Reaction
  • Expression vectors were constructed, expressing each of Lysl 68, Lysl70, and the chimeric lysin Lysl 70-168.
  • LB broth can be inoculated with competent E. coli and incubated overnight with agitation of 135 rpm at 37°C. The following day, 5 ml from the cultured LB broth can be added to 200 ml of fresh LB broth. The culture can be incubated with agitation at 37°C until an optical density (ODeoo) of 0.7-0.8 is reached. The optical density can be measured on a UV/Vis Spectrometer UVA (Unicam). The cells then can be centrifuged for 20 minutes at 5,000 rpm at 4°C.
  • the pellet After removal of the supernatant, the pellet can be re- suspended in 10 ml of a 10% glycerol solution previously cooled in ice. The volume then can be brought to 50 ml by adding more of the 10% glycerol solution and centrifuged again at 5,000 rpm for 20 minutes at 4°C. After removal of the supernatant, the pellet can be re-suspended as described before and centrifuged an additional time. The pellet then can be re-suspended in the residual 10% glycerol solution that remains in the tube after decantation. The samples then can be aliquoted and stored at -80°C.
  • an aliquot of competent cells can be removed from storage at -80°C and thawed at 4°C for 10-20 minutes. Then 1 ⁇ of plasmid DNA can be added to 25 ⁇ of competent cells. The suspended cells then can be transferred to an electroporation cuvette (Electroporation Cuvettes Plus model No. 610, BTX, Holliston, USA) and electroporated in a Gene Pulser Xcell System (Bio-Rad, Hertfordshire, U.K.). The parameters that can be used for a 1 mm cuvette are as follows: electric impulse - 10 ⁇ , Resistance - 600 Ohms and Voltage - 1800 V.
  • the cells can be resuspended in 1 ml of LB broth and incubated at 37°C for 1 hour with agitation at 135 rpm. Then the cells can be centrifuged at 13,000 rpm for 1 minute at room temperature. The cells then can be resuspended in 50 ⁇ of LB broth and plated on a Petri dish containing LB agar (50 ⁇ /plate) and the necessary selective markers. The plates can be incubated overnight at 37°C.
  • the construct can be used to transform E. coli BL21 for expression.
  • Transformed bacteria can be used to inoculate 5 ml of 2 x YT broth containing the appropriate selection markers. The cultures can be incubated overnight at 37°C with agitation until a OD (600nm) of 0.6 is obtained.
  • Expression of the lysin can be induced by the addition of 1 mM to 0.5 mM of IPTG. This can be done approximately after incubation for 4 hours at 37°C with agitation. .
  • lysis of the E. coli cells can be achieved by the addition of lysosyme (Sigma- Aldrich ) at a final concentration of 0.1 mg/ml and 1 ⁇ of Protease Inhibitor Cocktail Set I (Calbiochem, USA) followed by freezing and thawing.
  • the sample can be exposed to five cycles of freezing and thawing.
  • the sample then can be centrifuged at 14,0000 x g for 10 minutes at 4°C. After centrifugation, the supernatant can be removed and the pellet re-suspended in 500 to of PBS 1 x.
  • the liquid cultures can be centrifuged at 1 1 ,000 rpm for 40 minutes at 4 °C.
  • the supernatant can be removed and the pellet resuspended in 5 ml of BugBuster Master mix with Protease Inhibitor Cocktail Set I at a dilution of 1 : 1000.
  • the cells can be lysed according to the manufacturer's instructions.
  • the samples then can be centrifuged at 4°C and 14,000 x g for 10 minutes. After centrifugation, the supernatant can be removed and the pellet resuspended in 5 ml of PBS lx. The samples then can be centrifuged again at 4 °C and 14,000 x g for 1 0 minutes.
  • the pellet containing Lysl 68, Lys l 70, or Lys l 70-168 can be stored at 4 °C.
  • the samples can be analyzed and Lys l 68, Lys l 70, or Lys l 70- 168 purified by SDS-PAGE and Western Blot. 100139] Purification using a Ni-NTA column
  • Lys l 68, Lys l 70, or Lys 1 70- 1 68 can be purified using a Ni-NTA column
  • the Ni-NTA resin can be stored at 4 °C prior to be added to the column.
  • the column then can be washed with 50 ml of wash buffer (50 mM Na2HP0 4 , 300 mM NaCl, 20 mM imidazole in 1 L of distilled water at pH 8.0) using a peristaltic pump at medium speed.
  • the cellular extracts prepared according to the previously described protocols then can be loaded with a peristaltic pump set at low speed.
  • the column then can be washed with 50 ml of wash buffer to remove nonspecific proteins and other impurities.
  • the protein then can be eluted from the column using an elution buffer (50 mM Na 2 HP0 4. 300 mM NaCl, 250 mM imidazole in 1 L of distilled water at pH 8.0) and collected in 1 .5 ml fractions. All of the fractions can be analyzed by SDS-PAGE.
  • a Cellu.Sep H I High Grade Regenerated Cellulose Tubular Membrane can be prepared according to the manufacturer's instructions. The samples can be dialyzed against 1000 volumes of 50 mM Tris-HCl pH 7.5 overnight at 4 °C with slight agitation. The previous procedure can be repeated again until total incubation time reached 24 hours. After dialysis, the lysin can be removed from the interior of the membrane and stored at 4°C. A sample can be analyzed by SDS-PAGE and Western Blot and the amount of protein quantified using Bradford assay.
  • the proteins of interest can be concentrated on an Amicon Centrifugal Filter Device (Millipore, USA) according to the manufacturer's instructions.
  • the Amicon Centrifugal Filter Devices can also be used to exchange the buffer of the protein samples. In this mode, 12 ml of a 100 mM Tris-HCl buffer at pH 7.0 can be added together with a sample of Lys 1 68, Lys 1 70, or Lys 1 70- 1 68. The column then can be centrifuged for 30 minutes at 5,000 rpm at 4 °C.
  • Concentrated sample of the lysin can be analyzed by SDS-PAGE and/or Western Blot in order to confirm the integrity of the purified protein.
  • the concentration can be determined using a ND- 1000 spectrophotometer measuring the absorbance at 280 nm and using a Bradford assay.
  • polyacrylamide gels can be used.
  • the resolving gel can be prepared by adding 6.25 mi Protogel, 3.35 ml Protogel Resolving Buffer (National Diagnostics, Georgia, USA).
  • the stacking gel can be prepared using 650 Protogel, 1.25 ml Protogel Stacking Buffer (National Diagnostics), 3 ml distilled water, 50 41 APS 10%, and 7.5 TEMED.
  • Protein samples to be analyzed then can be denatured by placing them in 6 x denaturing buffer (0.35 M Tris-HCl at pH 6.8, 10.28% DS, 36% glycerol, 0.6 M DTT and 0.012 % bromophenol blue) and heated at 100 °C for 10 minutes.
  • the gel can be run on a Mini-PROTEAN Tetra Cell (Bio-Rad). While the samples are in the stacking gel, the voltage can be maintained at 140 V. After the samples enter the resolving gel, the voltage can be increased to 200 V. Precision Plus ProteinTM
  • the gel In order to visualize the protein bands on the SDS-PAGE gel, the gel can be immersed in a solution of Coomassie stain for 1 hour at ambient temperature. The gel then can be transferred to a destaining buffer (10% acetic acid, 10% methanol in 1 L of distilled water) to remove excess stain.
  • a destaining buffer (10% acetic acid, 10% methanol in 1 L of distilled water
  • the gel then can be placed in 1 x transfer buffer (48 mM Tris, 39 mM glycine, 0.04% SDS, 10% methanol, and 1L of distilled water) at ambient temperature.
  • the proteins then can be transferred from the gel to a nitrocellulose Hybond C (GE Healthcare, Germany) using a Mini Transblot Module (Bio-Rad). The transfer can take place at 200 mA for 1 hour.
  • the nitrocellulose membrane can be blocked in PBS 1 x. 5% milk protein, 0.05% TWEENTM 20 overnight at 4 °C. The membrane then can be washed 5 times in PBS lx, 0.05% Tween 20 at room temperature. The membrane then can be incubated for 1 hour with agitation at room temperature in a solution containing PBS Ix, 2% milk protein, 0.05 % Tween 20, and anti-His6 antibody conjugated to peroxidase diluted 1 :5000 (Roche). The membrane then can be washed three times for 15 minutes in a solution of PBS lx, 0.05% Tween 20 at room temperature. The protein of interest can be detected using the ECLTM Plus Western Blotting Detection System (GE Healthcare) following the manufacturer's instructions. The membrane then can be exposed to Amersham Hyperfilm ECL and developed in an AGFA Curix 60 processor.
  • ECLTM Plus Western Blotting Detection System GE Healthcare
  • Lytic activity of the lysins was tested on bacteria cells of different strains, including the phage host strains, by measuring optical density (OD).
  • a liquid medium 25 mM Phosphate-Na buffer pH 6.5, 250 mM NaCl.
  • the bacterial strains tested can be isolated from clinical samples (including blood, urine, pus, and medical devices) in different Portuguese hospitals and clinical settings or, in the case of the Micrococcus and Bacillus strains, obtained from ATCC strains.
  • Lysl 68 and Lysl 70 activity were tested as the percent reduction in turbidity for each of three E. faecalis strains, E. faecalis 926/05, E. faecalis 151 8/05, and E. faecalis 1915/05, after addition of ⁇ g/mL of each lysin.
  • C- indicaes a negative control where no lysin was added.
  • the results obtained with Lysl 68 and Lysl 70 show that strain E. faecalis 191 5/05 is much more sensitive to Lysl 68 activity compared with Lysl 70 (FIG. 2).
  • cell viability assays were conducted for each of the three E. faecalis strains, E. faecalis 926/05, E. faecalis 1518/05, and E. faecalis 1915/05, measured as CFU/mL at the initial (To) and end (T % ) of the turbidity reduction assay.
  • Lysl 68, Lysl 70, and Lysl 70-168 exhibit antibacterial activity towards numerous Enterococcus sp.
  • Lysl 70-168 appeared to show increased lytic spectrum compared to the native lysins.
  • Lysin activity of lysins Lysl 68 and Lysl 70 was tested in Wister rats, previously infected with 1.5xl 0'cells of E. faecalis 1915/05. 30min after the lysins were administered, their activity was measured as CFU/mL of E. faecalis 1915/05 occurring in the heart and blood of the rats (FIGs. 4-6).
  • FIG. 4 shows a therapeutic evaluation for Lysl 68 and Lysl 70 in the hearts of 3 female Wistar rats, where buffer was used as a negative control and the treatment was carried out after 24 hours of heart infection.
  • FIG. 5 shows a therapeutic evaluation of Lysl 68 and Lysl 70 in the blood of 3 female Wistar rats, where buffer was used as negative control and the treatment was carried out after 24 hours of heart infection.
  • FIG. 6 shows a therapeutic evaluation of Lysl 68 and Lysl 70 in the hearts of 1 male and 3 female Wistar rats, where the male heart and buffer were used as negative controls and the treatment was carried out after 19-24 hours of heart infection.

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Abstract

La présente invention concerne des polypeptides isolés et chimériques provenant d'un bactériophage entérococcique ayant une activité antibiotique et l'utilisation de ceux-ci dans le traitement et la lutte contre des infections bactériennes. Dans certains aspects spécifiques, la présente invention concerne l'utilisation d'un nouvel agent antibactérien issu du bactériophage 168 et de produits de constructions chimériques de celui-ci et leur utilisation pour le traitement et la lutte contre des infections provoquées par des bactéries à Gram positif, incluant Enterococcus faecalis .
PCT/PT2010/000050 2009-11-24 2010-11-24 Peptides de phage entérococcique et procédés d'utilisation de ceux-ci Ceased WO2011065854A1 (fr)

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WO2013141730A1 (fr) 2012-03-19 2013-09-26 Tecnifar-Indústria Técnica Farmacêutica, S.A. Compositions comprenant des cocktails de phages antibactériens et leurs utilisations pour le traitement d'infections bactériennes
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