EP1833844A1 - Molecules d'acides nucleiques, peptides signaux, et procedes de traitement - Google Patents

Molecules d'acides nucleiques, peptides signaux, et procedes de traitement

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Publication number
EP1833844A1
EP1833844A1 EP05814632A EP05814632A EP1833844A1 EP 1833844 A1 EP1833844 A1 EP 1833844A1 EP 05814632 A EP05814632 A EP 05814632A EP 05814632 A EP05814632 A EP 05814632A EP 1833844 A1 EP1833844 A1 EP 1833844A1
Authority
EP
European Patent Office
Prior art keywords
seq
streptococcus
csp
peptide
mutans
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05814632A
Other languages
German (de)
English (en)
Other versions
EP1833844A4 (fr
Inventor
Yi-Chen Cathy Huang
Celine Levesque
Dennis G. Cvitkovitch
Srinivasa Madhyastha
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kane Biotech Inc
Original Assignee
Kane Biotech Inc
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Filing date
Publication date
Application filed by Kane Biotech Inc filed Critical Kane Biotech Inc
Publication of EP1833844A1 publication Critical patent/EP1833844A1/fr
Publication of EP1833844A4 publication Critical patent/EP1833844A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • 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 (QS), 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.
  • QS quorum sensing
  • Examples include biofimi differentiation in Pseudomonas aeruginosa (Davies et at, 1998), swarming motility in Serratia Hquefaciens (Eberl et at, 1999), competence development in Streptococcus pneumoniae (Lee and Morrison, 1999) and Streptococcus mutatis (Li et at, 2001), and induction of virulence factors in Staphylococcus aureus (Ji et at, 1995).
  • 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.
  • 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 biofihns 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.
  • Caries and periodontal diseases are two of the most common chronic infectious diseases affecting centuries, 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.
  • 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 spp. such as Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis and Streptococcus mitis.
  • quorum-sensing systems to regulate several physiological processes, including the ability to incorporate foreign DNA, tolerate acid, form biofihn, 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 biofihn formation in S. mutatis (Cvitkovitch, et al., US Patent Application No. 20020081302, 2002).
  • a compound that competitively inhibits binding of CSP [SEQ ID NO:30] to S. mutans histidine kinase [SEQ ID NO:4].
  • the compound is a peptide or an antibody.
  • 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 TD NO:2].
  • an isolated polypeptide comprising a fragment of SEQ ID NO:2 or SEQ ID NO:30 and capable of inhibiting S. mutans genetic competence.
  • 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.
  • 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 NO:30 and capable of inhibiting S. mutans biofilm formation.
  • 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.
  • composition for reducing S. mutans biofilm formation comprising a peptide derivative of CSP.
  • composition for reducing S. mutans transformation efficiency comprising a peptide derivative of CSP.
  • 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 ED No: 47.
  • the peptide analog is a peptide having amino acid sequnece SEQ ID No:44.
  • the cell is bacterial.
  • the cell is S. mutans.
  • composition comprising the cell according to the invention and a pharmaceutically acceptable adjuvant.
  • CSP competence stimulating peptide
  • 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.
  • a pharmaceutical composition comprising at least one CSP inhibitor and a pharmaceutically acceptable carrier.
  • a method of treating or preventing a bacterial infection caused by a biofilm forming bacterium comprising administering a therapeutically effective amount of a pharmaceutical composition according to the invention.
  • a method of preventing dental plaque formation comprising administering a therapeutically effective amount of a pharmaceutical composition according to the invention.
  • 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.
  • a CSP inhibitor for the preparation of a medicament for treatment and prevention of an infection caused by a biofilm forming bacterium.
  • the CSP inhibitor is is a peptide analog ofS.mutans competence stimulating peptide (CSP) which inhibits biofilm formation.
  • 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.
  • the CSP inhibitor is a polypeptide having the amino acid sequence of SEQ ID No: 44.
  • the CSP inhibitor is an antibody specific for CSP or a fragment thereof.
  • the CSP inhibitor is an antisense oligonucleotide which inhibits CSP expression or transcription.
  • the CSP inhibitor is an antisense oligonucleotide which inhibits CSP peptide export.
  • compositions for inhibiting biofilm formation in Streptococcus spp comprising 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.
  • the composition further comprises one or more ingredients selected from a group consisting of: a surfactant, an antiseptic, and an antibiotic.
  • the composition comprises between 1 ⁇ g/ml and 100 ⁇ g/ml of the peptide.
  • 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 E ) No: 39, SEQ TD No: 42, SEQ TD No: 44, and SEQ TD No: 47.
  • 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: No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
  • 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.
  • the condition caused by dental plaque associated Streptococcus spp is selected from a group consisting of: dental caries, gingivitis, and endocarditis.
  • the peptide has the amino acid sequence of SEQ ID NO. 44.
  • 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.
  • the Streptococcus spp is
  • 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 NO:5].
  • ComC signal peptide precursor
  • ComD histidine kinase
  • ComE response regulator
  • Figure 2 shows the nucleic acid molecules SEQ ID Nos. 1, 3, 5, and
  • Figure 2 A S. mutans comC gene [SEQ ID NO: I]. 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 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 3 A 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].
  • 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 peptide!.
  • 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.
  • 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.
  • 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).
  • 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.
  • PCR polymerase chain reaction
  • Figure 9 shows the ComCDE local region [SEQ ID NO:21].
  • ComC first highlighted region; nucleotide 101 to 241
  • ComD second highlighted region; nucleotides 383 to 1708
  • ComE third highlighted region; nucleotides 1705 to 2457
  • 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 lOObp included both upstream and downstream (promoter is upstream).
  • Figure 1OA S. mutans comX gene [SEQ E) NO:22].
  • Figure 1OB S. mutans ComX protein [SEQ ID NO:23].
  • Figure 1OC S. mutans comX gene local region [SEQ ID NO:24].
  • Figure 11 shows the comA and comB nucleotide [SEQ ID NO:25] and
  • ComA and ComB are the components of the CSP exporter.
  • 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.
  • CSP synthetic signal peptide
  • Figure 13 is a schematic representation of quorum sensing circuit in S. mutans.
  • Figure 14 shows the effect of different concentrations of Hl on genetic transformation of S. mutans wild-type UAl 59. Results are expressed as the mean ⁇ SE of three independent experiments.
  • Figure 15 shows the effect of different concentrations of Hl on genetic transformation of S. mutans comD null mutant.
  • Figure 16 shows the effect of different concentrations ( ⁇ g/ml) of CSP and Hl on cell growth of S. mutatis wild-type UAl 59 in THYE at pH 5.5. Means OD 6OO values ⁇ SE. Results represent the average of three independent experiments.
  • Figure 17 shows the effect of different concentrations ( ⁇ g/ml) of CSP and Hl on cell growth of S. mutans wild-type UA159 in THYE at pH 7.5. Means OD 6O0 values ⁇ SE. Results represent the average of three independent experiments.
  • FIG 18 shows the effects of synthetic CSP analogues (B2, B3, C2,
  • Figure 19 shows the effect of E2 peptide on Streptococcus sobrinus biofilm formation.
  • Figure 20 shows the effect of E2 peptide on Streptococcus oralis biofilm formation.
  • Figure 21 shows the effect of E2 peptide on Streptococcus sanguis biofilm formation.
  • Figure 22 shows the effect of E2 peptide on Streptococcus mitis biofilm formation.
  • Figure 23 shows the effect of E2 peptide on Streptococcus gordonii biofilm formation.
  • Figure 24 shows the effect of E2 peptide on Streptococcus pneumoniae biofilm formation
  • 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.
  • CSP 21-amino acid peptide
  • SEQ ID NO:4 histidine kinase
  • SEQ ID NO:6 a response regulator
  • 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.
  • inhibitors 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.
  • 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.
  • Treatment or prevention of dental caries comprises addition of compounds that inhibit the stimulatory action of the 21-amino acid peptide [SEQ. ID NO:30] 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.
  • 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.
  • CSP Competence Stimulating Peptide
  • HK Histidine Kinase
  • RR Response Regulator
  • An isolated CSP from S. mutans is provided in accordance with certain embodiments of the present invention.
  • the peptide we work with is preferably chemically synthesized [SEQ ID NO: 14].
  • 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.
  • 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.
  • 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, hi another embodiment of the invention an isolated nucleic acid molecule encoding a HK polypeptide [SEQ ID NO:4] is disclosed.
  • 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, hi 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.
  • 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.
  • Certain embodiments of the invention include RR-encoding nucleic acid molecules and molecules having sequence identity or which hybridize to the RR- encoding 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 include RR [SE ID NO: 6] or polypeptides having sequence identity, which have RR activity, as prepared by the processes described in this application.
  • 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.
  • 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].
  • 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 ah, Nucleic Acid Res. 22:4673-4680.], described below, is most preferred.
  • Sequence A For example, if a nucleic acid molecule (called “Sequence A”) 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 10 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: I]. 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.
  • 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.
  • 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 non- conservative 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.
  • fragments and variants of CSP encompassed by the present invention should preferably have at least about 40%, 60%, 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: I]. Sequence identity is preferably measured with the ClustalW program (preferably using default parameters) (Thompson, JD et ah, Nucleic Acid Res. 22:4673-4680).
  • Nucleic acid molecules functionally equivalent to the CSP nucleic acid molecule in [SEQ ID NO:1] will be apparent from the following description.
  • 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 60%.
  • the mutated DNAs created in this manner should preferably encode a peptide having at least about 40%, preferably at least about 60%, at least about 80%, and more preferably at least about 90% 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.
  • [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: I].
  • 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.
  • CSP-encoding nucleic acids can be isolated using conventional DNA-DNA or DNA-RNA hybridization techniques.
  • 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, N.Y.).
  • 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 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: I]) 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 NO:1].). Similar methods are described in U.S. Patent No. 5,851,788, which is incorporated by reference in its entirety.
  • 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.
  • 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).
  • 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).
  • a detection reagent such as a detectable label
  • 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.
  • 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.
  • a peptide is capable of stimulating an acid tolerance response.
  • 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).
  • 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.
  • promoters can be inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-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).
  • 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.
  • 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.
  • 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-mediate ⁇ transformation, particle-bombardment- mediated 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.
  • 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.
  • 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.
  • 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.
  • 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 15 nucleotides of CSP in [SEQ ID NO: I].
  • Certain embodiments of the invention also include at least 15 consecutive nucleotides of [SEQ ID NO: I].
  • 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.
  • CSP nucleic acid molecules from other cells, or equivalent CSP-encoding cDNAs or synthetic DNAs can also be isolated by 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 (E. 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.
  • 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.
  • Peptide analogs were altered based on the amino acid sequence of S. mutans native CSP.
  • 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 nd , 3 rd , 4 th , 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 Hl is capable of inhibiting genetic competence.
  • mutans CSP (5 ⁇ g 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.
  • S. mutans peptide analogs are further tested for their ability to inhibit the formation 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 Fl [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
  • the S. mutans derived E2 [SEQ ID NO:44] peptide at a concentration as low as 5 ⁇ g/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, 20, 21, 22, 23, 24 and 25).
  • the anti-biofilm activity of E2 [SEQ TD 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).
  • 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 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.
  • 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.
  • 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 A”) has 90% identity to a portion of the peptide in [SEQ ID NO:30], 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.
  • 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 90% 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.
  • 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 amino acids.
  • 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.
  • 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.
  • peptide mimetics i.e., a modified peptide" or peptide or protein
  • a modified peptide or peptide or protein
  • characteristics such as solubility, stability, and/or susceptibility to hydrolysis and proteolysis. See for example, Morgan and Gainor, Ann. Rep. Med. Chem., 24:243-252 (1989).
  • 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.
  • 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 [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.
  • Variants produced by combinations of the techniques described above but which retain the same or similar CSP activity as naturally occurring CSP are also included in certain embodiments of the invention (for example, combinations of amino acid additions, and substitutions).
  • 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).
  • 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).
  • 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).
  • Variants of CSP encompassed by the present invention preferably have at least about 40% sequence identity, preferably at least about 60%, 75%, 80%, 90% 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.
  • 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.
  • 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 LD NO:27 and SEQ ID NO:28] may be prepared using the description in this application and techniques known in the art.
  • CSP variants and mutants are also applicable to the histidine kinase [SEQ ID NO:3 and SEQ ID NO:4], response regulator [SEQ ID NO:5 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 NO:25 and SEQ ID NO:26] or comB [SEQ ID NO:27 and SEQ ID NO:28] having activity.
  • 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.
  • U.S.E. Unique site elimination
  • 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.
  • Peptide analogs of S. mutans CSP peptide prepared by deleting and/or substitituting amino acids of the CSP peptides can be screened for biolf ⁇ lm formation inhibitions. Screening assays are described below.
  • the CSP inhibitors are also useful when combined with a carrier in a pharmaceutical composition.
  • the compositions are useful when administered in 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.
  • 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.
  • the pharmaceutical compositions are used to treat diseases caused by streptococcal infections such as dental caries, peridontal disease and endocarditis.
  • the pharmaceutical compositions are used to treat diseases caused by Actinomyces spp. and Streptococci spp.
  • 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.
  • compositions according to the invention may be prepared using a CSP inhibitor which is an antisense oligonucleotide.
  • CSP activity could be blocked by antisense mRNA which inhibits CSP expression or transcription.
  • the antisense oligonucleotide may be one which inhibits the activity of the exporter that secretes the CSP from the cell.
  • the CSP inhibitor is an antisense oligonucleotide which inhibits CSP expression or transcription.
  • 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.
  • the CSP inhibitor is an antisense oligonucleotide which" inhibits CSP peptide export.
  • 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.
  • 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.
  • compositions according to the invention may be prepared using an antibody or a fragment thereof, which selectively inhibits CSP activity.
  • an antibody or a fragment thereof which selectively inhibits CSP activity.
  • 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 Sfreptococci bacteria having impaired biofilm formation ability.
  • the inhibitory CSP peptide analog may be a synthetic peptide prepared using methods known in the art.
  • 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].
  • the pharmaceutical compositions are prepared using the E2 [SEQ ID NO:44] peptide.
  • compositions comprising CSP peptide analogs or pharmaceutically acceptable salts thereof, and in particular B3 [SEQ E) No:47], C2 [SEQ ID No:42], E2 [SEQ ID No:44], and Fl [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. sohrinus, 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.
  • pharmaceutically acceptable carrier or a “pharmaceutically acceptable salt” is meant ⁇ herein a material that is not biologically or otherwise undesirable, i.e., 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.
  • 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.
  • 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 described, for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA).
  • Carriers include saline and D5W (5% 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.
  • the excipients include serum albumin, glutamic or aspartic acid, phospholipids and fatty acids.
  • 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.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • 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.
  • 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.
  • 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.
  • 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.
  • disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • a process of producing cells genetically modified to produce a CSP derivative which inhibits transformation efficiency comprises administering to a patient S. niutans 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, WO02/44230.
  • a further aspect of the present invention provides the use in the preparation of a medicament for administration to a mammalian patient to alleviate dental caries, of viable, transfected S. mutans genetically modified to produce a CSP derivative which inhibits transformation efficiency.
  • a process of producing cells genetically modified to produce a CSP derivative which inhibits biofihn formation comprises administering to a patient cells genetically modified to produce a CSP derivative which inhibits biofihn formation.
  • 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.
  • compositions according to the invention can be administered by any suitable route known in the art.
  • the pharmaceutical composition can be administered topically to infected area.
  • the pharmaceutical composition may be administered orally, intravenously, or parenterally.
  • compositions 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.
  • a pharmaceutical composition comprising an antisense oligonucleotide with a pharmaceutical composition comprising a CSP peptide analog
  • a pharmaceutical composition comprising an antibody with a pharmaceutical composition comprising a CSP peptide analog
  • the pharmaceutical compositions may be prepared with one or more types of CSP inhibitors to yield a unitary dosage form.
  • compositions according to the invention may be desirable to administer with a known antibacterial agent such as an antibiotic.
  • a known antibacterial agent such as an antibiotic.
  • the pharmaceutical compositions which repress biofihn formation are also useful for rendering the bacterial cells more susceptible to antibiotics.
  • antibiotic refers to any compound known to one of ordinary skill in the art that will inhibit the growth of, or kill, bacteria.
  • the term “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, rifampichis, metronidazoles, quinolones, novobiocins, polyrnixins, gramicidins or any salts or variants thereof.
  • the antibiotic used will depend on the type of bacterial infection.
  • 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.
  • the present invention provides compositions useful for inhibiting and disrupting biofilms in Streptococcus spp.
  • the compositions comprise at least one peptide analogue of S. rnutans CSP which inhibits or disrupts biofilm formation in a Streptococcus spp.
  • the peptide analogues useful for practicing the invention include: Fl [SEQ ED NO. 37], Hl [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].
  • the compositions are prepared using the E2 [SEQ ID NO. 44] peptide.
  • 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.
  • 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.
  • 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 of Fl [SEQ ID NO. 37], Hl [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, hi a preferred embodiment of the invention, the pharmaceutical composition comprises the E2 [SEQ TD NO. 44] peptide analogue.
  • 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.
  • the pharmaceutical compositions according to the invention 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.
  • a known antibacterial agent such as an antibiotic
  • the antibiotic used will depend on the type of bacterial infection.
  • the biofilm inhibiting pharmaceutical compositions according to the invention also useful for rendering the bacterial cells more susceptible to antibiotics.
  • 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.
  • compositions according to the invention can be administered to humans or animals in the form of food, food additives, dentrif ⁇ ce gels, toothpaste, mouthwash, dental floss, denture wash, denture adhesive, chewing gum, candy, biscuits, soft drinks, or sports drinks.
  • 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).
  • additional ingredients including but not limited to: a surfactant, a chelating agent, an antibody, an antiseptic, and an antibiotic (see examples listed above).
  • 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], Hl [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].
  • the method comprises administering a therapeutically effective amount of E2 [SEQ ID NO. 44] peptide.
  • the peptide analogues maybe administered in the form of any of the pharmaceutical compositions encompassed by the present invention.
  • 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.
  • 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.
  • the method encompasses the treatment of infections caused by both oral and non-oral Streptococcus spp.
  • oral Streptococcus spp such as Streptococcus spp. including: Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis, sad 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.
  • the pharmaceutical compositions can be administered in the form of food, food additives, dentrif ⁇ ce gels, toothpaste, mouthwash, dental floss, denture wash, denture adhesive, chewing gum, candy, biscuits, soft drinks, or sports drinks.
  • the pharmaceutical compositions can be administered either locally or systemically depending on the nature and severity of the infection.
  • 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.
  • the therapeutically effective amount of the peptide analogue will be between 1 ⁇ g/ml and 1 mg/ml daily. In a further preferred embodiment, the therapeutically effective amount of the peptide analogue will be between 1 ⁇ g/ml and 100 ⁇ g/ml.
  • the peptide analogue administered is the E2 [SEQ ID NO. 44] peptide in an amount of between 1 ⁇ g/ml and 100 ⁇ g/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. Antimicrobial compositions
  • 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.
  • Antimicrobial compositions for inhibiting biofilm formation may comprise any of the peptide, antisense and antibody CSP inhibitors described above.
  • 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].
  • the antimicrobial compositions are prepared using the E2 [SEQ ID No:44] peptide.
  • the antimicrobial composition may further comprise additional ingredients including but not limited to: a surfactant, an antispetic and an antibiotic (see examples listed above).
  • 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 Fl [SEQ ID No:37], and the amount of the CSP inhibitor is preferrably between I ⁇ g/ml to 1 mg/ml.
  • 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 l ⁇ g/ml to lOO ⁇ g/ml.
  • Antibodies directed against the CSP would provide protection against caries.
  • Antibodies may be manufactured as described below.
  • a disclosed peptide [SEQ ID NO:2 or SEQ DD NO: 30] 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, LJ. et ah, "Monoclonal Antibody-Mediated Modulation of the Humoral Immune Response against Mucosally Applied Streptococcus mutans " (in press).
  • 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.
  • 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.
  • Vaccine compositions may comprise solid or liquid formulations such as gels, sprays, inhalants, tablets, toothpastes, mouthwashes or chewing gum.
  • cholera toxin can be used by coupling the peptide to its B-subunit to stimulate production of secretory antibody i.e., coupling to CTB.
  • Inhibitors are preferably directed towards CSP [SEQ ID NO:2 or SEQ
  • CSP [SEQ ID NO:2 or SEQ ID NO:30] with HK [SEQ ID NO:4] can include: contacting (i) 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 (i) 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 (i) and (ii).
  • 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 E) 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 E) NO:30] or CSP activity.
  • test compound levels may be either fixed or variable.
  • 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.
  • Monoclonal and polyclonal antibodies are prepared according to the description in this application and techniques known in the art.
  • 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-purif ⁇ cation of CSP or CSP- like peptides from crude extracts.
  • 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.
  • 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 immunolgical complex to ascertain the presence of CSP [SEQ ID NO:2] or a similar peptide.
  • kits 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 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.
  • the kit preferably includes 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.
  • [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 e ⁇ al. Infect Immun, 41:722-727 and Lindler and Macrina J Bacterid, 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.
  • DNA preferably plasmid DNA
  • S. mutans culture or culture of a bacteria expressing CSP [SEQ ID NO:1] or a variant thereof.
  • S. mutans is preferably grown in THYE plus 5% horse serum (THYE-HS). After 2-hr incubation, 1 ⁇ g/ml plasmid DNA or 10 ⁇ g/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 30- minute incubation equal amounts of DNA is added to each well (1 ⁇ g/ml plasmid or 10 ⁇ g/ml of chromosomal DNA) and incubation continued for another 2 hrs.
  • SCSP competence stimulating peptide
  • 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 (i.e., 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 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.
  • 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 N0:2]/HK [SEQ ID NO:4] /RR [SEQ ID NO: 6].
  • marker protein expression such as green fluorescent protein (GFP)
  • CSP green fluorescent protein
  • GFP green fluorescent protein
  • 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).
  • CSP [SEQ ID NO:2] 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.
  • 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 Svensater 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 37 0 C with 5% CO 2 for 2 hours.
  • 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 5% CO 2 and an aliquot of sample is taken to determine survival rate by viable cell counts.
  • 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.
  • histidine kinase [SEQ ID NO:3], CSP [SEQ ID NO:1] or response regulator [SEQ ID NO:5] are useful as research tools.
  • a cell or a cell line, such as an immortalized cell culture or a primary cell culture
  • 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.
  • histidine kinase [SEQ ID NO:3], CSP [SEQ ID NO:1] or response regulator [SEQ ID NO:5] nucleic acid molecules may be over- expressed 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.
  • 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 of polypeptides and peptides produced, its functionality and the phenotype of the cells.
  • the polypeptides and peptides are also useful for in vitro analysis of histidine kinase [SEQ ID NO:4], CSP [SEQ ID NO:2] or response regulator [SEQ ID NO:6] activity or structure.
  • the polypeptides and peptides produced can be used for microscopy or X-ray crystallography studies.
  • 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.
  • 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: (a) culturing cells containing a nucleic acid molecule which expresses histidine kinase [SEQ ID NO:4], CSP [SEQ ID NO.-l] 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
  • Suitable assays may be adapted from, for example, US patent no.
  • 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 37°C under an anaerobic condition.
  • biofilm medium 4X diluted Todd-Hewitt Yeast Extract supplemented with final concentration of 0.01% hog gastric mucin
  • 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.
  • SCSP synthetic competence stimulating peptide
  • 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 ⁇ l of fresh medium and are immediately spread on THYE agar plus appropriate antibiotics. Transformation frequency is determined after 48-h of incubation.
  • 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 genome database sequence and their nucleotide sequences determined. The deduced amino acid sequences are compared among the isolates by sequence alignment to confirm identity.
  • 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], comD [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 2-4), and transport and incorporate DNA.
  • the knockout mutants of com 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 ⁇ g/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 SO 4 , 35 mM NaCl, 2 mM MgSO 2 -7H 2 O, 0.2% (wt/vol) Casamino Acids and was supplemented with filter-sterilized vitamins, (0.04 mM nicotinic acid, 0.1 mM pyridoxine HCl, 0.01 mM pantothenic acid, 1 ⁇ M riboflavin, 0.3 ⁇ M thiamin HCl, and 0.05 ⁇ M D-biotin), amino acids (4 mM L-glutamic acid, 1 mM L-arginine HCl, 1.3 mM L-cysteine HCl, and 0.1 mM L-tryptophan) and 20 mM glucose.
  • filter-sterilized vitamins (0.04 mM nicotinic acid, 0.1 mM pyridox
  • S. mutans UAl 59 comD null mutant was constructed by a PCR- based 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 comD deletion mutant are P1-HK13 (5'- CACAACAACTTATTGACGCTATCCC-3'), P2-HK13 (5'-
  • GGCGCGCCAACTGGCAACAGGCAGCAGACC-3' P3-HK13 (5'-GGCC GGCCTCAAAACGATGCTGTCAAGGG-3'), P4-HK13 (5'-AGATTATCATTGGC GGAAGCG-3'X Erm-19 (5'-GGCGCGCCCCGGGCCCAAAATTTGTTTGAT-S'), and E ⁇ n-20 (5'- GGCCGGCCAGTCGGCAGCGACTCATAGAAT-3').
  • 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], Hl [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).
  • Fl and Hl 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.
  • 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.
  • the peptides were dissolved to 1 mg per ml in sterile distilled deionized water.
  • 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 ⁇ l of cell suspension into each well containing 200 ⁇ l of fresh THYE medium.
  • the cell suspensions were pre-adjusted to the same optical density at 0.D 600 before inoculation.
  • the plates were then placed in the Bioscreen system, which was set up to read optical density automatically every 15 minutes with shaking. The readings of optical density were automatically recorded and converted into growth curves.
  • Each assay was performed in quadruplicate.
  • strains include:
  • BM71, GB14, H7, JH1005, LTIl, 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, the medium was supplemented with either erythromycin (Em) (10 ⁇ g/ml) or kanamycin (Km) (500 ⁇ g/ml).
  • Em erythromycin
  • Km kanamycin
  • 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% CO2.
  • 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 ⁇ g erythromycin per ml or 500 ⁇ g kanamycin per ml for S. mutans, and 50 ⁇ g kanamycin per ml for E. coli.
  • 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 37 0 C in an anaerobic chamber (5% CO2). In liquid media, cultures were incubated in closed screw-cap tubes without agitation at 37°C in an anaerobic chamber (5% CO 2 ).
  • THYE yeast extract
  • 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 (BM).
  • BM 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 of biofihns. After cultures were incubated at 37 0 C for 20 h under an anaerobic condition, fluid medium was removed for viable cell counts.
  • 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 an anaerobic condition. Formation of biofilms was quantified by viable cell counts after 48 h of incubation.
  • Biofilms were developed in 96-well polystyrene microtiter plates. The growth of the biofilm was initiated by inoculating 10 ⁇ l of an overnight S. mutatis UAl 59 culture into 300 ⁇ l of SDM-glucose containing different concentrations (0, 0.1, 0.5, 2, and 5 ⁇ g per ml) of peptide analogs 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 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.
  • Biofilms were quantified by measuring the absorbance of stained biofilms at 490 nm with a microplate reader (model 3550; Bio-Rad Laboratories, Richmond, CA).
  • 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 ⁇ l of an overnight Streptococcus spp.
  • microtiter plates were then incubated at 37°C in an anaerobic chamber (5% 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% crystal violet for 10 minutes, rinsed with sterile distilled water and air dried for 15 minutes. Biofilm was quantified by measuring the absorbance of stained biofihn at 630 nm with a microplate reader.
  • S. mutans UAl 59 wild-type cells were assayed for genetic transformation. Overnight cultures of S. mutans UAl 59 were diluted (1:20) with prewarmed THYE broth and incubated at 37 0 C 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 ⁇ g/ml of plasmid pDL289 and different concentrations (0, 0.1, 0.5, 2, and 5 ⁇ g 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 0 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.
  • 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 fermentor 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 Partner 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 IM KOH or IM HCl.
  • 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.
  • glass rods were aseptically inserted into the chemostat for the initiation of biofilm formation.
  • biofilms of different ages were removed from the cultures for both genetic transformation and quantification of biofilms using viable cell counts.
  • SCSP competence stimulating peptide
  • Plasmid DNA included an integrative plasmid, pV AGTFA carrying an erythromycin resistance (Em r ) determinant and a fragment of the S. mutans gtfA gene.
  • the replicative plasmid, pDL289 carrying a kanamycin resistance gene (Km 1 ) was also used.
  • Chromosomal DNA harboring an Em r gene was prepared from a recombinant S. mutans strain harboring a chromosomally integrated copy of pVAGTFA.
  • Example 2 E2 CSP Peptide Analogue Inhibits Biofilm Formation in S. sobrinus, S. sanguis, S. sordonii, S. oralis and S. mitis [00229] An in vitro assay was performed as described in Materials and
  • 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 ⁇ g/ml showed inhibitory effects on both growth and biofilm formation in all the five organisms tested.
  • the percent inhibition of biofilm formation in these organisms varied from 40 to 75% (FIGS. 19, 20, 21, 22, and 23).
  • 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).
  • Example 3 E2 CSP Peptide Analogue Inhibits Biofilm Formation in Streptococcus pneumoniae
  • ComX is a unique link between multiple quorum sensing outputs and competence in Streptococcus pneumoniae. MoI. Microbiol. 50:623-633.

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Abstract

L'invention concerne des polypeptides isolés capables d'inhiber la compétence génétique de S. mutans et la formation de biofilm de S. mutans. On décrit aussi des analogues peptidiques de peptides stimulant la compétence de S. mutans, des compositions correspondantes, et des utilisations correspondantes qui permettent d'inhiber la formation de biofilm dans les streptocoques et de traiter et de prévenir les affections engendrées par les streptocoques associés à la plaque dentaire.
EP05814632A 2004-12-06 2005-12-06 Molecules d'acides nucleiques, peptides signaux, et procedes de traitement Withdrawn EP1833844A4 (fr)

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Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070178054A1 (en) * 2005-11-09 2007-08-02 Madhyastha Srinivasa Oral antimicrobial composition
GB0608966D0 (en) * 2006-05-05 2006-06-14 Norwegian University Of Life S Method and product
CA2661634C (fr) 2006-09-06 2017-03-28 The Regents Of The University Of California Peptides antimicrobiens cibles selectivement et leur utilisation
GB0618612D0 (en) * 2006-09-21 2006-11-01 Smith & Nephew Medical device
EP2248823A1 (fr) * 2009-04-28 2010-11-10 Institut National de la Recherche Agronomique Nouveau peptide de stimulation de compétences
WO2012159216A1 (fr) * 2011-05-25 2012-11-29 Kane Biotech Inc. Peptides régénérant le tissu osseux
WO2014033314A1 (fr) 2012-09-03 2014-03-06 Uab Bioseka Oligonucléotides antisens ciblant des glucosyltransférases bactériennes
LT6214B (lt) * 2013-10-07 2015-08-25 Uab "Bioseka" Priešprasminiai oligonukleotidai aterosklerozės ir kardiovaskulinių infekcijų prevencijai

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2332733C (fr) * 2000-04-10 2012-10-02 Dennis G. Cvitkovitch Sequences-signal, molecules d'acide nucleique et methode de traitement des caries
US6923962B2 (en) * 2000-04-10 2005-08-02 Dennis Cvitkovitch Signal peptides, nucleic acid molecules and methods for treatment of caries
US7087228B2 (en) * 2002-07-03 2006-08-08 University Of Southern California Preventing tooth decay and infective endocarditis using natural oligopeptides
US7625554B2 (en) * 2002-09-05 2009-12-01 Brod Staley A Treatment of alzheimer's disease

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CVITKOVITCH DENNIS G ET AL: "Quorum sensing and biofilm formation in Streptococcal infections." THE JOURNAL OF CLINICAL INVESTIGATION DEC 2003, vol. 112, no. 11, December 2003 (2003-12), pages 1626-1632, XP002522406 ISSN: 0021-9738 *
See also references of WO2006060903A1 *
SUNTHARALINGAM P ET AL: "Quorum sensing in streptococcal biofilm formation" TRENDS IN MICROBIOLOGY, ELSEVIER SCIENCE LTD., KIDLINGTON, GB, vol. 13, no. 1, 7 December 2004 (2004-12-07), pages 3-6, XP004705444 ISSN: 0966-842X *
VATS NEERAJ ET AL: "Active detachment of Streptococcus mutans cells adhered to epon-hydroxylapatite surfaces coated with salivary proteins in vitro" ARCHIVES OF ORAL BIOLOGY, vol. 45, no. 4, April 2000 (2000-04), pages 305-314, XP002522407 ISSN: 0003-9969 *

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AU2005313797A1 (en) 2006-06-15
JP2008522985A (ja) 2008-07-03
AU2005313797A2 (en) 2006-06-15
EP1833844A4 (fr) 2009-05-13
NZ556114A (en) 2010-02-26
JP4932731B2 (ja) 2012-05-16
CA2630088A1 (fr) 2006-06-15

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