AU2005313797A2 - Signal peptides, nucleic acid molecules and methods of treatment - Google Patents

Signal peptides, nucleic acid molecules and methods of treatment Download PDF

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AU2005313797A2
AU2005313797A2 AU2005313797A AU2005313797A AU2005313797A2 AU 2005313797 A2 AU2005313797 A2 AU 2005313797A2 AU 2005313797 A AU2005313797 A AU 2005313797A AU 2005313797 A AU2005313797 A AU 2005313797A AU 2005313797 A2 AU2005313797 A2 AU 2005313797A2
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streptococcus
peptide
csp
mutans
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Dennis G. Cvitkovitch
Yi-Chen Cathy Huang
Celine Levesque
Srinivasa Madhyastha
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Kane Biotech Inc
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04Antibacterial agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

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Description

WO 2006/060903 PCT/CA2005/001845 1 SIGNAL PEPTIDES, NUCLEIC ACID MOLECULES AND METHODS OF TREATMENT BACKGROUND OF INVENTION Field of the Invention [0001] The invention generally relates to compounds and methods that inhibit or disrupt microbial biofilms involved in infections in man and animals and in biofouling of surfaces susceptible to microbial accumulation.
Description of the Related Art [0002] Bacteria often attach and accumulate on surfaces, enabling them to resist removal and killing by mechanical and chemical means. This can result in persistent and chronic infections and fouling of devices that are in contact with liquids containing the colonizing bacteria. Bacteria respond to signals resulting from the proximity, density, and identity of microbial neighbors. Through the process of quorum sensing bacteria can indirectly determine population density by sensing concentration of a secreted signal molecule (Bassler, 2002). The ability of bacteria to communicate with one another by QS and behave collectively as a group confers significant advantages, including more efficient proliferation, better access to resources and niches, and a stronger defense against competitors (Jefferson, 2004).
Many QS systems having various effects on bacterial cell physiology have been studied. Examples include biofilm differentiation in Pseudomonas aeruginosa (Davies et al., 1998), swarming motility in Serratia liquefaciens (Eberl et al., 1999), competence development in Streptococcus pneumoniae (Lee and Morrison, 1999) and Streptococcus mutans (Li et al., 2001), and induction of virulence factors in Staphylococcus aureus (Ji et al., 1995).
[0003] Controlling bacterial biofilms is desirable for almost every human enterprise in which solid surfaces are introduced into non-sterile aqueous environments. U.S. Patent No. 6,024,958 describes peptides that attempt to control biofilm formation by preventing bacterial adherence to teeth. In addition to occurrence in dental caries, medical examples of biofilm growth include cases WO 2006/060903 PCT/CA2005/001845 2 involving indwelling medical devices, joint implants, prostatitis, endocarditis, and respiratory infections. In fact, the Centers for Disease Control and Prevention (CDC; Atlanta, GA) estimate that 65% of human bacterial infections involve biofilms. Nonmedical examples of biofilm colonization are water and beverage lines, cooling towers, radiators, aquaculture contamination, submerged pumps and impellers, hulls of commercial, fishing and military vessels and literally every situation where biofouling occurs. The potential benefits of basic research focused at biofilm physiology and genetics with the ultimate goal of controlling surface-mediated microbial growth are limitless.
[0004] Interest in the study of biofilm-grown cells has increased partly because biofilm growth provides a microenvironment for cells to exist in a physical and physiological state that can increase their resistance to antimicrobial compounds and mechanical forces (reviewed in Costerton and Lewandowski, Adv Dent Res, 11:192-195). Growth in biofilms can also facilitate the transfer of genetic information between different species (Christensen et al. Appl Environ Microbiol, 64:2247-2255).
Recent evidence suggests that biofilm-grown cells may display a dramatically different phenotype when compared with their siblings grown in liquid culture. In some, this altered physiological state has been shown to result from gene activation initiated by contact with surfaces (Finlay and Falkow. Microbiol Molec Rev, 61:136- 169) or from signal molecules produced by the bacteria allowing them to sense the cell density (quorum sensing) (Davies et al. Appl Environ Microbiol, 61:860-867).
Biofilms may also act as 'genotypic reservoirs', allowing persistence, transfer and selection of genetic elements conferring resistance to antimicrobial compounds.
[0005] Caries and periodontal diseases are two of the most common chronic infectious diseases affecting mankind, and are always associated with dental plaque formed as a biofilm on tooth surfaces. Thus the prevention of dental caries and periodontal diseases is targeted at the control of dental plaque.
[0006] Dental plaque is produced by sequential attachment of a variety of bacteria, which is dependent on both species involved and the surface composition (Kawashima et al., Oral. Microbiol. Immunol. 18: 220-225, 2003). Oral streptococci and Actinomyces spp. are the first to appear on the surface of the teeth. Streptococci account for approximately 20% of the salivary bacteria, which include Streptococcus WO 2006/060903 PCT/CA2005/001845 3 spp. such as Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis and Streptococcus mitis. Two "mutans group" streptococci, S. mutans and S. sobrinus, are directly involved in the initiation of dental caries, (Devulapalle et al., Carbohydr. Res.339:029-1034, 2004). As S.
mutans has evolved to depend on a biofilm lifestyle for survival and persistence in the oral cavity combined with its role as an opportunistic pathogen, it has become the best-studied example of a biofilm-forming, disease-causing Streptococcus (Burne, J. Dent. Res. 77:445-452, 1998).
[0007] Many Streptococci use quorum-sensing systems to regulate several physiological processes, including the ability to incorporate foreign DNA, tolerate acid, form biofilm, and become virulent. These quorum-sensing systems are primarily made of a small competence-stimulating peptide (CSP) that is detected by neighbouring cells via a histidine kinase/response regulator pair. It has been demonstrated that analogues of quorum-sensing peptides (analogues of CSP) can competitively inhibit biofilm formation in S. mutans (Cvitkovitch, et al., US Patent Application No. 20020081302, 2002).
[0008] None of the known types of S. mutans antibiotics has satisfactorily controlled caries. There is a need to identify new ways to control S. mutans induced caries.
SUMMARY OF THE INVENTION [0009] In accordance with certain embodiments of the present invention a compound is provided that competitively inhibits binding of CSP [SEQ ID NO:30] to S. mutans histidine kinase [SEQ ID NO:4]. In certain embodiments the compound is a peptide or an antibody. In some embodiments the compound is a derivative of [SEQ ID NO:2], a fragment of [SEQ ID NO:2] or a derivative of a fragment of [SEQ ID NO:2].
[0010] In accordance with certain embodiments of the present invention methods of making an above-described compound are provided. In still other embodiments of the invention methods are provided in which an above-described WO 2006/060903 PCT/CA2005/001845 4 compound is used for inhibiting the growth of S. mutans, for inhibiting dental caries, or for improving dental health.
[0011] In an aspect of the invention, provided is an isolated polypeptide comprising a fragment ofSEQ ID NO:2 or SEQ ID NO:30 and capable of inhibiting S. mutans genetic competence.
[0012] In an aspect of the invention, provided is an isolated polypeptide that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:30, and capable of inhibiting S.
mutans genetic competence.
[0013] In an embodiment of the invention, 1-5 amino acids have been removed from the COOH terminal of SEQ ID NO:2 or SEQ ID [0014] In an aspect of the invention, provided is an isolated polypeptide comprising a fragment of SEQ ID NO:2 or SEQ ID NO:30 and capable of inhibiting S. mutans biofilm formation.
[0015] In an aspect of the invention, provided is an isolated polypeptide that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:30 and capable of inhibiting S.
mutans biofilm formation.
[0016] In an embodiment of the invention, 1-5 amino acids of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:30 have been modified to include up to 1 amino acid substitution per each 10 amino acids.
[0017] In an aspect of the invention, provided is a composition for reducing S.
mutans biofilm formation comprising a peptide derivative of CSP.
[0018] In an aspect of the invention, provided is a composition for reducing S.
mutans transformation efficiency comprising a peptide derivative of CSP.
[0019] In an embodiment of the invention, the peptide analog is a peptide having an amino acid sequence selected from a group consisting of: SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 42 ,SEQ ID No: 44 and SEQ ID No: 47.
[0020] In an embodiment of the invention, the peptide analog is a peptide having amino acid sequnece SEQ ID No:44.
[0021] In an aspect of the invention, provided is a cell genetically engineered to produce the any one of the compositions of the invention set out above.
WO 2006/060903 PCT/CA2005/001845 [0022] In an embodiment of the invention, the cell is bacterial.
[0023] In an embodiment of the invention, the cell is S. mutans.
[0024] In an aspect of the invention, provided is use of the cell according to the invention to produce any one of the compositions of the invention set out above.
[0025] In an aspect of the invention, provided is a composition comprising the cell according to the invention and a pharmaceutically acceptable adjuvant.
[0026] In an aspect of the invention, provided is a method of inhibiting the growth ofS. mutans comprising administering a composition according to any one of claims 9 to 12 or 17.
[0027] In an aspect of the invention, provided is a method of inhibiting dental caries comprising administering any one of the compositions of the invention set out above.
[0028] In an aspect of the invention, provided is a method of improving dental health comprising administering any one of the compositions of the invention set out above.
[0029] In an aspect of the invention, provided is an isolated polypeptide having the amino acid sequence selected from a group consiting of: SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0030] In an aspect of the invention, provided is a peptide analog of S.mutans competence stimulating peptide (CSP) which inhibits biofilm formation.
[0031] In an aspect of the invention, provided is a composition for inhibiting biofilm formation comprising a polypeptide having an amino acid sequence selected from a group consisting of: SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47 and an orally acceptable excipient.
[0032] In an aspect of the invention, provided is a pharmaceutical composition comprising at least one CSP inhibitor and a pharmaceutically acceptable carrier.
[0033] In an aspect of the invention, provided is a method of treating or preventing a bacterial infection caused by a biofilm forming bacterium comprising WO 2006/060903 PCT/CA2005/001845 6 administering a therapeutically effective amount of a pharmaceutical composition according to the invention.
[0034] In an aspect of the invention, provided is a method of preventing dental plaque formation comprising administering a therapeutically effective amount of a pharmaceutical composition according to the invention.
[0035] In an aspect of the invention, provided is a method of treating or preventing a condition caused by a dental plaque associated bacterium comprising administrering a therapeutically effective amount of a pharmaceutical composition according to the invention.
[0036] In an aspect of the invention, provided is a use of a CSP inhibitor for the preparation of a medicament for treatment and prevention of an infection caused by a biofilm forming bacterium.
[0037] In an embodiment of the invention, the CSP inhibitor is is a peptide analog ofS.mutans competence stimulating peptide (CSP) which inhibits biofilm formation.
[0038] In another embodiment of the invention, the CSP inhibtior is a polypeptide having the amino acid sequence selected from a group consisting of: SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0039] In an embodiment of the invention, the CSP inhibitor is a polypeptide having the amino acid sequence of SEQ ID No: 44.
[0040] In an embodiment of the invention, the CSP inhibitor is an antibody specific for CSP or a fragment thereof.
[0041] In an embodiment of the invention, the CSP inhibitor is an antisense oligonucleotide which inhibits CSP expression or transcription.
[0042] In an embodiment of the invention, the CSP inhibitor is an antisense oligonucleotide which inhibits CSP peptide export.
[0043] In an aspect of the present invention, provided is a composition for inhibiting biofilm formation in Streptococcus spp, said composition comprising at WO 2006/060903 PCT/CA2005/001845 7 least one peptide having an amino acid sequence selected from a group consisting of: No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0044] In an embodiment of the invention, the composition further comprises one or more ingredients selected from a group consisting of: a surfactant, an antiseptic, and an antibiotic.
[0045] In an embodiment of the invention, the composition comprises between 1 tg/ml and 100 itg/ml of the peptide.
[0046] In an aspect of the present invention, provided is a use of at least one peptide having an amino acid sequence selected from a group consisting of: No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47., for the preparation of a medicament for inhibition of biofilm formation in Streptococcus spp.
[0047] In an aspect of the present invention, provided is a use of at least one peptide having an amino acid sequence selected from a group consisting of: No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47, for prevention or inhibition of biofilm formation in Streptococcus spp.
[0048] In an aspect of the present invention, provided is a use of at least one peptide having an amino acid sequence selected from a group consisting of: No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47, for treatment or prevention of a condition caused by dental plaque associated Streptococcus spp.
[0049] In an aspect of the present invention, provided is a method of treating or preventing a Streptococcus spp infection in a patient in need thereof, comprising administering a therapeutically effective amount of at least one peptide having an amino acid sequence selected from a group consisting of: No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0050] In an aspect of the present invention, provided is a method of inhibiting or preventing biofilm formation in Streptococcus spp in a patient in need thereof, comprising administering a therapeutically effective amount of at least one WO 2006/060903 PCT/CA2005/001845 8 peptide having an amino acid sequence selected from a group consisting of: No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0051] In an aspect of the present invention, provided is a method of treating or preventing a condition caused by dental plaque associated Streptococcus spp in a patient in need thereof comprising administering a therapeutically effective amount of at least one peptide having an amino acid sequence selected from a group consisting of: No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0052] In an embodiment of the invention, the condition caused by dental plaque associated Streptococcus spp is selected from a group consisting of: dental caries, gingivitis, and endocarditis.
[0053] In an embodiment of the invention, the peptide has the amino acid sequence of SEQ ID NO. 44.
[0054] In an embodiment of the invention, the Streptococcus spp is selected from a group consisting of: Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, Streptococcus cristatus, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae.
[0055] In an embodiment of the invention, the Streptococcus spp is Streptococcus pneumoniae.
[0056] These and other embodiments, features and advantages of the invention will be apparent with reference to the following description and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS [0057] Figure 1 shows the schematic layout of the arrangement of the genetic locus encoding the signal peptide precursor (ComC) [SEQ ID NO:1], the histidine kinase (ComD) [SEQ ID NO:3] and the response regulator (ComE) [SEQ ID This arrangement is different from other loci in related streptococci since the comC gene [SEQ ID NO:1] is transcribed from its own unique promoter, unlike the genes WO 2006/060903 PCT/CA2005/001845 9 thus far described in other streptococci that are arranged in an operon-like cluster with the comC/DE genes being transcribed from a single promoterand the comC gene [SEQ ID NO:1] is separated by 148 nucleotides from the comD gene [SEQ ID NO:3].
[0058] Figure 2 shows the nucleic acid molecules SEQ ID Nos. 1, 3, 5, and 29. Figure 2A. S. mutans comC gene [SEQ ID NO:1]. Encodes a precursor to a signal peptide [SEQ ID NO:2]. Figure 2B. S. mutans CSP encoding sequence [SEQ ID NO:29]. Encodes a Competence Stimulating Peptide [SEQ ID NO:30]. Figure 2C. S. mutans comD gene [SEQ ID NO:3]. Figure 2D. S. mutans comE gene [SEQ ID NO:5]. Encodes a response regulator that activates transcription of a number of genes [SEQ ID NO:6].
[0059] Figure 3. Sequence of the deduced amino acid sequence of the signal peptide [SEQ ID NO:2], histidine kinase [SEQ ID NO:4], and response regulator [SEQ ID NO:6]. Figure 3A. S. mutans ComC protein (CSP Precursor) [SEQ ID NO:2]. Figure 3B. S. mutans ComD protein (Histidine Kinase) [SEQ ID NO:4].
Figure 3C. S. mutans ComE protein (Response Regulator) [SEQ ID NO:6].
[0060] Figure 4. The deduced amino acid sequence of the signal peptide precursor in various strains and its predicted cleavage site. The original peptide is expressed as a 46 amino acid peptide that is cleaved after the glycine-glycine residues to generate an active signal peptidel.
[0061] Figure 5 shows the synthetic signal peptide [SEQ ID NO:14] that is effective at inducing competence, biofilm formation and acid tolerance in Streptococcus mutans.
[0062] Figure 6 shows the natural activity of the signal/receptor system functioning in vitro in model biofilms as determined by the ability of various strains of S. mutans to accept donor plasmid DNA conferring erythromycin resistance.
[0063] Figure 7 is a table illustrating the effect of synthetic peptide on genetic competence in S. mutans cells. Induction of genetic transformation in Streptococcus mutans by synthetic competence stimulating peptide (SCSP).
WO 2006/060903 PCT/CA2005/001845 [0064] Figure 8 is a list of the primers used to amplify the genes or internal regions of the target genes by polymerase chain reaction (PCR) for subsequent sequencing or inactivation.
[0065] Figure 9 shows the ComCDE local region [SEQ ID NO:21]. The ComC (first highlighted region; nucleotide 101 to 241), ComD (second highlighted region; nucleotides 383 to 1708) and ComE (third highlighted region; nucleotides 1705 to 2457) proteins are highlighted.
[0066] Figure 10 shows The comX DNA sequence [SEQ ID NO:22], protein sequence [SEQ ID NO:23], and the comX gene local region [SEQ ID NO:24] with 100bp included both upstream and downstream (promoter is upstream). Figure S. mutans comX gene [SEQ ID NO:22]. Figure 10B. S. mutans ComX protein [SEQ ID NO:23]. Figure 10C. S. mutans comX gene local region [SEQ ID NO:24].
[0067] Figure 11 shows the comA and comB nucleotide [SEQ ID NO:25] and [SEQ ID NO:27] and amino acid sequences [SEQ ID NO:26] and [SEQ ID NO:28].
ComA and ComB are the components of the CSP exporter. Figure 11A. S. mutans comA gene [SEQ ID NO:25]. Figure 11B. S. mutans ComA protein [SEQ ID NO:26]. Figure 11C. S. mutans comb gene [SEQ ID NO:27] Figure 11D. S. mutans ComB protein [SEQ ID NO:28].
[0068] Figure 12 illustrates the effect of synthetic peptide on acid resistance tolerance in S. mutans comC deficient cells. Addition of synthetic signal peptide (CSP) [SEQ ID NO:14] into the culture of the comC mutant restored the ability of the mutant to survive a low pH challenge when compared to the parent strain NG8.
[0069] Figure 13 is a schematic representation of quorum sensing circuit in S.
mutans.
[0070] Figure 14 shows the effect of different concentrations of H1 on genetic transformation of S. mutans wild-type UA159. Results are expressed as the mean SE of three independent experiments.
[0071] Figure 15 shows the effect of different concentrations of H1 on genetic transformation of S. mutans cormD null mutant.
WO 2006/060903 PCT/CA2005/001845 11 [0072] Figure 16 shows the effect of different concentrations (gg/ml) of CSP and H1 on cell growth of S. mutans wild-type UA159 in THYE at pH 5.5. Means
OD
6 0 o values SE. Results represent the average of three independent experiments.
[0073] Figure 17 shows the effect of different concentrations (pg/ml) of CSP and H1 on cell growth of S. mutans wild-type UA159 in THYE at pH 7.5. Means
OD
60 0 values SE. Results represent the average of three independent experiments.
[0074] Figure 18 shows the effects of synthetic CSP analogues (B2, B3, C2, E2, Fl and H1 peptides) on Streptococcus mutans biofilm formation.
[0075] Figure 19 shows the effect of E2 peptide on Streptococcus sobrinus biofilm formation.
[0076] Figure 20 shows the effect of E2 peptide on Streptococcus oralis biofilm formation.
[0077] Figure 21 shows the effect of E2 peptide on Streptococcus sanguis biofilm formation.
[0078] Figure 22 shows the effect of E2 peptide on Streptococcus mitis biofilm formation.
[0079] Figure 23 shows the effect of E2 peptide on Streptococcus gordonii biofilm formation.
[0080] Figure 24 shows the effect of E2 peptide on Streptococcus pneumoniae biofilm formation DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0081] In some Gram-positive bacteria (including Streptococcus mutans), when a specific histidine kinase receptor located in the cell membrane is disrupted, the cells become ineffective at developing a biofilm. The cells growing in this biofilm environment use a small peptide signal molecule to activate the receptor in surrounding cells, thereby communicating the message to form a biofilm. This same signal peptide and histidine kinase are also involved in the induction of genetic competence, the cell's ability to take up and incorporate DNA from its extracellular WO 2006/060903 PCT/CA2005/001845 12 environment, as well as that of acid tolerance, the cell's ability to survive pH levels as low as pH 3.0. A mechanism that blocks the signal molecule from activating the histidine kinase receptor molecule provides a novel method for controlling microbial biofilms, either alone or in combination with chemical or physical means.
[0082] We have identified a genetic locus in S. mutans consisting of three genes that encode: 1) a peptide precursor [SEQ ID NO:2] that is processed during export into a secreted 21-amino acid peptide (CSP) [SEQ ID NO:30]; 2) a histidine kinase [SEQ ID NO:4] that acts as a cell surface receptor activated by the peptide; 3) a response regulator [SEQ ID NO:6] that activates a number of other genes involved in genetic competence, biofilm formation, and acid tolerance of S. mutans. These properties have been attributed to the bacterium's ability to cause dental caries.
Inactivation of any of these three genes or impairment of interaction or activity of any of their encoded proteins will disrupt the bacterium's ability to take up foreign DNA, form biofilms, and tolerate acidic pH.
[0083] Streptococcus mutans is a resident of the biofilm environment of dental plaque, a matrix of bacteria and extracellular material that adheres to the tooth surface. Under appropriate environmental conditions populations of S. mutans and the pH of the surrounding plaque will drop. S. mutans, being among the most acid tolerant organisms residing in dental plaque, will increase it numbers in this acidic environment and eventually become a dominant member of the plaque community.
This situation eventually leads to dissolution of the tooth enamel, resulting in the development of dental caries. We control the accumulation and acid tolerance of this bacterium to make it less able to cause caries. We accomplish this by using inhibitors of an extracellular signal peptide that promotes the expression of genes involved in S.
mutans biofilm formation and acid tolerance. Compounds are disclosed that inhibit the action of the peptide. These inhibitors can include peptides, antibodies, or other agents that specifically inhibit the activation of the histidine kinase and the family of genes activated as a result of the histidine kinase activation by the signal molecule.
Inhibitors include: modified structures of the mature wild type CSP peptide where amino acids are removed from the N- and/or COOH terminal of the peptide and/or substitutions of internal amino acid residues. We delete, one, two to 5, 6 to 10 and to 15 amino acids from the mature wild type CSP peptide (for example at either WO 2006/060903 PCT/CA2005/001845 13 terminal) and measure competitive inhibition of signal peptide binding to histidine kinase 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids are deleted and inhibition measured). Inhibitors also include antibodies raised against the 21-amino acid CSP [SEQ ID NO:30] alone or coupled to a larger molecule to increase immunogenicity.
We also test inhibitors described in (Barrett et al. Proc. Natl. Acad. Sci USA 95:5317- 5322) and measure competitive inhibition of signal peptide binding to histidine kinase.
[0084] In addition to identifying the genes encoding this signaling/sensing system, we have identified and chemically synthesized a 21-amino acid peptide [SEQ ID NO:14] that promotes biofilm formation and acid tolerance of S. mutans. A survey of the literature and genome databases reveals that genes similar to this signalreceptor system are present in most Gram-positive bacteria, and therefore an inhibitor or family of related inhibitors may be effective at inhibiting biofilm formation among a large group of bacteria.
[0085] Treatment or prevention of dental caries comprises addition of compounds that inhibit the stimulatory action of the 21-amino acid peptide [SEQ.ID on biofilm formation and acid tolerance of S. mutans. This is accomplished by delivery of these compounds to the biofilm and/or to incorporate these inhibitors into materials to control growth on surfaces. This includes delivery by topical application, alone or in combination with other compounds including toothpaste, mouthwash, food or food additives.
[0086] Streptococcus mutans is also implicated in causing infective endocarditis. Inhibitors of biofilm formation, and hence aggregation are useful in the treatment of these bacterial infections as well.
[0087] We have also identified compounds which inhibit biofilm formation for other dental plaque baterial species such as Actinomyces spp. and other types of streptococci such as Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis and Streptococcus mitis. Streptococci account for approximately 20% of the salivary bacteria. The compounds comprise the modified structures of the S. mutans mature wild type CSP peptide having amino acids removed WO 2006/060903 PCT/CA2005/001845 14 from the N- and/or COOH terminal of the peptide and/or substitutions of internal amino acid residues.
Identification and Characterization of Competence Stimulating Peptide (CSP), Histidine Kinase (HK) and Response Regulator (RR) Competence Stimulating Peptide [0088] An isolated CSP from S. mutans is provided in accordance with certain embodiments of the present invention. Also provided in accordance with certain embodiments of the present invention is a recombinant isolated CSP [SEQ ID NO:2] peptide produced by a cell including a nucleic acid molecule encoding CSP [SEQ ID NO:1] operably linked to a promoter. Further provided in accordance with certain embodiments of the present invention is an isolated nucleic acid molecule encoding a CSP [SEQ ID NO:1]. The peptide we work with is preferably chemically synthesized [SEQ ID NO:14].
[0089] CSP-encoding nucleic acid molecules [SEQ ID NO:1] and molecules having sequence identity or which hybridize to the CSP-encoding sequence and which encode a peptide having CSP activity (preferred percentages for sequence identity are described below) as well as vectors including these molecules are provided in accordance with various embodiments of the present invention. In certain embodiments of the invention CSP [SEQ ID NO:2] or peptides having sequence identity (preferred percentages described below) or which have CSP activity are provided. The nucleic acid molecules and peptides disclosed herein may be from S.
mutans and they may be isolated from a native source, synthetic or recombinant. CSP [SEQ ID NO:2] or peptides having sequence identity, which have CSP activity, as prepared by the processes described in this application, are also provided in accordance with the present invention.
Histidine Kinase [0090] In accordance with certain embodiments of the present invention, an isolated HK [SEQ ID NO:4] from S. mutans is disclosed. Also disclosed is a recombinant isolated HK polypeptide produced by a cell including a nucleic acid molecule encoding HK [SEQ ID NO:3] operably linked to a promoter. In another WO 2006/060903 PCT/CA2005/001845 embodiment of the invention an isolated nucleic acid molecule encoding a HK polypeptide [SEQ ID NO:4] is disclosed.
[0091] HK-encoding nucleic acid molecules and molecules having sequence identity or which hybridize to the HK-encoding sequence [SEQ ID NO:3] and which encode a protein having HK activity (preferred percentages for sequence identity are described below) as well as vectors including these molecules are disclosed as part of the present invention. In accordance with some embodiments of the present invention, HK [SEQ ID NO:4] or polypeptides having sequence identity (preferred percentages described below) or which have HK activity are disclosed. The nucleic acid molecules and polypeptides disclosed herein may be from S. mutans and they may be isolated from a native source, synthetic or recombinant. Also provided according to certain embodiments of the present invention is HK [SEQ ID NO:4] or polypeptides having sequence identity, which have HK activity, as prepared by the processes described in this application.
Response Regulator [0092] In accordance with certain embodiments of the present invention an isolated RR [SEQ ID NO:6] from S. mutans is disclosed. A recombinant isolated RR [SEQ ID NO:6] polypeptide produced by a cell including a nucleic acid molecule encoding RR [SEQ ID NO:5] operably linked to a promoter is provided according to certain other embodiments of the present invention. Still other embodiments of the invention include an isolated nucleic acid molecule encoding a RR polypeptide.
[0093] Certain embodiments of the invention include RR-encoding nucleic acid molecules and molecules having sequence identity or which hybridize to the RRencoding sequence [SEQ ID NO:5] and which encode a polypeptide having RR activity (preferred percentages for sequence identity are described below) as well as vectors including these molecules. Some embodiments of the invention also include RR [SEQ ID NO:6] or polypeptides having sequence identity (preferred percentages described below) or which have RR activity. The nucleic acid molecules and polypeptides of the invention may be from S. mutans and they may be isolated from a native source, synthetic or recombinant. Certain embodiments of the invention WO 2006/060903 PCT/CA2005/001845 16 include RR [SE ID NO:6] or polypeptides having sequence identity, which have RR activity, as prepared by the processes described in this application.
[0094] The comA and comB nucleotide [SEQ ID NO:25 and SEQ ID NO:27] and amino acid sequences [SEQ ID NO:26 and SEQ ID NO:28] are also aspects of certain embodiments of the invention. ComA and ComB are components of the CSP exporter. The discussion of variants, sequence identity etc. for CSP, HK, RR applies to both the full sequences shown in the figures as well as bracketed portions of sequences (coding regions). The peptides and polypeptides may be natural, recombinantly produced or synthetic.
Functionally equivalent nucleic acid molecules [0095] Certain embodiments of the invention include nucleic acid molecules that are functional equivalents of all or part of the CSP sequence in [SEQ ID NO:1].
(A nucleic acid molecule may also be referred to as a DNA sequence or nucleotide sequence in this application. All these terms have the same meaning as nucleic acid molecule). Functionally equivalent nucleic acid molecules are DNA and RNA (such as genomic DNA, complementary DNA, synthetic DNA, and messenger RNA molecules) that encode peptides having the same or similar CSP activity as the CSP peptide shown in [SEQ ID NO:2]. Functionally equivalent nucleic acid molecules can encode peptides that contain a region having sequence identity to a region of a CSP peptide [SEQ ID NO:2] or more preferably to the entire CSP peptide. Identity is calculated according to methods known in the art. The ClustalW program (preferably using default parameters) [Thompson, JD et al., Nucleic Acid Res. 22:4673-4680.], described below, is most preferred. For example, if a nucleic acid molecule (called "Sequence has 90% identity to a portion of the nucleic acid molecule in [SEQ ID NO:1], then Sequence A will preferably be identical to the referenced portion of the nucleic acid molecule in [SEQ ID NO:1], except that Sequence A may include up to point mutations, such as substitutions with other nucleotides, per each 100 nucleotides of the referenced portion of the nucleic acid molecule in [SEQ ID NO: 1].
Mutations described in this application preferably do not disrupt the reading frame of the coding sequence. Nucleic acid molecules functionally equivalent to the CSP sequences can occur in a variety of forms as described below.
WO 2006/060903 PCT/CA2005/001845 17 [0096] Nucleic acid molecules may encode conservative amino acid changes in CSP peptide [SEQ ID NO:2]. Certain embodiments of the invention include functionally equivalent nucleic acid molecules that encode conservative amino acid changes within a CSP amino acid sequence and produce silent amino acid changes in
CSP.
[0097] Nucleic acid molecules may encode non-conservative amino acid substitutions, additions or deletions in CSP peptide. Some embodiments of the invention include functionally equivalent nucleic acid molecules that make nonconservative amino acid changes within the CSP amino acid sequence in [SEQ ID NO:2]. Functionally equivalent nucleic acid molecules include DNA and RNA that encode peptides, peptides and proteins having non-conservative amino acid substitutions (preferably substitution of a chemically similar amino acid), additions, or deletions but which also retain the same or similar CSP activity as the CSP peptide shown in [SEQ ID NO:2]. The DNA or RNA can encode fragments or variants of CSP. Fragments are useful as immunogens and in immunogenic compositions (U.S.
Patent No. 5,837,472). The CSP or CSP-like activity of such fragments and variants is identified by assays as described below. Fragments and variants of CSP encompassed by the present invention should preferably have at least about 80% or 95% sequence identity to the naturally occurring CSP nucleic acid molecule, or a region of the sequence, such as the coding sequence or one of the conserved domains of the nucleic acid molecule, without being identical to the sequence in [SEQ ID NO:1]. Sequence identity is preferably measured with the ClustalW program (preferably using default parameters) (Thompson, JD et al., Nucleic Acid Res. 22:4673-4680).
[0098] Nucleic acid molecules functionally equivalent to the CSP nucleic acid molecule in [SEQ ID NO:1] will be apparent from the following description. For example, the sequence shown in [SEQ ID NO:1] may have its length altered by natural or artificial mutations such as partial nucleotide insertion or deletion, so that when the entire length of the coding sequence within [SEQ ID NO:1], is taken as 100%, the functional equivalent nucleic acid molecule preferably has a length of about 60-120% thereof, more preferably about 80-110% thereof. Fragments may be less than WO 2006/060903 PCT/CA2005/001845 18 [0099] Nucleic acid molecules containing partial (usually 80% or less, preferably 60% or less, more preferably 40% or less of the entire length) natural or artificial mutations so that some codons in these sequences code for different amino acids, but wherein the resulting peptide retains the same or similar CSP activity as that of a naturally occurring CSP peptide [SEQ ID NO:2]. The mutated DNAs created in this manner should preferably encode a peptide having at least about preferably at least about 60%, at least about 80%, and more preferably at least about or 95% sequence identity to the amino acid sequence of the CSP peptide in [SEQ ID NO:2]. The ClustalW program preferably assesses sequence identity.
[00100] Since the genetic code is degenerate, the nucleic acid sequence in [SEQ ID NO:1] is not the only sequence which may code for a peptide having CSP activity. This invention includes nucleic acid molecules that have the same essential genetic information as the nucleic acid molecule described in [SEQ ID NO:1].
Nucleic acid molecules (including RNA) having one or more nucleic acid changes compared to the sequences described in this application and which result in production of a peptide shown in [SEQ ID NO:2] are within the scope of various embodiments of the invention.
[00101] Other functional equivalent forms of CSP-encoding nucleic acids can be isolated using conventional DNA-DNA or DNA-RNA hybridization techniques.
Thus, certain embodiments of the present invention also include nucleic acid molecules that hybridize to one or more of the sequences in [SEQ ID NO:1] or its complementary sequence, and that encode expression for peptides, peptides and proteins exhibiting the same or similar activity as that of the CSP peptide produced by the DNA in [SEQ ID NO:1] or its variants. Such nucleic acid molecules preferably hybridize to the sequence in [SEQ ID NO:1] under moderate to high stringency conditions (see Sambrook et al. Molecular Cloning: A Laboratory Manual, Most Recent Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, High stringency washes have low salt (preferably about 0.2% SSC), and low stringency washes have high salt (preferably about 2% SSC). A temperature of about 37 °C or about 42 °C is considered low stringency, and a temperature of about 50-65 °C is high stringency. Some embodiments of the invention also include a method of identifying nucleic acid molecules encoding a CSP activator peptide (preferably a WO 2006/060903 PCT/CA2005/001845 19 mammalian peptide), including contacting a sample containing nucleic acid molecules including all or part of [SEQ ID NO:1] (preferably at least about 15 or 20 nucleotides of [SEQ ID NO:1]) under moderate or high stringency hybridization conditions and identifying nucleic acid molecules which hybridize to the nucleic acid molecules including all or part of [SEQ ID Similar methods are described in U.S. Patent No. 5,851,788, which is incorporated by reference in its entirety.
[00102] Certain embodiments of the present invention also include methods of using all or part of the nucleic acid molecules which hybridize to all or part of [SEQ ID NO:1], for example as probes or in assays to identify antagonists or inhibitors of the peptides produced by the nucleic acid molecules (described below). Some embodiments of the present invention include methods of using nucleic acid molecules having sequence identity to the CSP nucleic acid molecule (as described below) in similar methods.
[00103] Certain embodiments of the invention also include a nucleic acid molecule detection kit including, preferably in a suitable container means or attached to a surface, a nucleic acid molecule as disclosed herein encoding CSP [SEQ ID NO:2] or a peptide having CSP activity and a detection reagent (such as a detectable label). Other variants of kits will be apparent from this description and teachings in patents such as U.S. Patent Nos. 5,837,472 and 5,801,233, which are incorporated by reference in their entirety.
[00104] A nucleic acid molecule described above is considered to have a function substantially equivalent to the CSP nucleic acid molecules [SEQ ID NO: 1] of the present invention if the peptide [SEQ ID NO:2] produced by the nucleic acid molecule has CSP activity. A peptide has CSP activity if it can stimulate genetic competence and acid tolerance in S. mutans. Activation of the HK [SEQ ID NO:4]/RR [SEQ ID NO:6] is shown where a peptide is capable of stimulating the uptake and incorporation of foreign DNA. We describe below how the activity of these peptide-mediated processes can be measured by determining the efficiency of plasmid uptake, which is a measure of genetic competence. Since the ability to transport and incorporate foreign DNA relies on activation of the HK [SEQ ID NO:4]/RR [SEQ ID NO:6] and subsequent genes activated by the signal cascade initiated by the signal peptide, measurement of the conferment of erythromycin WO 2006/060903 PCT/CA2005/001845 resistance by cells exposed to the peptide and plasmid DNA conferring erythromycin resistance indicates its level of function. Conversely if an inhibitor is capable of interfering with the action of the peptide the competence assay will indicate this by a corresponding decrease in the number of cells that acquire erythromycin resistance as described in the assays below (assays of genetic competence and assay of transformation of biofilms). Activation of the HK [SEQ ID NO:4]/RR [SEQ ID NO:6] is also shown where a peptide is capable of stimulating an acid tolerance response. We describe below how the activity of these peptide-mediated processes can be measured by determining the survival rate of cells in acidic pH conditions.
Since the ability to survive exposure to acidic pH depends on the activation of the HK/RR and subsequent genes activated by the signal peptide, measurement of the survival of S. mutans in low pH conditions indicates the level of function of the signal peptide. Conversely, if an inhibitor is capable of interfering with the signal peptide sensing system the assay for acid adaptation will indicate this by a corresponding decrease in the survival rate of cells grown in acidic pH conditions as described in the assay below (assay of acid adaptation).
Production of CSP in eukaryotic and prokaryotic cells [00105] The nucleic acid molecules disclosed herein may be obtained from a cDNA library. The nucleotide molecules can also be obtained from other sources known in the art such as expressed sequence tag analysis or in vitro synthesis. The DNA described in this application (including variants that are functional equivalents) can be introduced into and expressed in a variety of eukaryotic and prokaryotic host cells. A recombinant nucleic acid molecule for the CSP contains suitable operatively linked transcriptional or translational regulatory elements. Suitable regulatory elements are derived from a variety of sources, and they may be readily selected by one with ordinary skill in the art (Sambrook, J, Fritsch, E.E. Maniatis, T. (Most Recent Edition). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. New York; Ausubel et al. (Most Recent Edition). Current Protocols in Molecular Biology, John Wiley Sons, Inc.). For example, if one were to upregulate the expression of the nucleic acid molecule, one could insert a sense sequence and the appropriate promoter into the vector. Promoters can be inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue- WO 2006/060903 PCT/CA2005/001845 21 specific. Transcription is enhanced with promoters known in the art for expression.
The CMV and SV40 promoters are commonly used to express desired peptide in cells. Other promoters known in the art may also be used (many suitable promoters and vectors are described in the applications and patents referenced in this application).
[00106] If one were to downregulate the expression of the nucleic acid molecule, one could insert the antisense sequence and the appropriate promoter into the vehicle. The nucleic acid molecule may be either isolated from a native source (in sense or antisense orientations), synthesized, or it may be a mutated native or synthetic sequence or a combination of these.
[00107] Examples of regulatory elements include a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the vector employed, other genetic elements, such as selectable markers, may be incorporated into the recombinant molecule. Other regulatory regions that may be used include an enhancer domain and a termination region. The regulatory elements may bacterial, fungal, viral or avian in origin. Likewise the regulatory elements may originate from animal, plant, yeast, insect or other sources, including synthetically produced elements and mutated elements.
[00108] In addition to using the expression vectors described above, the peptide may be expressed by inserting a recombinant nucleic acid molecule in a known expression system derived from bacteria, viruses, yeast, mammals, insects, fungi or birds. The recombinant molecule may be introduced into the cells by techniques such as Agrobacterium tumefaciens-mediated transformation, particle-bombarcdnentmediated transformation, direct uptake, microinjection, coprecipitation, transfection and electroporation depending on the cell type. Retroviral vectors, adenoviral vectors, Adeno Associated Virus (AAV) vectors, DNA virus vectors and liposomes may be used. Suitable constructs are inserted in an expression vector, which may also include markers for selection of transformed cells. The construct may be inserted at a site created by restriction enzymes.
WO 2006/060903 PCT/CA2005/001845 22 [00109] In one.embodiment of the invention, a cell is transfected with a nucleic acid molecule of the invention inserted in an expression vector to produce cells expressing a peptide encoded by the nucleic acid molecule.
[00110] Another embodiment of the invention relates to a method of transfecting a cell with a nucleic acid molecule disclosed herein, inserted in an expression vector to produce a cell expressing the CSP peptide [SEQ ID NO:2] or other peptide of the invention. In accordance with certain embodiments of the invention a method is provided for expressing the disclosed peptides in a cell. A preferred process would include culturing a cell including a recombinant DNA vector including a nucleic acid molecule encoding CSP [SEQ ID NO:1] (or another nucleic acid molecule of the invention) in a culture medium so that the peptide is expressed.
The process preferably further includes recovering the peptide from the cells or culture medium.
Probes [00111] Certain embodiments of the present invention include oligonucleotide probes made from the cloned CSP nucleic acid molecules described in this application or other nucleic acid molecules disclosed herein (see Materials and Methods section).
The probes may be 15 to 20 nucleotides in length. A preferred probe is at least nucleotides of CSP in [SEQ ID NO:1]. Certain embodiments of the invention also include at least 15 consecutive nucleotides of [SEQ ID NO: The probes are useful to identify nucleic acids encoding CSP peptides as well as peptides functionally equivalent to CSP. The oligonucleotide probes are capable of hybridizing to the sequence shown in [SEQ ID NO:1] under stringent hybridization conditions. A nucleic acid molecule encoding a peptide disclosed herein may be isolated from other organisms by screening a library under moderate to high stringency hybridization conditions with a labeled probe. The activity of the peptide encoded by the nucleic acid molecule is assessed by cloning and expression of the DNA. After the expression product is isolated, the peptide is assayed for CSP activity as described in this application.
[00112] Functionally equivalent CSP nucleic acid molecules from other cells, or equivalent CSP-encoding cDNAs or synthetic DNAs, can also be isolated by WO 2006/060903 PCT/CA2005/001845 23 amplification using Polymerase Chain Reaction (PCR) methods. Oligonucleotide primers, such as degenerate primers, based on [SEQ ID NO:1] can be prepared and used with PCR and reverse transcriptase S. Kawasaki (1990), In Innis et al., Eds., PCR Protocols, Academic Press, San Diego, Chapter 3, p. 21) to amplify functional equivalent DNAs from genomic or cDNA libraries of other organisms. The oligonucleotides can also be used as probes to screen cDNA libraries.
Functionally equivalent peptides, peptides and proteins [00113] The present invention includes not only the peptides encoded by the sequences disclosed herein, but also functionally equivalent peptides, peptides and proteins that exhibit the same or similar CSP peptide activity.
[00114] We designed and synthesized peptide analogs based on the native sequence of the S. mutans CSP and assayed their ability to interfere with competence development, acid tolerance response, and biofilm formation.
Peptide analogs were altered based on the amino acid sequence of S. mutans native CSP.
[00115] A panel of 17 peptide analogs with modification in length and hydrophobicity were designed and synthesized. The first set of peptide analogs were generated by deleting the 1 st 2 n d 3 rd 4 h or 5 th residues from the N- and C-termini of the mature S. mutans CSP sequence [SEQ ID NO:30]. The second set included peptide analogs with substitutions of charged internal residues with neutral (valine) or hydrophobic (alanine) residues. The peptide analogs synthesized and tested in this study are listed at Table 1.
Peptide analog H1 is capable of inhibiting genetic competence.
[00116] All 17 peptide analogs designed and synthesized based on the sequence of the native S. mutans CSP were first screened for their ability to hinder transformation efficiency in the S. mutans wild-type UA159 strain. Among them, analog H1 [SEQ ID NO:39] caused a significant decrease (18-fold) in transformation efficiency compared to that of the natural transformation (without addition of exogenous CSP) of the S. mutans UA159 strain (Table 2 and Fig. These results demonstrate that H1 inhibited the S. mutans natural genetic transformation. The WO 2006/060903 PCT/CA2005/001845 24 competence regulon identified and characterized by our laboratory indicated that transformation in S. mutans is a comD-dependent process. To test the hypothesis that the peptide analog H1 is able to compete with the natural CSP produced by S. mutans for occupying the ComD histidine kinase receptor, we tested the ability of H1 to induce genetic competence in an S. mutans comD null mutant. As expected, the results showed that the effect of peptide analog H1 is indeed accomplished via the comD receptor, and therefore is a ComD-dependent process (Fig. 3).
[00117] In contrast, the peptide analogs IH-1 [SEQ ID NO:31], IH-2 [SEQ ID NO:32], B1 [SEQ ID NO:33], and C1 [SEQ ID NO:34] showed no significant effect on transformation efficiency compared to the wild-type S. mutans CSP (Table 2).
These results suggested that these peptide analogs have retained the native CSP activity despite the sequence modifications. However, the transformation efficiency of S. mutans UA159 in the presence of the peptide analogs D1 [SEQ ID NO:35], El [SEQ ID NO:36], F1 [SEQ ID NO:37], G1 [SEQ ID NO:38], A2 [SEQ ID B2 [SEQ ID NO:41], C2 [SEQ ID NO:42], D2 [SEQ ID NO:43], E2 [SEQ ID NO:44], F2 [SEQ ID NO:45], G2 [SEQ ID NO:46], or B3 [SEQ ID NO:47] is diminished compared to the wild-type S. mutans CSP (5 gig CSP). This suggested that these peptide analogs behave similarly to CSP in terms of competence stimulation but may not have the same affinity for the comD receptor as the native wild-type S.
mutans CSP.
[00118] TABLE 1. Modified versions of the mature S. mutans CSP peptide Peptide analog
CSP
IH-1 11-2 B1 C1 D1 El Fl Gl H1 A2 B2 C2 D2 Amino acid sequence
SGSLSTFFRLFNRSFTQALGK
SGSLSTFFRLFNRSFTQALGK
SGSLSTFFRLFNRSFTQALGK
SGSLSTFFRLFNRSFTQALGK
SGSLSTFFRLFNRSFTQALGK
SGSeSTFFRLFNRSFTQALGK
SGSLSTFFRLFNRSFTQALGK
SGSLSTFFRLFNRSFTQALGK
SGSLSTFFRLFNRSFTQALJGK
SGSLSTFFRLFNRSFTQALGK
SGSLSTFFRLFNRSFTQALGK
SGSLSTFFVLFNRSFTQALGK
SGSLSTFFALFNRSFTQALGK
SGSLSTFFRLFNVSFTQALGK
Modification mature wild-type CSP sequence 1 st residue removed from N' 1 st residue removed from C' 2 nd residue removed from N' 3 rd residue removed from N' 4 th residue removed from N' 5 th residue removed from N' 2 nd residue removed from C' 3 rd residue removed from C' 4 th residue removed from C' 5 th residue removed from C' Substitution of 1 st R residue with V Substitution of 1 st R residue with A Substitution of 2 nd R residue with V WO 2006/060903 PCT/CA2005/001845 E2 SGSLSTFFRLFNASFTQALGK Substitution of 2 nd R residue with A F2 SGSLSTFFRLFNRSFTQALGV Substitution of K residue with V G2 SGSLSTFFRLFNRSFTQALGA Substitution of K residue with A B3 SGTLSTFFRLFNRSFTQALGK JH1005 CSP sequence [00119] TABLE 2. Effect of 5 gg/ml of peptide analogs on competence of S.
mutans wild-type UA159 Peptide Transformation Transformation analog efficiency (vs no CSP) efficiency (vs 5 gg CSP) CSP 1554-fold increase IH-1 no effect" no effect IH-2 no effect no effect B1 no effect no effect C1 no effect no effect D1 275-fold increase 6-fold decrease El 791-fold increase 2-fold decrease Fl1 541-fold increase 3-fold decrease Gl 848-fold increase 2-fold decrease H1 18-fold decrease 28,000-fold decrease A2 125-fold increase 7-fold decrease B2 4-fold increase 414-fold decrease C2 32-fold increase 48-fold decrease D2 99-fold increase 16-fold decrease E2 252-fold increase 6-fold decrease F2 543-fold increase 3-fold decrease G2 56-fold increase 28-fold decrease B3 195-fold increase 8-fold decrease aNo effect: no significant difference by comparison with CSP.
[00120] TABLE 3. Effect of 5 gg/ml of peptide analogs on growth at pH acid resistance, and biofilm formation of S. mutans wild-type UA159 Peptide Growth Acid resistance Biofilm formation analog (pH 7.5) (pH 5.5) (SDM-glucose) CSP 4 growth growth no effect IH-1 4 growth growth no effect IH-2 4 growth growth no effect B1 growth growth no effect C1 4 growth growth no effect D1 growth growth no effect El 4 growth growth no effect Fl I growth I growth 1 36.7% biomass I growth growth 1124.4% biomass WO 2006/060903 PCT/CA2005/001845 H1 no effect A2 no effect B2 no effect C2 no effect D2 no effect E2 no effect F2 I growth G2 4 growth B3 no effect I growth no effect 4 growth no effect 4 growth no effect 1 growth no effect 4 growth no effect I growth 38.9% biomass I growth 4 38.7% biomass I growth 1 35.6% biomass 4 growth 1 34.4% biomass Multiple peptide analogs affect S. mutans cell growth in an acidic medium.
[00121] In order to determine if the peptide analogs were capable of inhibiting the acid tolerance mechanisms of S. mutans, the cells' ability to withstand acid challenge typically encountered in dental plaque, the S. mutans UA159 cells were grown in THYE medium at pH 7.5 and pH 5.5 in the presence of various concentrations of peptide analogs. The results presented at Table 3 showed that the peptide analogs Fl, F2, and G2 caused a diminution of cell growth at pH 7.5 and The peptide analogs H1, A2, B2, C2, D2, E2, and B3 have no effect on S. mutans cell growth at pH 7.5. Moreover, when the same peptide analogs were tested at pH 5.5, the results showed that there was a significant decrease in cell growth. Interestingly, the peptide analog H1 involved in the inhibition of genetic competence is also able to inhibit the S. mutans cell growth in an acidic medium (Fig. while the growth at neutral pH is unaffected (Fig. S. mutans peptide analogs inhibit Streptococci biofilm formation.
[00122] It has been demonstrated that the S. mutans comC null mutant unable to produce the CSP signal peptide forms a biofilm lacking the wild-type architecture.
Moreover, the exogenous addition of synthetic CSP restores the wild-type phenotype in the comC defective mutant (Li et al., 2002). Therefore, CSP seems to play an integral part in S. mutans biofilm formation. Consequently, the S. mutans peptide analogs were tested for their ability to inhibit the formation of S. mutans biofilms. In a first study, the results of which are presented at Table 3, S. mutans peptide analogs Fl WO 2006/060903 PCT/CA2005/001845 27 [SEQ ID NO:37] G1 [SEQ ID NO:38], E2 [SEQ ID NO:44], F2 [SEQ ID G2 [SEQ ID NO:46] and B3 [SEQ ID NO:47] significantly reduced biomass ranging from 24.4% to 38.9% compared to the S. mutans biofilm grown in the presence of wild-type CSP suggesting that these peptide analogs are able to hinder the signal pathway regulating the formation of biofilm by S. mutan [00123] In a second study, the anti-biofilm activity of B2 [SEQ ID NO:41], B3 [SEQ ID NO:47], C2 [SEQ ID NO:42], E2 [SEQ ID NO:44], Fl [SEQ ID NO:37], and HI [SEQ ID NO:39] was investigated. The anti-biofilm activity of the peptide analogues against S. mutans in terms of percentage inhibition varied from 0 to (Figure 18). Peptide analog E2 [SEQ ID NO:44] showed the highest anti-biofilm activity (80% inhibition) among the six synthetic CSP analogues tested. Peptide analog E2 could be a potent S. mutans QS inhibitor as it elicited a significant decrease in biofilm formation as well as inhibited cell growth at pH 5.5 without affecting the cell growth at neutral pH.
[00124] In additional studies, the S. mutans peptide analogs are further tested for their ability to inhibit the fornation of biofilms by other types of bacteria, and in particular, other dental plaque associated bacteria. The peptide analogs B3 [SEQ ID NO:47], C2 [SEQ ID NO:42], E2 [SEQ ID NO:44] and F1 [SEQ ID NO:37], are found to signifantly reduce biofilm formation in dental plaque associated streptococci including S. sobrinus, S. sanguis, S: gordonii, S. oralis, S. mitis and non-dental plaque associated Streptococci such as S. pneumoniae [00125] The S. mutans derived E2 [SEQ ID NO:44] peptide at a concentration as low as 5 tig/ml showed inhibitory effects on both growth and biofilm formation in S. sobrinus, S. sanguis, S. gordonii, S. oralis, S. mitis and S. pneumoniae.. The percent inhibition of biofilm formation in these organisms varied from 40 to 75% (Figures 19, 21, 22, 23, 24 and 25). Furthermore, the anti-biofilm activity of E2 [SEQ I) NO:44] peptide was tested against mixed culture of the above Streptococcus spp. It also showed a significant inhibitory effect on the mixed culture biofilm formation (data not shown).
[00126] A peptide is considered to possess a function substantially equivalent to that of the CSP peptide [SEQ ID NO:2] if it has CSP activity. CSP activity means WO 2006/060903 PCT/CA2005/001845 28 that it is able to confer genetic competence to S. mutans, as measured by an increased ability to incorporate and express foreign genetic material, when added to cells as described in the assay of genetic competence below. CSP activity also means that the peptide is able to confer an acid tolerance response in S. mutans as measured by an increase in cell survival under acidic pH conditions when added to cells as described in the assay for acid adaptation below. Functionally equivalent peptides, peptides and proteins include peptides, peptides and proteins that have the same or similar protein activity as CSP when assayed, i.e. they are able to stimulate genetic competence and low pH tolerance (the ability to withstand acid challenges ofpH 3.5 -pH 3.0 for up to 3 hours) in S. mutans. A peptide has CSP activity if it is capable of increasing the frequency of uptake and expression of foreign DNA as described in the following assay for genetic competence and if the peptide can promote an acid tolerance response as described in the assay for acid adaptation.
[00127] Identity refers to the similarity of two peptides or proteins that are aligned so that the highest order match is obtained. Identity is calculated according to methods known in the art, such as the ClustalW program. For example, if a peptide (called "Sequence has 90% identity to a portion of the peptide in [SEQ ID then Sequence A will be identical to the referenced portion of the peptide in [SEQ ID NO:30], except that Sequence A may include up to 1 point mutations, such as substitutions with other amino acids, per each 10 amino acids of the referenced portion of the peptide in [SEQ ID NO:30]. Peptides, peptides and proteins functional equivalent to the CSP peptides can occur in a variety of forms as described below.
[00128] Peptides biologically equivalent in function to CSP peptide include amino acid sequences containing amino acid changes in the CSP sequence [SEQ ID NO:2]. The functional equivalent peptides have at least about 40% sequence identity, preferably at least about 60%, at least about 75%, at least about 80%, at least about or at least about 95% sequence identity, to the natural CSP peptide [SEQ ID NO:2] or a corresponding region. The ClustalW program preferably determines sequence identity. Most preferably, 1, 2, 3, 4, 5, 5-10, 10-15 amino acids are modified.
[00129] Variants of the CSP peptide may also be created by splicing. A combination of techniques known in the art may be used to substitute, delete or add WO 2006/060903 PCT/CA2005/001845 29 amino acids. For example, a hydrophobic residue such as methionine can be substituted for another hydrophobic residue such as alanine. An alanine residue may be substituted with a more hydrophobic residue such as leucine, valine or isoleucine.
An aromatic residue such as phenylalanine may be substituted for tyrosine. An acidic, negatively-charged amino acid such as aspartic acid may be substituted for glutamic acid. A positively-charged amino acid such as lysine may be substituted for another positively-charged amino acid such as arginine. Modifications of the peptides disclosed herein may also be made by treating such peptide with an agent that chemically alters a side group, for example, by converting a hydrogen group to another group such as a hydroxy or amino group.
[00130] Peptides having one or more D-amino acids are contemplated in certain embodiments of the present invention. Also contemplated are peptides where one or more amino acids are acetylated at the N-terminus. Those skilled in the art recognize that a variety of techniques are available for constructing peptide mimetics a modified peptide, or peptide or protein) with the same or similar desired biological activity as the corresponding disclosed peptide but with more favorable activity than the peptide with respect to characteristics such as solubility, stability, and/or susceptibility to hydrolysis and proteolysis. See for example, Morgan and Gainor, Ann. Rep. Med. Chemn., 24:243-252 (1989).
[00131] Certain embodiments of the invention also include hybrid nucleic acid molecules and peptides, for example where a nucleic acid molecule from the nucleic acid molecule disclosed herein is combined with another nucleic acid molecule to produce a nucleic acid molecule which expresses a fusion peptide. One or more of the other domains of CSP described in this application could also be used to make fusion peptides. For example, a nucleotide domain from a molecule of interest may be ligated to all or part of a nucleic acid molecule encoding CSP peptide (or a molecule having sequence identity) described in this application. Fusion nucleic acid molecules and peptides can also be chemically synthesized or produced using other known techniques. Certain embodiments of the invention include a nucleic acid molecule encoding a fusion peptide or a recombinant vector including the nucleic acid molecule WO 2006/060903 PCT/CA2005/001845 [00132] The variants preferably retain the same or similar CSP activity as the naturally occurring CSP [SEQ ID NO:2]. The CSP activity of such variants can be assayed by techniques described in this application and known in the art.
[00133] Variants produced by combinations of the techniques described above but which retain the same or similar CSP activity as naturally occurring CSP [SEQ ID NO:2] are also included in certain embodiments of the invention (for example, combinations of amino acid additions, and substitutions).
[00134] Variants of CSP produced by techniques described above which competitively inhibit CSP activity are also included in certain embodiments of the invention (for example, combinations of amino acid additions, and substitutions).
[00135] Variants of CSP produced by techniques described above which decrease transformation efficiency of bacteria are also included in the invention (for example, combinations of amino acid additions, and substitutions).
[00136] Variants of CSP produced by techniques described above which decrease biofilm formation are also included in certain embodiments of the invention (for example, combinations of amino acid additions, and substitutions).
[00137] Variants of CSP encompassed by the present invention preferably have at least about 40% sequence identity, preferably at least about 60%, 75%, 80%, or 95% sequence identity, to the naturally occurring peptide, or corresponding region or moiety of the peptide, or corresponding region. Sequence identity is preferably measured with the ClustalW.
Histidine Kinase Response Regulator [00138] Certain embodiments of the invention also include sequences having identity with the histidine kinase, response regulator of the invention and comA and comB. Preferred percentages of identity (nucleic acid molecule and polypeptide) are the same as those described for the CSP.
[00139] As well, probes and antibodies for a histidine kinase [SEQ ID NO:3 and SEQ ID NO:4], response regulator [SEQ ID NO:5 and SEQ ID NO:6] comA [SEQ ID NO:25 and SEQ ID NO:26] or comb [SEQ ID NO:27 and SEQ ID NO:28] WO 2006/060903 PCT/CA2005/001845 31 may be prepared using the description in this application and techniques known in the art. The description for preparation of CSP variants and mutants is also applicable to the histidine kinase [SEQ ID NO:3 and SEQ ID NO:4], response regulator [SEQ ID and SEQ ID NO:6] or comA [SEQ ID NO:25 and SEQ ID NO:26] and comB [SEQ ID NO:27 and SEQ ID NO:28] of the invention. Certain embodiments of the invention also include fragments of HK having HK activity, fragments of RR [SEQ ID NO:5 and SEQ ID NO:6] having RR activity and fragments of comA [SEQ ID and SEQ ID NO:26] or comB [SEQ ID NO:27 and SEQ ID NO:28] having activity.
Design of CSP peptide competitive inhibitors [00140] The activity of the CSP peptide [SEQ ID NO:2] may be varied by carrying out selective site-directed mutagenesis. We characterize the binding domain and other critical amino acid residues in the peptide that are candidates for mutation, insertion and/or deletion. Sequence variants may be synthesized. A DNA plasmid or expression vector containing the CSP nucleic acid molecule [SEQ ID NO:1] or a nucleic acid molecule having sequence identity may be used for these studies using the U.S.E. (Unique site elimination) mutagenesis kit from Pharmacia Biotech or other mutagenesis kits that are commercially available, or using PCR. Peptide analogs of S.
mutans CSP peptide can be prepared by deleting and/or substitituting amino acids at the C' or N' terminus of the CSP peptides using mutagenesis methods known in the art. Once the mutation is created and confirmed by DNA sequence analysis, the mutant peptide is expressed using an expression system and its activity is monitored.
This approach is useful to identify CSP inhibitors. All these modifications of the CSP DNA sequences [SEQ ID NO:1] presented in this application and the peptides produced by the modified sequences are encompassed by the present invention.
[00141] Peptide analogs of S. mutans CSP peptide prepared by deleting and/or substitituting amino acids of the CSP peptides can be screened for biolfilm formation inhibitions. Screening assays are described below.
Pharmaceutical compositions [00142] The CSP inhibitors are also useful when combined with a carrier in a pharmaceutical composition. The compositions are useful when administered in WO 2006/060903 PCT/CA2005/001845 32 methods of medical treatment or prophylaxis of a disease, disorder or abnormal physical state caused by S. mutans. Certain embodiments of the invention also include methods of medical treatment of a disease, disorder or abnormal physical state characterized by excessive S. mutans or levels or activity of CSP peptide [SEQ ID NO:2], for example by administering a pharmaceutical composition including a carrier and a CSP inhibitor. Caries is one example of a disease, which can be treated or prevented by antagonizing CSP [SEQ ID NO:2]. The compositions are also useful when administered in methods of medical treatment or prophylaxis of a disease, disorder or abnormal physical state caused by other dental plaque causing bacteria including but not limited to Actinomyces spp. and other Streptococci spp.
[00143] The pharmaceutical compositions can be administered to humans or animals by methods such as food, food additives, dentrifice gels, toothpaste, mouthwash, dental floss, denture wash, denture adhesives, chewing gum, candies, biscuits, soft drinks or sports drinks in methods of medical treatment. The CSP inhibitors of the invention may be coupled to lipids or carbohydrates. This increases their ability to adhere to teeth, either by prolonging the duration of the adhesion or by increasing its affinity, or both. They may also be coupled to polymers, for example in dental work (eg. crowns, braces, fillings) or dental floss. The pharmaceutical compositions can be administered to humans or animals. Dosages to be administered depend on individual patient condition, indication of the drug, physical and chemical stability of the drug, toxicity of the desired effect and the chosen route of administration (Robert Rakel, ed., Conn's Current Therapy (1995, W.B. Saunders Company, USA)). The pharmaceutical compositions are used to treat diseases caused by streptococcal infections such as dental caries, peridontal disease and endocarditis.
In a preferred embodiment, the pharmaceutical compositions are used to treat diseases caused by Actinomyces spp. and Streptococci spp. In further preferred embodiment, the pharmaceutical compositions are used to treat Streptococci infections caused by but not limited to: S. mutans, S. sobrinus, S. oralis, S. sanguis, S. mitis, S. gordonii, S.
pneumoniae, S. pyogenes, and S. agalactiae.
[00144] The pharmaceutical compositions according to the invention may be prepared using a CSP inhibitor which is an antisense oligonucleotide. For example, CSP activity could be blocked by antisense mRNA which inhibits CSP expression or WO 2006/060903 PCT/CA2005/001845 33 transcription. Alternatively, the antisense oligonucleotide may be one which inhibits the activity of the exporter that secretes the CSP from the cell. We have the sequence of these exporters. There are two copies of the genes (comAB) [SEQ ID NO:25 and SEQ ID NO:27] that are involved in export. The prepartion of antisense technology is well known in the art.
[00145] In one embodiment, the CSP inhibitor is an antisense oligonucleotide which inhibits CSP expression or transcription. Preferrably, the antisense oligonucleotide is an oligonucleotide complementary to at least 10 consecutive nucleotides of an oligonucleotide encoding CSP, said oligonucleotide having the nucleic acid sequence of SEQ ID NO:1.
[00146] In another embodiment, the CSP inhibitor is an antisense oligonucleotide which inhibits CSP peptide export. Preferably the antisense oligonucleotide is an oligonucleotide complementary to at least 10 consecutive nucleotides of an oligonucleotide encoding a CSP exporter, said oligonucleotide having the nucleic acid sequence of SEQ ID. NO: 25 or 27.
[00147] Nucleic acid molecules (antisense inhibitors of CSP) [SEQ ID NO:1] and competitive inhibitors of CSP [SEQ ID NO:2] or the peptide analogs of S. mutans CSP, may be introduced into cells using in vivo delivery vehicles such as liposomes.
They may also be introduced into these cells using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation or using liposomes. In some instances it will be desirable to employ liposomes targeted to the bacteria of interest.
[00148] The pharmaceutical compositions according to the invention may be prepared using an antibody or a fragment thereof, which selectively inhibits CSP activity. A more detailed discussion of the preparation of CSP specific antibodies is set out below.
[00149] In a preferred embodiment, the pharmaceutical compositions according to the invention are prepared using one or more CSP peptide analogs capable of inhibiting biofilm formation in dental plaque associated bacteria. The inhibitory CSP peptide analog may be a naturally occurring mutant CSP peptide obtained from Streptococci bacteria having impaired biofilm formation ability. The inhibitory CSP WO 2006/060903 PCT/CA2005/001845 34 peptide analog may be a synthetic peptide prepared using methods known in the art. In a more preferred embodiment, the inhibitory CSP peptide analog is one or more of the following modified S. mutans CSP peptides: B3 [SEQ ID No:47], C2 [SEQ ID No:42], E2 [SEQ ID No:44], and Fl [SEQ ID No:37]. In further preferred embodiment, the pharmaceutical compositions are prepared using the E2 [SEQ ID NO:44] peptide.
[00150] Pharmaceutical compositions comprising CSP peptide analogs or pharmaceutically acceptable salts thereof, and in particular B3 [SEQ ID No:47], C2 [SEQ ID No:42], E2 [SEQ ID No:44], and F1 [SEQ ID No:37] peptides are particularly useful for methods of treatment or prophylaxis of a disease, disorder or abnormal physical state caused by Streptococci infection. Such pharmaceutical compositons are especially useful for treating infections caused by one or more of one or more oral Streptococci bacteria such as S. mutans, S. sobrinus, S. oralis, S. sanguis, S. mitis, S.
gordonii. The pharmaceutical compositions are also useful for treating and preventing other types of Streptococci infections such as S. pneumoniae, S. pyogenes, and S. agalactiae.
[00151] By "pharmaceutically acceptable," such as in the recitation of a "pharmaceutically acceptable carrier," or a "pharmaceutically acceptable salt," is meant herein a material that is not biologically or otherwise undesirable, the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
[00152] "Carriers" or "vehicles" as used herein refer to conventional pharmaceutically acceptable carrier materials suitable for drug administration, and include any such materials known in the art that are nontoxic and do not interact with other components of a pharmaceutical composition or drug delivery system in a deleterious manner.
[00153] The pharmaceutical compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the nucleic acid molecule or peptide is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable carriers are WO 2006/060903 PCT/CA2005/001845 described, for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA). Carriers include saline and D5W dextrose and water). Excipients include additives such as a buffer, solubilizer, suspending agent, emulsifying agent, viscosity controlling agent, flavor, lactose filler, antioxidant, preservative or dye. There are preferred excipients for stabilizing peptides for parenteral and other administration. The excipients include serum albumin, glutamic or aspartic acid, phospholipids and fatty acids.
[00154] On this basis, the pharmaceutical compositions could include an active compound or substance, such as a CSP inhibitor, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and isoosmotic with the physiological fluids. The pharmaceutical carrier will depend on the intended route of administration. The methods of combining the active molecules with the vehicles or combining them with diluents are well known to those skilled in the art. The compositions may also contain additives such as antioxidants, buffers, bacteriostatis, bactericidal antibiotics and solutes which render the formulation isotonic in the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The composition could include a targeting agent for the transport of the active compound to specified sites.
[00155] The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
[00156] Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
WO 2006/060903 PCT/CA2005/001845 36 [00157] The pharmaceutical compositions according to the invention can be administered by any suitable route known in the art. In cases where the infection is localized, the pharmaceutical composition can be administered topically to infected area. In cases where the infection is systemic, the pharmaceutical composition may be administered orally, intravenously, or parenterally.
[00158] For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
[00159] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, or cellulose preparations such as, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone. If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
[00160] According to another aspect of the present invention, there is provided a process of producing cells genetically modified to produce a CSP derivative which inhibits transformation efficiency. Another aspect comprises administering to a patient S. mutans genetically modified to produce a CSP derivative which inhibits transformation efficiency. Methods of producing and administering genetically engineered cells are known in the art, see, for example, W002/44230.
[00161] A further aspect of the present invention provides the use in the preparation of a medicament for administration to a mammalian patient to alleviate WO 2006/060903 PCT/CA2005/001845 37 dental caries, of viable, transfected S. mutans genetically modified to produce a CSP derivative which inhibits transformation efficiency.
[00162] According to another aspect of the present invention, there is provided a process of producing cells genetically modified to produce a CSP derivative which inhibits biofilm formation. Another aspect comprises administering to a patient cells genetically modified to produce a CSP derivative which inhibits biofilm formation.
[00163] A further aspect of the present invention provides the use in the preparation of a medicament for administration to a mammalian patient to improve oral health or to alleviate dental caries, of viable, transfected cells genetically modified to produce a CSP derivative which inhibits biofihn formation.
[00164] The phannaceutical compositions according to the invention can be administered by any suitable route known in the art. In cases where the infection is localized, the pharmaceutical composition can be administered topically to infected area.
In cases where the infection is systemic, the pharmaceutical composition may be administered orally, intravenously, or parenterally.
[00165] In some instances, it may be desirable to administer one or more pharmaceutical compositions according to the invention to treat an infection caused by more than one type of bacteria. For example, it may be desirable to administer a pharmaceutical composition comprising an antisense oligonucleotide with a pharmaceutical composition comprising a CSP peptide analog. In another instance, it may be desirable to administer a pharmaceutical composition comprising an antibody with a pharmaceutical composition comprising a CSP peptide analog. Alternatively, the pharmaceutical compositions may be prepared with one or more types of CSP inhibitors to yield a unitary dosage form.
[00166] In some instances, it may be desirable to administer the pharmaceutical compositions according to the invention with a known antibacterial agent such as an antibiotic. In some instances, the pharmaceutical compositions which repress biofilm formation are also useful for rendering the bacterial cells more susceptible to antibiotics.
[00167] The term "antibiotic" as used herein refers to any compound known to one of ordinary skill in the art that will inhibit the growth of, or kill, bacteria. The term WO 2006/060903 PCT/CA2005/001845 38 "antibiotic" includes, but is not limited to, beta-lactams (penicillins and cephalosporins), vancomycins, bacitracins, macrolides (erythromycins), lincosamides (clindomycin), chloramphenicols, tetracyclines, aminoglycosides (gentamicins), amphotericins, cefazolins, clindamycins, mupirocins, sulfonamides and trimethoprim, rifampicins, metronidazoles, quinolones, novobiocins, polymixins, gramicidins or any salts or variants thereof. The antibiotic used will depend on the type of bacterial infection.
[00168] The therapeutically effective dosage of the pharmaceutical compositions according to the invention will depend on the CSP inhibitor, the type and severity of the infection and whether the pharmaceutical composition comprises a further active ingredient such as an antibiotic. Generally, the therapeutically effective dose is the minimal amount sufficient for controlling biofilm formation and which is not toxic to the human or animal treated. Methods for determining effective dosages and toxicity are known in the art.
Anti-biofilm Compositions and Uses Thereof [00169] In a further aspect, the present invention provides compositions useful for inhibiting and disrupting biofilms in Streptococcus spp. The compositions comprise at least one peptide analogue of S. mutans CSP which inhibits or disrupts biofilm formation in a Streptococcus spp. The peptide analogues useful for practicing the invention include: Fl [SEQ ID NO. 37], H1 [SEQ ID NO. 39], B2 [SEQ ID NO.
41], C2 [SEQ ID NO. 42], E2 [SEQ ID NO. 44], and B3 [SEQ ID NO. 47]. In a preferred embodiment of the invention, the compositions are prepared using the E2 [SEQ ID NO. 44] peptide.
[00170] The compositions according to the invention are effective for inhibiting and disrupting biofilm formation in Streptococci which employ a quorum sensing system. The compositions are effective for both oral and non-oral Streptococcus spp. Examples of oral streptococci infections which can be modulated using the compositions of the invention include, but are not limited to: Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis and Streptococcus mitis. Examples of non-oral streptococci infections which can be modulated using the compositions of the invention, include, but are not limited to: Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae.
WO 2006/060903 PCT/CA2005/001845 39 [00171] In an embodiment of the invention, the composition may be formulated as a pharmaceutical composition suitable for administration to a human or animal patient suffering an infection with one or more Streptococcus spp. The pharmaceutical composition may comprise one or more ofF1 [SEQ ID NO. 37], HI [SEQ ID NO. 39], B2 [SEQ ID NO. 41], C2 [SEQ ID NO. 42], E2 [SEQ ID NO. 44], and B3 [SEQ ID NO. 47] peptide analogues or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In a preferred embodiment of the invention, the pharmaceutical composition comprises the E2 [SEQ ID NO. 44] peptide analogue.
[00172] The peptide analogues may be coupled to lipids or carbohydrates. This increases their ability to adhere to the infected surface such as teeth, either by prolonging the duration of the adhesion or by increasing its affinity, or both.
[00173] In some instances, it may be desirable to administer the pharmaceutical compositions according to the invention with a known antibacterial agent such as an antibiotic, examples of which are listed above. The antibiotic used will depend on the type of bacterial infection. For example, in some instances the biofilm inhibiting pharmaceutical compositions according to the invention also useful for rendering the bacterial cells more susceptible to antibiotics. Alternatively, the pharmaceutical compositions may be prepared with one or more active ingredients such as an antibiotic, in addition to the, CSP peptide analogue, to yield a unitary dosage form.
[00174] In addition to conventional oral pharmaceutical formulations such tablets, capsules and syrups, the compositions according to the invention can be administered to humans or animals in the form of food, food additives, dentrifice gels, toothpaste, mouthwash, dental floss, denture wash, denture adhesive, chewing gum, candy, biscuits, soft drinks, or sports drinks.
[00175] 1 The antimicrobial composition may further comprise additional ingredients including but not limited to: a surfactant, a chelating agent, an antibody, an antiseptic, and an antibiotic (see examples listed above).
WO 2006/060903 PCT/CA2005/001845 [00176] In a further aspect, the present invention provides a method of treating or preventing a Streptococcus spp infection in a patient in need thereof, comprising administering a therapeutically effective amount of at least one peptide having an amino acid sequence selected from a group consisting of: Fl [SEQ ID NO. 37], H1 [SEQ ID NO. 39], B2 [SEQ ID NO. 41], C2 [SEQ ID NO. 42], E2 [SEQ ID NO. 44], and B3 [SEQ ID NO. 47]. In a preferred embodiment, the method comprises administering a therapeutically effective amount of E2 [SEQ ID NO. 44] peptide.
The peptide analogues may be administered in the form of any of the pharmaceutical compositions encompassed by the present invention. In some instances, the method of treatment or prevention of a Streptococcus spp infection further comprises the administration of a therapeutically effective amount of an additional active agent such as an antibiotic (see examples above). The antibiotic may be administered concurrently with the peptide analogue. In circumstances where it is desirable to enhance penetration of the antibiotic into the biofilm, it is preferably to administer the peptide analogue first to disrupt the biofilm.
[00177] The "therapeutically effective amount" or "therapeutically effective dosage" of the pharmaceutical compositions according to the invention will depend on the peptide analogue, the type and severity of the infection and whether the pharmaceutical composition comprises a further active ingredient such as an antibiotic. Generally, the therapeutically effective dose is the minimal amount sufficient for controlling biofilm formation and/or eliminating the infection, which is not toxic to the human or animal treated. Methods for determining effective dosages and toxicity are known in the art.
[00178] The method encompasses the treatment of infections caused by both oral and non-oral Streptococcus spp. In the case of oral Streptococcus spp, such as Streptococcus spp. including: Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus mitis, the pharmaceutical compositions are useful for reducing and controlling dental plaque.
The compositions are also use for the treatment and prophylaxis of a condition caused by dental plaque, including for example, dental caries, periodontal disease, such as gingivitis, and endocarditis. In such cases, it is generally desirable to topically administer the pharmaceutical compositions to the infected areas. For example, the WO 2006/060903 PCT/CA2005/001845 41 pharmaceutical compositions can be administered in the form of food, food additives, dentrifice gels, toothpaste, mouthwash, dental floss, denture wash, denture adhesive, chewing gum, candy, biscuits, soft drinks, or sports drinks.
[00179] In the case of infections caused by non-oral Streptococcus spp. such as, but not limited to: Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae, the pharmaceutical compositions can be administered either locally or systemically depending on the nature and severity of the infection.
[00180] The precise dose for any of the pharmaceutical compositions of the present invention will depend on a number of factors which will be apparent to those skilled in the art and in light of the disclosure herein. In particular these factors include: the identity of the compounds to be administered, the formulation, the route of administration employed, the patient's gender, age, and weight, and the severity of the condition being treated. Methods for determining dosage and toxicity are well known in the art with studies generally beginning in animals and then in humans if no significant animal toxicity is observed. For example, in instances where the pharmaceutical compositions are used to treat an infection, the appropriateness of the dosage can be assessed by monitoring the severity of the infection using conventional methods known in the art. Where the dose provided does not cause bacterial levels to decline to normal or tolerable levels, following at least three to fourteen days of treatment, the dose can be increased. The patient should be monitored for signs of adverse drug reactions and toxicity.
[00181] In a preferred embodiment of the invention, the therapeutically effective amount of the peptide analogue will be between 1 jtg/ml and 1 mg/nml daily.
In a further preferred embodiment, the therapeutically effective amount of the peptide analogue will be between 1 jig/ml and 100 gig/ml. In a still further preferred embodiment, the peptide analogue administered is the E2 [SEQ ID NO. 44] peptide in an amount of between 1 jig/ml and 100 uig/ml. In instances which include the concurrent administration of an additional active ingredient such as an antibiotic, the amount of peptide analogue may be reduced. Alternatively, it may be appropriate to reduce the amount of the conventional dose of the antibiotic.
WO 2006/060903 PCT/CA2005/001845 42 Antimicrobial compositions [00182] The CSP inhibitors described above for use in the preparation of pharmaceutical compositions can also be used to prepare antimicrobial compositions such as disinfectants useful for inhibiting biofilm formation on various surfaces.
[00183] Antimicrobial compositions for inhibiting biofilm formation may comprise any of the peptide, antisense and antibody CSP inhibitors described above.
In a preferred embodiment of the invention, the CSP inhibitor is used to prepare the antimicrobial composition is a peptide analogue of S. mutans CSP which inhibits biofilm formation. More preferably, the CSP inhibitor is one or more of the following S. mutans CSP peptide analogues: B3 [SEQ ID No:47], C2 [SEQ ID No:42], E2 [SEQ ID No:44], and Fl [SEQ ID No:37]. In further preferred embodiment, the antimicrobial compositions are prepared using the E2 [SEQ ID No:44] peptide.
[00184] The antimicrobial composition may further comprise additional ingredients including but not limited to: a surfactant, an antispetic and an antibiotic (see examples listed above).
[00185] Where the CSP inhibitor is one or more of S. mutans CSP peptide analogues B3 [SEQ ID No:47], C2 [SEQ ID No:42], E2 [SEQ ID No:44], and F1 [SEQ ID No:37], and the amount of the CSP inhibitor is preferrably between 1Ig/ml to 1 mg/ml. In preferred embodiments, wherein the CSP inhibitor is the E2 [SEQ ID No:44] peptide, the amount of E2 [SEQ ID NO:44] peptide is preferably is preferrably between g/ml to 100 pg/ml.
Vaccines [00186] Antibodies directed against the CSP [SEQ ID NO:2 or SEQ ID would provide protection against caries. Antibodies may be manufactured as described below. Alternatively, a disclosed peptide [SEQ ID NO:2 or SEQ ID or a fragment thereof may be used with a carrier to make a vaccine. The peptide or fragment may also be conjugated to another molecule to increase its antigenicity. Antibodies can also be coupled to the peptide (Brady, L.J. et al., "Monoclonal Antibody-Mediated Modulation of the Humoral Immune Response against Mucosally Applied Streptococcus mutans (in press). In order to enhance the WO 2006/060903 PCT/CA2005/001845 43 immune response the peptide can be coupled to KLH, ovalbumin, or thyroglobulin prior to immunization. The vaccine composition will trigger the mammal's immune system to produce antibodies. Certain embodiments of the invention include vaccine compositions and methods of vaccinating a mammal, preferably a human, against dental caries by administering to the mammal an effective amount of a vaccine composition. Techniques for preparing and using vaccines are known in the art. To prepare the vaccine, the peptide, or a fragment of the peptide, may be mixed with other antigens (of different immunogenicity), a vehicle or an excipient. Examples of peptide vaccines are found in U.S. Patent Nos. 5,679,352, 5,194,254 and 4,950,480.
Techniques for preparing vaccines involving site-directed mutagenesis are described in U.S. Patent Nos. 5,714,372, 5,543,302, 5,433,945, 5,358,868, 5,332,583, 5,244,657, 5,221,618, 5,147,643, 5,085,862 and 5,073,494. Vaccines may be administered by known techniques, such as topical or parenteral administration. Vast changes are taking place in vaccinology consequent to the introduction of new technologies. Acellular purified fractions devoid of side effects, non-pathogenic but immunogenic mutants, recombinant technology, conjugated vaccines, combination vaccines (to limit the number of injections). Vaccine delivery systems can deliver multiple doses of the vaccine at a single contact point. A genetically engineered oral vaccine is useful to impart better and longer duration of immunity. Oral vaccines are useful. The nose as a route for immunization is also useful. DNA alone can constitute the vaccines, inducing both humoral and cell-mediated immune responses. Live recombinant vaccines are also useful. Potent adjuvants add to the efficacy of the vaccines. One can also 'humanize' mouse monoclonals by genetic engineering and express these efficiently in plants. These recombinant antibodies are opening out an era of highly specific and safe therapeutic interventions. An advantage of preformed antibodies directed at a defined target and given in adequate amounts is the certainty of efficacy in every recipient, in contrast to vaccines, where the quality and quantum of immune response varies from individual to individual. For example, nasal immunization may be done as described in C. Jespersgaard et al. "Protective Immunity against Streptococcus mutans Infection in Mice after Intranasal Immunization with the Glucan-Binding Region of S. mutans Glucosyltransferase" Infection and Immunity, December 1999, p. 6543-6549, Vol. 67, No. 12. Vaccine compositions may comprise solid or liquid formulations such as gels, sprays, inhalants, tablets, toothpastes, mouthwashes or chewing gum.
WO 2006/060903 PCT/CA2005/001845 44 [00187] For vaccine application, cholera toxin can be used by coupling the peptide to its B-subunit to stimulate production of secretory antibody coupling to
CTB.
Screening for inhibitors of CSP [00188] Inhibitors are preferably directed towards CSP [SEQ ID NO:2 or SEQ ID NO:30] to block S. mutans competence, low pH tolerance and biofilm formation.
[00189] A method of identifying a compound which reduces the interaction of CSP [SEQ ID NO:2 or SEQ ID NO:30] with IIK [SEQ ID NO:4], can include: contacting CSP [SEQ ID NO:2 or SEQ ID NO:30] with (ii) HK [SEQ ID NO:4], a CSP-binding fragment of HK [SEQ ID NO:4] or a derivative of either of the foregoing in the presence of the compound; and b) determining whether the interaction between and (ii) is reduced, thereby indicating that the compound reduces the interaction of CSP [SEQ ID NO:2 or SEQ ID NO:30] and HK [SEQ ID NO:4]. A CSP inhibitor (caries treating or preventing compound) inhibits the interaction between and By way of example, one can screen a synthetic peptide library. One could also screen small non-peptide organic molecules.
[00190] In one embodiment, the invention includes an assay for evaluating whether test compounds are capable of acting as agonists or antagonists for CSP [SEQ ID NO:2], or a peptide having CSP functional activity, including culturing cells containing DNA which expresses CSP [SEQ ID NO:1], or a peptide having CSP activity so that the culturing is carried out in the presence of at least one compound whose ability to modulate CSP activity is sought to be determined and thereafter monitoring the cells for either an increase or decrease in the level of CSP [SEQ ID NO:2 or SEQ ID NO:30] or CSP activity. Other assays (as well as variations of the above assay) will be apparent from the description of this invention and techniques such as those disclosed in U.S. Patent No. 5,851,788, 5,736,337 and 5,767,075 which are incorporated by reference in their entirety. For example, the test compound levels may be either fixed or variable.
Preparation of antibodies WO 2006/060903 PCT/CA2005/001845 [00191] The CSP [SEQ ID NO:2 or SEQ ID NO:30] peptide is also useful as an antigen for the preparation of antibodies that can be used to purify or detect other CSP-like peptides. Antibodies may also block CSP [SEQ ID NO:2] binding to HK [SEQ ID NO:4]. Antibodies are preferably targeted to the entire CSP [SEQ ID NO:2] sequence. The CSP peptide [SEQ ID NO:2 or SEQ ID NO:30] may be conjugated to other compounds, in order to increase immunogenicity.
[00192] We generate polyclonal antibodies against the CSP [SEQ ID NO:2 or SEQ ID NO:30], which is a unique sequence. Monoclonal and polyclonal antibodies are prepared according to the description in this application and techniques known in the art. For examples of methods of preparation and uses of monoclonal antibodies, see U.S. Patent Nos. 5,688,681, 5,688,657, 5,683,693, 5,667,781, 5,665,356, 5,591,628, 5,510,241, 5,503,987, 5,501,988, 5,500,345 and 5,496,705, which are incorporated by reference in their entirety. Examples of the preparation and uses of polyclonal antibodies are disclosed in U.S. Patent Nos. 5,512,282, 4,828,985, 5,225,331 and 5,124,147 which are incorporated by reference in their entirety.
Antibodies recognizing CSP [SEQ ID NO:2 or SEQ ID NO:30] can be employed to screen organisms or tissues containing CSP peptide [SEQ ID NO:2] or CSP-like peptides. The antibodies are also valuable for immuno-purification of CSP or CSPlike peptides from crude extracts.
[00193] An antibody (preferably the antibody described above) may be used to detect CSP [SEQ ID NO:2] or a similar peptide, for example, by contacting a biological sample with the antibody under conditions allowing the formation of an immunological complex between the antibody and .a peptide recognized by the antibody and detecting the presence or absence of the immunological complex whereby the presence of CSP [SEQ ID NO:2] or a similar peptide is detected in the sample. Certain embodiments of the invention also include compositions preferably including the antibody, a medium suitable for the formation of an immunological complex between the antibody and a peptide recognized by the antibody and a reagent capable of detecting the inununolgical complex to ascertain the presence of CSP [SEQ ID NO:2] or a similar peptide. Certain embodiments of the invention also include a kit for the in vitro detection of the presence or absence of CSP [SEQ ID NO:2] or a similar peptide in a biological sample, wherein the kit preferably includes WO 2006/060903 PCT/CA2005/001845 46 an antibody, a medium suitable for the formation of an immunological complex between the antibody and a peptide recognized by the antibody and a reagent capable of detecting the immunological complex to ascertain the presence of CSP [SEQ ID NO:2] or a similar peptide in a biological sample. Further background on the use of antibodies is provided, for example in U.S. Patent Nos. 5,695,931 and 5,837,472, which are incorporated by reference in their entirety.
Assay of genetic competence [00194] The ability of the peptide to activate the HK [SEQ ID NO:4] and RR [SEQ ID NO:6] and the subsequent genes involved in the conferral of the properties of genetic competence, acid tolerance and biofilm formation can be determined by measuring the efficiency of uptake and expression of DNA (preferably plasmid DNA) in S. mutans when exposed to signal peptide and/or inhibitor. Two methods modified based on the protocols described by Perry et al. Infect Immun, 41:722-727 and Lindler and Macrina J Bacteriol, 166:658-665 are used to assay genetic competence.
The method involves adding DNA and CSP [SEQ ID NO:1] (preferably plasmid DNA) to a S. mutans culture (or culture of a bacteria expressing CSP [SEQ ID NO:1] or a variant thereof). The rate of transformation is then determined. S. mutans is preferably grown in THYE plus 5% horse serum (THYE-HS). After 2-hr incubation, 1 p g/ml plasmid DNA or 10 ig/ml of chromosomal DNA is added to the culture. To assay induction of competence, synthetic competence stimulating peptide, (SCSP) [SEQ ID NO:14] is then added to the cultures, incubation continued for 30 minutes with a final concentration of 500 ng/ml of SCSP added to each sample. After the minute incubation equal amounts of DNA is added to each well (1 [tg/ml plasmid or pg/ml of chromosomal DNA) and incubation continued for another 2 hrs. Cell dilutions were immediately spread on THYE agar plates plus appropriate antibiotics.
Transformation frequency was expressed as the number of transformants (antibiotic resistant cells) per number of viable recipients. This is determined by comparing the number of cells able to grow in the presence of antibiotic (conferred by the applied plasmid or chromosomal DNA) relative to the total number of cells present that grow in the absence of antibiotic). A higher value indicates a higher rate of transformation and thus is reflective of a stimulatory effect by the peptide.
Consequently, addition of a molecule that successfully acts as an inhibitor results in a WO 2006/060903 PCT/CA2005/001845 47 lower ratio of transformants/recipients, indicating that the inhibitor is effective at blocking activity of the CSP [SEQ ID NO:2]. CSP deficient cells [SEQ ID NO:1 or SEQ ID NO:2] may also be used in a variation of these assays. One can identify compounds that inhibit CSP [SEQ ID NO:2] or variants thereof by adding a test compound to the mixture to determine if the rate of transformation is decreased by the addition of the test compound.
[00195] The activity of the system can also be measured by an in vitro assay that relies on the measurement of marker protein expression (such as green fluorescent protein (GFP)) via expression from a fusion to a promoter controlled by the signal cascade initiated by CSP [SEQ ID NO:2]/HK [SEQ ID NO:4] /RR [SEQ ID NO:6]. One such promoter occurs immediately 5' proximal to the S. mutans comX gene. S. mutans cells grown in microtiter wells are exposed to the CSP [SEQ ID NO:2] and/or inhibitor and the level of fluorescence of the comX::GFP strain is measured to give a quantitative measure of CSP [SEQ ID NO:2] stimulation (and conversely inhibitor activity). One can identify compounds that inhibit CSP [SEQ ID NO:2] or variants thereof by adding a test compound to the mixture to determine if the quantitative measure of CSP [SEQ ID NO:2] stimulation is decreased by the addition of the test compound.
Assay of acid resistance tolerance [00196] The ability of CSP [SEQ ID NO:2] to promote acid resistance tolerance is determined by measuring the cell survival rate of S. mutans when exposed to acidic pH. In one example, S. mutans are first grown in batch culture to assay acid tolerance response in 'standard' log- and stationary-phase cells by using a modification of methods described previously by Svensiter et al. Oral Microbiol. Immunol., 12:266-73. Mid-log-phase cells are obtained by transferring one volume of overnight culture into nine volumes (1:10) of fresh TYG medium (pH 7.5) and incubated at 370C with 5% CO 2 for 2 hours. These cells are then collected by centrifugation at 8,000 x g for 10 min and resuspended in 2 ml of fresh TYG (pH 5.5) at various cell densities as determined by O.D 600 The cells are induced for acid adaptation by incubation at pH 5.5 for 2 h at 37 0 C with 5% CO 2 The adapted log-phase cells are then exposed to the killing pH. Killing pH is pre-determined by incubating unadapted, mid-log phase cells in TYG medium at pH values from 6.0 to WO 2006/060903 PCT/CA2005/001845 48 Stationary-phase cells are prepared by re-suspending late-log phase cells in TY medium (tryptone-yeast extract) without glucose. The culture is incubated at 37 0 C for 2 h to allow the cells to fully enter into stationary phase. Induction of acid adaptation in stationary-phase cells follows a similar procedure to that for log-phase cells.
Adaptation of both log- and stationary-phase cells to acidic pH is determined by measuring the ability of bacterial cells to survive a killing pH for 3 h. Acid killing is initiated by resuspending cells in the same volume of fresh TYG (pH 3.5) and an aliquot of cell suspension is taken immediately from each sample to determine total viable cell number at zero time. The cells are then incubated for 3 h at 37 0 C with
CO
2 and an aliquot of sample is taken to determine survival rate by viable cell counts.
Addition of a molecule that successfully acts as an inhibitor results in a decrease in the acid resistance tolerance of S mutans resulting in a corresponding decrease in cell survival indicating that the inhibitor is effective at blocking activity of CSP [SEQ ID NO:2]. CSP [SEQ ID NO:1 or SEQ ID NO:2] deficient cells may also be used in a variation of these assays wherein addition of the signal peptide can complement the acid-adaptation-defective phenotype of a comC [SEQ ID NO:1 or SEQ ID NO:2] deficient cell. One can identify compounds that inhibit CSP [SEQ ID NO:1 or SEQ ID NO:2] or variants thereof by adding a test compound to the mixture to determine if the survival rate of cells is decreased by the addition of the test compound [00197] Cells transformed with a nucleic acid molecule disclosed herein (histidine kinase [SEQ ID NO:3], CSP [SEQ ID NO:1] or response regulator [SEQ ID are useful as research tools. For example, one may obtain a cell (or a cell line, such as an immortalized cell culture or a primary cell culture) that does not express histidine kinase [SEQ ID NO:3], CSP [SEQ ID NO:1] or response regulator [SEQ ID insert a histidine kinase [SEQ ID NO:3], CSP [SEQ ID NO:1] or response regulator [SEQ ID NO:5] nucleic acid molecule in the cell, and assess the level of expression and activity. Alternatively, histidine kinase [SEQ ID NO:3], CSP [SEQ ID NO:1] or response regulator [SEQ ID NO:5] nucleic acid molecules may be overexpressed in a cell that expresses a histidine kinase [SEQ ID NO:3], CSP [SEQ ID NO:1] or response regulator [SEQ ID NO:5] nucleic acid molecule. In another example, experimental groups of cells may be transformed with vectors containing different types of histidine kinase [SEQ ID NO:3], CSP [SEQ ID NO:1] or response regulator [SEQ ID NO:5] nucleic acid molecules to assess the levels ofpolypeptides WO 2006/060903 PCT/CA2005/001845 49 and peptides produced, its functionality and the phenotype of the cells. The polypeptides and peptides are also useful for in vitro analysis ofhistidine kinase [SEQ ID NO:4], CSP [SEQ ID NO:2] or response regulator [SEQ ID NO:6] activity or structure. For example, the polypeptides and peptides produced can be used for microscopy or X-ray crystallography studies.
[00198] The histidine kinase [SEQ ID NO:3 and SEQ ID NO:4], CSP [SEQ ID NO:1 and SEQ ID NO:2] or response regulator [SEQ ID NO:5 and SEQ ID NO:6] nucleic acid molecules and polypeptides are also useful in assays for the identification and development of compounds to inhibit and/or enhance polypeptide or peptide.
function directly. For example, they are useful in an assay for evaluating whether test compounds are capable of acting as antagonists for histidine kinase [SEQ ID NO:4], CSP [SEQ ID NO:2] or response regulator [SEQ ID NO:6] by: culturing cells containing a nucleic acid molecule which expresses histidine kinase [SEQ ID NO:4], CSP [SEQ ID NO:1] or response regulator peptides [SEQ ID NO:5] (or fragments or variants thereof having histidine kinase CSP or response regulator activity) wherein the culturing is carried out in the presence of increasing concentrations of at least one test compound whose ability to inhibit histidine kinase [SEQ ID NO:4], CSP [SEQ ID NO:2] or response regulator [SEQ ID NO:6] is sought to be determined; and (b) monitoring in the cells the level of inhibition as a function of the concentration of the test compound, thereby indicating the ability of the test compound to inhibit histidine kinase [SEQ ID NO:4], CSP [SEQ ID NO:2] or response regulator [SEQ ID NO:6] activity.
[00199] Suitable assays may be adapted from, for example, US patent no.
5,851,788.
EXAMPLES
Materials and Methods Growth conditions of cells [00200] Cells are grown in Todd Hewitt yeast extract medium at various dilutions with and without 5% horse serum and 0.01% hog gastric mucin.
Protocol for Transformation of Biofilm-grown Cells WO 2006/060903 PCT/CA2005/001845 [00201] Biofilms are developed on polystyrene microtiter plates to provide a rapid and simple method for assaying biofilm formation, and hence activity of the peptide [SEQ ID NO:2]/receptor [SEQ ID NO:6]/kinase [SEQ ID NO:4] system.
Formation of biofilms is initiated by inoculating 20 ul of cell suspension into each well containing 2 ml of biofilm medium (4X diluted Todd-Hewitt Yeast Extract supplemented with final concentration of 0.01% hog gastric mucin) for overnight incubation at 37C under an anaerobic condition. After 20-h incubation, fluid medium is removed and added with 2 ml of pre-warmed, fresh THYE plus 5% horse serum.
The cultures are incubated for 30 minutes and each well is supplemented with a final concentration of 200 ng/ml of synthetic competence stimulating peptide (SCSP) and varying concentrations of the inhibitor and the incubation is continued. After minutes, plasmid DNA (1 mg/ml) or chromosomal DNA (10 mg/ml) is added to each well and the cultures are incubated for an additional 2 hr. Planktonic cells are then removed and the wells are washed once with PBS buffer. Biofilm cells are collected into 2 ml fresh medium by a gentle sonication or washing the wells using a pipette.
The samples are centrifuged at 12,000 x g for 5 min. Both biofilm and planktonic cells are resuspended into 200 Al of fresh medium and are immediately spread on THYE agar plus appropriate antibiotics. Transformation frequency is determined after 48-h of incubation.
Genome database analysis [00202] Homologues of the Streptococcus pneumoniae comD [SEQ ID NO:3]/E [SEQ ID NO:5] genes encoding a histidine kinase [SEQ ID NO:4]/ response regulator [SEQ ID NO:6] system were identified. This sequence was used to design primers to amplify the region from a number of S. mutans isolates. An open reading frame consisting of 138 nucleotides was located 148 nucleotides 5' proximal from the end of the comD homolog in the opposite orientation (Fig This ORF was found to encode a peptide of 46-amino acid [SEQ ID NO:2] in length, the precursor of the 21amino acid CSP [SEQ ID PCR amplification and nucleotide sequencing [00203] The comCDE genes [SEQ ID NO:21] were amplified from the genomes of several S. mutans isolates by PCR using primers designed based on the WO 2006/060903 PCT/CA2005/001845 51 genome database sequence and their nucleotide sequences determined. The deduced amino acid sequences are compared among the isolates by sequence alignment to confirm identity.
Gene inactivations [00204] Genes are inactivated by integration of internal homologous fragments into the suicide vector pVA8912. Mutants defective in each of the individual genes (comC [SEQ ID NO:1], cormD [SEQ ID NO:3], comE [SEQ ID NO:5] are inactivated and their phenotypes are compared to the parent strain NG8 for their abilities to form biofilms, tolerate acidic pH (pH and transport and incorporate DNA. The knockout mutants of corn D [SEQ ID NO:3] and E [SEQ ID NO:5] were constructed by insertion-duplication mutagenesis, whereas the knockout comC [SEQ ID NO:1] mutant was created by allelic exchange via insertion of an erythromycin resistance determinant into the comC [SEQ ID NO:1] locus (Li et al, 2001). All mutant strains were therefore resistant to erythromycin. The wild-type strain was subcultured routinely on Todd-Hewitt-Yeast Extract (THYE) agar plates (BBL; Becton Dickinson, Cockeysville, MD), whereas the mutants were maintained on THYE agar plus 10 jtg/ml of erythromycin. A minimal medium (DMM) was prepared to grow biofilms by a modification of the method described previously (Loo et al, 2000). The medium contained 58 mM K 2
HPO
4 15 mM KH 2
PO
4 10 mM (NH 4 2 S0 4 mM NaC1, 2 mM MgS02-7H 2 0, 0.2% (wt/vol) Casamino Acids and was supplemented with filter-sterilized vitamins, (0.04 mM nicotinic acid, 0.1 mM pyridoxine HC1, 0.01 mM pantothenic acid, 1 gM riboflavin, 0.3 gM thiamin HC1, and 0.05 giM D-biotin), amino acids (4 mM L-glutamic acid, 1 mM L-arginine HC1, 1.3 mM L-cysteine HC1, and 0.1 mM L-tryptophan) and 20 mM glucose.
Creation of comD deletion mutant.
[00205] An S. mutans UA159 comD null mutant was constructed by a PCRbased deletion strategy involving restriction-ligation and allelic replacement as described previously (Lau et al., 2002). The primers used to construct and confirm the S. mutans cormD deletion mutant are P1-HK13 CACAACAACTTATTGACGCTATCCC-3'), P2-HK13 GGCGCGCCAACTGGCAACAGGCAGCAGACC-3'), P3-HK13 WO 2006/060903 PCT/CA2005/001845 GGCCTCAAAACGATGCTGTCAAGGG-3'), P4-HK13 GGAAGCG-3'), Erm-19 (5'-GGCGCGCCCCGGGCCCAAAATTTGTTTGAT-3'), and Erm-20 GGCCGGCCAGTCGGCAGCGACTCATAGAAT-3').
Synthesis of syntheticpeptide [00206] The sequence of the processed peptide was deduced by determining the cleavage site to be located beside the gly-gly amino acid residues (numbers 24 and (fig. A peptide was synthesized corresponding to amino acid sequence of residues 26-46 inclusive.
Synthesis ofpeptide analogs [00207] The sequences of the peptide analogs used in this study are listed in Table 4. The peptides were synthesized by methods known in the art.
TABLE 4: Synthetic CSP Analogues Peptide Amino acid sequence F1 SGSLSTFFRLFNRSFTQALK (SEQ ID NO. 37) H1 SGSLSTFFRLFNRSFTQLGK (SEQ ID NO. 39) B2 SGSLSTFFVLFNRSFTQALGK (SEQ ID NO. 41) C2 SGSLSTFFALFNRSFTQALGK (SEQ ID NO. 42) E2 SGSLSTFFRLFNASFTQALGK (SEQ ID NO. 44) B3 SGTLSTFFRLFNRSFTQA (SEQ ID NO. 47) [00208] Competence stimulating peptide (CSP) analogues were synthesized based on the sequence of the mature 21 amino acids CSP (SGSLSTFFRLFNRSFTQALGK). The CSP peptide analogues (Fl [SEQ ID No:37], H1 [SEQ ID No:39], B2 [SEQ ID No:41], C2 [SEQ ID No:42], E2 [SEQ ID No:44], and B3 [SEQ ID No:47]) were synthesized by the Advanced Protein Technology Centre, Peptide Synthesis Facility of Hospital for Sick Children (Toronto, ON) and Mimotopes (San Diago, CA). While the F1 and H1 analogues were generated by deleting the 2nd and 4th residues from the C' termini, separately, the B2 and C2 analogues in which the charged residues were substituted with neutral (alanine) or hydrophobic (valine) residues. In E2 analogue, second arginine (from the C' terminus) was substituted with neutral alanine. The B3 analogue was generated by substituting 3rd residue from the N' terminus with threonine and by deleting 1st, 2nd and 3rd residues from the C' terminus.
WO 2006/060903 PCT/CA2005/001845 53 [00209] The peptides were dissolved to 1 mg per ml in sterile distilled deionized water. To any insoluble peptides, 10% (vol/vol) acetic acid, 20% (vol/vol) acetonitrile or 100% (vol/vol) dimethylformamide (DMF) was subsequently added.
Peptides were stored at -20°C until used.
Restoration ofphenotypic defects by addition of CSP [00210] To determine if the synthetic peptide [SEQ ID NO:14] could restore defective phenotypes of the comC [SEQ ID NO:2] mutants, a chemically synthesized 21-amino acid competence-stimulating peptide (CSP) [SEQ ID NO:14] (Li et al, 2001) was used in complementary experiments. The peptide was freshly dissolved in sterile distilled water to a concentration of 1 mg/ml. The CSP solution was then added to the cultures at a final concentration of 2 jg/ml 2 h after inoculation of bacterial cells.
Growth rates [00211] The parent and mutant strains were grown in THYE medium for assaying their growth curves using a Bioscreen Microbiology Reader incorporating a multi-well disposable microtiter plate (Bioscreen C, Helsinki, Finland). The Bioscreen Reader was equipped with Biolink software program that allowed us to record and display the growth curves and growth rate calculations automatically. The growth of the strains was initiated by inoculating 5 tl of cell suspension into each well containing 200 pl of fresh THYE medium. The cell suspensions were pre-adjusted to the same optical density at O.D 600 before inoculation. The plates were then placed in the Bioscreen system, which was set up to read optical density automatically every minutes with shaking. The readings of optical density were automatically recorded and converted into growth curves. Each assay was performed in quadruplicate.
Bacterial strains and growth conditions [00212] Seven strains of S. mutans were used in this study (strains include: BM71, GB14, H7, JH1005, LT11, NG8, and UAB159. All the strains were cultured from freeze-dried ampoules and routinely maintained on Todd-Hewitt Yeast Extract (THYE) plates. For selection of antibiotic resistant colonies following transformation, WO 2006/060903 PCT/CA2005/001845 54 the medium was supplemented with either erythromycin (Em) (10 ig/ml) or kanamycin (Km) (500 jig/ml).
[00213] S. mutans strain wild-type UA159 and its comD null mutant were routinely grown on Todd-Hewitt supplemented with 0.3% (wt/vol) yeast extract (THYE) agar plates and incubated at 37 0 C in air with 5% C02. For biofilm experiments, S. mutans strains were grown in a semidefined minimal medium (SDM) supplemented with 5 mM glucose as described previously (Li et al., 2002). The replicative plasmid pDL289 (Buckley et al., 1995) was used as donor DNA for genetic transformation experiments. Plasmid DNA was prepared from Escherichia coli cultures by using a commercial plasmid preparation kit (Qiagen). When needed, antibiotics were added as follows: 10 jg erythromycin per ml or 500 gg kanamycin per ml for S. mutans, and 50 gg kanamycin per ml for E. coli.
[00214] Streptococcus spp. including S. sobrinus, S. sanguis, S. gordonii, S.
oralis, S. mitis and Streptococcus pneumoniae were also used to study the inhibitory effects of the synthetic peptide analogues. They were grown in Todd-Hewitt broth containing 0.3% yeast extract (THYE) at pH 5.5 or 7.5. They were subcultured routinely on THYE agar plates and incubated at 37C in an anaerobic chamber C02). In liquid media, cultures were incubated in closed screw-cap tubes without agitation at 37 0 C in an anaerobic chamber CO2).
Assay for S. mutans biofilms formed on polystyrene microtiter plates (a) [00215] Biofilms were developed on polystyrene microtiter plates to provide a rapid and simple method for assaying genetic transformation. A 4X diluted THYE medium supplemented with final concentration of 0.01% hog gastric mucin was used as biofilm medium Formation of biofilms was initiated by inoculating 20 .l of cell suspension into each well containing 2 ml of BM and four wells were set up: two for assaying transformation and two for quantification ofbiofilms. After cultures were incubated at 37 0 C for 20 h under an anaerobic condition, fluid medium was removed for viable cell counts. The wells were rinsed once with 10mM PBS buffer (pH 7.2) and biofilm cells were collected in 2 ml PBS by a gentle sonication for 15 seconds.
Both biofilm and the planktonic cells were immediately spread on THYE plates using a spiral system (Spriral Plater, Model D, Cincinnati, OH) and incubated at 37 0 C under WO 2006/060903 PCT/CA2005/001845 an anaerobic condition. Formation of biofilms was quantified by viable cell counts after 48 h of incubation.
Assayfor S. mutans biofihns formed on polystyrene microtiter plates (b) [00216] Biofilms were developed in 96-well polystyrene microtiter plates. The growth of the biofilm was initiated by inoculating 10 jil of an overnight S. mutans UA159 culture into 300 pl of SDM-glucose containing different concentrations (0, 0.1, 0.5, 2, and 5 gg per ml) of peptide analogs in the individual wells of a 96-well nmicrotiter plate. Wells without cells were used as blank controls. The microtiter plates were then incubated at 37 0 C in air with 5% CO 2 for 16 h without agitation. After the incubation, the planktonic cells were carefully removed and the plates were air dried overnight. The plates were then stained with 0.01% (wt/vol) safranin for 10 min, rinsed with sterile distilled water and air dried. Biofilms were quantified by measuring the absorbance of stained biofilms at 490 nm with a microplate reader (model 3550; Bio-Rad Laboratories, Richmond, CA).
Assay for S. mutans, S. sobrinus, S. sanguis, S. gordonii, S. oralis, S. mitis and S.
pneumonaie biofilms formed on polystyrene microtiter plates [00217] To determine the anti-biofilm activity of synthetic E2 peptide against Streptococcus spp. including S. sobrinus, S. sanguis, S. gordonii, S. oralis and S.
mitis, the growth of biofilms on 96-well polystyrene microtiter plate was initiated by inoculating 10 /d of an overnight Streptococcus spp. culture into 300 /l of semidefined minimal medium (58 mM K 2
HPO
4 15 mM KH 2
PO
4 10 mM (NH 4 2 S0 4 mM NaC1, and 2mM MgSO 4 .7H 2 0) supplemented with filter-sterilized vitamins (0.04 mM nicotinic acid, 0.1 mM pyridoxine HC1, 0.1 mM pantothenic acid, 1 ttM riboflavin, 0.3 jM thiamine HC1, 0.05 jM D-biotin), amino acids (4 mM L-glutamic acid, 1 mM L-arginine HC1, 1.3 mM L-cysteine HC1, 0.1 mM L-tryptophan), 0.2% casamino acids, and 20 mM glucose containing E2 peptide (0 and 5 ml) in the individual wells of a 96-well microtiter plate. Wells without cells were used as blank controls. The microtiter plates were then incubated at 37 0 C in an anaerobic chamber
CO
2 for 24 hours without agitation. After the incubation, the growth was measured at 600 nm with a microplate reader. The planktonic cells were carefully removed and plates were air dried overnight. The plates were then stained with 0.4% WO 2006/060903 PCT/CA2005/001845 56 crystal violet for 10 minutes, rinsed with sterile distilled water and air dried for minutes. Biofilm was quantified by measuring the absorbance of stained biofilm at 630 nm with a microplate reader.
S. mutans competence assay [00218] To determine if the p.eptide analogs had any impact on the development of genetic competence, S. mutans UA159 wild-type cells were assayed for genetic transformation. Overnight cultures of S. mutans UA159 were diluted (1:20) with prewarmed THYE broth and incubated at 37C in air with 5% CO 2 until an optical density (OD) of approximately 0.1 at 600 nm was reached. The culture was then divided into six aliquots containing 1 jug/ml of plasmid pDL289 and different concentrations 0.1, 0.5, 2, and 5 gg per ml) of peptide analogs. The cultures were incubated at 37 0 C in air with 5% CO 2 for 2.5 h, gently sonicated for 10s to disperse the streptococcal chains, and spread on THYE plates containing kanamycin. Plates were incubated at 37°C in air with 5% CO 2 for 48 h. Total recipient cells were counted by spreading serial dilutions on THYE agar plates without antibiotic.
Transformation efficiency was expressed as the percentage of kanamycin resistant transformants over the total number of recipient cells.
S. mutans acid resistance assay [00219] The effect of peptides on acid tolerance was evaluated by assessment of growth in THYE at pH 7.5 and pH 5.5. Overnight S. mutans wild-type UA159 cells were diluted (1:20) with prewarmed THYE broth and incubated at 37 0 C in air with
CO
2 until an OD 600 of approximately 0.4 was reached. A 20-fold dilution was made into 400 pl of either THYE pH 7.5 or THYE pH 5.5 broth containing different concentrations 0.1, 0.5, 2, and 5 gg per ml) of peptide analogs and added in the individual wells of a 100-well Bioscreen C plate in triplicate. Wells without cells were used as blank controls. A Bioscreen microbiology reader (Labsystems, Helsinki, Finland) was employed to continuously grow cells and measure cell growth for 16 h at 37 0 C. Measurements were taken every 20 min with shaking to prevent cell aggregation.
Assay for "steady-state" biofilms WO 2006/060903 PCT/CA2005/001845 57 [00220] Biofilms were also grown in a chemostat-based biofilm fermentor to define and optimize the conditions for genetic competence of biofilm-grown cells.
The biofilm fennentor was modified in the Mechanical Engineering and Glass Blowing Shops, University of Toronto, based on a similar system described previously (Li and Bowden, 1994). The vessel was made of glass with a working volume of 400 ml. The vessel lip was constructed of stainless steel with 10 sampling ports, which allowed sterile insertion and retrieval of glass rods (0.5 cm in diameter, approximately 4.0 cm 2 area immersed in fluid medium), providing abiotic surfaces for accumulation of biofilms. Temperature in the chemostat vessel was maintained at 37 0 C 0.1 by a temperature controller (Model R-600F, Cole Parmer Instrument Cop., Vernon Hill, IL). The culture pH was controlled by a pH control unit (Digital pH Meter/Controller, Model 501-3400, Barnant Corp. Barrington, IL) through the addition of 1M KOH or 1M HC1. The vessel was placed on a magnetic stirrer (Fisher Scientific) and the culture was stirred at 200 rpm by a polypropylene coated magnetic stirrer bar (3 cm in length). Continuous cultures were obtained by pumping fresh 4X diluted THYE medium supplemented with a final concentration of 0.01% hog gastric mucin (Type III, Sigma) into the vessel (400 ml) at the desired dilution rates. Daily maintenance of the chemostat included optical density reading, viable cell counts and pH measurement in fluid cultures. When the cultures reached "steady-state" (at least mean generation times), glass rods were aseptically inserted into the chemostat for the initiation of biofilm formation. Then, biofilms of different ages were removed from the cultures for both genetic transformation and quantification of biofilms using viable cell counts.
Scanning electron microscopy (SEM) [00221] To examine spatial distribution and biofilm thickness by scanning electron microscopy, biofilms of different ages were removed by slicing off the bottom of the microtiter wells that were then washed once with 10 mM KPO 4 and fixed with 2 ml of 3.7% formaldehyde in 10 mM KP04 buffer overnight. The samples were then dehydrated with a series of alcohol baths 50%, 70%, 95% and 100%), critical point dried with liquid C0 2 mounted and sputter coated with gold.
The samples were then examined using a scanning electron microscope (Model S- 2500, Hitachi Instruments, San Jose, CA).
WO 2006/060903 PCT/CA2005/001845 58 Transformation protocol [00222] Two methods modified based on the protocols described by Perry et al (Infect Immun, 41:722-727) and Lindler and Macrina (I Bacteriol, 166:658-665) were used to assay natural transformation of biofilm cells. Biofilms formed on polystyrene microtiter plates were added with 2 ml of pre-warmed, fresh THYE plus 5% horse serum (THYE-HS) immediately following removal of the BM medium, and the incubation continued at 37 0 C. After 2h incubation, a final concentration of 1 pg/ml plasmid DNA or 10 ug/ml of chromosomal DNA was added to each well. The cultures were incubated for an additional 2 h before collection of the cells for plating.
To assay induction of competence by synthetic competence stimulating peptide (SCSP) [SEQ ID NO:14], the cultures were incubated for 30 min and a final concentration of 500 ng/ml of SCSP [SEQ ID NO:14] was added to each well. After a min incubation, equal amounts of DNA was added to each well (1 tg/ml plasmid or 10 ptg/ml of chromosomal DNA) and incubation continued for another 2 h. Fluid medium was then removed from individual wells and the wells were washed once with PBS buffer. Biofilm cells were collected into 2 ml PBS buffer by gentle sonication or by washing the wells using a pipette. The samples were centrifuged at 12,000 X g for 5 min. Both biofilm and planktonic cells were resuspended into 200 jl of fresh medium and were immediately spread on THYE agar plates plus appropriate antibiotics. For the biofilms developed in the chemostat, rods with biofilm cells were removed and placed into 2 ml of pre-warmed, fresh THYE-HS medium for 30 min incubation. Transformation was then initiated by using the same methods as described above. The planktonic cells were also removed to compare the transformation frequency. After completion of the transformation procedures, both biofilm and planktonic cells were spread on THYE agar plus appropriate antibiotic.
Transformation frequency was assessed after 48-h incubation. Transformation frequency was expressed as the number of transformants per pLg DNA per viable recipient at the time of DNA added.
Donor DNA [00223] Both plasmid and chromosomal DNA were used as donor DNA to assay genetic transformation in this study. Plasmid DNA included an integrative WO 2006/060903 PCT/CA2005/001845 59 plasmid, pVAGTFA carrying an erythromycin resistance (Em t determinant and a fragment of the S. mutans gtfA gene. The replicative plasmid, pDL289 carrying a kanamycin resistance gene (Km) was also used. Chromosomal DNA harboring an Emr gene was prepared from a recombinant S. mutans strain harboring a chromosomally integrated copy ofpVAGTFA.
[00224] The present invention has been described in detail and with particular reference to the preferred embodiments; however, it will be understood by one having ordinary skill in the art that changes can be made without departing from the spirit and scope thereof. For example, where the application refers to peptides, it is clear that polypeptides may often be used. Likewise, where a gene is described in the application, it is clear that nucleic acid molecules or gene fragments may often be used.
[00225] All publications (including GenBank entries), patents and patent applications are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
[00226] Example 1 CSP Peptide Analogues Inhibit Biofilm Formation in Streptococcus mutans [00227] An in vitro assay was performed as described in Materials and Methods to determine the effects of six synthetic analogues of CSP (FL, H1, B2, C2, E2 and B3; TABLE 4) on biofilm formation in Streptococcus mutans. The analogues were synthesized as described in Materials and Methods. The anti-biofilm activity of analogues against S. mutans in terms of percentage inhibition varied from 0 to (FIG 18). The E2 peptide was selected for further in vitro studies as it showed the highest anti-biofilm activity (80% inhibition) among six synthetic CSP analogues tested.
[00228] Example 2 E2 CSP Peptide Analogue Inhibits Biofilm Formation in S. sobrinus, S. sanguis, S. gordonii, S. oralis and S. mitis WO 2006/060903 PCT/CA2005/001845 [00229] An in vitro assay was performed as described in Materials and Methods to determine whether E2 analogue of CSP could inhibit biofilm formation in Streptococcus spp. such as S. sobrinus, S. sanguis, S. gordonii, S. oralis and S. mitis.
E2 peptide at a concentration as low as 5 |pg/ml showed inhibitory effects on both growth and biofilm formation in all the five organisms tested. The percent inhibition of biofihn formation in these organisms varied from 40 to 75% (FIGS. 19, 20, 21, 22, and 23). Furthermore, the anti-biofilm activity of E2 peptide was tested against mixed culture of the above Streptococcus spp. It also showed a significant inhibitory effect on the mixed culture biofilm formation (data not shown).
[00230] Example 3 E2 CSP Peptide Analogue Inhibits Biofilm Formation in Streptococcus pneumoniae [00231] This example illustrates the effect of S. mutans CSP analogue on the CSP-mediated quorum-sensing signaling system in a non-oral streptococcal pathogen was studied. An in vitro assay was performed as described in Materials and Methods to determine whether E2 analogue of CSP could inhibit biofilm formation in Streptococcus pneumoniae. E2 peptide at a concentration as low as 5 tg/ml showed a considerable antibiofilm activity against S. pneumoniae (25% inhibition, FIG. 24).
WO 2006/060903 PCT/CA20051001845 61
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Claims (12)

  1. 2. An isolated polypeptide that is at least 95% identical to SEQ ID NO:2 or SEQ ID and capable of inhibiting S. mutans genetic competence.
  2. 3. The isolated polypeptide of claim 1 or 2 wherein I-5 amino acids have been removed 0from the COOHl- terminal of SEQ ID NO:2 or SEQ ID C(l
  3. 4. An isolated polypeptide comprising a fragment of SEQ ID NO:2 or SEQ ID NO:30 and capable of inhibiting S. mutans biofilm formation. An isolated polypeptide that is at least 95% identical to SEQ ID NO:2 or SEQ ID and capable of inhibiting S. mutans biofilm formation.
  4. 6. The isolated polypeptide of claim 5 or 6 wherein 1-5 amino acids of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:30 have been modified to include up to I amino acid substitution per each 10 amino acids.
  5. 7. A composition for reducing S. mutans biofilm formation or S. mulans transformation efficiency comprising a peptide analog of CSP.
  6. 8. The composition according to claim 7, wherein the peptide analog is a peptide having an amino acid sequence selected from a group consisting of: SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 42 ,SEQ ID No: 44 and SEQ ID No: 47.
  7. 9. A cell genetically engineered to produce the composition of any one of claims 7 to 8. A cell of claim 9, wherein said cell is S. mutans. II. Use of the cell according to any one of claims 9 to 10 to produce the composition of any one of claims 7 to 9. I-:\terrvr\Keep\Retype\P64057 Amended Claims June 2007.doc
  8. 12. A composition comprising the cell according to any one of claims 9 to 10 and a pharmaceutically acceptable adjuvant.
  9. 13. A method of inhibiting the growth ofS. mutans comprising administering a composition S according to any one of claims 7 to 8 or 12.
  10. 14. A method of inhibiting dental caries comprising administering a composition according to any one of claims 7 to 8 or 12. 0 15. A method of improving dental health comprising administering a composition according C- to any one of claims 7 to 8 or 12.
  11. 16. An isolated polypeptide having the amino acid sequence selected from the group consisting of SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44 and SEQ ID No:
  12. 47. 17. A peptide analog ofS.mutans competence stimulating peptide (CSP) which inhibits biofilm formation. 18. The peptide analog according to claim 17, wherein the peptide analog is naturally occurring. 19. The peptide analog according to claim 17, wherein the peptide analog is synthetic. The peptide analog according to claim 19, wherein the synthetic peptide analog is prepared by deleting and/or substituting amino acids at C' or N' terminus of the S. mutans CSP. 21. The peptide according to claim 17, wherein the S. mutans CSP has the amino acid sequence of SEQ ID No. 22. The peptide according to claim 17, wherein the peptide analogs inhibit biofilms formed by dental plaque associated bacterium. -H:\tcrrvr\Kccp\Rctypc\P64057 Amended Claims June 2007.doc -66- O O 23. The peptide analog according to claim 17, wherein the dental plaque associated bacteria 0 is Streptococci or Actinomyces. l 24. The peptide analog according to claim 23, wherein the Streptococci bacteria is selected Sfrom a group consisting of: S. mutans, S. sorbinus. S. sanguis, S. gordonni, S. oralis and S. mitis. 25. The peptide according to claim 17, wherein the peptide analogs inhibit biofilms formed S by Streptococci bacteria selected from a group consisting of: S. pneumonaie, S. pyogenes, and S. S agalactiae. 26. A pharmaceutical composition comprising at least one CSP inhibitor and a pharmaceutically acceptable carrier. 27. The pharmaceutical composition according to claim 26, wherein the CSP inhibitor is a peptide analog of S.mutans competence stimulating peptide (CSP) which inhibits biofilm formation. 28. The pharmaceutical composition according to claim 26 or 27, wherein the CSP inhibtior is a polypeptide sequence having the amino acid selected from a group consisting of: a polypeptide of SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47. 29. A method of treating or preventing a bacterial infection caused by a biofilm forming bacterium comprising administering a therapeutically effective amount of the pharmaceutical composition according to any one of claims 26 to 28. A method of preventing dental plaque formation comprising administering a therapeutically effective amount of the pharmaceutical composition according to any one of claims 26 to 28. I :\terryr\Kccp\Retype\P64057 Amended Claims June 2007.doc -67- 31. A method of treating or preventing a condition caused by a dental plaque associated bacterium comprising administering a therapeutically effective amount of the pharmaceutical N composition according to any one of claims 26 to 28. r 32. The method according to claim 31, wherein the condition is selected from a group S consisting of: dental caries, peridontal disease, gingivitis, and endocarditis. S 33. Use of a CSP inhibitor for the preparation of a medicament for treatment or inhibitor of S an infection caused by a biofilm forming bacterium. 34. The use according to claim 33, wherein the CSP inhibitor is a peptide analog of S.mutans competence stimulating peptide (CSP) which inhibits biofilm formation. The use according to claim 33 or 34, wherein the CSP inhibtior is a polypeptide has an amino acid sequence selected from a group consisting of: SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44 and SEQ ID No: 47. 36. A composition for inhibiting biofilm formation in Streptococcus spp, said composition comprising at least one peptide having an amino acid sequence selected from a group consisting of: SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 44, and SEQ ID NO. 47 and wherein the Streptococcus spp is selected from a group consisting of: Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, Streptococcus cristatus. Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae. 37. The composition according to claim 36, wherein the composition further comprises one or more ingredients selected from a group consisting of: a surfactant, an antiseptic, and an antibiotic. 38. The composition according to any one of claims 36 to 37, wherein, the composition comprises between 1 pg/ml and 100 pg/ml of the peptide. I :\tcrryr\Kccp\Retypc\P64057 Amended Claims June 2007.doc -68- O O 39. Use of at least one peptide having an amino acid sequence selected from a group consisting of: SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 44, and SEQ ID NO. 47, for the preparation of a medicament for inhibition of biofilm formation in Streptococcus spp, wherein the Streptococcus spp is selected from a group consisting of: Streptococcus sobrinus, Streptococcus sanguis. Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, Streptococcus crislatus. Streptococcus S pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae. C 40. A method of treating or preventing a Streptococcus spp infection in a patient in need thereof, comprising administering a therapeutically effective amount of at least one peptide having an amino acid sequence selected from a group consisting of: SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 44, and SEQ ID NO. 47. 41. A method of inhibiting or preventing biofilm formation in Streptococcus spp in a patient in need thereof, comprising administering a therapeutically effective amount of at least one peptide having an amino acid sequence selected from a group consisting of: SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 44, and SEQ ID NO. 47. 42. The method according claim to 40 or 41 wherein the Streptococcus spp is selected from a group consisting of: Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, Streptococcus crislatus, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae. 43. A method of treating or preventing a condition caused by dental plaque associated Streptococcus spp in a patient in need thereof comprising administering a therapeutically effective amount of at least one peptide having an amino acid sequence selected from a group consisting of: SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 44, and SEQ ID NO. 47. I l:\terryr\Kccp\Rclypc\lP64057 Amended Claims June 2007.doc -69- 44. The method according to claim 43, wherein the condition is selected from a group consisting of: dental caries, periodontal disease, gingivitis, and endocarditis. The method according to claim 42 or 43, wherein the Streptococcus spp is selected from a group consisting of: Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, and Streptococcus cristatus. I :\lcrryr\Keep\Retype\P64057 Amended Claims June 2007.doc
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