WO2009059054A2 - Bacteria strains an d bacteriocin produced therefrom - Google Patents

Bacteria strains an d bacteriocin produced therefrom Download PDF

Info

Publication number
WO2009059054A2
WO2009059054A2 PCT/US2008/081866 US2008081866W WO2009059054A2 WO 2009059054 A2 WO2009059054 A2 WO 2009059054A2 US 2008081866 W US2008081866 W US 2008081866W WO 2009059054 A2 WO2009059054 A2 WO 2009059054A2
Authority
WO
WIPO (PCT)
Prior art keywords
gly
leu
bacteria
ala
htra
Prior art date
Application number
PCT/US2008/081866
Other languages
French (fr)
Other versions
WO2009059054A3 (en
Inventor
Jeffrey Weiser
Suzanne Dawid
Original Assignee
The Trustees Of The University Of Pennsylvania
Children's Hospital Of Philadelphia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Trustees Of The University Of Pennsylvania, Children's Hospital Of Philadelphia filed Critical The Trustees Of The University Of Pennsylvania
Publication of WO2009059054A2 publication Critical patent/WO2009059054A2/en
Publication of WO2009059054A3 publication Critical patent/WO2009059054A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention is directed to the control or treatment of bacterial infection or colonization through the use of novel bacteriocin producing Streptococcus strains. Specifically, the invention relates to methods and compositions comprising bacteriocins produced by upregulating the blp locus in strains of Streptococcus bacteria.
  • Streptococcus pneumoniae colonizes a majority of children by 1 year of age. Colonization with a single strain can last for weeks to months but is eventually cleared. Serial culturing of samples from the nasopharynxes of children has demonstrated that the population of pneumococci is constantly in flux, with one strain replacing another over time. In addition, some children appear to be colonized with two or more pneumococcal strains at any given time. The recent introduction of the seven-valent conjugate pneumococcal vaccine to the standard infant vaccination schedule has dramatically reduced the incidence of both colonization and invasive disease caused by the pneumococcus.
  • the vaccine contains only 7 of the >90 serotypes of pneumococci, there have been concerns that some of the remaining serotypes may fill the void left by the vaccine-targeted organisms. Over the intervening years since its release, reports have suggested that serotype replacement may be occurring at the levels of both colonization and disease. For example, the incidence of the previously uncommon nonvaccine serotype 19A has increased significantly in the postvaccine era as a cause of invasive pneumococcal disease in children of less than 5 years of age. Whether infections with these formerly infrequent serotypes will eventually result in a similar disease spectrum with respect to numbers and severity has yet to be determined.
  • Bacteriocins are small antimicrobial peptides produced by many bacterial species that have been implicated in intra- and interspecies competition. Bacteriocins typically target organisms that are either closely related to or within the same species as the producer bacteria. Producer bacteria are protected from the effects of their own bacteriocins via production of a specific immunity protein. This protein is typically cotranscribed with the genes encoding the bacteriocins.
  • the invention provides a method for generating bacteriocins in a bacteria comprising the step of contacting with an agent capable of upregulating expression of the blp locus.
  • the blp locus is represented by the nucleotide sequence set forth in SEQ. ID. NO l.
  • the invention provides a method for treating a bacterial infection or colonization in a subject, comprising the step of administering to said subject a bacteriocin producing bacteria, wherein the bateriocin is produced by upregulating the expression of the blp locus, thereby treating an infection in said subject.
  • the invention provides a composition for preventing bacterial growth or colonization, comprising an effective amount of a bacteriocin, wherein the bateriocin is produced by upregulating the expression of the blp locus.
  • the invention provides a method for generating bacteriocins in a bacteria comprising the step of contacting the bacteria with an agent capable of upregulating expression of the blp locus, wherein the agent comprises an amino acid sequence of blpC.
  • the invention provides a vaccine for treating, preventing or ameliorating a subject against pneumococcal infection or colonization, comprising a pharmaceutically acceptable carrier and an immunologically effective amount of a recombinant bacteria that produces a bacteriocin, wherein the vaccine comprises an amino acid sequence of blpC.
  • the invention provides a composition for inhibiting bacterial growth or colonization, comprising an effective amount of a blpC to generate bacteriocins in a bacteria, wherein the blpC peptide comprises the amino acid sequence of GLWEDILYSLNIIKHNNTKGLHHPIQL.
  • the invention provides a method for generating bacteriocins in a bacteria comprising the step of down-regulating the expression of htrA.
  • the invention provides a method for generating bacteriocins in a bacteria comprising the step of down-regulating the expression of ciaRH.
  • the invention provides a method for generating bacteriocins in a bacteria comprising the steps of upregulating expression of the blp locus; down- regulating the expression of htrA; and down-regulating the expression of ciaRH.
  • the invention provides a method for generating bacteriocins in a strain of bacteria comprising the steps of upregulating expression of the blp locus, wherein the upregulation of expression of the blp locus is caused by down-regulating the expressions of htrA and ciaRH.
  • the invention provides a method for treating an infection, the method comprising the steps of inhibiting or reducing bacterial growth or colonization in a subject comprising the steps of administering to said subject a bacteriocin producing bacteria, thereby treating an infection in said subject, wherein the bacteriocin is produced by down- regulating the expression of htrA.
  • the invention provides a method for treating an infection, the method comprising the steps of inhibiting or reducing bacterial growth or colonization in a subject comprising the steps of administering to said subject a bacteriocin producing bacteria, thereby treating an infection in said subject, wherein the bacteriocin is produced by down- regulating the expression of ciaRH.
  • the invention provides a composition for inhibiting bacterial growth or colonization, comprising an effective amount of a bacteriocin, wherein the bacteriocin is produced by down-regulating the expression of htrA.
  • the invention provides a composition for inhibiting bacterial growth or colonization, comprising an effective amount of a bacteriocin, wherein the bacteriocin is produced by down-regulating the expression of ciaRH.
  • the invention provides a composition for inhibiting bacterial growth or colonization, comprising an effective amount of a bacteriocin, wherein the bacteriocin is produced by upregulating the expression of the blp locus and down-regulating the expressions of htrA and ciaRH.
  • the invention provides a mutated Streptococcus pneumoniae bacteria, wherein said bacteria exhibits elevated production pneumocinMN relative to a non- mutant bacteria, and wherein the mutation is in htrA, ciaR, ciaH or their combination.
  • the invention provides a vaccine for treating, preventing or ameliorating a subject against pneumococcal infection or colonization, comprising a pharmaceutically acceptable carrier and an immunologically effective amount of a mutant bacteria, wherein said mutant bacteria exhibits elevated production pneumocinMN relative to a non-mutant bacteria, and wherein the mutation is in htrA, ciaR, ciaH or their combination.
  • Figure 1 (A) Table summarizing results of agar overlay assays with deletion mutants of the blp locus. Plus signs designate definite zones of inhibition, and empty cells designate combinations that were not tested. (B) Alignment of BIpM and BIpN amino acid sequences from type 6A and TIGR4 strains and the chimeric proteins from 6AblpMN0 T1GR . Shaded amino acids are nonconserved, and arrows designate putative cleavage sites of preproteins. (C) Photographs of results of selected overlay assays. Pictures a to g demonstrate test strains with zones of inhibition, while pictures h to m demonstrate test strains lacking inhibition.
  • Pictures a and b are shown with a TIGR4 overlay, and pictures c to m are shown with an overlay of 6A ⁇ bIpMNO.
  • FIG. 1 Graphical demonstration of the blp locus in a type 6A strain and comparison with the corresponding portion of the TIGR4 genome.
  • Solid arrows represent coding sequences for double glycine-containing proteins, vertically striped arrows represent genes of unknown function, checked arrows represent transport genes, the white box represents an insertion sequence element, and gray boxes represent the conserved putative BIpR binding sites designating the start sites of operons.
  • the gap in 6A designates an unsequenced region;
  • FIG. 3 Amino acid alignment of sequences of BIpM and BIpN from a selection of clinical isolates of the serotypes indicated. Shaded amino acids are areas of nonconservation. Arrows designate putative cleavage sites of preproteins; and
  • FIG. 19 AAbIpMNO is outcompeted by TIGR4 (A) or its parent type 19A strain (B) during mouse nasopharyngeal colonization.
  • Six-week-old BALB/c mice were challenged intranasally with single or dual inoculations of the type 19A parental strain (19A; open circles), the 19AblpMN0 WT corrected mutant (19A; closed circles), 19A ⁇ bIpMNO (closed diamonds), and TIGR4 (19AbIp-; closed triangles) (A) or with single or dual inoculations of the type 19A strain andl9 A ⁇ bIpMNO (B).
  • the colonizing strain is depicted on the x axis and was detected in lavage fluid at 4 days postinoculation at the density indicated (y axis). Coinoculated strains are shown in parentheses. Statistical analysis was done by the Mann- Whitney test, and horizontal lines indicate median values. Dashed lines denote the limit of detection.
  • FIG. 1 Organization of the blp locus of the type 6A strain. Open reading frames within the locus are designated by arrows as follows: regulatory proteins by white arrows, transporter genes by hatched arrows, immunity genes by grey arrows, double glycine containing preptides by black arrows and genes of unknown function by horizontal stripes. Letters above the arrows identify the blp gene designation. Black rectangles identify the putative response regulator binding sites. The designated region upstream of blpC has not been directly sequenced from this strain and is derived from the published TIGR4 sequence.
  • FIG. 6 Overlay assay demonstrating the effect of deletion of ciaRH or htrA on pneumocinMN mediated growth inhibition. Plate grown organisms were inoculated into TS plates containing catalase and allowed to grow for 6 hours before an overlay containing 0.5% TS agar, catalase and approx 10 15 CFU/ml of the overlay strain 6AoObIpMNPO, containing a deletion in the putative immunity protein BIpP, was carefully applied to the plate. Results of the overlay assay were recorded after overnight growth for strains: A. 6At, B. 6AtDaVuH, C. 6Atciareplaced, D. 6AtDhtrA298-1152 , E. 6At htrAs234A , F. 6AthtrAreplaced, G. 6AtDhtrAblpMNO, ⁇ . 6Ao, I. 6AociaH ⁇ 230P .
  • FIG. 7 Miller assays on broth grown organisms demonstrating the transcriptional activity of opacity variants with and without the addition of synthetic BIpC. - A. Variants were transformed with plasmid pEVPblpM resulting in duplication/insertion of the lacZ reporter gene behind the blpM promoter to create 6AoblpMlacZ, 6AtblpMlacZ. 6AoblpMlacZDbgaA (circles) and 6AtblpMlacZDbgaA (squares) were grown from single colonies in TS broth. Samples were taken at hourly intervals after reaching an OD620 of 0.1 and promoter activity determined by Miller assay (closed symbols).
  • FIG. 8 Dose response of opacity variants with and without HtrA to synthetic BIpC. 6AtblpMlacZDbgaA (closed squares) and 6AoblpMlacZDbgaA (closed circles) and their corresponding htrA deletions (open symbols) were grown from single colonies to an OD620 of 0.1. Increasing concentrations of synthetic BIpC were added to ImI of culture for each isolate and allowed to incubate for 2hrs at 37° C. Miller assays were then performed on each culture after determining the end point ODOOO. Miller Units are the mean of three determinations +/- SE. The table contains derived ECso and 95% confidence intervals for all four strains denoted above following non-linear regression analysis.
  • Figure 9 Variation in HtrA levels in opacity variants of the type 6A strain.
  • - A Reporter fusions of the htrA promoter to the lacZ gene were introduced into opaque and transparent variants lacking endogenous ⁇ -galactosidase activity with and without a deletion in ciaH. Cultures were grown from a single colony to an OD620 of 0.1 then OD620 was read and samples were taken every hour for four hours and used in Miller assay to determine promoter activity. OD620 is designated by open symbols and dotted lines. Miller Units are shown as closed symbols. Miller Units are the mean of three determinations +/- SE. - B. Organisms were grown in broth culture to OD620 of -0.500 and pelleted.
  • FIG. 10 Western blots detecting FLAG® epitope tag.
  • Bacterial lysates derived from plate grown organisms were loaded with equal amount of total protein and separated using 15% SDS-PAGE. The transferred membrane was probed with anti-FLAG® monoclonal antibody M2 or anti -pneumolysin antibody as a loading control. Samples were loaded as follows: 1. 6AtDhtrA, 2. 6AoObIpMNPO, 3, 6AoblpMFiAG, 4. 6AtblpMFiAG, 5. 6AoblpMFLAoOhtrA, 6. 6AtblpMFLAGDhtrA, 7. 6AtblpNFiAG, 8.
  • provided herein is methods and compositions for the control or treatment of infection through the use of novel bacteriocin producing Streptococcus strains and/or novel bacteriocins produced by this bacteria.
  • novel bacteriocins amino acid sequences of the novel bacteriocins, and to the strains of Streptococcus producing the novel bacteriocins.
  • therapeutic compositions containing the novel bacteriocins and/or the strains of Streptococcus producing them and to uses of the therapeutic compositions.
  • the blp locus of pneumococcus encodes a number of bacteriocin- like peptides. Upstream of the bacteriocin genes, the locus contains open reading frames for a typical two-component regulatory system (blpR and blpH), a small peptide pheromone Q)IpC), and a dedicated ABC transporter (blpA and -B). In another embodiment the ABC transporter recognizes the N termini of both the pheromone and the bacteriocins and transports these peptides across the cytoplasmic membrane, concurrent with cleavage at a conserved double-glycine motif.
  • Cleaved extracellular BIpC can bind in one embodiment, to the sensor kinase, BIpH. This interaction results, in another embodiment, in the activation of BIpR and, in another embodiment, upregulation of the entire gene cluster via binding to consensus sequences within each promoter.
  • Transcriptional analysis of the locus in the two fully sequenced pneumococcal strains R6 and TIGR4 demonstrate that application of chemically synthesized BIpC results in one embodiment, in upregulation of a number of operons only within the locus in one embodiment, comprising those encoding the regulatory proteins in one embodiment, or transport apparatus, and putative bacteriocins in certain other discrete embodiments of the methods and compositions described herein.
  • the transcript level of a downstream operon encoding BIpXY and -Z was upregulated in another embodiment, by the addition of BIpC.
  • this operon encodes proteins involved in immunity. Analysis of a number of pneumococcal strains demonstrated that there are at least four different pheromones produced and that each is specific for its cognate BlpR/H protein.
  • the blp locus is characterized in a clinical isolate of pneumococcus demonstrating an in-vitro phenotype consistent with bacteriocin activity and in another embodiment defines the importance of the locus in competition during nasopharyngeal pneumococal colonization.
  • bacteriocin genes of a number of clinical isolates are sequenced to determine which amino acids are important in dictating inhibition in vitro.
  • a method for generating bacteriocins in a bacteria strain comprising the step of contacting the bacteria with an agent capable of upregulating expression of the blp locus in the bacterial genome.
  • the blp locus is represented by the nucleotide sequence set forth in SEQ. ID. NO 1:
  • the blp locus is represented by a fragment of the nucleotide sequence set forth in SEQ. ID. NO 1.
  • said bacteria is Streptococcus.
  • said Streptococcus is Streptococcus pneumoniae.
  • "contacting" a bacteria with a substance refers to (a) providing the substance to the environment of the bacteria (e.g., solution, in vitro culture medium, anatomic fluid or tissue) or (b) applying or providing the substance directly to the surface of the bacteria, in either case, so that the substance comes in contact with the surface of the cell in a manner allowing for biological interactions between the bacteria and the substance.
  • the substance e.g., solution, in vitro culture medium, anatomic fluid or tissue
  • said agent used in the methods and compositions described herein for the upregulation of the blp locus expression is encoded by the blpC gene.
  • said agent used in the methods and compositions described herein for the upregulation of the blp locus expression is an mRNA.
  • the agent is a protein.
  • the agent is a synthetic protein.
  • the agent is a synthetic peptide.
  • the agent is a peptido-mimetic molecule.
  • the agent is a small molecule, or in another embodiment, the agent is a combination thereof.
  • the agent used in the methods and compositions described herein for the upregulation of the blp locus expression is BIpC, or its functional fragment in another discrete embodiment.
  • BIpC comprise the amino-acid sequence set forth in SEQ. ID. No.
  • pneumococcus colonizes the nasopharynx as the initial step in its pathogenesis.
  • human nasopharyngeal carriage is a major reservoir of pneumococci and the source of horizontal spread of this pathogen within the community in another embodiment.
  • Factors that contribute to clearance of colonization affect in one embodiment, the frequency of transmission of the pneumococcus and the overall incidence of pneumococcal disease in the population.
  • both host and bacterial factors that contribute to clearance remain incompletely characterized.
  • colonization is cleared in between about 4 to 8 weeks after a new strain is acquired, but the length of carriage is highly variable both between individuals and among different serotypes.
  • the methods and compositions provided herein are effective in the prevention or clearance of pneumococal colonization in a subject's nasopharyngeal passages.
  • the BIpC protein, or its functional fragment are between about 67 to about 99% homologous to SEQ ID No. 2.
  • the BIpC protein, or its functional fragment are between about 67 to about 75% homologous to SEQ ID No. 2.
  • the BIpC protein, or its functional fragment are between about 76 to about 85% homologous to SEQ ID No. 2.
  • the BIpC protein, or its functional fragment are between about 86 to about 95% homologous to SEQ ID No. 2.
  • the BIpC protein, or its functional fragment are between about 96 to about 99% homologous to SEQ ID No. 2.
  • the expression of blp locus is upregulated by an agent that comprises an amino acid sequence of blpC.
  • the amino acid sequence of blpC comprises the sequence of GLWEDILYSLNIIKHNNTKGLHHPIQL.
  • the invention provides a vaccine for treating, preventing or ameliorating a subject against pneumococcal infection or colonization.
  • the vaccine comprises a pharmaceutically acceptable carrier and an immunologically effective amount of a recombinant Streptococcus bacteria that produces a bacteriocin, wherein the vaccine comprises a synthetic amino acid sequence of blpC.
  • the synthetic amino acid sequence of blpC comprises the sequence of GLWEDILYSLNIIKHNNTKGLHHPIQL.
  • a bacteriocin is pneumocinMN that coprises the amino acid sequences of blpM, blpN, or fragments thereof.
  • the term "Homologous” refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. In another embodiment, if 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
  • the DNA sequences ATTGCC and TATGGC share 50% homology. In one embodiment, a comparison is made when two sequences are aligned to give maximum homology.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology # of identical positions/total # of positions times 100).
  • the determination of percent homology between two sequences can be accomplished using a mathematical algorithm.
  • a non-limiting example of a mathematical algorithim utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • a non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • the agent used in the methods and compositions described herein for the upregulation of the blp locus expression has about 75%, 80%, 85%, 90%, or 95% homology.
  • the addition of the agent used in the methods and compositions described herein for the upregulation of the blp locus expression results in the upregulation of a blp locus operon.
  • said operon encodes a regulator protein, a transport protein, an immunity protein, a bacteriocin, or a combination thereof.
  • said regulator protein is encoded by the blpR gene.
  • said regulator protein is encoded by the blpH gene.
  • said regulator protein is encoded by a combination blpR and blpH genes.
  • said regulator protein is encoded by amino acid sequences set forth in SEQ ID NO's 3-4:
  • the BIpR protein, BIpH protein, or its functional fragment are between about 67 to about 99% homologous to SEQ ID No. 3-4, respectively. In another embodiment, the BIpR protein, the BIpH protein or its functional fragment are between about 67 to about 75% homologous to SEQ ID No. 3-4, respectively. In another embodiment, the the BIpR protein, the BIpH protein, or its functional fragment are between about 76 to about 85% homologous to SEQ ID No. 3-4, respectively. In another embodiment, the BIpR protein, the BIpH protein, or its functional fragment are between about 86 to about 95% homologous to SEQ ID No. 3-4, respectively. In another embodiment, the the BIpR protein, the BIpH protein, or its functional fragment are between about 96 to about 99% homologous to SEQ ID No. 3-4, respectively.
  • said transport protein is encoded by the blpA, or blpB gene, or a combination thereof.
  • said transport protein is BIpA.
  • said transport protein is BIpB.
  • said transport protein is encoded by amino acid sequences set forth in SEQ ID NO 5-6: Met-Lys-Gly-Phe-Gly-Met-Phe-Arg-Phe-Arg-Arg-Thr-Phe-Val-Pro-Gln-Ile-Asp-Met-Arg- Asn-Cys-Gly-Val-Ala-Ala-Leu-Ala-Leu-Val-Ala-Lys-Tyr-Tyr-Gly-Ser-Asp-Tyr-Ser-Leu- Ala-His-Leu-Arg-Glu-Leu-Ala-Lys-Thr-Asn-Lys-Glu-Gly-Thr-Thr-Ala-Leu-Gly-
  • said bacteriocin is encoded by the blpM gene, the blpN gene, allelic variants or a combination thereof. In another embodiment, said bacteriocin is encoded by blpM gene. In another embodiment, said bacteriocin is encoded by blpN gene. In another embodiment, said bacteriocin is encoded by a functional fragment or otherwise a combination of amino acid sequences set forth in SEQ ID NO's 7-19:
  • the BIpM protein, BIpN protein, or its functional fragment are between about 67 to about 99% homologous to SEQ ID No. 7-19. In another embodiment, the BIpR protein, the BIpH protein or its functional fragment are between about 67 to about 75% homologous to SEQ ID No. 7-19. In another embodiment, the the BIpR protein, the BIpH protein, or its functional fragment are between about 76 to about 85% homologous to SEQ ID No. 7-19. In another embodiment, the BIpR protein, the BIpH protein, or its functional fragment are between about 86 to about 95% homologous to SEQ ID No. 7-19. In another embodiment, the the BIpR protein, the BIpH protein, or its functional fragment are between about 96 to about 99% homologous to SEQ ID No. 7-19.
  • the term "Operon" refers to a cluster of contiguous genes transcribed from one promoter that gives rise to a polycistronic mRNA.
  • the agent used in the methods and compositions described herein for the upregulation of the blp locus expression activates the blp operon.
  • the term "regulator protein” refers to an initial protein in the pathway that is a transmembrane histidine kinase that dimerizes in response to an external signal.
  • one partner of the dimer phosphorylates a specific histidine residue of the other partner and the phosphoryl group is subsequently transferred to an aspartyl residue of a response-regulator protein. This results, in one embodiment, in the activation of a pathway leading to the up-regulation of specific genes.
  • the histidine kinase sensor and the response regulator are separate proteins.
  • transport proteins are multi-pass transmembrane proteins, which either actively transport molecules across the membrane or passively allow them to cross.
  • Active transport involves directional pumping of a solute across the membrane, against an electrochemical gradient in certain embodiments.
  • active transport is tightly coupled to a source of metabolic energy, such as ATP hydrolysis in one embodiment, or an electrochemically favorable ion gradient in another embodiment.
  • passive transport involves the movement of a solute down its electrochemical gradient.
  • Transport proteins can be classified in other embodiments, as either carrier proteins or channel proteins.
  • Carrier proteins which can function in active or passive transport, bind in one embodiment to a specific solute to be transported and undergo a conformational change which transfers the bound solute across the membrane.
  • Channel proteins which only function in passive transport, form hydrophilic pores across the membrane.
  • solutes such as inorganic ions
  • Transported substrates range from nutrients and ions to a broad variety of drugs, peptides and proteins.
  • the agents used in the methods and compositions described herein result in the upregulation of the expression of genes encoding for transport proteins.
  • the invention provides a method for generating bacteriocins in a bacteria comprising the step of down-regulating the expression of htrA.
  • the pneumococcal gene htrA encodes a putative serine protease that is localized on the surface of Streptococcus pneumoniae.
  • the expression of htrA is controlled by the QaRH two-component system.
  • htrA is represented by amino acid sequence set forth is SEQ ID NO: 20: mkhlktfykk wfqllvvivi sffsgalgsf sitqltqkss vnnsnnsti tqtayknens ttqavnkvkd avvsvitysa nrqnsvfgnd dtdtdsqris segsgviykk ndkeayivtn nhvingaskv dirlsdgtkv pgeivgadtf sdiavvkiss ekvttvaefg dsskltvget aiaigsplgs eyantvtqgi vsslnrnvsl ksedgqaist kaiqtdtain pgnsggplin iqgqvigits
  • htrA is represented by an amino acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:20.
  • htrA is represented by a fragment of an amino acid sequence setforth in SEQ ID NO:20. The fragment may comprise one or more functional domains or conserved regions.
  • htrA is represented by an amino acid sequence that is orthologous or homologous to SEQ ID NO: 20.
  • htrA is represented by nucleic acid sequence set forth is SEQ ID NO: 21: atgaaacatctgaaaccttttataaaaatggtttcagctgctggtggtgattgtgattagcttttttagcggcgcgctgggca gctttagcattacccagctgacccagaaaagcagcgtgaacaacagcaacaacagcaccattacccagaccgcgaciaaaac gaaaacagcaccacccaggcggtgaacaaagtgaaagatgcggtggtgagcgtgattacctatagcgcgaaccgccagaacagc gtgttggcaacgatgataccgataccgatagccagcgcattagcagcgaaggcagcggcgtgatttataaaaaaaaaa
  • htrA is represented by a nucleic acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:21.
  • htrA is represented by a fragment of a nucleic acid sequence setforth in SEQ ID NO:21. The fragment may comprise one or more functional regions.
  • the hrtA is downregulated by a method known to one of skill in the art.
  • the down-regulation of the expression of htrA is caused by mutations in one or more functional domains of htrA.
  • the down-regulation of the expression of htrA is caused by an antisense oligonucleotide complementary to all or a portion of a messenger RNA encoding htrA, wherein said antisense oligonucleotide inhibits the production of htrA.
  • the down-regulation of the expression of htrA is caused by siRNA that inhibits the production of htrA.
  • the down-regulation of the expression of htrA is caused by an antibody that inhibits the production of htrA.
  • the invention provides a method for generating bacteriocins in a bacteria comprising the step of down-regulating the expression of ciaRH.
  • ciaRH refers to a bacterial two-component signal-transducing systems (TCSTS) that mediate adaptive responses to environmental signals in Streptococcus pneumoniae.
  • TSTS signal-transducing systems
  • two membrane-spanning regions in QaH separate the N-terminal external sensor domain from the cytoplasmic kinase domain.
  • htrA, ciaR, and ciaH genes are arranged in an operon with a 8-bp overlap.
  • mutations in the histidine protein kinase of ciaH conferred increased resistance to beta-lactam antibiotics, indicating, that in one embodiment ciaR controls genes that are involved in the biochemistry of the bacterial cell wall. In one embodiment, ciaH mutants are affected in the development of genetic competence as well.
  • ciaR is represented by amino acid sequence set forth is SEQ ID NO: 22: mqrvefflrq iwynsfnkee flmikillve ddlglsnsvf dflddfadvm qvfdgeegly eaesgvydli lldlmlpekn gfqvlkelre kgittpvlim takeslddkg hgfelgaddy ltkpfyleel kmriqallkr sgkfnentlt ygnivvnlst ntvkvedtpv ellgkefdll vyflqnqnvi lpktqifdrl wgfdsdttis vvevyvskvr kklkgttfae nlqtlrsvgy llkdvq
  • ciaR is represented by an amino acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:22.
  • ciaR is represented by a fragment of an amino acid sequence setforth in SEQ ID NO:22. The fragment may comprise one or more functional domains or conserved regions.
  • ciaR is represented by an amino acid sequence that is orthologous or homologous to SEQ ID NO: 22.
  • ciaR is represented by nucleic acid sequence set forth is SEQ ID NO: 23: atgcagcgcgtggaattttttctgcgccagatttggtataacagctttaacaaagaagaatttctgatgattaaaattctgctggt ggaagatgatctgggcctgagcaacagcgtgtttgattttctggatgattttgcggatgtgatgcaggtgttttgatggcgaagaaggcct gtatgaagcggcgtgtatgatctgattctgctggatctgatgctgcggaaaaaacgctttcaggtgctgctgctgctggaaaaaacgctttcaggtgctgaaagaactgc gcgaaaaaggcattaccaccccgg
  • ciaR is represented by a nucleic acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:23.
  • ciaR is represented by a fragment of a nucleic acid sequence setforth in SEQ ID NO:23. The fragment may comprise one or more functional regions.
  • ciaH is represented by amino acid sequence set forth is SEQ ID NO: 24: mfsklkktwy addfsyfirn fgvftlifst mtliilqvmh sslytsvddk lhglsenpqa viqlainrat eeikdlenar adaskveikp nvssntevil fdkdftqlls gnrflgldki klekkelghi yqiqvfnsyg qeeiyrvilm etnissvstn ikyaavlint sqleqasqkh eqlivvvmas fwilsllasl ylarvsvrpl lesmqkqqsf venashelrt plavlqnrle tlfrkpeati mdvsesiass leev
  • ciaH is represented by an amino acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:24.
  • ciaH is represented by a fragment of an amino acid sequence setforth in SEQ ID NO:24. The fragment may comprise one or more functional domains or conserved regions.
  • ciaH is represented by an amino acid sequence that is orthologous or homologous to SEQ ID NO: 24.
  • ciaH is represented by amino acid sequence set forth is SEQ ID NO: 25: atgtttagcaaactgaaaaaacctggtatgcggatgattttagctattttattcgcaactttggcgtgtttaccctgatttttagc accatgaccctgattattctgcaggtgatgcatagcagcctgtataccagcgtggatgataaactgcatggcctgagcgaaaacccgc aggcggtgattcagctggcgattaaccgcgcgaccgaagaaattaaagatctggaaaacgcgcgcgcggatgcgagcaaagtgga aattaaaccgaacgtgtgagcagcaacaccgaagtgattctgtgttgaagattttacccagctgctga
  • the ciaRH may be downregulated by a method known to one of skill in the art.
  • the down-regulation of ciaRH is caused by mutations in one or more functional domains of ciaR.
  • the down-regulation of ciaRH is caused by mutations in one or more functional domains of ciaH.
  • the down- regulation of ciaRH is caused by an antisense oligonucleotide complementary to all or a portion of a messenger RNA encoding ciaR, wherein said antisense oligonucleotide inhibits the production of ciaR.
  • the down-regulation of ciaRH is caused by an antisense oligonucleotide complementary to all or a portion of a messenger RNA encoding ciaH, wherein said antisense oligonucleotide inhibits the production of ciaH.
  • the down-regulation of ciaRH is caused by siRNA that inhibits the production of ciaR.
  • the down-regulation of ciaRH is caused by siRNA that inhibits the production of ciaH.
  • the down-regulation of ciaRH is caused by an antibody that inhibits the production of ciaR.
  • the down-regulation of ciaRH is caused by an antibody that inhibits the production of ciaH.
  • the invention provides a method for generating bacteriocins in a bacteria comprising the steps of upregulating expression of the blp locus; down- regulating the expression of htrA; and down-regulating the expression of ciaRH.
  • the invention provides a method for generating bacteriocins in a strain of bacteria comprising the steps of upregulating expression of the blp locus, wherein the upregulation of expression of the blp locus is caused by down-regulating the expressions of htrA and ciaRH.
  • bacteriocin is BIpM, BIpN, a functional fragment or a combination thereof.
  • the bacteriocin is pneumocinMN.
  • said bacteria is Streptococcus.
  • said Streptococcus is Streptococcus pneumoniae.
  • pneumocinMN is produced by the blp locus, upregulated by an agent.
  • pneumocinMN comprises an amino acid sequence of blpM or its functional fragment.
  • pneumocinMN comprises an amino acid sequence of blpN or its functional fragment.
  • pneumocinMN comprises an amino acid sequences of both blpM and blpN or their functional fragments.
  • pneumocinMN is capable of preventing pneumococcal infection or colonization in a subject.
  • the invention provides a vaccine for treating, preventing or ameliorating a subject against pneumococcal infection or colonization.
  • the vaccine comprises a pharmaceutically acceptable carrier and an immunologically effective amount of a recombinant Streptococcus bacteria that produces pneumocinMN, wherein the recombinant bacteria comprises an amino acid sequence of blpC.
  • the amino acid sequence of blpC comprises the sequence of GLWEDILYSLNIIKHNNTKGLHHPIQL
  • the bacteriocin is produced by upregulating the expression of the blp locus. In another embodiment, the bacteriocin is produced by upregulating the expression of blpM. In another embodiment, the bacteriocin is produced by upregulating the expression of blpN. In another embodiment, the bacteriocin is produced by down-regulating the expression of hrtA. In another embodiment, the bacteriocin is produced by down- regulating the expression of ciaRH.
  • the term "allele” which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for that gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. In one embodiment, alleles of a specific gene differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and/or insertions of nucleotides.
  • an allele of a gene is a form of a gene containing mutations.
  • the term "bacteriocin” is used to describe inhibitory agents produced by bacteria that meet the minimum criteria of (1) being a peptide and (2) possessing bactericidal activity.
  • the term "bacteriocin” refers to a polypeptide produced, by ribosome synthesis, from microorganisms capable of inhibiting specifically the growth of other bacteria.
  • bacteriocin is pneumocinMN.
  • pneumocinMN comprises amino acid sequences of blpM and blpN.
  • fragment refers to a portion of a protein or peptide that has been enzymatically or chemically truncated or cleaved. Such a fragment may encompass any portion of the native amino acid sequence of the protein.
  • the term “treatment” refers to any process, action, application, therapy, or the like, wherein a subject, including a human being, is subjected to medical aid with the object of improving the subject's condition, directly or indirectly.
  • the term “treating” refers to reducing incidence, or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, improving symptoms, improving prognosis or combination thereof in other embodiments.
  • said infection is a lower respiratory infection, upper respiratory infection, invasive infection, or a combination thereof.
  • said upper respiratory infection is Sinusitis, Otitis media, Tracheobronchitis, or a combination thereof.
  • said lower respiratory infection is Pneumonia, Broncho-pneumonia, or a combination thereof.
  • said invasive infection is Primary bacteremia, Meningitis, Spontaneous bacterial peritonitis, Sepsis with tissue seeding, or a combination thereof.
  • the type of otitis media is recurrent acute otitis media (RAOM), chronic otitis media with effusion (COME), acute post-tympanostomy otorrhea (APTO), or chronic suppurative otitis media (CSOM) in another discreet embodiment.
  • RAOM acute acute otitis media
  • COMP chronic otitis media with effusion
  • APTO acute post-tympanostomy otorrhea
  • CSOM chronic suppurative otitis media
  • the treatment of the individual to be treated is determined based on the bacterial profile of the otitis media.
  • Acute otitis media refers to a condition characterized by fluid in the middle ear accompanied by signs or symptoms of ear infection (bulging eardrum usually accompanied by pain; or perforated eardrum, often with drainage of purulent or infectious material).
  • a patient with recurrent acute otitis media has had either more than three acute episodes in a period of six months or four or more acute episodes in a period of 12 months.
  • Otitis media with effusion refers to a condition characterized by fluid in the middle ear without signs or symptoms of ear infection. Otitis media with effusion is defined as chronic (COME) when middle ear effusion has been present for at least 3 months.
  • CSOM Chronic suppurative otitis media
  • CSOM Chronic suppurative otitis media
  • Chronic otitis media with effusion may be defined as a middle ear effusion, without perforation of the tympanic membrane, which is reported to persist for 3 months.
  • Chronic suppurative otitis media is a perforated tympanic membrane with persistent drainage from the middle ear.
  • Acute post-tympanostomy otorrhea refers to a condition characterized by the presence of purulent fluid or inflamed middle ear mucosa occurs following tympanostomy tubes placement. Drainage following tube placement that persists for less than 8 weeks, is classified as acute.
  • the term "sinusitis" is the result of undrained mucous in one or more of the sinus cavities.
  • sinus inflammation is caused by the presence of fungi, bacterial and viral infection, or allergens.
  • obstructions due to deviated septum and nasal polyps which form in the nasal passages and which obstruct breathing lead to sinus inflammation. Regardless of the cause, inflammation of the sinus cavities causes the swelling and congestion of membranes associated with the sinuses. Pain results from the congestion and mucous production increases and the mucous itself becomes thicker.
  • the term "upper respiratory infection” refers to the predominant colonization or growth of a disease causing microbe, pathogen, bacteria, virus, fungi, or live particle taking place in, on, around or within the throat, nasopharynx, the eustachian tube, the nasal passages, or the sinuses.
  • ther term "lower respiratory infection” refers to the predominant colonization or growth of a disease causing microbe, pathogen, bacteria, virus, fungi, or live particle taking place in, on, around, or within the bronchi, bronchioles, the alveoli, and the lungs.
  • the bacteria involved in the pathogenesis of the diseases described herein and for which the methods of treatment described herein is effective is S. pneumoniae.
  • each one of the above anatomical features has mucous and mucous membranes associated with each such anatomical feature.
  • the sinus cavities have mucous membranes and those membranes have their related mucous.
  • the alveoli of the lungs also posses mucous and mucous membranes.
  • the human respiratory system comprises all the above anatomical features and all the associated mucous membranes and the associated mucous within, and in contact with, those mucous membranes. So, for the purposes herein, the human respiratory system includes the pulmonary anatomy, the sinus anatomy, the nasal anatomy, the ear anatomy, and all the associated mucous membranes and the associated mucous within, and in contact with, those mucous membranes.
  • a composition for inhibiting bacterial growth or colonization comprising an effective amount of a bacteriocin, produced by an upregulation of expression of the blp locus of a bacteria.
  • said bacteria is Streptococcus.
  • said Streptococcus is Streptococcus pneumoniae.
  • said bacteriocin further comprising of a pharmaceutically acceptable carrier.
  • an effective amount of compounds of the present invention or pharmaceutical preparations thereof, as defined herein, are administered via any of the usual and acceptable methods known in the art, either singly or in combination with another compound or compounds of the present invention or other pharmaceutical agents, such as antibiotic agents, antiviral agents or a combination thereof.
  • the methods of administering the active ingredients of the present invention con comprise of but is not limited to nasal sprays, topical solutions, patches or a combination thereof.
  • the compounds may be formulated as ointments, gels, creams or lotions, or as the active ingredient of a transdermal patch.
  • the administration can be carried out in one embodiment, in single unit dosage form with continuous therapy or in another embodiment, in single dose therapy ad libitum.
  • subject refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae.
  • the subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans.
  • subject does not exclude an individual that is normal in all respects.
  • S. pneumoniae strains used in this study are described in Table . 1. An array of clinical strains were analyzed and considered unrelated based on differences in capsular type and time and location of isolation. All strains were grown in tryptic soy broth (TS) or on tryptic soy agar (TSA) supplemented with catalase (4,741 U/plate; Worthington, Lakewood, NJ), except where indicated. TSA was supplemented with streptomycin (100 ⁇ g/ml), kanamycin (500 ⁇ g/ml), or erythromycin (1 ⁇ g/ml) where indicated.
  • TS tryptic soy broth
  • TSA tryptic soy agar
  • streptomycin 100 ⁇ g/ml
  • kanamycin 500 ⁇ g/ml
  • erythromycin 1 ⁇ g/ml
  • Cultures were grown on agar plates at 37°C in 5% CO 2 or anaerobically using a BBL gas pack system. Broth cultures were grown at 37°C without agitation. For transformation, bacteria were grown from plates at low inocula in C+Y (pH 8.0) at 37°C until the optical density at 620 nm reached 0.150. One-hundred-microliter aliquots were removed and placed at 30°C with 10 ng/ml of purified competence-stimulating peptides 1 and 2. After 10 min, approximately 100 pg/ml of DNA was added to the mixture and incubated at 30°C for an additional 40 min. The culture was then transferred to 37°C and incubated for 2 h before being plated on selective medium.
  • Bacteriocin assay Pneumococci grown on TSA plates overnight were resuspended in phosphate -buffered saline (PBS) to an optical density at 595 nm of 0.700. Test strains were then stabbed into TSA plates and allowed to grow anaerobically at 37 °C for 6 h. Plates were carefully overlaid with 10 CFU/ml of a mid- log-phase broth- grown overlay strain in 7 ml of TS containing 0.5% agar which had been maintained at 37°C before application and returned to an anaerobic environment for overnight growth at 37 °C. Test strains that scored positive for bacteriocin activity had a clear zone of complete inhibition of the overlay strain surrounding the area of test strain growth.
  • PBS phosphate -buffered saline
  • blp sequence analysis Primers 1 and 2 (Table J) were used to PCR amplify and sequence the region of DNA likely to contain blpM and blpN based on sequence comparison.
  • the blp locus from the type 6 A (and 6B) strain was amplified using primers 14 and 15, which amplified a 6,600-bp fragment.
  • PCR was performed with Pfx high-fidelity polymerase (Invitrogen, Carlsbad, CA), using the following cycling parameters: 30 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 1 min/kb.
  • PCR products were purified and sequenced using a BigDye Terminator v3.1 cycle sequencing kit from Applied Biosystems. Sequencing products were analyzed on a 3730 DNA analyzer from Applied Biosystems.
  • mice were purchased from Taconic and were housed in accordance with Institutional Animal Care and Use Committee protocols. Five to 7- week- old BALB/c mice were inoculated intranasally with 10 ⁇ l containing 2 x 10 7 to 4 x 10 7 CFU of a recently animal-passaged pneumococcus strain resuspended in PBS. All suspensions were plated for colony counts following inoculation to ensure that no inhibition had occurred in suspension prior to intranasal instillation.
  • mice were sacrificed by CO 2 asphyxiation, the trachea of each was exposed, 200 ⁇ l of sterile PBS was instilled into it, and the lavage fluid exiting the nares was collected. The lavage fluid was then serially diluted in PBS and plated on TSA. Plates were supplemented with neomycin (5 ⁇ g/ml) to prevent the growth of contaminants or with neomycin plus streptomycin to select for growth of the 19A derivatives. Results of antibiotic selection were verified using colony immunoblotting with a rabbit polyclonal antibody against capsular serotype 4 on neomycin-only plates. The lower limit of detection of this assay was 100 CFU/ml of lavage fluid.
  • Pneumococcal mutants were created as follows.
  • the blpMNO region was cloned into Escherichia coli plasmid pUC19 at the Smal site, using primers 1 and 11.
  • the resulting plasmid was designated pBlpAL.
  • the janus insertion was created by amplifying the cassette from strain CP1296 by PCR and engineering B stBI and Nhel sites into the 5' and 3' regions, respectively (primers 12 and 13). This product was ligated into pBlpAL cut with BstBI and Nhel at unique sites that span blpM through blpO.
  • the resulting plasmid was transformed into 6ASm r and selected for kanamycin resistance and streptomycin sensitivity.
  • This strain was named 6AAMNOjanus.
  • the remaining blpMNO mutants were constructed by replacing the janus cassette in this strain.
  • the blpM and -N deletions were constructed by performing inverse PCR on pBlpAL, using primers 3 and 4 for the blpM deletion and primers 5 and 6 for the blpN deletion. These primers were engineered to create a unique ⁇ sil site between the stop and start codons of the respective genes.
  • the resulting PCR products were then cut with ⁇ sil, ligated, and transformed into E.
  • coli ToplO cells (Invitrogen, Carlsbad, CA).
  • the blpO deletion was created by performing inverse PCR on pBlpAL, using primers 7 and 2.
  • the resulting product was phosphorylated using T4 D ⁇ A kinase, blunt end ligated, and transformed into E. coli ToplO cells.
  • the chimeric protein was created by amplification of D ⁇ A from TIGR4, using primers 8 and 9, which introduced a BstBI site at the 5' end.
  • This product was digested with BstBI and Nhel and ligated to the 1,022-kb BstBI/Nhel fragment of pBlpAL. All plasmids were verified by restriction digestion.
  • the janus cassette was replaced in strain 6AAMNOjanus by transforming the strain with the PCR product produced by primers 1 and 11 and selecting colonies on streptomycin plates. Deletion of the blpMN operon in the serotype 19A strain was performed by amplifying the janus cassette insertion in strain 6AAMNOjanus, using primers 1 and 11, and transforming the product into a streptomycin-resistant derivative of 19A. An unmarked mutation deleting the entire blpMNO region was created in this strain as described above. The janus cassette in ⁇ 9AAMN0janus was replaced with the wild-type locus by transforming cells with the plasmid pBlpAL.
  • 6AAbIpR was created by isolating DNA from the type 3 isolate containing an erythromycin cassette, replacing the blpR gene, and transforming the construct into the type 6A strain. This mutation was backcrossed three times to reduce the possibility of transformation occurring with unlinked DNA.
  • the integrative plasmid pEVP3 was used to create a transcriptional fusion to the blpMNPO and htrA promoters.
  • the blp reporter fusions one kb of DNA including the promoter region of blpMNPO and a small portion of upstream and downstream sequence was cloned between the Xbal and Nsil sites of the plasmid using primers 1 and 2 (Table 4) and maintained in ToplO cells.
  • htrA reporter fusions For the htrA reporter fusions, an 1100 bp fragment including the promoter region of htrA including 72bp of coding sequence were cloned between the Xbal and Nsil sites of the plasmid using primers 9 and 10 (Table 4).
  • the transparent type 6A variant was transformed with plasmid DNA and transformants were selected for by plating on TS plates supplemented with 3 ⁇ g/ml of chloramphenicol. For all transformations, bacteria were grown from plates at low inocula in C + Y pH 8.0 at 37° C until OD620 reached 0.150.
  • CSP 1 and 2 were purchased from Genscript as the following peptides: CSPl : EMRLSKFFRDFILQRKK and CSP2: EMRISRIILDFLFLRKK. After lOmin, approximately 100pg/ml of either purified genomic or plasmid DNA was added to the mixture and incubated at 30° C for an additional 40 min. The culture was then transferred to 37° C and incubated for 2 hours before plating on selective media.
  • Plasmid integration was confirmed using a primer to the upstream flanking region (primer 3 for blp insertion, 11 for htrA insertion) and a reverse primer internal to the lacZ gene (primer 4).
  • genomic DNA was isolated from the corresponding transparent transformant and used to transform the opaque variant of the type 6A strain. This approach was required due to the low transformation efficiency of this strain.
  • the native bgaA gene was deleted by transforming pneumococcus with plamid pE668 and selecting for erythromycin resistance, ⁇ -galactosidase activity was determined using a modified Miller assay as follows.
  • Pneumococcal strains were streaked for isolated colonies on TSA overnight. Single colonies were picked and inoculated into 10ml of TS broth. Cultures were allowed to grow for ⁇ 8 hours at 37° C to reach an OD620 of 0.1. Samples were then removed as indicated and assayed for ⁇ -galactosidase activity. For growth analysis, ImI of culture was removed and lO ⁇ l of 10% Triton X-100 added to this volume.
  • Synthetic active BIpC peptide determined by DNA sequencing of the 6A strain locus was found to be GLWEDILYSLNIIKHNNTKGLHHPIQL. This peptide was synthesized and purified to 95% purity by Genscript (Piscataway, NJ). Transcriptional activity was then determined for samples with and without the addition of BIpC. Assays were performed in triplicate. For dose response calculations, increasing amounts of BIpC were added to identical cultures and allowed to incubate for 2 hours. ECso and 95% confidence intervals were determined using Prism as follows. Peptide concentrations were transformed to their natural log values, then data points were then normalized such that the lowest number of each set was made equal to 0% and the highest number to 100%.
  • Nonlinear regression (curve fit) analysis was then performed without constraining the Hill Slope.
  • ECso was defined as the concentration of BIpC required to reach 50% of maximal activity.
  • the htrA gene was deleted in blp reporter constructs by insertion of the Janus cassette as described below.
  • the ciaH mutations were created in htrA reporter constructs using Janus insertion as described below.
  • the blpA gene was disrupted by insertion of the erythromycin cassette into a unique EcoRV site within the coding sequence of the gene using the plasmid pBlpAL, creating pBlp ALbIpA: :erm. This plasmid was then used to transform the 6At strain containing the blpMNPO lacZ fusion. Transformants were selected on media containing erythromycin and insertions confirmed by PCR.
  • Bands were visualized using enhanced chemiluminescence plus Western blotting detection system (GE Healthcare). Band intensities were determined for each dilution using gel analysis tools of ImageJ (Wayne Rasband; N1H [http://rsb.info.nih.gOv/j/]) and only dilutions within the linear range used for densitometry determination. Equal loading of comparable dilutions of the variants was confirmed using the monoclonal anti-pneumolysin antibody (Novocastra, UK) followed by anti-mouse HRP secondary. The amount of HtrA in variants was determined by equalizing for pneumolysin density in the same lane.
  • htrA and ciaH mutations in type 6A derivatives were created using exchange of the Janus cassette. DNA from pneumococcal isolates containing the Janus cassette replacing the internal portions of either htrA or ciaH genes was used to transform a streptomycin-resistant derivative of the type 6A variants. This mutation was backcrossed once to remove unlinked DNA. An in-frame, unmarked deletion of htrA was derived by transforming the isolates containing the Janus cassette with DNA from strain P1544.
  • the serine to alanine mutation in htrA was created in the 6A background by exchanging the Janus cassette with the mutated htrA gene using a plasmid with a fragment of DNA containing the mutated htrA gene.
  • the wildtype htrA gene was used to replace the Janus cassette by transforming strain P 1720 with genomic DNA from the type 6 A strain.
  • Constitutively active CiaH mutation CiaH ⁇ 230P was created by exchanging the Janus cassette in ciaH with DNA from strain Pl 386.
  • the wildtype ciaH locus was used to replace the Janus cassette and assessed for recovery of the wildtype phenotype.
  • Janus replacements were screened for loss of kanamycin resistance and recovery of streptomycin resistance. Appropriate mutations were confirmed by PCR. Identical methods were used to separately introduce the mutation in htrA and the deleted ciaH into the 6A derivative ⁇ - galactosidase reporter strains.
  • the BIpN FLAG fusion was created by amplifying the blpN gene with primers 7 and 8 (Table 4) introducing a C-terminal FLAG® tag in frame into the coding sequence followed by a stop codon and an Nsil site.
  • the PCR product was digested with BstBI and Nsil and ligated into vector pBlpALDN also digested with BstBI and Nsil creating plasmid pB IpALNFLAG.
  • the resultant plasmids were sequenced to ensure in-frame insertion and used to transform Pl 573 containing a Janus cassette disrupting the blpMNPO locus.
  • Transformants were selected on streptomycin containing plates and confirmed for the proper insertion by loss of kanamycin resistance and by PCR.
  • a streptomycin resistant isolate of the strain was first transformed with DNA derived from the opaque variant Pl 573 containing the Janus cassette in the blpMNPO locus. The isolate was selected on kanamycin plates and confirmed for the correct insertion by PCR.
  • the FLAG® epitope was inserted into the BIpM and BIpN sequences as described above.
  • Deletions in htrA in FLAG® strains were produced by transformation with DNA from strain Pl 180 and selection on erythromycin containing plates followed by a single back transformation. Deletions were confirmed by Western blotting using HtrA polyclonal antiserum for loss of protein expression. Detection of BIPMFLAG and BIPNFLAG was carried out by growing cells to confluence on TS plates supplemented with catalase. One hour prior to harvesting, 250 ⁇ g of synthetic BIpC were spread onto the surface of select plates. Cells were scraped from the surface of the plate and resuspended in PBS with 0.1% Triton XlOO for lysis. Equal quantities of lysates were denatured and separated on a 15% polyacrylamide gel. The gel was transferred and the membrane blocked in TBS with 5% NFDM for 2 hours.
  • the membrane was probed with ANTI-FLAG® M2 antibodies (Sigma-Aldrich), washed and detected with anti-mouse secondary antibody conjugated to alkaline phosphatase. The membrane was then stripped and re-probed with anti-pneumolysin antibody as a loading control followed by detection with anti mouse secondary antibody.
  • Example 1 Bacteriocin-like activity in a clinical isolate of the pneumococcus
  • Example 2 BIpR r ⁇ 2ulates expression of in vitro bacteriocin activity and immunity
  • the blpR regulatory gene homologue of the type 6A strain was deleted by replacement of an internal fragment of the gene with the erythromycin resistance cassette.
  • the resulting strain, 6AJbIpR was analyzed for loss of its inhibitory activity and immunity, using the plate overlay method.
  • 6AAbIpR was deficient in in vitro intraspecies inhibition when tested against TIGR4.
  • 6AAbIpR was killed by its parent strain, suggesting a deficiency in expression of its immunity phenotype (Fig. IA). Therefore, deletion of the BIpR response regulator led to a defect in both killing and immunity.
  • Example 3 Sequence analysis of the 6A blp locus [000102] Given the preliminary results for the type 6A strain, the blp locus was sequenced from the N terminus of the blpA gene to the end of the previously defined locus, SP0547 (Fig. 2). This region was predicted to contain the genes encoding the bacteriocins based on the arrangement of the locus in other previously sequenced strains. As in the TIGR4 locus, putative bacteriocin genes in the type 6 A locus were preceded by a highly conserved consensus sequence for BIpR binding. The type 6A strain's blp locus contains homologues for the predicted bacteriocin genes blpM, -N, and -O.
  • genes would be predicted to encode three proteins, with each containing a conserved N-terminal signal sequence followed by a double-glycine motif, consistent with the sequences of previously described bacteriocins. Surprisingly, similar genes were also found in the TIGR4 genome, although TIGR4 did not inhibit the growth of the type 6A strain.
  • the type 6A strain encodes BIpM, - N, and -O proteins that have 6 of 84, 2 of 67, and 2 of 49 residues, respectively, that differ from the TIGR4 sequence.
  • the type 6A locus contains two operons downstream of the putative BIpM, -N, and -O bacteriocins, preceded by two additional BIpR consensus binding sequences that contain open reading frames (ORFs) encoding proteins of unknown function.
  • the final operon contains homologous ORFs for the genes blpX, -Y, and -Z and, unlike TIGR4, is predicted to include the downstream ORF SP0547 due to deletion of a transcriptional terminator sequence.
  • BIpX, -Y, and -Z and SP0547 are 100%, 96%, 98%, and 99% identical to the TIGR4 sequence, respectively, at the amino acid level.
  • Example 4 Deletional analysis of blpM, -N, and -O
  • deletions were created in blpM, -N, and -O by deleting each gene in its entirety, leaving only its predicted stop and start codons.
  • the deletion of the entire blpMNO locus was created by introduction of a deletion spanning from the 5' end of the blpM ORF through the 3' end of the blpO ORF.
  • a chimeric gene was created by exchanging the type 6A strain blpM and -N with TIGR4 blpM and -N.
  • the chimeric protein contains the ⁇ terminus of 6A blpM with all three amino acid changes in the C terminus found in the TIGR4 locus and the entire blpN locus from TIGR4 (Fig. YB). This chimeric construct was used to determine if the difference in killing between the two strains was the result of the differences in these amino acids. To confirm this result and to demonstrate an absence of additional mutations outside the locus explaining the phenotype, a PCR product containing the corresponding original parental type 6A locus was used to replace the janus cassette. All strains were tested for a loss of in vitro inhibitory activity by the plate overlay method against strains TIGR4 and 6AAbIpMNO and used as an overlay against the type 6A isolate to look for a loss of immunity (Fig. IA and C).
  • Bacteriocins tend to have a significant degree of divergence when different strains within the same species are compared. This divergence may allow for intraspecies competition. Small changes in the bacteriocin often require reciprocal changes in the immunity protein so that organisms expressing similar but not identical bacteriocins are not protected from each other by their own immunity proteins.
  • blpM and -N coding sequences for the nine clinical isolates were analyzed. These strains include an array of clinical isolates of diverse capsular types that were isolated in different locations at different times. Seven of the nine isolates had sequences homologous to blpM and -N.
  • the remaining two isolates contained coding regions for other bacteriocin- like peptides Q)IpI and blpK) homologous to those found in the TIGR4 locus.
  • the BlpM and - ⁇ sequences were aligned and analyzed for conserved amino acids (Fig. 3). Interestingly, the seven BlpM sequences seemed to be divided into two groups. Group 1 contains those with 100% identity to the TIGR4 sequence. Group 2 comprises those with 98 to 100% identity to the type 6A strain's sequence. In comparing the Blp ⁇ sequences, the RL amino acid sequence at amino acids 40 and 41 was seen in all strains containing the group 1 BlpM sequence, while the KI sequence was seen in strains containing the group 2 BlpM sequence.
  • Example 6 The blp locus is functional in vivo during colonization
  • strain 19 AAbIpMNO was deficient in growth inhibition when tested against TIGR4 and had an immunity defect when tested against the parent strain (Fig. IA and C).
  • the corrected mutant, 19 AblpMNO w ⁇ had the expected wild-type phenotype in both inhibition and immunity.
  • the type 19A strain, TIGR4, 19 AbIpMNO , and 19AAbIpMNO were inoculated intranasally either alone or in pairs. Singly inoculated mice were colonized with TIGR4, 19A, 19 AAbIpMNO, or ⁇ 9AblpMN0 w ⁇ at equivalent levels (Fig. 4A and B).
  • Dually colonized mice given 19A and 19 AAbIpMNO were colonized predominantly with the type 19A strain (Fig. 4B).
  • Dually colonized mice given TIGR4 and 19 AblpMN0 WT were colonized primarily with the 19A strain, mimicking our in vitro inhibition results (Fig. 4A).
  • the competitive advantage of the 19A strain was eliminated when TIGR4 was coinoculated with 19 AAbIpMNO. In fact, these animals were colonized primarily with TIGR4, with little detectible colonization by the mutant strain.
  • CiaRH is involved in the regulation of a large number of disparate loci including a number of genes involved in pneumococcal competence.
  • the inventors of the instant application found that CiaRH is involved in pneumocinMN expression.
  • mutants of the type 6A strain with either a deletion in CiaH or a constitutively active version of CiaH were created and blp mediated inhibition using overlay assays were determined.
  • HtrA is differentially regulated opacity variants
  • CiaRH activity results in the up regulation of the serine protease HtrA. Many of the important roles in bacterial growth and survival attributed to the CiaRH system are mediated by its upregulation of HtrA. It is also known that expression of HtrA is required for pneumococcal growth at elevated temperatures, resistance to H2O2 and virulence in a pneumonia model of infection. CiaRH mediated increases in HtrA expression in a CiaRH overexpressing strain were recently shown to repress the pneumococcal competence pathway, likely as a consequence of protease mediated digestion of a regulatory element controlling the com regulon.
  • the inventors of the instant application evaluated the opacity variants of the type 6A strain for differential expression of HtrA at the level of transcription and translation.
  • Opaque and transparent variants of the type 6A strain lacking endogenous ⁇ -galactosidase activity were transformed with an integrative plasmid derivative of pEVP3 containing the htrA promoter fused to the ⁇ -galactosidase gene.
  • Miller assays on these constructs grown in TS broth demonstrated that, in late exponential phase growth, opaque variants produce ⁇ 4 fold less htrA transcript than transparent strains (Figure 9A).
  • Example 10 HtrA is required for the post-transcriptional regulation of bacteriocin expression
  • HtrA expression affects pneumocin MN mediated activity
  • the inventors moved an in-frame, unmarked deletion of the htrA gene as well as a protease deficient htrA gene encoding a serine to alanine mutation in the active site into the transparent type 6A variant. Deletion mutants were confirmed by PCR and Western blot using anti-HtrA antiserum. Serine to alanine mutants were confirmed by PCR and western blotting with anti-HtrA antiserum to confirm recovery of the HtrA specific band.
  • the type 6A transparent variant with either a deletion or inactivating mutation in the htrA gene had an increased zone of inhibition when tested in the plate inhibition assay and compared to the transparent parent ( Figure 6A, D, E).
  • Figure 6G A complemented transparent strain in which the mutated htrA gene was replaced with the wildtype locus also had no detectable activity, verifying that no new deletions impacting the inhibitory phenotype had been introduced during the transformation steps required for insertion of the Janus cassette ( Figure 6F).
  • HtrA can modify the bacterial response to peptide pheromone, an additional regulator functioning downstream of BIpC signaling is involved in controlling blp expression in opacity variants.
  • HtrA activity plays a role in transcriptional regulation of the blp locus
  • the combined observations that peak activities of blpMNPO transcript were unaffected by a deletion in htrA and saturating levels of BIpC in plates could not restore pneumocinMN inhibitory activity in transparent strains show that HtrA also participates in the post transcriptional regulation of pneumocinMN.
  • Example 11 Deletion of htrA results in increased levels of epitope tagged pneumocinMN
  • the transparent variant expressing pneumocinMFLAG produced no detectable product even after addition of synthetic BIpC pheromone while the opaque variant had a small amount of tagged peptide detectable following pheromone induction ( Figure 10 Lanes 3 and 4).
  • transparent variants with a deletion in htrA produced large amounts of pneumocinMFLAG independent of BIpC addition ( Figure 10 Lane 6).
  • Deletion of htrA in the opaque variants also resulted in an increase in pneumocinMFLAG over that seen in the opaque variant expressing wildtype htrA.
  • a FLAG tagged version of blpN was transformed into the opacity variants with and without a deletion in htrA.
  • the BIpNFLAG expressing organisms retained inhibitory activity when tested in plate overlay assays.
  • Introduction of a FLAG tag onto the C -terminus of the blpN coding sequence resulted in an identical pattern of expression as the BIpMFLAG expressing strains after Western blotting with anti-flag antibody.
  • the NFLAG was undetectable in transparent isolates, detectable at low levels in opaque variants and at high levels in transparent variants lacking htrA expression ( Figure 10 Lanes 7-9). This implies that HtrA plays a role in post-transcriptional regulation which is the major determinant controlling pneumocinMN activity.

Abstract

This invention is relates to the control or treatment of bacterial infection or colonization through the use of novel bacteriocin producing Streptococcus strains. Specifically, the invention relates to methods and compositions comprising bacteriocins produced by upregularting the expression of the blp locus and downregulating the expressions of HtrA and CiaRH in a Streptococcus bacteria.

Description

BACTERIA STRAINS AND BACTERIOCIN PRODUCED THEREFROM
FIELD OF INVENTION
[0001] This invention is directed to the control or treatment of bacterial infection or colonization through the use of novel bacteriocin producing Streptococcus strains. Specifically, the invention relates to methods and compositions comprising bacteriocins produced by upregulating the blp locus in strains of Streptococcus bacteria.
BACKGROUND OF THE INVENTION
[0002] Streptococcus pneumoniae colonizes a majority of children by 1 year of age. Colonization with a single strain can last for weeks to months but is eventually cleared. Serial culturing of samples from the nasopharynxes of children has demonstrated that the population of pneumococci is constantly in flux, with one strain replacing another over time. In addition, some children appear to be colonized with two or more pneumococcal strains at any given time. The recent introduction of the seven-valent conjugate pneumococcal vaccine to the standard infant vaccination schedule has dramatically reduced the incidence of both colonization and invasive disease caused by the pneumococcus. Because the vaccine contains only 7 of the >90 serotypes of pneumococci, there have been concerns that some of the remaining serotypes may fill the void left by the vaccine-targeted organisms. Over the intervening years since its release, reports have suggested that serotype replacement may be occurring at the levels of both colonization and disease. For example, the incidence of the previously uncommon nonvaccine serotype 19A has increased significantly in the postvaccine era as a cause of invasive pneumococcal disease in children of less than 5 years of age. Whether infections with these formerly infrequent serotypes will eventually result in a similar disease spectrum with respect to numbers and severity has yet to be determined. The fact that serotype replacement has occurred suggests that competition between the predominant vaccine strains and the replacement strains was occurring at some level prior to vaccination of the population. Understanding the dynamics that occur between distinct strains of pneumococci within the polymicrobial environment of the human nasopharynx may help to better predict the outcome of vaccination. These interactions are likely to include bacterial factors that allow one strain to gain a foothold in the competitive environment of the nasopharynx.
[0003] Bacteriocins are small antimicrobial peptides produced by many bacterial species that have been implicated in intra- and interspecies competition. Bacteriocins typically target organisms that are either closely related to or within the same species as the producer bacteria. Producer bacteria are protected from the effects of their own bacteriocins via production of a specific immunity protein. This protein is typically cotranscribed with the genes encoding the bacteriocins.
SUMMARY OF THE INVENTION
[0004] In one embodiment, the invention provides a method for generating bacteriocins in a bacteria comprising the step of contacting with an agent capable of upregulating expression of the blp locus. In one embodiment, the blp locus is represented by the nucleotide sequence set forth in SEQ. ID. NO l.
[0005] In another embodiment, the invention provides a method for treating a bacterial infection or colonization in a subject, comprising the step of administering to said subject a bacteriocin producing bacteria, wherein the bateriocin is produced by upregulating the expression of the blp locus, thereby treating an infection in said subject.
[0006] In another embodiment, the invention provides a composition for preventing bacterial growth or colonization, comprising an effective amount of a bacteriocin, wherein the bateriocin is produced by upregulating the expression of the blp locus.
[0007] In another embodiment, the invention provides a method for generating bacteriocins in a bacteria comprising the step of contacting the bacteria with an agent capable of upregulating expression of the blp locus, wherein the agent comprises an amino acid sequence of blpC.
[0008] In another embodiment, the invention provides a vaccine for treating, preventing or ameliorating a subject against pneumococcal infection or colonization, comprising a pharmaceutically acceptable carrier and an immunologically effective amount of a recombinant bacteria that produces a bacteriocin, wherein the vaccine comprises an amino acid sequence of blpC.
[0009] In another embodiment, the invention provides a composition for inhibiting bacterial growth or colonization, comprising an effective amount of a blpC to generate bacteriocins in a bacteria, wherein the blpC peptide comprises the amino acid sequence of GLWEDILYSLNIIKHNNTKGLHHPIQL.
[00010] In another embodiment, the invention provides a method for generating bacteriocins in a bacteria comprising the step of down-regulating the expression of htrA.
[00011] In another embodiment, the invention provides a method for generating bacteriocins in a bacteria comprising the step of down-regulating the expression of ciaRH.
[00012] In another embodiment, the invention provides a method for generating bacteriocins in a bacteria comprising the steps of upregulating expression of the blp locus; down- regulating the expression of htrA; and down-regulating the expression of ciaRH.
[00013] In another embodiment, the invention provides a method for generating bacteriocins in a strain of bacteria comprising the steps of upregulating expression of the blp locus, wherein the upregulation of expression of the blp locus is caused by down-regulating the expressions of htrA and ciaRH.
[00014] In another embodiment, the invention provides a method for treating an infection, the method comprising the steps of inhibiting or reducing bacterial growth or colonization in a subject comprising the steps of administering to said subject a bacteriocin producing bacteria, thereby treating an infection in said subject, wherein the bacteriocin is produced by down- regulating the expression of htrA.
[00015] In another embodiment, the invention provides a method for treating an infection, the method comprising the steps of inhibiting or reducing bacterial growth or colonization in a subject comprising the steps of administering to said subject a bacteriocin producing bacteria, thereby treating an infection in said subject, wherein the bacteriocin is produced by down- regulating the expression of ciaRH. [00016] In another embodiment, the invention provides a composition for inhibiting bacterial growth or colonization, comprising an effective amount of a bacteriocin, wherein the bacteriocin is produced by down-regulating the expression of htrA.
[00017] In another embodiment, the invention provides a composition for inhibiting bacterial growth or colonization, comprising an effective amount of a bacteriocin, wherein the bacteriocin is produced by down-regulating the expression of ciaRH.
[00018] In another embodiment, the invention provides a composition for inhibiting bacterial growth or colonization, comprising an effective amount of a bacteriocin, wherein the bacteriocin is produced by upregulating the expression of the blp locus and down-regulating the expressions of htrA and ciaRH.
[00019] In another embodiment, the invention provides a mutated Streptococcus pneumoniae bacteria, wherein said bacteria exhibits elevated production pneumocinMN relative to a non- mutant bacteria, and wherein the mutation is in htrA, ciaR, ciaH or their combination.
[00020] In another embodiment, the invention provides a vaccine for treating, preventing or ameliorating a subject against pneumococcal infection or colonization, comprising a pharmaceutically acceptable carrier and an immunologically effective amount of a mutant bacteria, wherein said mutant bacteria exhibits elevated production pneumocinMN relative to a non-mutant bacteria, and wherein the mutation is in htrA, ciaR, ciaH or their combination.
[00021] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[00022] The invention will be better understood from a reading of the following detailed description taken in conjunction with the drawings in which like reference designators are used to designate like elements, and in which:
Figure 1. (A) Table summarizing results of agar overlay assays with deletion mutants of the blp locus. Plus signs designate definite zones of inhibition, and empty cells designate combinations that were not tested. (B) Alignment of BIpM and BIpN amino acid sequences from type 6A and TIGR4 strains and the chimeric proteins from 6AblpMN0T1GR. Shaded amino acids are nonconserved, and arrows designate putative cleavage sites of preproteins. (C) Photographs of results of selected overlay assays. Pictures a to g demonstrate test strains with zones of inhibition, while pictures h to m demonstrate test strains lacking inhibition. Pictures a and b are shown with a TIGR4 overlay, and pictures c to m are shown with an overlay of 6AΔbIpMNO. Test strains: a and c, type 6A; b and f, type 19A; d, 6AblpMN0WT; e, 6AΔbIpO; g, 19AblpMN0WT; h, 6AΔbIpMNO; i, 6AΔbIpM; j, 6AΔbIpN; k, TIGR4; 1, 6AblpMNOTIGR4; m, 19 AΔbIpMNO;
Figure 2. Graphical demonstration of the blp locus in a type 6A strain and comparison with the corresponding portion of the TIGR4 genome. Solid arrows represent coding sequences for double glycine-containing proteins, vertically striped arrows represent genes of unknown function, checked arrows represent transport genes, the white box represents an insertion sequence element, and gray boxes represent the conserved putative BIpR binding sites designating the start sites of operons. The gap in 6A designates an unsequenced region;
Figure 3. Amino acid alignment of sequences of BIpM and BIpN from a selection of clinical isolates of the serotypes indicated. Shaded amino acids are areas of nonconservation. Arrows designate putative cleavage sites of preproteins; and
Figure 4. 19 AAbIpMNO is outcompeted by TIGR4 (A) or its parent type 19A strain (B) during mouse nasopharyngeal colonization. Six-week-old BALB/c mice were challenged intranasally with single or dual inoculations of the type 19A parental strain (19A; open circles), the 19AblpMN0WT corrected mutant (19A; closed circles), 19AΔbIpMNO (closed diamonds), and TIGR4 (19AbIp-; closed triangles) (A) or with single or dual inoculations of the type 19A strain andl9 AΔbIpMNO (B). The colonizing strain is depicted on the x axis and was detected in lavage fluid at 4 days postinoculation at the density indicated (y axis). Coinoculated strains are shown in parentheses. Statistical analysis was done by the Mann- Whitney test, and horizontal lines indicate median values. Dashed lines denote the limit of detection.
Figure 5. Organization of the blp locus of the type 6A strain. Open reading frames within the locus are designated by arrows as follows: regulatory proteins by white arrows, transporter genes by hatched arrows, immunity genes by grey arrows, double glycine containing preptides by black arrows and genes of unknown function by horizontal stripes. Letters above the arrows identify the blp gene designation. Black rectangles identify the putative response regulator binding sites. The designated region upstream of blpC has not been directly sequenced from this strain and is derived from the published TIGR4 sequence.
Figure 6. Overlay assay demonstrating the effect of deletion of ciaRH or htrA on pneumocinMN mediated growth inhibition. Plate grown organisms were inoculated into TS plates containing catalase and allowed to grow for 6 hours before an overlay containing 0.5% TS agar, catalase and approx 10 15 CFU/ml of the overlay strain 6AoObIpMNPO, containing a deletion in the putative immunity protein BIpP, was carefully applied to the plate. Results of the overlay assay were recorded after overnight growth for strains: A. 6At, B. 6AtDaVuH, C. 6Atciareplaced, D. 6AtDhtrA298-1152 , E. 6At htrAs234A , F. 6AthtrAreplaced, G. 6AtDhtrAblpMNO, Η. 6Ao, I. 6AociaHτ230P .
Figure 7. Miller assays on broth grown organisms demonstrating the transcriptional activity of opacity variants with and without the addition of synthetic BIpC. - A. Variants were transformed with plasmid pEVPblpM resulting in duplication/insertion of the lacZ reporter gene behind the blpM promoter to create 6AoblpMlacZ, 6AtblpMlacZ. 6AoblpMlacZDbgaA (circles) and 6AtblpMlacZDbgaA (squares) were grown from single colonies in TS broth. Samples were taken at hourly intervals after reaching an OD620 of 0.1 and promoter activity determined by Miller assay (closed symbols). Growth curves derived from the same experiment are shown as open symbols with dotted lines. - B. Kinetics of transcriptional response to synthetic BIpC in a reporter fusion deficient in 30 pheromone secretion. 6AtblpMlacZDblpA was grown to OD620 of ~ 0.1 and 100ng/ml of synthetic BIpC was added to half of the culture volume. Samples were taken from each culture at half hour intervals up to three hours (open squares no BIpC added, close squares BIpC added). Peak activity was seen between 2 and 2.5 hours after addition of pheromone. Miller Units are the mean of three determinations +/- SE.
Figure 8. Dose response of opacity variants with and without HtrA to synthetic BIpC. 6AtblpMlacZDbgaA (closed squares) and 6AoblpMlacZDbgaA (closed circles) and their corresponding htrA deletions (open symbols) were grown from single colonies to an OD620 of 0.1. Increasing concentrations of synthetic BIpC were added to ImI of culture for each isolate and allowed to incubate for 2hrs at 37° C. Miller assays were then performed on each culture after determining the end point ODOOO. Miller Units are the mean of three determinations +/- SE. The table contains derived ECso and 95% confidence intervals for all four strains denoted above following non-linear regression analysis.
Figure 9. Variation in HtrA levels in opacity variants of the type 6A strain. - A. Reporter fusions of the htrA promoter to the lacZ gene were introduced into opaque and transparent variants lacking endogenous β-galactosidase activity with and without a deletion in ciaH. Cultures were grown from a single colony to an OD620 of 0.1 then OD620 was read and samples were taken every hour for four hours and used in Miller assay to determine promoter activity. OD620 is designated by open symbols and dotted lines. Miller Units are shown as closed symbols. Miller Units are the mean of three determinations +/- SE. - B. Organisms were grown in broth culture to OD620 of -0.500 and pelleted. Pelleted organisms were resuspended in PBS and sonicated. Equal concentrations and 1:1 dilutions of total protein were separated by SDS-PAGE and transferred for western blotting. Polyclonal anti-HtrA antibody was used to determine HtrA levels in opaque (6Ao) and transparent (6At) backgrounds. Monoclonal anti pneumolysin antibody was used as a loading control.
Figure 10. Western blots detecting FLAG® epitope tag. Bacterial lysates derived from plate grown organisms were loaded with equal amount of total protein and separated using 15% SDS-PAGE. The transferred membrane was probed with anti-FLAG® monoclonal antibody M2 or anti -pneumolysin antibody as a loading control. Samples were loaded as follows: 1. 6AtDhtrA, 2. 6AoObIpMNPO, 3, 6AoblpMFiAG, 4. 6AtblpMFiAG, 5. 6AoblpMFLAoOhtrA, 6. 6AtblpMFLAGDhtrA, 7. 6AtblpNFiAG, 8. 6AtblpNFiAGDhtrA, 9. 6AoblpNFiAG. Sequences above blots designates constructed FLAG insertions (underlined) into the predicted secreted form of the BIpM and BIpN peptides. DETAILED DESCRIPTION OF THE INVENTION
[00023] In one embodiment, provided herein is methods and compositions for the control or treatment of infection through the use of novel bacteriocin producing Streptococcus strains and/or novel bacteriocins produced by this bacteria. In another embodiment, provided herein are novel bacteriocins, amino acid sequences of the novel bacteriocins, and to the strains of Streptococcus producing the novel bacteriocins. In one embodiment, provided herein are therapeutic compositions containing the novel bacteriocins and/or the strains of Streptococcus producing them and to uses of the therapeutic compositions.
[00024] In one embodiment the blp locus of pneumococcus encodes a number of bacteriocin- like peptides. Upstream of the bacteriocin genes, the locus contains open reading frames for a typical two-component regulatory system (blpR and blpH), a small peptide pheromone Q)IpC), and a dedicated ABC transporter (blpA and -B). In another embodiment the ABC transporter recognizes the N termini of both the pheromone and the bacteriocins and transports these peptides across the cytoplasmic membrane, concurrent with cleavage at a conserved double-glycine motif. Cleaved extracellular BIpC can bind in one embodiment, to the sensor kinase, BIpH. This interaction results, in another embodiment, in the activation of BIpR and, in another embodiment, upregulation of the entire gene cluster via binding to consensus sequences within each promoter. Transcriptional analysis of the locus in the two fully sequenced pneumococcal strains R6 and TIGR4 demonstrate that application of chemically synthesized BIpC results in one embodiment, in upregulation of a number of operons only within the locus in one embodiment, comprising those encoding the regulatory proteins in one embodiment, or transport apparatus, and putative bacteriocins in certain other discrete embodiments of the methods and compositions described herein. The transcript level of a downstream operon encoding BIpXY and -Z was upregulated in another embodiment, by the addition of BIpC. In one embodiment, this operon encodes proteins involved in immunity. Analysis of a number of pneumococcal strains demonstrated that there are at least four different pheromones produced and that each is specific for its cognate BlpR/H protein.
[00025] In one embodiment, the blp locus is characterized in a clinical isolate of pneumococcus demonstrating an in-vitro phenotype consistent with bacteriocin activity and in another embodiment defines the importance of the locus in competition during nasopharyngeal pneumococal colonization. In one embodiment, bacteriocin genes of a number of clinical isolates are sequenced to determine which amino acids are important in dictating inhibition in vitro.
[00026] In one embodiment, provided herein is a method for generating bacteriocins in a bacteria strain comprising the step of contacting the bacteria with an agent capable of upregulating expression of the blp locus in the bacterial genome. In another embodiment, the blp locus is represented by the nucleotide sequence set forth in SEQ. ID. NO 1:
Figure imgf000010_0001
Figure imgf000011_0001
[00027] In another embodiment, the blp locus is represented by a fragment of the nucleotide sequence set forth in SEQ. ID. NO 1. In another embodiment said bacteria is Streptococcus. In one embodiment, said Streptococcus is Streptococcus pneumoniae.
[00028] In one embodiment, "contacting" a bacteria with a substance refers to (a) providing the substance to the environment of the bacteria (e.g., solution, in vitro culture medium, anatomic fluid or tissue) or (b) applying or providing the substance directly to the surface of the bacteria, in either case, so that the substance comes in contact with the surface of the cell in a manner allowing for biological interactions between the bacteria and the substance.
[00029] In another embodiment, said agent used in the methods and compositions described herein for the upregulation of the blp locus expression, is encoded by the blpC gene. In one embodiment, said the agent used in the methods and compositions described herein for the upregulation of the blp locus expression, is an mRNA. In another embodiment, the agent is a protein. In another embodiment, the agent is a synthetic protein. In another embodiment, the agent is a synthetic peptide. In another embodiment, the agent is a peptido-mimetic molecule. In another embodiment, the agent is a small molecule, or in another embodiment, the agent is a combination thereof.
[00030] In one embodiment, the agent used in the methods and compositions described herein for the upregulation of the blp locus expression, is BIpC, or its functional fragment in another discrete embodiment. In one embodiment, BIpC comprise the amino-acid sequence set forth in SEQ. ID. No. 2: Met-Asp-Lys-Lys-Gln-Asn-Leu-Thr-Ser-Phe-Gln-Glu-Leu-Thr-Thr-Thr-Glu-Leu-Asn-Gln- Ile-Thr-Gly-Gly-Gly-Leu-Trp-Glu-Asp-Leu-Leu-Tyr-Asn-Ile-Asn-Arg-Tyr-Ala-His-Tyr-Ile- Thr (SEQ ID. No. 2).
[00031] In one embodiment, pneumococcus colonizes the nasopharynx as the initial step in its pathogenesis. In another embodiment, human nasopharyngeal carriage is a major reservoir of pneumococci and the source of horizontal spread of this pathogen within the community in another embodiment. Factors that contribute to clearance of colonization affect in one embodiment, the frequency of transmission of the pneumococcus and the overall incidence of pneumococcal disease in the population. In one embodiment, both host and bacterial factors that contribute to clearance remain incompletely characterized. In one embodiment, colonization is cleared in between about 4 to 8 weeks after a new strain is acquired, but the length of carriage is highly variable both between individuals and among different serotypes. In one embodiment, the methods and compositions provided herein, are effective in the prevention or clearance of pneumococal colonization in a subject's nasopharyngeal passages. [00032] In one embodiment, the BIpC protein, or its functional fragment are between about 67 to about 99% homologous to SEQ ID No. 2. In another embodiment, the BIpC protein, or its functional fragment are between about 67 to about 75% homologous to SEQ ID No. 2. In another embodiment, the BIpC protein, or its functional fragment are between about 76 to about 85% homologous to SEQ ID No. 2. In another embodiment, the BIpC protein, or its functional fragment are between about 86 to about 95% homologous to SEQ ID No. 2. In another embodiment, the BIpC protein, or its functional fragment are between about 96 to about 99% homologous to SEQ ID No. 2.
[00033] In one embodiment, the expression of blp locus is upregulated by an agent that comprises an amino acid sequence of blpC. In another embodiment, the amino acid sequence of blpC comprises the sequence of GLWEDILYSLNIIKHNNTKGLHHPIQL.
[00034] In another embodiment, the invention provides a vaccine for treating, preventing or ameliorating a subject against pneumococcal infection or colonization. The vaccine comprises a pharmaceutically acceptable carrier and an immunologically effective amount of a recombinant Streptococcus bacteria that produces a bacteriocin, wherein the vaccine comprises a synthetic amino acid sequence of blpC. In another embodiment, the synthetic amino acid sequence of blpC comprises the sequence of GLWEDILYSLNIIKHNNTKGLHHPIQL. In an exemplarary embodiment, a bacteriocin is pneumocinMN that coprises the amino acid sequences of blpM, blpN, or fragments thereof.
[00035] In one embodiment the term "Homologous", refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. In another embodiment, if 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. In another embodiment, the DNA sequences ATTGCC and TATGGC share 50% homology. In one embodiment, a comparison is made when two sequences are aligned to give maximum homology.
[00036] In one embodiment, to determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology # of identical positions/total # of positions times 100). In another embodiment, the determination of percent homology between two sequences can be accomplished using a mathematical algorithm. In one embodiment a non-limiting example of a mathematical algorithim utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. MoI. Biol. 215:403-10. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. In another embodiment a non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used, in one embodiment. In another embodiment, programs which are equivalent in terms of the results they produce can be used. In one embodiment, the agent used in the methods and compositions described herein for the upregulation of the blp locus expression, has about 75%, 80%, 85%, 90%, or 95% homology. [00037] In another embodiment, the addition of the agent used in the methods and compositions described herein for the upregulation of the blp locus expression, results in the upregulation of a blp locus operon. In one embodiment, said operon encodes a regulator protein, a transport protein, an immunity protein, a bacteriocin, or a combination thereof. In another embodiment, said regulator protein is encoded by the blpR gene. In another embodiment, said regulator protein is encoded by the blpH gene. In another embodiment, said regulator protein is encoded by a combination blpR and blpH genes. In another embodiment, said regulator protein is encoded by amino acid sequences set forth in SEQ ID NO's 3-4:
Met-Arg-Ile-Phe-Val-Leu-Glu-Asp-Asp-Phe-Ser-Gln-Gln-Thr-Arg-Ile-Glu-Thr-Thr-Ile- Glu-Lys-Leu-Leu-Lys-Ala-His-His-Ile-Ile-Pro-Ser-Ser-Phe-Glu-Val-Phe-Gly-Lys-Pro-Asp- Gln-Leu-Leu-Ala-Glu-Val-His-Glu-Lys-Gly-Ala-His-Gln-Leu-Phe-Phe-Leu-Asp-Ile-Glu- Ile-Arg-Asn-Glu-Glu-Met-Lys-Gly-Leu-Glu- VaI- Ala- Arg-Lys-Ile-Arg- Asp- Arg-Asp-Pro- Tyr-Ala-Leu-Ile-Val-Phe-Val-Thr-Thr-His-Ser-Glu-Phe-Met-Pro-Leu-Ser-Phe-Arg-Tyr-Gln- Val-Ser-Ala-Leu-Asp-Tyr-Ile-Asp-Lys-Ala-Leu-Ser-Ala-Glu-Glu-Phe-Glu-Ser-Arg-Ile-Glu- Thr-Ala-Leu-Leu-Tyr-Ala-Asn-Ser-Gln-Asp-Ser-Lys-Ser-Leu-Ala-Glu-Asp-Cys-Phe-Tyr- Phe-Lys-Ser-Lys-Phe-Ala-Gln-Phe-Gln-Tyr-Pro-Phe-Lys-Glu-Val-Tyr-Tyr-Leu-Glu-Thr- Ser-Pro-Arg-Ala-His-Arg-Val-Ile-Leu-Tyr-Thr-Lys-Thr-Asp-Arg-Leu-Glu-Phe-Thr-Ala-Ser- Leu-Glu-Glu-Val-Phe-Lys-Gln-Glu-Pro-Arg-Leu-Leu-Gln-Cys-His-Arg-Ser-Phe-Leu-Ile- Asn- Pro-Ala- Asn-Val-Val-His-Leu-Asp-Lys-Lys-Glu-Lys-Leu-Leu-Phe-Phe-Pro-Asn-Gly- Gly-Ser-Cys-Leu-Ile-Ala-Arg-Tyr-Lys-Val-Arg-Glu-Val-Ser-Glu-Ala-Ile-Asn-Lys-Leu-His (SEQ ID NO 3)
Met-Asn-Ile-Ala-Trp-Ile-Leu-Leu-Tyr-Thr-Leu-Val-Thr-Asn-Gly-Leu-Glu-Ile-Val-Ile-Phe- Phe-Lys-Val-Asp-Gly-Ile-Asp-Leu-Thr-Phe-Glu-Arg-Ile-Phe-Lys-Ala-Phe-Leu-Leu-Lys-Ile- Leu-Leu-Ala-Phe-Val-Phe-Val-Met-Ile-Ser-Tyr-Ile-Val-Gly-Asn-Val-Tyr-Leu-Ser-Tyr-Phe- Met-Glu-Pro-Leu-Tyr-Gly-Ile-Gly-Leu-Ser-Phe-Leu-Leu-Leu-Arg-Gly-Leu-Pro-Lys-Lys- Leu-Leu-Phe-Phe-Tyr-Gly-Leu-Phe-Pro-Met-Ile-Leu-Val-Asn-Leu-Phe-Tyr-Arg-Gly-Val- Ser-Tyr-Phe-Val-Leu-Pro-Phe-Leu-Gly-Gln-Gly-Gln-Val-Tyr-Asp-Gly-Tyr-Ser-Phe-Thr- Gly-Leu-Cys-Ile-Ile-Ile-Phe-Asn-Phe-Phe-Ile-Ser-Leu-Ala-Phe-Leu-Lys-Trp-Leu-Asp-Tyr- Asp-Phe-Thr-Ser-Leu-Arg-Lys-Glu-Ile-Leu-Asp-Lys-Ala-Phe-Gln-Lys-Ser-Leu-Thr-Gln- Ile-Asn-Trp-Ile-Met-Gly-Gly-Tyr-Tyr-Leu-Val-Met-Glu-Ser-Leu-Ser-Phe-Phe-Glu-Tyr-Glu- Gln-Ser-Ile-Gln-Ser-Lys-Thr-Val-Arg-His-Leu-Ile-Leu-Val-Phe-Tyr-Leu-Leu-Phe-Phe-Met- Gly-Val-Ile-Lys-Lys-Leu-Asp-Thr-Tyr-Leu-Lys-Glu-Lys-Leu-Tyr-Glu-Arg-Leu-Glu-Gln- Glu-Gln-Ala-Leu-Arg-Tyr-Arg-Asp-Met-Glu-Arg-Tyr-Ser-Arg-His-Ile-Glu-Glu-Leu-Tyr- Lys-Glu-Val-Arg-Ser-Phe-Arg-His-Asp-Tyr-Thr-Asn-Leu-Leu-Thr-Ser-Leu-Arg-Leu-Gly- Ile-Glu-Glu-Glu-Asp-Met-Glu-Gln-Ile-Lys-Glu-Val-Tyr-Gly-Ser-Val-Leu-Lys-Asp-Ser-Ser- Gln-Lys-Leu-Gln-Asn-Asn-Lys-Tyr-Asp-Leu-Gly-Arg-Leu-Val-Asn-Ile-Arg-Asp-Lys-Ala- Leu-Lys-Ser-Leu-Leu-Ala-Gly-Lys-Phe-Leu-Lys-Ala-Arg-Asp-Lys-Asn-Ile-Val-Phe-Asn- Val-Glu-Val-Pro-Glu-Glu-Ile-Gln-Val-Glu-Gly-Met-Ser-Leu-Leu-Asp-Phe-Leu-Thr-Ile-Val- Ser-Ile-Leu-Cys-Asp-Asn-Ala-Ile-Glu-Ala-Ser-Val-Glu-Ala-Ser-Gln-Pro-His-Val-Ser-Ile- Ala-Phe-Leu-Lys-Asn-Gly-Ala-Gln-Glu-Thr-Phe-Ile-Ile-Glu-Asn-Ser-Ile-Lys-Glu-Glu-Gly- Ile-Asp-Ile-Ser-Glu-Ile-Phe-Ser-Phe-Gly-Ala-Ser-Ser-Lys-Gly-Glu-Glu-Arg-Gly-Val-Gly- Leu-Tyr-Thr-Val-Met-Lys-Ile-Val-Glu-Ser-His-Pro-Asn-Thr-Ser-Leu-Asn-Thr-Thr-Cys- Gln-Asn-Gln-Val-Phe-Arg-Gln-Val-Leu-Thr-Val-Ile-His-Thr-Glu (SEQ ID NO 4).
[00038] In one embodiment, the BIpR protein, BIpH protein, or its functional fragment are between about 67 to about 99% homologous to SEQ ID No. 3-4, respectively. In another embodiment, the BIpR protein, the BIpH protein or its functional fragment are between about 67 to about 75% homologous to SEQ ID No. 3-4, respectively. In another embodiment, the the BIpR protein, the BIpH protein, or its functional fragment are between about 76 to about 85% homologous to SEQ ID No. 3-4, respectively. In another embodiment, the BIpR protein, the BIpH protein, or its functional fragment are between about 86 to about 95% homologous to SEQ ID No. 3-4, respectively. In another embodiment, the the BIpR protein, the BIpH protein, or its functional fragment are between about 96 to about 99% homologous to SEQ ID No. 3-4, respectively.
[00039] In another embodiment, said transport protein is encoded by the blpA, or blpB gene, or a combination thereof. In one embodiment, said transport protein is BIpA. In another embodiment, said transport protein is BIpB. In another embodiment, said transport protein is encoded by amino acid sequences set forth in SEQ ID NO 5-6: Met-Lys-Gly-Phe-Gly-Met-Phe-Arg-Phe-Arg-Arg-Thr-Phe-Val-Pro-Gln-Ile-Asp-Met-Arg- Asn-Cys-Gly-Val-Ala-Ala-Leu-Ala-Leu-Val-Ala-Lys-Tyr-Tyr-Gly-Ser-Asp-Tyr-Ser-Leu- Ala-His-Leu-Arg-Glu-Leu-Ala-Lys-Thr-Asn-Lys-Glu-Gly-Thr-Thr-Ala-Leu-Gly-Leu-Val- Glu-Ala-Ala-Lys-Lys-Ile-Gly-Phe-Glu-Thr-Arg-Ala-Ile-Lys-Ala-Glu-Met-Ser-Leu-Phe-Glu- Met-Glu-Asp-Val-Pro-Tyr-Pro-Phe-Ile-Ala-His-Val-Asn-Lys-Asp-Gly-Lys-Leu-Gln-His- Tyr-Tyr-Val-Ile-Tyr-Lys-Ser-Thr-Lys-Asp-Tyr-Leu-Ile-Ile-Gly-Asp-Pro-Asp-Pro-Ser-Val- Lys-Val-Thr-Lys-Met-Thr-Lys-Glu-Arg-Phe-Glu-Lys-Glu-Trp-Thr-Gly-Val-Thr-Leu-Phe- Met-Gly-Pro-Glu-Pro-Ser-Tyr-Lys-Pro-His-Lys-Asp-Lys-Lys-Asn-Gly-Leu-Trp-Asp-Phe- Leu-Pro-Leu-Ile-Phe-Lys-Gln-Arg-Ser-Leu-Ile- Ala- Asn- lie- Val-Phe-Ala-Ser- Leu-Leu- VaI- Thr-Leu-Ile-Asn-Ile-Leu-Gly-Ser-Tyr-Tyr-Leu-Gln-Gly-Ile-Leu-Asp-Glu-Tyr-Val-Pro-Asn- Gln-Met-Lys-Ser-Thr-Leu-Gly-Ile-Ile-Ser-Ile-Gly-Leu-Val-Val-Thr-Tyr-Val-Leu-Gln-Gln- Met-Met-Thr-Phe-Ala-Arg-Asp-Tyr-Leu-Leu-Thr-Ile-Leu-Ser-Gln-Arg-Leu-Thr-Ile-Asp- Val-Ile-Leu-Ser-Tyr-Ile-Arg-His-Ile-Phe-Glu-Leu-Pro-Met-Ser-Phe-Phe-Ala-Thr-Arg-Arg- Thr-Gly-Glu-Val-Ile-Ser-Arg-Phe-Ser-Asp-Ala-Asn-Ser-Ile-Ile-Asp-Ala-Leu-Ala-Ser-Thr- Ile-Leu-Ser-Leu-Phe-Leu-Asp-Phe-Ser-Ile-Val-Ile-Ile-Val-Gly-Gly-Val-Leu-Leu-Ile-Gln- Asn-Ser-Asn-Leu-Phe-Lys-Leu-Val-Leu-Cys-Ser-Val-Pro-Ile-Tyr-Thr-Leu-Ile-Val-Phe-Ala- Phe-Met-Lys-Pro-Phe-Glu-Leu-Met-Asn-His-Asp-Val-Met-Gln-Ser-Asn-Ala-Met-Val-Asn- Ser-Ala-Ile-Ile-Glu-Asp-Ile-Asn-Gly-Ile-Glu-Thr-Ile-Lys-Ser-Leu-Thr-Ser-Glu-Glu-Val- Cys-Tyr-Gln-Lys-Ile-Asp-Gly-Glu-Phe-Val-Asp-Tyr-Leu-Asp-Asn-Ser-Phe-Arg-Leu-Ser- Lys-Leu-Ser-Ile-Leu-Gln-Thr-Ser-Leu-Lys-Gln-Gly-Ala-Gln-Leu-Ile-Leu-Asn-Val-Leu-Ile- Leu-Trp-Thr-Gly-Ala-Gln-Leu-Val-Met-Gly-Asn-Thr-Ile-Ser-Ile-Gly-Gln-Leu-Ile-Thr-Phe- Asn-Met-Leu-Leu-Ser-Tyr-Phe-Thr-Asn-Pro-Leu-Glu-Asn-Ile-Ile-Asn-Leu-Gln-Thr-Lys- Leu-Gln-Ser-Ala-Lys-Val-Ala-Asn-Thr-Arg-Leu-Asn-Glu-Val-Tyr-Leu-Ile-Glu-Ser-Glu- Phe-Gly-Gln-Ser-Asp-Asp-Thr-Tyr-Gln-Glu-Gly-Ile-Ala-Asp-Gly-Asp-Ile-Thr-Val-Thr- Asp-Leu-Ser-Tyr-Lys-Tyr-Gly-Phe-Gly-Arg-Asp-Thr-Leu-Thr-Asp-Val-Ser-Leu-Thr-Ile- Arg-Gln-Gly-Glu-Lys-Ile-Ser-Phe-Val-Gly-Val-Ser-Gly-Ser-Gly-Lys-Thr-Thr-Leu-Ala-Lys- Met-Leu-Val-Asn-Phe-Tyr-Gln-Pro-Tyr-Lys-Gly-His-Ile-Asp-Phe-Asn-Gly-Gln-Asn-Ile- Ser-Arg-Ile-Asp-Lys-Lys-Thr-Leu-Arg-Gln-His-Ile-Asn-Tyr-Leu-Pro-Gln-Gln-Ser-Tyr-Ile- Phe-Ser-Gly-Ser-Val-Leu-Glu-Asn-Leu-Thr-Leu-Gly-Ala-Ala-Arg-Gly-Ile-Thr-Gln-Ala- Asp-Ile-Leu-Lys-Ala-Cys-Glu-Ile-Ala-Glu-Ile-Arg-Gln-Asp-Ile-Glu-Asn-Met-Pro-Met-Gly- Phe-Gln-Thr-Glu-Leu-Ser-Asp-Gly-Ala-Gly-Leu-Ser-Gly-Gly-Gln-Lys-Gln-Arg-Ile-Ala- Leu-Ala-Arg-Ala-Leu-Leu-Thr-Lys-Ser-Pro-Val-Leu-Ile-Leu-Asp-Glu-Ala-Thr-Ser-Gly- Leu-Asp-Val-Leu-Thr-Glu-Lys-Gln-Val-Ile-Asp-Asn- Leu-Leu- Ala- Leu-Lys-Asp-Lys-Thr- Ile-Ile-Phe-Val-Ala-His-Arg-Leu-Ser-Ile-Ala-Glu-Arg-Thr-Asp-Arg-Ile-Val-Val-Ile-Asp- Gln-Gly-Arg-Val-Val-Glu-Thr-Gly-Ser-His-Gln-Asp-Leu-Met-Ser-Asn-Pro-Gly-Phe-Tyr- Tyr-Gln-Leu-Phe-Arg-Lys (SEQ ID NO 5)
Met-Asn-Pro-Asn-Leu-Phe-Arg-Ser-Val-Glu-Phe-Tyr-Gln-Arg-Arg-Tyr-His-Asn-Tyr-Ala-
Thr-Val-Leu-Ile-Ile-Pro-Leu-Ser-Leu-Leu-Phe-Thr-Phe-Ile-Leu-Ile-Phe-Ser-Leu-Val-Ala-
Thr-Lys-Glu-Ile-Thr-Val-Thr-Ser-Gln-Gly-Glu-Ile-Ala-Pro-Thr-Ser-Val-Ile-Ala-Ser-Ile-Gln- Ser-Thr-Ser- Asp- Asn-Pro-Ile-Leu-Ala-Asn-His-Leu-Val- Ala- Asn-Gln- VaI- Val-Glu-Lys- Gly-Asp-Leu-Leu-Ile-Lys-Tyr-Ser-Glu-Thr-Met-Glu-Glu-Ser-Gln-Lys-Thr-Ala-Leu-Ala- Thr-Gln-Leu-Gln-Arg-Leu-Glu-Lys-Gln-Lys-Glu-Gly-Leu-Gly-Ile-Leu-Lys-Gln-Ser-Leu- Glu-Lys-Ala-Thr-Asp-Leu-Phe-Ser-Gly-Glu-Asp-Glu-Phe-Gly-Tyr-His-Asn-Thr-Phe-Met- Asn-Phe-Thr-Lys-Gln-Ser-His-Asp-Ile-Glu-Leu-Gly-Ile-Thr-Lys-Thr-Asn-Thr-Glu-Val-Ser- Asn-Gln-Ala-Asn-Leu-Ser-Asn-Ser-Ser-Ser-Ser-Ala-Ile-Glu-Gln-Glu-Ile-Thr-Lys-Val-Gln- Gln-Gln-Ile-Gly-Glu-Tyr-Gln-Glu-Leu-Arg-Asp-Ala-Ile-Ile-Asn-Asn-Arg-Ala-Arg-Leu-Pro- Thr-Gly-Asn-Pro-His-Gln-Ser-Ile-Leu-Asn-Arg-Tyr-Leu-Val-Ala-Ser-Gln-Gly-Gln-Thr- Gln-Gly-Thr-Ala-Glu-Glu-Pro-Phe-Leu-Ser-Gln-Ile-Asn-Gln-Ser-Ile-Ala-Gly-Leu-Glu-Ser- Ser-Ile-Ala-Ser-Leu-Lys-Ile-Gln-Gln-Ala-Gly-Ile-Gly-Ser-Val-Ala-Thr-Tyr-Asp-Asn-Ser- Leu-Ala-Thr-Lys-Ile-Glu-Val-Leu-Arg-Thr-Gln-Phe-Leu-Gln-Thr-Ala-Ser-Gln-Gln-Gln- Leu-Thr-Val-Glu-Asn-Gln-Leu-Thr-Glu-Leu-Lys-Val-Gln-Leu-Asp-Gln-Ala-Thr-Gln-Arg- Leu-Glu-Asn-Asn-Thr-Leu-Thr-Ser-Pro-Ser-Lys-Gly-Ile-Val-His-Leu-Asn-Ser-Glu-Phe- Glu-Gly-Lys-Asn-Arg-Ile-Pro-Thr-Gly-Thr-Glu-Ile-Ala-Gln-Ile-Phe-Pro-Val-Ile-Thr-Asp- Thr-Arg-Glu-Val-Leu-Ile-Thr-Tyr-Tyr-Val-Ser-Ser-Asp-Tyr-Leu-Pro-Leu-Leu-Asp-Lys- Gly-Gln-Thr-Val-Arg-Leu-Lys-Leu-Glu-Lys-Ile-Gly-Asn-His-Gly-Thr-Thr-Ile-Ile-Gly-Gln- Leu-Gln-Thr-Ile-Asp-Gln-Thr-Pro-Thr-Arg-Thr-Glu-Gln-Gly-Asn-Leu-Phe-Lys-Leu-Thr- Ala-Leu- Ala-Lys-Leu-Ser- Asn-Glu-Asp-Ser- Lys-Leu-Ile-Gln- Tyr-Gly-Leu-Gln-Gly- Arg- Val-Thr-Ser-Val-Thr-Thr-Lys-Lys-Thr-Tyr-Phe-Asp-Tyr-Phe-Lys-Asp-Lys-Ile-Leu-Thr- His-Ser-Asp (SEQ ID NO 6)
[00040] In one embodiment, said bacteriocin is encoded by the blpM gene, the blpN gene, allelic variants or a combination thereof. In another embodiment, said bacteriocin is encoded by blpM gene. In another embodiment, said bacteriocin is encoded by blpN gene. In another embodiment, said bacteriocin is encoded by a functional fragment or otherwise a combination of amino acid sequences set forth in SEQ ID NO's 7-19:
Met-Asp-Thr-Lys-Ile-Met-Glu-Gln-Phe-His-Glu-Met-Asp-Ile-Thr-Met-Leu-Ser-Ser-Ile-Glu- Gly-Gly-Lys-Asn-Asn-Trp-Gln-Thr-Asn-Val-Leu-Glu-Gly-Gly-Gly-Ala-Ala-Phe-Gly-Gly- Trp-Gly-Leu-Gly-Thr-Ala-Ile-Cys-Ala-Ala-Ser-Gly-Val-Gly-Ala-Pro-Phe-Met-Gly-Ala- Cys-Gly-Tyr-Ile-Gly-Ala-Lys-Phe-Gly-Val-Asp-Leu-Trp-Ala-Gly-Val-Thr-Gly-Ala-Thr- Gly-Gly-Phe (SEQ ID 7)
Met-Asp-Thr-Lys-Ile-Met-Glu-Gln-Phe-His-Glu-Met-Asp-Ile-Thr-Met-Leu-Ser-Ser-Ile-Glu- Gly-Gly-Lys-Asn-Asn-Trp-Gln-Thr-Asn-Val-Leu-Glu-Gly-Gly-Gly-Ala-Ala-Phe-Gly-Gly- Trp-Gly-Leu-Gly-Thr-Ala-Ile-Cys-Ala-Ala-Ser-Gly-Val-Gly-Ala-Pro-Phe-Met-Gly-Ala- Cys-Gly-Tyr-Ile-Gly-Ala-Lys-Phe-Gly-Val-Asp-Leu-Trp-Ala-Gly-Val-Thr-Gly-Ala-Thr- Gly-Gly-Phe (SEQ ID 8)
Met-Asp-Thr-Lys-Ile-Met-Glu-Gln-Phe-His-Glu-Met-Asp-Ile-Thr-Met-Leu-Ser-Ser-Ile-Glu- Gly-Gly-Lys-Asn-Asn-Trp-Gln-Thr-Asn-Val-Leu-Glu-Gly-Gly-Gly-Ala-Ala-Phe-Gly-Gly- Trp-Gly-Leu-Gly-Thr-Ala-Ile-Cys-Ala-Ala-Ser-Gly-Val-Gly-Ala-Pro-Phe-Met-Gly-Ala- Cys-Gly-Tyr-Ile-Gly-Ala-Lys-Phe-Gly-Val-Ala-Leu-Trp-Ala-Gly-Val-Thr-Gly-Ala-Thr- Gly-Gly-Phe (SEQ ID 9)
Met-Asp-Thr-Lys-Ile-Met-Glu-Gln-Phe-His-Glu-Met-Asp-Ile-Thr-Met-Leu-Ser-Ser-Ile-Glu- Gly-Gly-Lys-Asn-Asn-Trp-Gln-Thr-Asn-Val-Leu-Glu-Gly-Gly-Gly-Ala-Ala-Phe-Gly-Gly- Trp-Gly-Leu-Gly-Thr-Ala-Ile-Cys-Ala-Ala-Ser-Gly-Val-Gly-Ala-Pro-Phe-Met-Gly-Ala- Cys-Gly-Tyr-Ile-Gly-Ala-Lys-Phe-Gly-Val-Asp-Leu-Trp-Ala-Gly-Val-Thr-Gly-Ala-Thr- Gly-Gly-Phe (SEQ ID 10)
Met-Asp-Thr-Lys-Ile-Met-Glu-Gln-Phe-His-Glu-Met-Asp-Ile-Thr-Met-Leu-Ser-Ser-Ile-Glu- Gly-Gly-Lys-Asn-Asn-Trp-Gln-Thr-Asn-Val-Leu-Glu-Gly-Gly-Gly-Ala-Ala-Phe-Gly-Gly- Trp-Gly-Leu-Gly-Thr-Ala-Ile-Cys-Ala-Ala-Ser-Gly-Val-Gly-Ala-Pro-Phe-Met-Gly-Ala- Cys-Gly-Tyr-Ile-Gly-Ala-Lys-Phe-Gly-Val-Asp-Leu-Trp-Ala-Gly-Val-Thr-Gly-Ala-Thr- Gly-Gly-Phe (SEQ ID 11)
Met-Asp-Thr-Lys-Met-Met-Glu-Gln-Phe-His-Glu-Met-Asp-Ile-Ala-Met-Leu-Ser-Ser-Ile- Glu-Gly-Gly-Lys-Asn-Asn-Trp-Gln-Thr-Asn-Val-Phe-Glu-Gly-Gly-Ser-Ala-Ala-Phe-Gly- Gly-Trp-Gly-Leu-Gly-Thr-Ala-Ile-Cys-Ala-Ala-Ser-Gly-Val-Gly-Ala-Pro-Phe-Met-Gly- Ala-Cys-Gly-Tyr-Ile-Gly-Ala-Lys-Phe-Gly-Val-Ala-Leu-Trp-Ala-Gly-Val-Thr-Gly-Ala- Thr-Gly-Gly-Phe (SEQ ID 12)
Met-Asn-Thr-Lys-Met-Met-Glu-Gln-Phe-His-Glu-Met-Asp-Ile-Ala-Met-Leu-Ser-Ser-Ile- Glu-Gly-Gly-Lys-Asn-Asn-Trp-Gln-Thr-Asn-Val-Phe-Glu-Gly-Gly-Ser-Ala-Ala-Phe-Gly- Gly-Trp-Gly-Leu-Gly-Thr-Ala-Ile-Cys-Ala-Ala-Ser-Gly-Val-Gly-Ala-Pro-Phe-Met-Gly- Ala-Cys-Gly-Tyr-Ile-Gly-Ala-Lys-Phe-Gly-Val-Ala-Leu-Trp-Ala-Gly-Val-Thr-Gly-Ala- Thr-Gly-Gly-Phe (SEQ ID 13). Ala-Ala- VaI- Val-Ala-Ala-Leu-Gly-Cys-Ala-Ala-Gly-Gly- Val-Lys-Tyr-Gly-Arg-Leu-Leu- Gly-Pro-Trp-Gly-Ala-Ala-Ile-Gly-Gly-Ile-Gly-Gly-Ala-Val-Val-Cys-Gly-Tyr-Leu-Ala-Tyr- Thr-Ala-Thr-Ser (SEQ ID 14).
Ala-Ala- VaI- Val-Ala-Ala-Leu-Gly-Cys-Ala-Ala-Gly-Gly- Val-Lys-Tyr-Gly-Lys-Ile-Leu- Gly-Pro-Trp-Gly-Ala-Ala-Ile-Gly-Gly-Ile-Gly-Gly-Ala-Val-Val-Cys-Gly-Tyr-Leu-Ala-Tyr- Thr-Ala-Thr-Ser (SEQ ID 15).
Ala-Ala- VaI- Val-Ala-Ala-Leu-Gly-Cys-Ala-Ala-Gly-Gly-Val-Lys-Tyr-Gly-Arg-Leu-Leu- Gly-Pro-Trp-Gly-Ala-Ala-Ile-Gly-Gly-Ile-Gly-Gly-Ala-Val-Val-Cys-Gly-Tyr-Leu-Ala-Tyr- Thr-Ala-Thr-Ser (SEQ ID 16).
Lys-Asn-Asn-Trp-Gln-Thr-Asn-Val-Leu-Glu-Gly-Gly-Gly-Ala-Ala-Phe-Gly-Gly-Trp-Gly- Leu-Gly-Thr-Ala-Ile-Cys-Ala-Ala-Ser-Gly-Val-Gly-Ala-Pro-Phe-Met-Gly-Ala-Cys-Gly- Tyr-Ile-Gly-Ala-Lys-Phe-Gly-Val-Ala-Leu-Trp-Ala-Gly-Val-Thr-Gly-Ala-Thr-Ser-Gly-Phe (SEQ ID 17).
Lys-Asn-Asn-Trp-Gln-Thr-Asn-Val-Phe-Glu-Gly-Gly-Ser-Ala-Ala-Phe-Gly-Gly-Trp-Gly- Leu-Gly-Thr-Ala-Ile-Cys-Ala-Ala-Ser-Gly-Val-Gly-Ala-Pro-Phe-Met-Gly-Ala-Cys-Gly- Tyr-Ile-Gly-Ala-Lys-Phe-Gly-Val-Ala-Leu-Trp-Ala-Gly-Val-Thr-Gly-Ala-Thr-Gly-Gly-Phe (SEQ ID 18).
Lys-Asn-Asn-Trp-Gln-Thr-Asn-Val-Leu-Glu-Gly-Gly-Gly-Ala-Ala-Phe-Gly-Gly-Trp-Gly- Leu-Gly-Thr-Ala-Ile-Cys-Ala-Ala-Ser-Gly-Val-Gly-Ala-Pro-Phe-Met-Gly-Ala-Cys-Gly- Tyr-Ile-Gly-Ala-Lys-Phe-Gly-Val-Asp-Leu-Trp-Ala-Gly-Val-Thr-Gly-Ala-Thr-Gly-Gly-Phe (SEQ ID 19).
[00041] In one embodiment, the BIpM protein, BIpN protein, or its functional fragment are between about 67 to about 99% homologous to SEQ ID No. 7-19. In another embodiment, the BIpR protein, the BIpH protein or its functional fragment are between about 67 to about 75% homologous to SEQ ID No. 7-19. In another embodiment, the the BIpR protein, the BIpH protein, or its functional fragment are between about 76 to about 85% homologous to SEQ ID No. 7-19. In another embodiment, the BIpR protein, the BIpH protein, or its functional fragment are between about 86 to about 95% homologous to SEQ ID No. 7-19. In another embodiment, the the BIpR protein, the BIpH protein, or its functional fragment are between about 96 to about 99% homologous to SEQ ID No. 7-19.
[00042] In one embodiment, the term "Operon", refers to a cluster of contiguous genes transcribed from one promoter that gives rise to a polycistronic mRNA. In another embodiment, the agent used in the methods and compositions described herein for the upregulation of the blp locus expression, activates the blp operon.
[00043] In one embodiment, the term "regulator protein" refers to an initial protein in the pathway that is a transmembrane histidine kinase that dimerizes in response to an external signal. In another embodiment, one partner of the dimer phosphorylates a specific histidine residue of the other partner and the phosphoryl group is subsequently transferred to an aspartyl residue of a response-regulator protein. This results, in one embodiment, in the activation of a pathway leading to the up-regulation of specific genes. In bacteria, the histidine kinase sensor and the response regulator are separate proteins.
[00044] In one embodiment, In one embodiment, transport proteins are multi-pass transmembrane proteins, which either actively transport molecules across the membrane or passively allow them to cross. Active transport involves directional pumping of a solute across the membrane, against an electrochemical gradient in certain embodiments. In another embodiment, active transport is tightly coupled to a source of metabolic energy, such as ATP hydrolysis in one embodiment, or an electrochemically favorable ion gradient in another embodiment. In one embodiment, passive transport involves the movement of a solute down its electrochemical gradient. Transport proteins can be classified in other embodiments, as either carrier proteins or channel proteins. Carrier proteins, which can function in active or passive transport, bind in one embodiment to a specific solute to be transported and undergo a conformational change which transfers the bound solute across the membrane. Channel proteins, which only function in passive transport, form hydrophilic pores across the membrane. When the pores open, specific solutes, such as inorganic ions, pass through the membrane and down the electrochemical gradient of the solute. Examples include facilitative transporters, the secondary active symporters and antiporters driven by ion gradients, and active ATP binding cassette transporters involved in multiple-drug resistance and targeting of antigenic peptides to MHC Class I molecules. Transported substrates range from nutrients and ions to a broad variety of drugs, peptides and proteins. In one embodiment, the agents used in the methods and compositions described herein, result in the upregulation of the expression of genes encoding for transport proteins.
[00045] In another embodiment, the invention provides a method for generating bacteriocins in a bacteria comprising the step of down-regulating the expression of htrA. In one embodiment, the pneumococcal gene htrA encodes a putative serine protease that is localized on the surface of Streptococcus pneumoniae. In another embodiment, the expression of htrA, is controlled by the QaRH two-component system.
[00046] In another embodiment, htrA is represented by amino acid sequence set forth is SEQ ID NO: 20: mkhlktfykk wfqllvvivi sffsgalgsf sitqltqkss vnnsnnnsti tqtayknens ttqavnkvkd avvsvitysa nrqnsvfgnd dtdtdsqris segsgviykk ndkeayivtn nhvingaskv dirlsdgtkv pgeivgadtf sdiavvkiss ekvttvaefg dsskltvget aiaigsplgs eyantvtqgi vsslnrnvsl ksedgqaist kaiqtdtain pgnsggplin iqgqvigits skiatnggts veglgfaipa ndainiieql ekngkvtrpa lgiqmvnlsn vstsdirrln ipsnvtsgvi vrsvqsnmpa nghlekydvi tkvddkeias stdlqsalyn hsigdtikit yyrngkeett siklnkssgd les.
[00047] In another embodiment, htrA is represented by an amino acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:20. In another embodiment, htrA is represented by a fragment of an amino acid sequence setforth in SEQ ID NO:20. The fragment may comprise one or more functional domains or conserved regions. In another embodiment, htrA is represented by an amino acid sequence that is orthologous or homologous to SEQ ID NO: 20.
[00048] In another embodiment, htrA is represented by nucleic acid sequence set forth is SEQ ID NO: 21: atgaaacatctgaaaaccttttataaaaaatggtttcagctgctggtggtgattgtgattagcttttttagcggcgcgctgggca gctttagcattacccagctgacccagaaaagcagcgtgaacaacagcaacaacaacagcaccattacccagaccgcgtataaaaac gaaaacagcaccacccaggcggtgaacaaagtgaaagatgcggtggtgagcgtgattacctatagcgcgaaccgccagaacagc gtgtttggcaacgatgataccgataccgatagccagcgcattagcagcgaaggcagcggcgtgatttataaaaaaaacgataaagaa gcgtatattgtgaccaacaaccatgtgattaacggcgcgagcaaagtggatattcgcctgagcgatggcaccaaagtgccgggcgaa attgtgggcgcggatacctttagcgatattgcggtggtgaaaattagcagcgaaaaagtgaccaccgtggcggaatttggcgatagca gcaaactgaccgtgggcgaaaccgcgattgcgattggcagcccgctgggcagcgaatatgcgaacaccgtgacccagggcattgt gagcagcctgaaccgcaacgtgagcctgaaaagcgaagatggccaggcgattagcaccaaagcgattcagaccgataccgcgatt aacccgggcaacagcggcggcccgctgattaacattcagggccaggtgattggcattaccagcagcaaaattgcgaccaacggcg gcaccagcgtggaaggcctgggctttgcgattccggcgaacgatgcgattaacattattgaacagctggaaaaaaacggcaaagtga cccgcccggcgctgggcattcagatggtgaacctgagcaacgtgagcaccagcgatattcgccgcctgaacattccgagcaacgtg accagcggcgtgattgtgcgcagcgtgcagagcaacatgccggcgaacggccatctggaaaaatatgatgtgattaccaaagtggat gataaagaaattgcgagcagcaccgatctgcagagcgcgctgtataaccatagcattggcgataccattaaaattacctattatcgcaa cggcaaagaagaaaccaccagcattaaactgaacaaaagcagcggcgatctggaaagc
[00049] In another embodiment, htrA is represented by a nucleic acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:21. In another embodiment, htrA is represented by a fragment of a nucleic acid sequence setforth in SEQ ID NO:21. The fragment may comprise one or more functional regions.
[00050] According to one aspect of the invention, the hrtA is downregulated by a method known to one of skill in the art. In one embodiment, the down-regulation of the expression of htrA is caused by mutations in one or more functional domains of htrA. In another embodiment, the down-regulation of the expression of htrA is caused by an antisense oligonucleotide complementary to all or a portion of a messenger RNA encoding htrA, wherein said antisense oligonucleotide inhibits the production of htrA. In another embodiment, the down-regulation of the expression of htrA is caused by siRNA that inhibits the production of htrA. In another embodiment, the down-regulation of the expression of htrA is caused by an antibody that inhibits the production of htrA.
[00051] In another embodiment, the invention provides a method for generating bacteriocins in a bacteria comprising the step of down-regulating the expression of ciaRH. In one embodiment, ciaRH refers to a bacterial two-component signal-transducing systems (TCSTS) that mediate adaptive responses to environmental signals in Streptococcus pneumoniae. In another embodiment, two membrane-spanning regions in QaH separate the N-terminal external sensor domain from the cytoplasmic kinase domain. In another embodiment, htrA, ciaR, and ciaH genes are arranged in an operon with a 8-bp overlap. In one embodiment, mutations in the histidine protein kinase of ciaH conferred increased resistance to beta-lactam antibiotics, indicating, that in one embodiment ciaR controls genes that are involved in the biochemistry of the bacterial cell wall. In one embodiment, ciaH mutants are affected in the development of genetic competence as well. [00052] In another embodiment, ciaR is represented by amino acid sequence set forth is SEQ ID NO: 22: mqrvefflrq iwynsfnkee flmikillve ddlglsnsvf dflddfadvm qvfdgeegly eaesgvydli lldlmlpekn gfqvlkelre kgittpvlim takeslddkg hgfelgaddy ltkpfyleel kmriqallkr sgkfnentlt ygnivvnlst ntvkvedtpv ellgkefdll vyflqnqnvi lpktqifdrl wgfdsdttis vvevyvskvr kklkgttfae nlqtlrsvgy llkdvq
[00053] In another embodiment, ciaR is represented by an amino acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:22. In another embodiment, ciaR is represented by a fragment of an amino acid sequence setforth in SEQ ID NO:22. The fragment may comprise one or more functional domains or conserved regions. In another embodiment, ciaR is represented by an amino acid sequence that is orthologous or homologous to SEQ ID NO: 22.
[00054] In another embodiment, ciaR is represented by nucleic acid sequence set forth is SEQ ID NO: 23: atgcagcgcgtggaattttttctgcgccagatttggtataacagctttaacaaagaagaatttctgatgattaaaattctgctggt ggaagatgatctgggcctgagcaacagcgtgtttgattttctggatgattttgcggatgtgatgcaggtgtttgatggcgaagaaggcct gtatgaagcggaaagcggcgtgtatgatctgattctgctggatctgatgctgccggaaaaaaacgctttcaggtgctgaaagaactgc gcgaaaaaggcattaccaccccggtgctgattatgccgcgaaagaaagcctggatgataaaggccatggctttgaactgggcgcgg atgattatctgaccaaaccgttttatctggaagaactgaaaatgcgcattcaggcgctgctgaaacgcgcggcaaatttaacgaaaaca ccctgacctatggcaacattgtggtgaacctgagcaccacaccgtgaaagtggaagataccccggtggaactgctgggcaaagaatt tgatctgctggtgtattttctgcagaaccagaacgtgattctgccgaaaacccagatttttgatcgcctgtggggctttgatagcgatacca ccattagcgtggtggaagtgtatgtgagcaaagtgcgcaaaaaactgaaaggcaccacctttgcggaaaacctgcagaccctgcgca gcgtgggctat ctgctgaaagatgtgcag
[00055] In another embodiment, ciaR is represented by a nucleic acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:23. In another embodiment, ciaR is represented by a fragment of a nucleic acid sequence setforth in SEQ ID NO:23. The fragment may comprise one or more functional regions.
[00056] In another embodiment, ciaH is represented by amino acid sequence set forth is SEQ ID NO: 24: mfsklkktwy addfsyfirn fgvftlifst mtliilqvmh sslytsvddk lhglsenpqa viqlainrat eeikdlenar adaskveikp nvssntevil fdkdftqlls gnrflgldki klekkelghi yqiqvfnsyg qeeiyrvilm etnissvstn ikyaavlint sqleqasqkh eqlivvvmas fwilsllasl ylarvsvrpl lesmqkqqsf venashelrt plavlqnrle tlfrkpeati mdvsesiass leevrnmrfl ttsllnlarr ddgikpelae vptsffnttf tnyemiasen nrvfrfenri hrtivtdqll lkqlmtilfd navkyteedg eidflisatd rnlyllvsdn gigistedkk kifdrfyrvd kartrqkggf glglslakqi vdalkgtvtv kdnkpkgtif evkiaiqtps kkkk
[00057] In another embodiment, ciaH is represented by an amino acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:24. In another embodiment, ciaH is represented by a fragment of an amino acid sequence setforth in SEQ ID NO:24. The fragment may comprise one or more functional domains or conserved regions. In another embodiment, ciaH is represented by an amino acid sequence that is orthologous or homologous to SEQ ID NO: 24.
[00058] In another embodiment, ciaH is represented by amino acid sequence set forth is SEQ ID NO: 25: atgtttagcaaactgaaaaaaacctggtatgcggatgattttagctattttattcgcaactttggcgtgtttaccctgatttttagc accatgaccctgattattctgcaggtgatgcatagcagcctgtataccagcgtggatgataaactgcatggcctgagcgaaaacccgc aggcggtgattcagctggcgattaaccgcgcgaccgaagaaattaaagatctggaaaacgcgcgcgcggatgcgagcaaagtgga aattaaaccgaacgtgagcagcaacaccgaagtgattctgtttgataaagattttacccagctgctgagcggcaaccgctttctgggcct ggataaaattaaactggaaaaaaaagaactgggccatatttatcagattcaggtgtttaacagctatggccaggaagaaatttatcgcgt gattctgatggaaaccaacattagcagcgtgagcaccaacattaaatatgcggcggtgctgattaacaccagccagctggaacaggc gagccagaaacatgaacagctgattgtggtggtgatggcgagcttttggattctgagcctgctggcgagcctgtatctggcgcgcgtg agcgtgcgcccgctgctggaaagcatgcagaaacagcagagctttgtggaaaacgcgagccatgaactgcgcaccccgctggcgg tgctgcagaaccgcctggaaaccctgtttcgcaaaccggaagcgaccattatggatgtgagcgaaagcattgcgagcagcctggaa gaagtgcgcaacatgcgctttctgaccaccagcctgctgaacctggcgcgccgcgatgatggcattaaaccggaactggcggaagt gccgaccagcttttttaacaccacctttaccaactatgaaatgattgcgagcgaaaacaaccgcgtgtttcgctttgaaaaccgcattcat cgcaccattgtgaccgatcagctgctgctgaaacagctgatgaccattctgtttgataacgcggtgaaatataccgaagaagatggcga aattgattttctgattacgcgaccgatcgcaacctgtatctgctggtgagcgataacggcattggcattagcaccgaagataaaaaaaaa atttttgatcgcttttatcgcgtggataaagcgcgcacccgccagaaaggcggctttggcctgggcctgagcctggcgaaacagattgt ggatgcgctgaaaggcaccgtgaccgtgaaagataacaaaccgaaaggcaccatttttgaagtgaaaattgcgattcagaccccgag caaaaaaaaaaaa [00059] In another embodiment, ciaH is represented by a nucleic acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:25. In another embodiment, ciaH is represented by a fragment of a nucleic acid sequence setforth in SEQ ID NO:25. The fragment may comprise one or more functional regions.
[00060] The ciaRH may be downregulated by a method known to one of skill in the art. In one embodiment, the down-regulation of ciaRH is caused by mutations in one or more functional domains of ciaR. In another embodiment, the down-regulation of ciaRH is caused by mutations in one or more functional domains of ciaH. In another embodiment, the down- regulation of ciaRH is caused by an antisense oligonucleotide complementary to all or a portion of a messenger RNA encoding ciaR, wherein said antisense oligonucleotide inhibits the production of ciaR. In another embodiment, the down-regulation of ciaRH is caused by an antisense oligonucleotide complementary to all or a portion of a messenger RNA encoding ciaH, wherein said antisense oligonucleotide inhibits the production of ciaH. In another embodiment, the down-regulation of ciaRH is caused by siRNA that inhibits the production of ciaR. In another embodiment, the down-regulation of ciaRH is caused by siRNA that inhibits the production of ciaH. In another embodiment, the down-regulation of ciaRH is caused by an antibody that inhibits the production of ciaR. In another embodiment, the down-regulation of ciaRH is caused by an antibody that inhibits the production of ciaH.
[00061] In another embodiment, the invention provides a method for generating bacteriocins in a bacteria comprising the steps of upregulating expression of the blp locus; down- regulating the expression of htrA; and down-regulating the expression of ciaRH. In another embodiment, the invention provides a method for generating bacteriocins in a strain of bacteria comprising the steps of upregulating expression of the blp locus, wherein the upregulation of expression of the blp locus is caused by down-regulating the expressions of htrA and ciaRH.
[00062] In one embodiment, provided herein is a method for treating an infection in a subject, comprising the steps of inhibiting or reducing bacterial growth or colonization in a subject comprising the steps of administering to said subject a bacteriocin producing bacteria. In another embodiment, the bacteriocin is BIpM, BIpN, a functional fragment or a combination thereof. In another embodiment, the bacteriocin is pneumocinMN. In one embodiment, said bacteria is Streptococcus. In another embodiment, said Streptococcus is Streptococcus pneumoniae.
[00063] In one embodiment, pneumocinMN is produced by the blp locus, upregulated by an agent. In another embodiment, pneumocinMN comprises an amino acid sequence of blpM or its functional fragment. In another embodiment, pneumocinMN comprises an amino acid sequence of blpN or its functional fragment. In another embodiment, pneumocinMN comprises an amino acid sequences of both blpM and blpN or their functional fragments. In another embodiment, pneumocinMN is capable of preventing pneumococcal infection or colonization in a subject.
[00064] In another embodiment, the invention provides a vaccine for treating, preventing or ameliorating a subject against pneumococcal infection or colonization. The vaccine comprises a pharmaceutically acceptable carrier and an immunologically effective amount of a recombinant Streptococcus bacteria that produces pneumocinMN, wherein the recombinant bacteria comprises an amino acid sequence of blpC. In another embodiment, the amino acid sequence of blpC comprises the sequence of GLWEDILYSLNIIKHNNTKGLHHPIQL
[00065] In one embodiment, the bacteriocin is produced by upregulating the expression of the blp locus. In another embodiment, the bacteriocin is produced by upregulating the expression of blpM. In another embodiment, the bacteriocin is produced by upregulating the expression of blpN. In another embodiment, the bacteriocin is produced by down-regulating the expression of hrtA. In another embodiment, the bacteriocin is produced by down- regulating the expression of ciaRH.
[00066] In one embodiment, the term "allele" which is used interchangeably herein with "allelic variant" refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for that gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. In one embodiment, alleles of a specific gene differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and/or insertions of nucleotides. In another embodiment, an allele of a gene is a form of a gene containing mutations. [00067] In one embodiment, the term "bacteriocin" is used to describe inhibitory agents produced by bacteria that meet the minimum criteria of (1) being a peptide and (2) possessing bactericidal activity. In another embodiment the term "bacteriocin" refers to a polypeptide produced, by ribosome synthesis, from microorganisms capable of inhibiting specifically the growth of other bacteria. In an exemplarary embodiment, bacteriocin is pneumocinMN. In one embodiment, pneumocinMN comprises amino acid sequences of blpM and blpN.
[00068] In one embodiment, the term "fragment" refers to a portion of a protein or peptide that has been enzymatically or chemically truncated or cleaved. Such a fragment may encompass any portion of the native amino acid sequence of the protein.
[00069] In one embodiment, the term "treatment" refers to any process, action, application, therapy, or the like, wherein a subject, including a human being, is subjected to medical aid with the object of improving the subject's condition, directly or indirectly. In another embodiment, the term "treating" refers to reducing incidence, or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, improving symptoms, improving prognosis or combination thereof in other embodiments.
[00070] "Treating" embraces in another embodiment, the amelioration of an existing condition. The skilled artisan would understand that treatment does not necessarily result in the complete absence or removal of symptoms. Treatment also embraces palliative effects: that is, those that reduce the likelihood of a subsequent medical condition. The alleviation of a condition that results in a more serious condition is encompassed by this term. [00071] In one embodiment, said infection is a lower respiratory infection, upper respiratory infection, invasive infection, or a combination thereof. In one embodiment, said upper respiratory infection is Sinusitis, Otitis media, Tracheobronchitis, or a combination thereof. In another embodiment, said lower respiratory infection is Pneumonia, Broncho-pneumonia, or a combination thereof. In another embodiment, said invasive infection is Primary bacteremia, Meningitis, Spontaneous bacterial peritonitis, Sepsis with tissue seeding, or a combination thereof.
[00072] In one embodiment, the type of otitis media is recurrent acute otitis media (RAOM), chronic otitis media with effusion (COME), acute post-tympanostomy otorrhea (APTO), or chronic suppurative otitis media (CSOM) in another discreet embodiment. In another embodiment of the invention, the treatment of the individual to be treated is determined based on the bacterial profile of the otitis media.
[00073] In one embodiment the term, "Acute otitis media" (AOM), as used herein, refers to a condition characterized by fluid in the middle ear accompanied by signs or symptoms of ear infection (bulging eardrum usually accompanied by pain; or perforated eardrum, often with drainage of purulent or infectious material). A patient with recurrent acute otitis media (RAOM) has had either more than three acute episodes in a period of six months or four or more acute episodes in a period of 12 months.
[00074] In one embodiment the term, "Otitis media with effusion" (OME), as used herein, refers to a condition characterized by fluid in the middle ear without signs or symptoms of ear infection. Otitis media with effusion is defined as chronic (COME) when middle ear effusion has been present for at least 3 months.
[00075] In one embodiment the term, "Chronic suppurative otitis media" (CSOM), as used herein, differs from "chronic otitis media with effusion" (COME) with respect to the state of the tympanic membrane. Chronic otitis media with effusion (COME) may be defined as a middle ear effusion, without perforation of the tympanic membrane, which is reported to persist for 3 months. Chronic suppurative otitis media is a perforated tympanic membrane with persistent drainage from the middle ear.
[00076] In one embodiment the term, "Acute post-tympanostomy otorrhea" (APTO), as used herein, refers to a condition characterized by the presence of purulent fluid or inflamed middle ear mucosa occurs following tympanostomy tubes placement. Drainage following tube placement that persists for less than 8 weeks, is classified as acute.
[00077] In one embodiment, the term "sinusitis" is the result of undrained mucous in one or more of the sinus cavities. In one embodiment, sinus inflammation, is caused by the presence of fungi, bacterial and viral infection, or allergens. In another embodiment obstructions due to deviated septum and nasal polyps which form in the nasal passages and which obstruct breathing lead to sinus inflammation. Regardless of the cause, inflammation of the sinus cavities causes the swelling and congestion of membranes associated with the sinuses. Pain results from the congestion and mucous production increases and the mucous itself becomes thicker.
[00078] In one embodiment, the term "upper respiratory infection" refers to the predominant colonization or growth of a disease causing microbe, pathogen, bacteria, virus, fungi, or live particle taking place in, on, around or within the throat, nasopharynx, the eustachian tube, the nasal passages, or the sinuses.
[00079] In another embodiment, ther term "lower respiratory infection" refers to the predominant colonization or growth of a disease causing microbe, pathogen, bacteria, virus, fungi, or live particle taking place in, on, around, or within the bronchi, bronchioles, the alveoli, and the lungs.
[00080] In one embodiment, the bacteria involved in the pathogenesis of the diseases described herein and for which the methods of treatment described herein is effective, is S. pneumoniae.
[00081] Each one of the above anatomical features has mucous and mucous membranes associated with each such anatomical feature. For example, the sinus cavities have mucous membranes and those membranes have their related mucous. The alveoli of the lungs also posses mucous and mucous membranes. For the purposes herein, the human respiratory system comprises all the above anatomical features and all the associated mucous membranes and the associated mucous within, and in contact with, those mucous membranes. So, for the purposes herein, the human respiratory system includes the pulmonary anatomy, the sinus anatomy, the nasal anatomy, the ear anatomy, and all the associated mucous membranes and the associated mucous within, and in contact with, those mucous membranes. [00082] In one embodiment, provided herein is a composition for inhibiting bacterial growth or colonization, comprising an effective amount of a bacteriocin, produced by an upregulation of expression of the blp locus of a bacteria. In another embodiment, said bacteria is Streptococcus. In one embodiment said Streptococcus is Streptococcus pneumoniae. In one embodiment, said bacteriocin further comprising of a pharmaceutically acceptable carrier. In the practice of the embodiments of methods as described herein, an effective amount of compounds of the present invention or pharmaceutical preparations thereof, as defined herein, are administered via any of the usual and acceptable methods known in the art, either singly or in combination with another compound or compounds of the present invention or other pharmaceutical agents, such as antibiotic agents, antiviral agents or a combination thereof. The methods of administering the active ingredients of the present invention con comprise of but is not limited to nasal sprays, topical solutions, patches or a combination thereof.
[00083] For topical administration to the epidermis, the compounds may be formulated as ointments, gels, creams or lotions, or as the active ingredient of a transdermal patch. The administration can be carried out in one embodiment, in single unit dosage form with continuous therapy or in another embodiment, in single dose therapy ad libitum.
[00084] The term "about" as used herein means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.
[00085] The term "subject" refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans. The term "subject" does not exclude an individual that is normal in all respects.
[00086] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.
EXAMPLES
Materials and Methods:
[00087] Bacterial strains and growth conditions. The S. pneumoniae strains used in this study are described in Table .1. An array of clinical strains were analyzed and considered unrelated based on differences in capsular type and time and location of isolation. All strains were grown in tryptic soy broth (TS) or on tryptic soy agar (TSA) supplemented with catalase (4,741 U/plate; Worthington, Lakewood, NJ), except where indicated. TSA was supplemented with streptomycin (100 μg/ml), kanamycin (500 μg/ml), or erythromycin (1 μg/ml) where indicated. Cultures were grown on agar plates at 37°C in 5% CO2 or anaerobically using a BBL gas pack system. Broth cultures were grown at 37°C without agitation. For transformation, bacteria were grown from plates at low inocula in C+Y (pH 8.0) at 37°C until the optical density at 620 nm reached 0.150. One-hundred-microliter aliquots were removed and placed at 30°C with 10 ng/ml of purified competence-stimulating peptides 1 and 2. After 10 min, approximately 100 pg/ml of DNA was added to the mixture and incubated at 30°C for an additional 40 min. The culture was then transferred to 37°C and incubated for 2 h before being plated on selective medium.
Figure imgf000033_0001
[00089] Bacteriocin assay. Pneumococci grown on TSA plates overnight were resuspended in phosphate -buffered saline (PBS) to an optical density at 595 nm of 0.700. Test strains were then stabbed into TSA plates and allowed to grow anaerobically at 37 °C for 6 h. Plates were carefully overlaid with 10 CFU/ml of a mid- log-phase broth- grown overlay strain in 7 ml of TS containing 0.5% agar which had been maintained at 37°C before application and returned to an anaerobic environment for overnight growth at 37 °C. Test strains that scored positive for bacteriocin activity had a clear zone of complete inhibition of the overlay strain surrounding the area of test strain growth.
[00090] blp sequence analysis. Primers 1 and 2 (Table J) were used to PCR amplify and sequence the region of DNA likely to contain blpM and blpN based on sequence comparison. The blp locus from the type 6 A (and 6B) strain was amplified using primers 14 and 15, which amplified a 6,600-bp fragment. An extension of the 3' region of the locus, including the downstream gene SP0547, was amplified by primers 16 and 17. PCR was performed with Pfx high-fidelity polymerase (Invitrogen, Carlsbad, CA), using the following cycling parameters: 30 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 1 min/kb. PCR products were purified and sequenced using a BigDye Terminator v3.1 cycle sequencing kit from Applied Biosystems. Sequencing products were analyzed on a 3730 DNA analyzer from Applied Biosystems.
[00091] TABLE 2
Primers used in blp study
Primer Sequence and description
1 TTCCTTTCATATAGTGGATAGGTC ATG (SEQ ID NO 14); 275 bp upstream of blpM
2 CAGTTTACGGAACAAGTTTTAATAT ATG (SEQ ID NO 15) ; 40 bp upstream of blpO
3 CCAATGCATTAACAAAAGGAGACTTGTATG (SEQ ID NO 16) ; construction of blpM mutation, forward primer containing Nsil site separating start and stop codons of blpM
4 CCAATGCATAACAAATACTCCTTTTTTA (SEQ ID NO 17); construction of blpM mutation, reverse primer containing Nsil site separating start and stop codons of blpM
5 CCAATGCATTAAAAATGAAAGCTAAATTTT (SEQ ID NO 18); construction of blpN mutation, forward primer containing Nsil site separating start and stop codons of blpN
6 CCAATGCATACAAGTCTCCTTTTGTTA (SEQ ID NO 19); construction of blpN mutation, reverse primer with Nsil site separating start and stop codons of blpN
7 TTAATTTACAGGGGAGTTTCTTT; forward primer 56 bp from 3 ' end of blpO ORF
8 TGTCTTCGAAGGTGGTGGTGCTGCTTTTG (SEQ ID NO 20); forward primer engineering BstBI site into TIGR4 blpM for construction of chimera
9 CCCTGTAAATTAAGCTAGCAAATAC (SEQ ID NO 21); reverse primer for amplification of blpMNO from TIGR4 for construction of chimera
10 GAAGAGATTAGGGTTTTGTGCC (SEQ ID NO 22); forward primer 35 bp upstream of stop codon of blpA
11 TCTCGCAAGGAAGATGTTCCG (SEQ ID NO 23); reverse primer 22 bp upstream of stop codon of SP0535
12 GGCCGCTTTCGAAGGATCCGTTTGATTTTTAATGGATAAT (SEQ ID NO 24); forward primer for amplification of the janus cassette with engineered BstBI site
13 ACCTGCTAGCGGGCCCCTTTCCTTATGCTTTTGGAC (SEQ ID NO 25); reverse primer for amplification of janus cassette with engineered Nhel site
14 GTGAGCGACTTTATAGTTTCAATCC (SEQ ID NO 26); forward primer for sequencing of type 6A blp locus 240 bp upstream of the stop codon of blpA
15 CTGAAAATGAGTTCCTCCTGG (SEQ ID NO 27); reverse primer for sequencing of type 6A blp locus within SP0547
16 CTGAAAATGAGTTCCTCCTGG (SEQ ID NO 28); forward primer for sequencing of entire SP0547 locus 427 bp downstream of start codon of blpY Primer Sequence and description
17 GCCTCTGGATTGGCTTGGGTATCA (SEQ ID NO 29) ; reverse primer for sequencing of SPO547 within SP0548
[00092] Mouse colonization assay. All mice were purchased from Taconic and were housed in accordance with Institutional Animal Care and Use Committee protocols. Five to 7- week- old BALB/c mice were inoculated intranasally with 10 μl containing 2 x 107 to 4 x 107 CFU of a recently animal-passaged pneumococcus strain resuspended in PBS. All suspensions were plated for colony counts following inoculation to ensure that no inhibition had occurred in suspension prior to intranasal instillation. At 4 days postinoculation, a time point shown in pilot studies to provide a stable level of colonization, the mice were sacrificed by CO2 asphyxiation, the trachea of each was exposed, 200 μl of sterile PBS was instilled into it, and the lavage fluid exiting the nares was collected. The lavage fluid was then serially diluted in PBS and plated on TSA. Plates were supplemented with neomycin (5 μg/ml) to prevent the growth of contaminants or with neomycin plus streptomycin to select for growth of the 19A derivatives. Results of antibiotic selection were verified using colony immunoblotting with a rabbit polyclonal antibody against capsular serotype 4 on neomycin-only plates. The lower limit of detection of this assay was 100 CFU/ml of lavage fluid.
[00093] Generation of defined blp mutants. Pneumococcal mutants were created as follows. The blpMNO region was cloned into Escherichia coli plasmid pUC19 at the Smal site, using primers 1 and 11. The resulting plasmid was designated pBlpAL. The janus insertion was created by amplifying the cassette from strain CP1296 by PCR and engineering B stBI and Nhel sites into the 5' and 3' regions, respectively (primers 12 and 13). This product was ligated into pBlpAL cut with BstBI and Nhel at unique sites that span blpM through blpO. The resulting plasmid, called pBlpALAMNOjanus, was transformed into 6ASmr and selected for kanamycin resistance and streptomycin sensitivity. This strain was named 6AAMNOjanus. The remaining blpMNO mutants were constructed by replacing the janus cassette in this strain. The blpM and -N deletions were constructed by performing inverse PCR on pBlpAL, using primers 3 and 4 for the blpM deletion and primers 5 and 6 for the blpN deletion. These primers were engineered to create a unique Νsil site between the stop and start codons of the respective genes. The resulting PCR products were then cut with Νsil, ligated, and transformed into E. coli ToplO cells (Invitrogen, Carlsbad, CA). The blpO deletion was created by performing inverse PCR on pBlpAL, using primers 7 and 2. The resulting product was phosphorylated using T4 DΝA kinase, blunt end ligated, and transformed into E. coli ToplO cells. The chimeric protein was created by amplification of DΝA from TIGR4, using primers 8 and 9, which introduced a BstBI site at the 5' end. This product was digested with BstBI and Nhel and ligated to the 1,022-kb BstBI/Nhel fragment of pBlpAL. All plasmids were verified by restriction digestion. The janus cassette was replaced in strain 6AAMNOjanus by transforming the strain with the PCR product produced by primers 1 and 11 and selecting colonies on streptomycin plates. Deletion of the blpMN operon in the serotype 19A strain was performed by amplifying the janus cassette insertion in strain 6AAMNOjanus, using primers 1 and 11, and transforming the product into a streptomycin-resistant derivative of 19A. An unmarked mutation deleting the entire blpMNO region was created in this strain as described above. The janus cassette in \9AAMN0janus was replaced with the wild-type locus by transforming cells with the plasmid pBlpAL. All constructs were verified for a double-crossover event by a loss of kanamycin resistance and by PCR. 6AAbIpR was created by isolating DNA from the type 3 isolate containing an erythromycin cassette, replacing the blpR gene, and transforming the construct into the type 6A strain. This mutation was backcrossed three times to reduce the possibility of transformation occurring with unlinked DNA.
Figure imgf000039_0001
Figure imgf000039_0002
Figure imgf000040_0001
[00094] Construction and analysis of reporter constructs. The integrative plasmid pEVP3 was used to create a transcriptional fusion to the blpMNPO and htrA promoters. For the blp reporter fusions, one kb of DNA including the promoter region of blpMNPO and a small portion of upstream and downstream sequence was cloned between the Xbal and Nsil sites of the plasmid using primers 1 and 2 (Table 4) and maintained in ToplO cells. For the htrA reporter fusions, an 1100 bp fragment including the promoter region of htrA including 72bp of coding sequence were cloned between the Xbal and Nsil sites of the plasmid using primers 9 and 10 (Table 4). For both plasmids, the transparent type 6A variant was transformed with plasmid DNA and transformants were selected for by plating on TS plates supplemented with 3μg/ml of chloramphenicol. For all transformations, bacteria were grown from plates at low inocula in C + Y pH 8.0 at 37° C until OD620 reached 0.150. lOOμl aliquots were removed and placed at 30° C with 10ng/ml of a 1:1 mixture of purified competence stimulating peptides 1 and 2 (CSP). CSP 1 and 2 were purchased from Genscript as the following peptides: CSPl : EMRLSKFFRDFILQRKK and CSP2: EMRISRIILDFLFLRKK. After lOmin, approximately 100pg/ml of either purified genomic or plasmid DNA was added to the mixture and incubated at 30° C for an additional 40 min. The culture was then transferred to 37° C and incubated for 2 hours before plating on selective media.
[00095] Plasmid integration was confirmed using a primer to the upstream flanking region (primer 3 for blp insertion, 11 for htrA insertion) and a reverse primer internal to the lacZ gene (primer 4). For derivation of the opaque isolate with either the blpMNPO or htrA promoter reporter fusions, genomic DNA was isolated from the corresponding transparent transformant and used to transform the opaque variant of the type 6A strain. This approach was required due to the low transformation efficiency of this strain. The native bgaA gene was deleted by transforming pneumococcus with plamid pE668 and selecting for erythromycin resistance, β-galactosidase activity was determined using a modified Miller assay as follows. Pneumococcal strains were streaked for isolated colonies on TSA overnight. Single colonies were picked and inoculated into 10ml of TS broth. Cultures were allowed to grow for ~8 hours at 37° C to reach an OD620 of 0.1. Samples were then removed as indicated and assayed for β-galactosidase activity. For growth analysis, ImI of culture was removed and lOμl of 10% Triton X-100 added to this volume. The mixture was incubated for 10 minutes at 37° C at which point 250μl of 5X Z buffer (5mM MgCl, 5OmM KCl, 0.3M Na2HPO4, 0.2M NaHzPO4, 25OmM BME, 4mg/ml ONPG) was added. The tubes were incubated at room temperature until solutions were visibly yellow and the reaction was stopped with 500μl of 1.0M NaCθ3 and results were read at OD420 and OD550. Miller Units were determined. Samples were blanked against media alone. In order to determine the effect of exogenous BIpC on focZ-expressing strains, either water or the indicated amount of synthetic BIpC was added to cultures at an OD620 of ~ 0.100. Synthetic active BIpC peptide determined by DNA sequencing of the 6A strain locus was found to be GLWEDILYSLNIIKHNNTKGLHHPIQL. This peptide was synthesized and purified to 95% purity by Genscript (Piscataway, NJ). Transcriptional activity was then determined for samples with and without the addition of BIpC. Assays were performed in triplicate. For dose response calculations, increasing amounts of BIpC were added to identical cultures and allowed to incubate for 2 hours. ECso and 95% confidence intervals were determined using Prism as follows. Peptide concentrations were transformed to their natural log values, then data points were then normalized such that the lowest number of each set was made equal to 0% and the highest number to 100%. Nonlinear regression (curve fit) analysis was then performed without constraining the Hill Slope. ECso was defined as the concentration of BIpC required to reach 50% of maximal activity. The htrA gene was deleted in blp reporter constructs by insertion of the Janus cassette as described below. The ciaH mutations were created in htrA reporter constructs using Janus insertion as described below. The blpA gene was disrupted by insertion of the erythromycin cassette into a unique EcoRV site within the coding sequence of the gene using the plasmid pBlpAL, creating pBlp ALbIpA: :erm. This plasmid was then used to transform the 6At strain containing the blpMNPO lacZ fusion. Transformants were selected on media containing erythromycin and insertions confirmed by PCR.
[00096] Quantitation of HtrA levels in opacity variants. The type 6 A opaque and transparent variants were grown in TS broth at 37° C to OD620 0.5. Cells were pelleted and resuspended in PBS and sonicated. Equal amounts of protein were boiled in SDS/BME containing buffer for 5 minutes and separated on a Tris-HCl 10% polyacrylamide gel at sequential 1:1 dilutions. The gel was transferred and blocked with TBS with 5% non fat dry milk for 2 hours. The blot was first probed with polyclonal htrA antiserum, washed and binding detected with anti-rabbit HRP secondary antibody. Bands were visualized using enhanced chemiluminescence plus Western blotting detection system (GE Healthcare). Band intensities were determined for each dilution using gel analysis tools of ImageJ (Wayne Rasband; N1H [http://rsb.info.nih.gOv/j/]) and only dilutions within the linear range used for densitometry determination. Equal loading of comparable dilutions of the variants was confirmed using the monoclonal anti-pneumolysin antibody (Novocastra, UK) followed by anti-mouse HRP secondary. The amount of HtrA in variants was determined by equalizing for pneumolysin density in the same lane.
[00097] Construction of htrA and ciaH mutations in type 6A derivatives. Both htrA and ciaH mutations were created using exchange of the Janus cassette. DNA from pneumococcal isolates containing the Janus cassette replacing the internal portions of either htrA or ciaH genes was used to transform a streptomycin-resistant derivative of the type 6A variants. This mutation was backcrossed once to remove unlinked DNA. An in-frame, unmarked deletion of htrA was derived by transforming the isolates containing the Janus cassette with DNA from strain P1544. The serine to alanine mutation in htrA was created in the 6A background by exchanging the Janus cassette with the mutated htrA gene using a plasmid with a fragment of DNA containing the mutated htrA gene. As a control, the wildtype htrA gene was used to replace the Janus cassette by transforming strain P 1720 with genomic DNA from the type 6 A strain. Constitutively active CiaH mutation CiaHτ230P was created by exchanging the Janus cassette in ciaH with DNA from strain Pl 386. As a control, the wildtype ciaH locus was used to replace the Janus cassette and assessed for recovery of the wildtype phenotype. Janus replacements were screened for loss of kanamycin resistance and recovery of streptomycin resistance. Appropriate mutations were confirmed by PCR. Identical methods were used to separately introduce the mutation in htrA and the deleted ciaH into the 6A derivative β- galactosidase reporter strains.
[00098] Construction of epitope tagged BIpM and BIpN. The pUC19 derivative plasmid pBlpAL containing a cloned region of the type 6A blp locus spanning from the 5' end of blpA to downstream of blpO was digested with BstBI. Complementary primers 5 and 6 (Table 4) encoding an in- frame insertion of the FLAG® epitope with BstBI sticky ends were ligated into the plamid and used to transform E. coli Top 10 cells creating plasmid pB IpALMFLAG. The BIpN FLAG fusion was created by amplifying the blpN gene with primers 7 and 8 (Table 4) introducing a C-terminal FLAG® tag in frame into the coding sequence followed by a stop codon and an Nsil site. The PCR product was digested with BstBI and Nsil and ligated into vector pBlpALDN also digested with BstBI and Nsil creating plasmid pB IpALNFLAG. The resultant plasmids were sequenced to ensure in-frame insertion and used to transform Pl 573 containing a Janus cassette disrupting the blpMNPO locus. Transformants were selected on streptomycin containing plates and confirmed for the proper insertion by loss of kanamycin resistance and by PCR. In order to make the epitope tag in the transparent variant of the type 6A strain, a streptomycin resistant isolate of the strain was first transformed with DNA derived from the opaque variant Pl 573 containing the Janus cassette in the blpMNPO locus. The isolate was selected on kanamycin plates and confirmed for the correct insertion by PCR. The FLAG® epitope was inserted into the BIpM and BIpN sequences as described above.
[00099] Deletions in htrA in FLAG® strains were produced by transformation with DNA from strain Pl 180 and selection on erythromycin containing plates followed by a single back transformation. Deletions were confirmed by Western blotting using HtrA polyclonal antiserum for loss of protein expression. Detection of BIPMFLAG and BIPNFLAG was carried out by growing cells to confluence on TS plates supplemented with catalase. One hour prior to harvesting, 250μg of synthetic BIpC were spread onto the surface of select plates. Cells were scraped from the surface of the plate and resuspended in PBS with 0.1% Triton XlOO for lysis. Equal quantities of lysates were denatured and separated on a 15% polyacrylamide gel. The gel was transferred and the membrane blocked in TBS with 5% NFDM for 2 hours.
The membrane was probed with ANTI-FLAG® M2 antibodies (Sigma-Aldrich), washed and detected with anti-mouse secondary antibody conjugated to alkaline phosphatase. The membrane was then stripped and re-probed with anti-pneumolysin antibody as a loading control followed by detection with anti mouse secondary antibody.
Example 1: Bacteriocin-like activity in a clinical isolate of the pneumococcus
[000100] .In order to characterize the functional significance of the blp locus in pneumococci, an array of clinical isolates (n = 9) was screened for their relative abilities to inhibit growth of an unrelated strain in an agar overlay assay. In order to remove the inhibitory effects of pneumococcal hydrogen peroxide production, assays were done either aerobically with catalase added to both agar layers or under anaerobic conditions, with equivalent results. This screening procedure identified isolates of type 6A and 19A with inhibitory activity in vitro when tested against a number of other pneumococcal isolates, including the fully sequenced type 4 strain TIGR4 (Table 5 and Fig. 1C). The TIGR4 strain had no reciprocal activity against type 6A or 19A or any other clinical isolate tested under these assay conditions (Table 5).
Figure imgf000045_0001
Figure imgf000046_0001
Example 2: BIpR rβ2ulates expression of in vitro bacteriocin activity and immunity
[000101] In order to confirm that the blp locus was responsible for intraspecies inhibition, the blpR regulatory gene homologue of the type 6A strain was deleted by replacement of an internal fragment of the gene with the erythromycin resistance cassette. The resulting strain, 6AJbIpR, was analyzed for loss of its inhibitory activity and immunity, using the plate overlay method. As predicted, 6AAbIpR was deficient in in vitro intraspecies inhibition when tested against TIGR4. In addition, as expected, 6AAbIpR was killed by its parent strain, suggesting a deficiency in expression of its immunity phenotype (Fig. IA). Therefore, deletion of the BIpR response regulator led to a defect in both killing and immunity. These observations provided the first direct functional demonstration that the blp locus is involved in intraspecies competition in vitro.
Example 3: Sequence analysis of the 6A blp locus [000102] Given the preliminary results for the type 6A strain, the blp locus was sequenced from the N terminus of the blpA gene to the end of the previously defined locus, SP0547 (Fig. 2). This region was predicted to contain the genes encoding the bacteriocins based on the arrangement of the locus in other previously sequenced strains. As in the TIGR4 locus, putative bacteriocin genes in the type 6 A locus were preceded by a highly conserved consensus sequence for BIpR binding. The type 6A strain's blp locus contains homologues for the predicted bacteriocin genes blpM, -N, and -O. These genes would be predicted to encode three proteins, with each containing a conserved N-terminal signal sequence followed by a double-glycine motif, consistent with the sequences of previously described bacteriocins. Surprisingly, similar genes were also found in the TIGR4 genome, although TIGR4 did not inhibit the growth of the type 6A strain. The type 6A strain encodes BIpM, - N, and -O proteins that have 6 of 84, 2 of 67, and 2 of 49 residues, respectively, that differ from the TIGR4 sequence. The type 6A locus contains two operons downstream of the putative BIpM, -N, and -O bacteriocins, preceded by two additional BIpR consensus binding sequences that contain open reading frames (ORFs) encoding proteins of unknown function. The final operon contains homologous ORFs for the genes blpX, -Y, and -Z and, unlike TIGR4, is predicted to include the downstream ORF SP0547 due to deletion of a transcriptional terminator sequence. BIpX, -Y, and -Z and SP0547 are 100%, 96%, 98%, and 99% identical to the TIGR4 sequence, respectively, at the amino acid level. Example 4: Deletional analysis of blpM, -N, and -O
[000103] Using chromosomal allelic replacement, the 6A blpM, -N, and -O ORFs were deleted both individually and in combination. In order to create in- frame, unmarked deletions in these small, closely approximated genes, the janus cassette was inserted between unique restriction sites, replacing the entire blpMNO operon. A type 6A strain derivative made streptomycin resistant was used for insertion of the janus cassette in the blpMNO locus. The resulting isolate was resistant to kanamycin and sensitive to streptomycin, confirming the insertion of the janus cassette. The cassette was then replaced by transforming this strain with fragments containing altered versions of the blpMNO operon. Separate deletions were created in blpM, -N, and -O by deleting each gene in its entirety, leaving only its predicted stop and start codons. The deletion of the entire blpMNO locus was created by introduction of a deletion spanning from the 5' end of the blpM ORF through the 3' end of the blpO ORF. In order to determine the significance of the small number of amino acid differences between BlpM and BIpN in the type 6A isolate and TIGR4, a chimeric gene was created by exchanging the type 6A strain blpM and -N with TIGR4 blpM and -N. The chimeric protein contains the Ν terminus of 6A blpM with all three amino acid changes in the C terminus found in the TIGR4 locus and the entire blpN locus from TIGR4 (Fig. YB). This chimeric construct was used to determine if the difference in killing between the two strains was the result of the differences in these amino acids. To confirm this result and to demonstrate an absence of additional mutations outside the locus explaining the phenotype, a PCR product containing the corresponding original parental type 6A locus was used to replace the janus cassette. All strains were tested for a loss of in vitro inhibitory activity by the plate overlay method against strains TIGR4 and 6AAbIpMNO and used as an overlay against the type 6A isolate to look for a loss of immunity (Fig. IA and C).
[000104] In vitro assays for bacteriocin activity demonstrated that both the blpM and blpN genes are required for wild-type intraspecies inhibitory activity but not immunity. Unlike the blpM and -N deletions, the blpO deletion had wild-type levels of activity in both inhibition and immunity. Strain 6AJbIpMNO, containing a deletion of the entire locus, had a deficiency in both inhibition and immunity, suggesting that a gene in this locus contributes to the immunity phenotype. The construct containing the corrected wild-type locus, 6AblpMN0WT , had parental levels of activity, confirming that mutations outside blpMN could not account for the observed phenotypes. The type 6A strain expressing the chimeric form of BIpM and -N, 6AbIpMNO , was deficient in intraspecies inhibition, similar to the phenotype of wild-type TIGR4. This strain retained the parent strain immunity phenotype. These data suggest that both BIpM and -N are necessary for the bacteriocin activity seen in vitro. Moreover, the difference in activity between the TIGR4 and type 6A strains could be attributed to the five amino acids that differ between the two strains in the mature, processed forms of BIpM and -N.
Example 5: Conservation of BIpM and -N sequences among pneumococcal strains
[000105] Bacteriocins tend to have a significant degree of divergence when different strains within the same species are compared. This divergence may allow for intraspecies competition. Small changes in the bacteriocin often require reciprocal changes in the immunity protein so that organisms expressing similar but not identical bacteriocins are not protected from each other by their own immunity proteins. In order to determine the relative conservation of the BIpM and -N proteins, blpM and -N coding sequences for the nine clinical isolates were analyzed. These strains include an array of clinical isolates of diverse capsular types that were isolated in different locations at different times. Seven of the nine isolates had sequences homologous to blpM and -N. The remaining two isolates contained coding regions for other bacteriocin- like peptides Q)IpI and blpK) homologous to those found in the TIGR4 locus. The BlpM and -Ν sequences were aligned and analyzed for conserved amino acids (Fig. 3). Interestingly, the seven BlpM sequences seemed to be divided into two groups. Group 1 contains those with 100% identity to the TIGR4 sequence. Group 2 comprises those with 98 to 100% identity to the type 6A strain's sequence. In comparing the BlpΝ sequences, the RL amino acid sequence at amino acids 40 and 41 was seen in all strains containing the group 1 BlpM sequence, while the KI sequence was seen in strains containing the group 2 BlpM sequence. In vitro inhibition assays with the seven clinical isolates demonstrated that only strains in group 2 had detectible activity. One strain in this group, a type 12F strain, showed no detectible inhibitory activity on overlay assays against any strain tested, and the five strains in group 1 also had no detectible inhibitory activity.
Example 6: The blp locus is functional in vivo during colonization
[000106] In order for the blp locus to play a role in intra- and interspecies competition, not only must it be expressed in the polymicrobial environment of the nasopharynx, but organisms must be in close enough proximity to be affected by secreted antimicrobial proteins. To determine if the blp locus is both expressed and functional during colonization, we performed competition experiments in BALB/c mice. Because the type 6A strain was highly virulent in mice in the nasal colonization model, competition experiments were performed with the serotype 19A strain from group 2. An unmarked mutation of the blpMNO operon and a replacement of the wild-type operon were created in this strain and analyzed by the overlay assay for phenotype. Like the type 6A strain, strain 19 AAbIpMNO was deficient in growth inhibition when tested against TIGR4 and had an immunity defect when tested against the parent strain (Fig. IA and C). The corrected mutant, 19 AblpMNOwτ , had the expected wild-type phenotype in both inhibition and immunity. The type 19A strain, TIGR4, 19 AbIpMNO , and 19AAbIpMNO were inoculated intranasally either alone or in pairs. Singly inoculated mice were colonized with TIGR4, 19A, 19 AAbIpMNO, or \9AblpMN0wτ at equivalent levels (Fig. 4A and B). Dually colonized mice given 19A and 19 AAbIpMNO were colonized predominantly with the type 19A strain (Fig. 4B). Dually colonized mice given TIGR4 and 19 AblpMN0WT were colonized primarily with the 19A strain, mimicking our in vitro inhibition results (Fig. 4A). The competitive advantage of the 19A strain was eliminated when TIGR4 was coinoculated with 19 AAbIpMNO. In fact, these animals were colonized primarily with TIGR4, with little detectible colonization by the mutant strain. These experiments suggest that production of bacteriocins by the wild-type strain was able to inhibit growth of immunity-deficient strains during colonization, verifying the role of these peptides in vivo.
Example 7: Opacity variants have different levels of pneumocinMN inhibitory activity
[000107] In order to evaluate the expression of pneumocinMN inhibitory activity in opacity variants, overlay experiments with the opaque and transparent variants of a type 6A isolate were performed. This type 6A strain was chosen for analysis because the activity of the bacteriocin produced by its blpMNPO locus has been well characterized. Opaque or transparent variants were tested for pneumocinMN production by overlay assay against a type 6A mutant lacking the blpMNPO operon including the putative immunity gene, blpP (Figure 6A, H). Unexpectedly, the opaque variants produced wide zones of inhibition when tested against a sensitive strain while the transparent variants had no detectable inhibition. Identical results were seen when the sensitive TIGR4 strain was used as the overlay strain. Similar findings were noted for opacity variants of an unrelated type 19A strain which contains a blp locus with activity attributable to blpMN homologues. The deletion of the blpMNPO operon in the type 6A strain or deletion of the regulator blpRH completely abolished inhibition by the opaque variant demonstrating that inhibitory activity requires the presence of an intact blpMNPO transcript.
[000108] In order to determine if the difference in pneumocinMN activity in opacity variants was due to transcriptional regulation of the locus, reporter fusions to the blpMNPO promoter in the type 6A strain were created. The strain backgrounds contained a deletion in the native β-galactosidase gene to remove endogenous enzyme activity. Reporter integrants were grown in TS broth from single colonies to eliminate the possibility of spontaneous induction by pheromone present on the plate. Once cultures reached OD620 of 0.100, samples were taken every hour to determine β-galactosidase activity throughout the growth phase. Surprisingly, both opaque and transparent variants had very low levels of transcript through early stationary phase without any significant induction (Figure 7A). When plated on X-gal containing plates, both opaque and transparent variants produced blue colonies in areas where organisms were plated in high density.
[000109] In order to determine if the opacity variants grown in broth culture produce equivalent amounts of transcript when exposed to synthetic pheromone BIpC, the kinetics of response to exogenous synthetic BIpC in the 6A strain containing a deletion in blpA was first determined. This strain is unable to transport and process endogenous BIpC such that the locus is only activated in the presence of exogenous BIpC. This strain showed peak transcriptional activity 2 hours after addition of 100ng/ml of BIpC to organisms grown in culture (Figure 7B). Similar kinetics of response to BIpC was noted when using reporter fusions with an intact blpA gene. When opaque and transparent variants of the type 6A strain containing the reporter construct were stimulated with increasing concentrations of synthetic BIpC and sampled after two hours of stimulation, the variants had nearly identical maximal levels of transcriptional activity (Figure 8). Additionally, dose response analysis of these data showed that, although peak levels were similar, the calculated ECso of the opaque variant was 3.7 fold lower than that of the transparent variant that lacked inhibitory activity (Figure 8). In order to determine if subtle transcriptional differences due to altered sensitivity to pheromone were accounting for the differential expression of pneumocinMN, overlay assays were performed on plates containing 100ng/ml of synthetic BIpC. When transparent strains containing blpMNPO reporter fusions were plated for single colonies on TS/X-gal with 100ng/ml of synthetic BIpC, all colonies were blue indicating that the locus was activated under these conditions even at the single colony level. When transparent variants were exposed to saturating quantities of synthetic BIpC in overlay assays, they still had no detectable pneumocinMN activity indicating that some degree of post transcription regulation was responsible for the differential expression of pneumocinMN activity in opacity variants.
Example 8: The role of CiaRH in pneumocin expression
[000110] It is known that the two-component system CiaRH is involved in the regulation of a large number of disparate loci including a number of genes involved in pneumococcal competence. The inventors of the instant application found that CiaRH is involved in pneumocinMN expression. In order to determine the effect of CiaRH on pneumocinMN activity, mutants of the type 6A strain with either a deletion in CiaH or a constitutively active version of CiaH (CiaHτ230P mutant) were created and blp mediated inhibition using overlay assays were determined. Although transparent variants of the type 6A strain had no zone of inhibition when tested against a sensitive overlay, transparent variants with a deletion in CiaH had a large zone of inhibition (Figure 6B). The transparent strain regained the original null phenotype when the deletion was replaced with the wildtype locus, making it unlikely that the observed phenotype was due to a new mutation introduced elsewhere in the chromosome during transformation (Figure 6C). When opaque variants of the type 6A were transformed with DNA encoding the constitutively active CiaHτ230P, the resultant construct lost all detectable inhibition consistent with a negative regulatory role of CiaH on the expression of pneumocin MN activity (Figure 61).
Example 9: HtrA is differentially regulated opacity variants
[000111] It is known that CiaRH activity results in the up regulation of the serine protease HtrA. Many of the important roles in bacterial growth and survival attributed to the CiaRH system are mediated by its upregulation of HtrA. It is also known that expression of HtrA is required for pneumococcal growth at elevated temperatures, resistance to H2O2 and virulence in a pneumonia model of infection. CiaRH mediated increases in HtrA expression in a CiaRH overexpressing strain were recently shown to repress the pneumococcal competence pathway, likely as a consequence of protease mediated digestion of a regulatory element controlling the com regulon. Given the similarities between the com locus and the blp locus, and the increase in pneumocinMN inhibition in the CiaH deletion strain, the inventors of the instant application evaluated the opacity variants of the type 6A strain for differential expression of HtrA at the level of transcription and translation. Opaque and transparent variants of the type 6A strain lacking endogenous β-galactosidase activity were transformed with an integrative plasmid derivative of pEVP3 containing the htrA promoter fused to the β-galactosidase gene. Miller assays on these constructs grown in TS broth demonstrated that, in late exponential phase growth, opaque variants produce ~4 fold less htrA transcript than transparent strains (Figure 9A). In order to confirm that the regulation of htrA in the type 6A strain was similar to what had been previously reported, CiaH deletions were introduced into these isolates. Only low levels htrA transcription were detectable in either the opaque or transparent 1 reporter strain containing a deletion in ciaH (Figure 9A). In order to verify that transcriptional differences correlate with differences in detectable protein levels, semi-quantitative Western blotting was performed on lysates from broth grown opacity variants of the type 6A strain using anti-HtrA antiserum. Pneumolysin was used as a loading control because its levels do not vary between opacity variants. The transparent variant produced more HtrA than the opaque variant (Figure 9B). The increased expression of HtrA in the transparent variant was estimated at ~3-fold using densitometry, similar to the differences noted in transcriptional activity.
Example 10: HtrA is required for the post-transcriptional regulation of bacteriocin expression
[000112] In order to determine if the level of HtrA expression affects pneumocin MN mediated activity, the inventors moved an in-frame, unmarked deletion of the htrA gene as well as a protease deficient htrA gene encoding a serine to alanine mutation in the active site into the transparent type 6A variant. Deletion mutants were confirmed by PCR and Western blot using anti-HtrA antiserum. Serine to alanine mutants were confirmed by PCR and western blotting with anti-HtrA antiserum to confirm recovery of the HtrA specific band. Similar to the CiaH deletion mutant, the type 6A transparent variant with either a deletion or inactivating mutation in the htrA gene had an increased zone of inhibition when tested in the plate inhibition assay and compared to the transparent parent (Figure 6A, D, E). When the blpMNPO locus was deleted from the htrA mutant strain, the zone of inhibition was lost, confirming that inhibitory activity requires the presence of an intact blpMNPO locus 1 (Figure 6G). A complemented transparent strain in which the mutated htrA gene was replaced with the wildtype locus also had no detectable activity, verifying that no new deletions impacting the inhibitory phenotype had been introduced during the transformation steps required for insertion of the Janus cassette (Figure 6F). [000113] To determine if the mutations in htrA affected pneumocin MN activity at the transcriptional or post-transcriptional level, the blpMNPO promoter activity was assessed in transparent variants with and without an intact htrA gene using the β-galactosidase reporter fusion. Dose-response curves as well as determination of maximal activities after addition of synthetic BIpC were determined for transparent strains with and without a deletion in htrA. Deletion of htrA from the transparent variant of the type 6A strain had no effect on maximal transcription from the blpMNPO promoter following addition of synthetic BIpC (Figure 8). Dose response curves of the reporter strains to increasing amounts of BIpC demonstrated a 10 fold increase in ECso when comparing the transparent strain with the OhtrA mutant (Figure 8) demonstrating that HtrA can affect signaling via the peptide pheromone BIpC. Similarly, opaque variants with a deletion in htrA had a 10 fold decrease in ECso compared with the wildtype opaque strain. These data show that HtrA activity decreases responsiveness to the peptide pheromone BIpC. Specifically, although the presence of htrA had a dramatic effect on the dose response curve in both variants, the ratio of ECso values of opaque and transparent variants remained constant (-3.7). This observation shows that, although HtrA can modify the bacterial response to peptide pheromone, an additional regulator functioning downstream of BIpC signaling is involved in controlling blp expression in opacity variants. Although HtrA activity plays a role in transcriptional regulation of the blp locus, the combined observations that peak activities of blpMNPO transcript were unaffected by a deletion in htrA and saturating levels of BIpC in plates could not restore pneumocinMN inhibitory activity in transparent strains show that HtrA also participates in the post transcriptional regulation of pneumocinMN.
Example 11: Deletion of htrA results in increased levels of epitope tagged pneumocinMN
[000114] In order to visualize the effect of HtrA expression on pneumocinMN activity, a FLAG® tag was introduced into the predicted secreted fragment of the pneumocinM peptide. The wildtype blpM locus was exchanged with the tagged blpM gene in the type 6A opaque and transparent variants. Although the hydrophilic FLAG® tag was fused within the relatively hydrophilic N-terminus of the largely hydrophobic pneumocin M peptide where it would be least likely to disrupt the overall structure of the peptide, the resultant tagged peptides were deficient in inhibition in overlay assays. Using Western blotting with ANTI- FLAG® M2 antibodies (Sigma- Aldrich), it was detected that pneumocinMFLAG in organisms grown to high density on plates and not in broth grown organisms (Figure 10). This observation is consistent with a lack of detectable pneumocinMN mediated inhibitory activity in the broth of liquid grown organisms, an observation that is likely due to a combination of a lack of induction of the locus in broth culture and the relative hydrophobicity of the peptides. In order to determine the effect of htrA expression on pneumocin M expression, both opacity variants with a tagged blpM were created in an htrA negative background. Consistent with the lack of plate inhibition, the transparent variant expressing pneumocinMFLAG produced no detectable product even after addition of synthetic BIpC pheromone while the opaque variant had a small amount of tagged peptide detectable following pheromone induction (Figure 10 Lanes 3 and 4). Again, consistent with the plate inhibition data, transparent variants with a deletion in htrA produced large amounts of pneumocinMFLAG independent of BIpC addition (Figure 10 Lane 6). Deletion of htrA in the opaque variants also resulted in an increase in pneumocinMFLAG over that seen in the opaque variant expressing wildtype htrA. In order to determine if HtrA had the same effect on BIpN production, a FLAG tagged version of blpN was transformed into the opacity variants with and without a deletion in htrA. In contrast to the MFLAG expressing constructs, the BIpNFLAG expressing organisms retained inhibitory activity when tested in plate overlay assays. Introduction of a FLAG tag onto the C -terminus of the blpN coding sequence resulted in an identical pattern of expression as the BIpMFLAG expressing strains after Western blotting with anti-flag antibody. The NFLAG was undetectable in transparent isolates, detectable at low levels in opaque variants and at high levels in transparent variants lacking htrA expression (Figure 10 Lanes 7-9). This implies that HtrA plays a role in post-transcriptional regulation which is the major determinant controlling pneumocinMN activity.
[000115] Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

Claims

What is claimed is:
1. A method for generating bacteriocins in a bacteria comprising the step of contacting the bacteria with an agent capable of upregulating expression of the blp locus.
2. The method of claim 1, whereby said bacteria is Streptococcus.
3. The method of claim 2 whereby said Streptococcus is Streptococcus pneumoniae .
4. The method of claim 1, whereby said agent is blpC.
5. The method of claim 1, whereby said agent is an mRNA, a protein, a synthetic protein, a synthetic peptide, a peptide-mimetic, a small molecule, or a combination thereof.
6. The method of claim 1 , whereby addition of said agent results in the upregulation of a blp locus operon.
7. The method of claim 6, whereby said operon encodes a regulator protein, a transport protein, an immunity protein, a bacteriocin, or a combination thereof.
8. The method of claim 7, whereby said regulator protein is encoded by a polypeptide sequence of blpR, blpH, or a functional fragment thereof.
9. The method of claim 7, whereby said transport protein is blpA, blpB, or a functional fragment thereof.
10. The method of claim 7, whereby said bacteriocin is blpM, blpN, or functional fragment a thereof.
11. A method for treating an infection, the method comprising the steps of inhibiting or reducing bacterial growth or colonization in a subject comprising the steps of administering to said subject a bacteriocin producing bacteria, wherein the bacteriocin is produced by upregulating the expression of the blp locus, thereby treating an infection in said subject
12. The method of claim 11, whereby said bacteriocin is blpM, blpN, or a functional fragment thereof.
13. The method of claim 11, whereby said bacteria is Streptococcus.
14. The method of claim 11, whereby said Streptococcus is Streptococcus pneumoniae .
15. The method of claim 11, whereby said infection is a lower respiratory infection, upper respiratory infection, invasive infection, or a combination thereof.
16. The method of claim 15, whereby said upper respiratory infection is Sinusitis, Otitis media, Tracheobronchitis, or a combination thereof.
17. The method of claim 15, whereby said lower respiratory infection is Pneumonia, Broncho-pneumonia, or a combination thereof.
18. The method of claim 15, whereby said invasive infection is Primary bacteremia, Meningitis, Spontaneous bacterial peritonitis, Sepsis with tissue seeding, or a combination thereof.
19. The method of claim 11, further comprising contacting the bacteria with an agent capable of inhibiting the expression or function of blpR, or its encoded proteins.
20. A composition for inhibiting bacterial growth or colonization, comprising an effective amount of a bacteriocin, produced by an upregulation of expression of the blp locus of a bacteria.
21. The composition of claim 20, wherein said bacteria is Streptococcus.
22. The composition of claim 20, wherein said Streptococcus is Streptococcus pneumoniae.
23. The composition of claim 20, wherein said bacteriocin is pneumocinMN.
24. The composition of claim 20, further comprising of a pharmaceutically acceptable carrier.
25. The composition of claim 20, further comprising another antibiotic agent, antiviral agent or a combination thereof.
26. The composition of claim 20, further comprising a topical application agent.
27. The composition of claim 20, further comprising a nasal spray agent.
28. The composition of claim 20, further comprising an agent capable of inhibiting the expression, or function of blpR.
29. A method for generating bacteriocins in a bacteria comprising the step of down- regulating the expression of htrA.
30. The method of claim 29, whereby said bacteria is Streptococcus.
31. The method of claim 30 whereby said Streptococcus is Streptococcus pneumoniae.
32. The method of claim 29, whereby the down-regulation of the expression of htrA is caused by mutations in one or more functional domains of htrA.
33. The method of claim 29, whereby the down-regulation of the expression of htrA is caused by an antisense oligonucleotide complementary to all or a portion of a messenger
RNA encoding htrA, wherein said antisense oligonucleotide inhibits the production of htrA.
34. The method of claim 29, whereby the down-regulation of the expression of htrA is caused by siRNA that inhibits the production of htrA.
35. The method of claim 29, whereby the down-regulation of the expression of htrA is caused by an antibody that inhibits the production of htrA.
36. A method for generating bacteriocins in a bacteria comprising the step of down- regulating the ciaRH.
37. The method of claim 36, whereby said bacteria is Streptococcus.
38. The method of claim 37, whereby said Streptococcus is Streptococcus pneumoniae .
39. The method of claim 36, whereby the down-regulation of ciaRH is caused by mutations in one or more functional domains of ciaR or ciaH.
40. The method of claim 36, whereby the down-regulation of ciaRH is caused by an antisense oligonucleotide complementary to all or a portion of a messenger RNA encoding ciaR or CiaH.
41. The method of claim 36, whereby the down-regulation of ciaRH is caused by siRNA that inhibits the production of ciaR or ciaH.
42. The method of claim 36, whereby the down-regulation of ciaRH is caused by an antibody that inhibits the production of ciaR or ciaH.
43. A method for generating bacteriocins in a bacteria comprising the steps of upregulating expression of the blp locus; down-regulating the expression of htrA; and down- regulating the expression of ciaRH.
44. A method for generating bacteriocins in a strain of bacteria comprising the steps of upregulating expression of the blp locus, wherein the upregulation of expression of the blp locus is caused by down-regulating the expressions of htrA and ciaRH.
45. A method for treating an infection, the method comprising the steps of inhibiting or reducing bacterial growth or colonization in a subject comprising the steps of administering to said subject a bacteriocin producing bacteria, thereby treating an infection in said subject, wherein the bacteriocin is produced by down-regulating the expression of htrA.
46. A method for treating an infection, the method comprising the steps of inhibiting or reducing bacterial growth or colonization in a subject comprising the steps of administering to said subject a bacteriocin producing bacteria, thereby treating an infection in said subject, wherein the bacteriocin is produced by down-regulating the expression of ciaRH.
47. A composition for inhibiting bacterial growth or colonization, comprising an effective amount of a bacteriocin, wherein the bacteriocin is produced by down-regulating the expression of htrA.
48. A composition for inhibiting bacterial growth or colonization, comprising an effective amount of a bacteriocin, wherein the bacteriocin is produced by down-regulating the expression of ciaRH.
49. A method for generating bacteriocins in a bacteria comprising the step of contacting the bacteria with an agent capable of upregulating expression of the blp locus, wherein the agent comprises an amino acid sequence of blpC.
50. The method of claim 49, wherein the amino acid sequence comprises the sequence of GLWEDILYSLNIIKHNNTKGLHHPIQL.
51. The method of claim 49, wherein the bacteriocin in pneumocinMN.
52. The method of claim 49, wherein pneumocinMN comprises an amino acid sequence of blpM.
53. The method of claim 49, wherein pneumocinMN comprises an amino acid sequence of blpN.
54. The method of claim 49, wherein said bacteria is Streptococcus.
55. The method of claim 49, wherein said Streptococcus is Streptococcus pneumoniae .
56. A vaccine for treating, preventing or ameliorating a subject against pneumococcal infection or colonization, comprising a pharmaceutically acceptable carrier and an immunologically effective amount of a recombinant bacteria that produces a bacteriocin, wherein the recombinant bacteria comprises an amino acid sequence of blpC.
57. The vaccine of claim 56, wherein the amino acid sequence comprises the sequence of GLWEDILYSLNIIKHNNTKGLHHPIQL.
58. The vaccine of claim 56, wherein the bacteriocin in pneumocinMN.
59. The vaccine of claim 56, wherein pneumocinMN comprises an amino acid sequence of blpM.
60. The vaccine of claim 56, wherein pneumocinMN comprises an amino acid sequence of blpN.
61. The vaccine of claim 56, wherein said bacteria is Streptococcus.
62. The vaccine of claim 56, wherein said Streptococcus is Streptococcus pneumoniae .
63. A composition for inhibiting bacterial growth or colonization, comprising an effective amount of a blpC to generate bacteriocins in a bacteria.
64. The composition of claim 63, wherein the blpC comprises the amino acid sequence of GLWEDILYSLNIIKHNNTKGLHHPIQL.
65. The composition of claim 63, wherein the bacteriocin is pneumocinMN.
66. The composition of claim 63, wherein said bacteria is Streptococcus.
67. The composition of claim 63, wherein said Streptococcus is Streptococcus pneumoniae.
68. A mutated Streptococcus pneumoniae bacteria, wherein said bacteria exhibits elevated production pneumocinMN relative to a non-mutant bacteria, and wherein the mutation is in htrA, ciaR, ciaH or their combination.
69. A vaccine for treating, preventing or ameliorating a subject against pneumococcal infection or colonization, comprising a pharmaceutically acceptable carrier and an immunologically effective amount of a mutant bacteria of claim 68.
PCT/US2008/081866 2007-10-30 2008-10-30 Bacteria strains an d bacteriocin produced therefrom WO2009059054A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US89607P 2007-10-30 2007-10-30
US61/000,896 2007-10-30

Publications (2)

Publication Number Publication Date
WO2009059054A2 true WO2009059054A2 (en) 2009-05-07
WO2009059054A3 WO2009059054A3 (en) 2009-09-17

Family

ID=40591762

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/081866 WO2009059054A2 (en) 2007-10-30 2008-10-30 Bacteria strains an d bacteriocin produced therefrom

Country Status (1)

Country Link
WO (1) WO2009059054A2 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3439693A4 (en) * 2016-04-05 2020-05-27 The Research Foundation For The State University Of New York University at Buffalo Novel pneumococcal vaccine formulations
CN111601894A (en) * 2018-01-16 2020-08-28 花王株式会社 Method for producing protease of M23A family
WO2021201699A1 (en) * 2020-04-03 2021-10-07 Blis Technologies Limited Antiviral treatment comprising blis containing probiotic products

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6207411B1 (en) * 1995-04-12 2001-03-27 Teagasc Bacteriocins
US6448224B1 (en) * 1998-05-06 2002-09-10 St. Jude Children's Research Hospital Antibiotics and methods of using the same
US20020136712A1 (en) * 1997-10-31 2002-09-26 Fischetti Vincent Bacterial phage associated lysing enzymes for the prophylactic and therapeutic treatment of colonization and infections caused by streptococcus pneumoniae
US20030021811A1 (en) * 2000-03-16 2003-01-30 Weiser Jeffrey N. Modulating production of pneumococcal capsular polysaccharide
US20050249706A1 (en) * 1999-10-04 2005-11-10 Vion Pharmaceuticals, Inc. Compositions and methods for tumor-targeted delivery of effector molecules
US20060229244A1 (en) * 2004-09-27 2006-10-12 Robert Dorit Engineered bacteriocins and bacteriocin combinations and methods for treating bacterial based infections
US20070135339A1 (en) * 2005-12-08 2007-06-14 Stern Norman J Bacteriocin inducer peptides

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6207411B1 (en) * 1995-04-12 2001-03-27 Teagasc Bacteriocins
US20020136712A1 (en) * 1997-10-31 2002-09-26 Fischetti Vincent Bacterial phage associated lysing enzymes for the prophylactic and therapeutic treatment of colonization and infections caused by streptococcus pneumoniae
US6448224B1 (en) * 1998-05-06 2002-09-10 St. Jude Children's Research Hospital Antibiotics and methods of using the same
US20050249706A1 (en) * 1999-10-04 2005-11-10 Vion Pharmaceuticals, Inc. Compositions and methods for tumor-targeted delivery of effector molecules
US20030021811A1 (en) * 2000-03-16 2003-01-30 Weiser Jeffrey N. Modulating production of pneumococcal capsular polysaccharide
US20060229244A1 (en) * 2004-09-27 2006-10-12 Robert Dorit Engineered bacteriocins and bacteriocin combinations and methods for treating bacterial based infections
US20070135339A1 (en) * 2005-12-08 2007-06-14 Stern Norman J Bacteriocin inducer peptides

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DAWID ET AL: 'The blp Bacteriocins of Streptococcus pneumoniae Mediate Intraspecies Competition both In Vitro and in Vivo' INFECTION AND IMMUNITY vol. 75, January 2007, pages 443 - 451 *
DE SAIZIEU ET AL: 'Microarray-Based Identification of a Novel Streptococcus pneumoniae Regulon Controlled by an Autoinduced Peptide' JOUMAL OF BACTERIOLOGY vol. 182, September 2000, pages 4696 - 4703 *
KNUTSEN ET AL: 'Two Separate Quorum-Sensing Systems Upregulate Transcription of the Same ABC Transporter in Streptococcus pneumoniae' JOUMAL OF BACTERIOLOGY vol. 186, May 2004, pages 3078 - 3085 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3439693A4 (en) * 2016-04-05 2020-05-27 The Research Foundation For The State University Of New York University at Buffalo Novel pneumococcal vaccine formulations
US11103568B2 (en) 2016-04-05 2021-08-31 The Research Foundation For The State University Of New York Pneumococcal vaccine formulations
CN111601894A (en) * 2018-01-16 2020-08-28 花王株式会社 Method for producing protease of M23A family
WO2021201699A1 (en) * 2020-04-03 2021-10-07 Blis Technologies Limited Antiviral treatment comprising blis containing probiotic products

Also Published As

Publication number Publication date
WO2009059054A3 (en) 2009-09-17

Similar Documents

Publication Publication Date Title
Ricca et al. Characterization of bofA, a gene involved in intercompartmental regulation of pro-sigma K processing during sporulation in Bacillus subtilis
Heaton et al. Mobilization of vancomycin resistance by transposon-mediated fusion of a VanA plasmid with an Enterococcus faecium sex pheromone-response plasmid
Boland et al. Status of YopM and YopN in the Yersinia Yop virulon: YopM of Y. enterocolitica is internalized inside the cytosol of PU5–1.8 macrophages by the YopB, D, N delivery apparatus.
Hossain et al. An extracelluar protease, SepM, generates functional competence-stimulating peptide in Streptococcus mutans UA159
Jiang et al. Identification of six type III effector genes with the PIP box in Xanthomonas campestris pv. campestris and five of them contribute individually to full pathogenicity
Majchrzykiewicz et al. Generic and specific adaptive responses of Streptococcus pneumoniae to challenge with three distinct antimicrobial peptides, bacitracin, LL-37, and nisin
Chimalapati et al. Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo
AU2013327059B2 (en) Immunomodulatory minicells and methods of use
Hossain et al. Motility is an important determinant for pathogenesis of Erwinia carotovora subsp. carotovora
Ramakrishnan et al. Multiple structural proteins are required for both transcriptional activation and negative autoregulation of Caulobacter crescentus flagellar genes
Dennis et al. Identification and characterization of the tolQRA genes of Pseudomonas aeruginosa
Jiang et al. The type III secretion effector XopXccN of Xanthomonas campestris pv. campestris is required for full virulence
US20140286904A1 (en) Anitmicrobial peptide produced by intestinal lactobacillus salivarius
Dawid et al. Bacteriocin activity of Streptococcus pneumoniae is controlled by the serine protease HtrA via posttranscriptional regulation
US20200323926A1 (en) Bacteria for Targeting Tumors and Treating Cancer
PT1867714E (en) 5-fluorouracil-resistant bacteria and method for production thereof
Fang et al. Transcriptional regulation of virulence factors Hla and phenol-soluble modulins α by AraC-type regulator Rbf in Staphylococcus aureus
Zu et al. ComDE two-component signal transduction systems in oral Streptococci: Structure and function
WO2009059054A2 (en) Bacteria strains an d bacteriocin produced therefrom
US20210205431A1 (en) Compositions comprising recombinant probiotic bacteria and methods of use thereof
US6699970B2 (en) Mutacin I biosynthesis genes and proteins
Gagnon et al. Cloning, sequencing and expression in Escherichia coli of the ptsI gene encoding enzyme I of the phosphoenolpyruvate: sugar phosphotransferase transport system from Streptococcus salivarius
US6852835B2 (en) DNA molecules and polypeptides of Pseudomonas syringae Hrp pathogenicity island and their uses
ES2278737T3 (en) MUTANTS OF LACTOBACILUS CASEI WITH DEFECTIVE REGULATION OF CARBON CATABOLISM.
Burucoa et al. Nucleotide sequence and characterization of peb4A encoding an antigenic protein in Campylobacter jejuni

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08846169

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08846169

Country of ref document: EP

Kind code of ref document: A2