JP2008522985A - Signal peptide, nucleic acid molecule and therapeutic method - Google Patents

Abstract

  The present invention provides isolated polypeptides that can inhibit the genetic capacity of S. mutans and biofilm formation of S. mutans. The present invention further provides peptide analogs of the S. mutans receptivity stimulating peptides, the compositions described above, and the above described treatment and prevention of streptococcal biofilm formation and treatment of symptoms caused by plaque-associated streptococcus Provide use.

Description

  The present invention relates generally to compounds and methods for inhibiting or destroying bacterial biofilms that are primarily responsible for human and animal infections and biological contamination of surfaces susceptible to bacterial accumulation.

Bacteria often adhere to and accumulate on surfaces, making themselves resistant to removal or sterilization by mechanical and chemical means. This can result in persistent and chronic infections, as well as soiling of the device in contact with fluids containing colonization bacteria. Bacteria respond to signals coming from the proximity, density, and bacterial entities of neighboring microorganisms. Through the quorum sensing process, bacteria determine population density indirectly by sensing the concentration of secreted signal molecules (Bassler, 2002). The ability of bacteria to communicate with each other through QS and act collectively as a group has significant advantages, including more efficient growth, better access to resources and the right place, and stronger protection against competitors. (Jefferson, 2004). Many QS systems have been studied that have diverse effects on the physiology of bacterial cells. Examples include Pseudomonas aeruginosa biofilm differentiation (Davies et al. 1998), Serratia Liquefaciens swarming motility (Eberl et al. 1999), Streptococcus pneumoniae (Streptococcus pneumoniae) (Lee & Morrison, 1999) and Streptococcus mutans (Li et al. 2001) receptive capacity development and induction of virulence factors of Staphylococcus aureus (Ji et al. 1995).
Control of bacterial biofilms is desirable for almost all human businesses where solid surfaces are introduced into non-sterile aqueous environments. US Pat. No. 6,024,958 describes peptides that attempt to control biofilm formation by preventing bacterial adhesion to the teeth. Besides the emergence in caries, medical examples of biofilm growth include cases involving endogenous medical devices, joint implants, prostatitis, endocarditis and respiratory infections. In fact, the Centers for Disease Control and Prevention (CDC) estimates that 65% of human bacterial infections require biofilm. Non-medical examples of biofilm settlement formation include water and beverage lines, cooling towers, radiators, aquaculture contamination, submersible pumps and impellers, hulls of civilian, fishing and military ships, and literally all situations where biological contamination occurs. is there. The potential benefits of basic research focused on biofilm physiology and genetics with the ultimate goal of controlling surface-mediated bacterial growth are endless.

The interest of biofilm-proliferating cell research is that, in part, the growth of biofilms can exist in physical and physiological states that can increase the cell's resistance to antimicrobial compounds and mechanical forces It is growing because it provides a micro environment (reviewed in Costerton & Lewandowski (Adv Dent Res, 11: 192-195)). Biofilm growth can also facilitate the transfer of genetic information between different species (Christensen et al. Appl Environ Microbiol, 64: 2247-2255). Recently obtained evidence suggests that biofilm-grown cells can display a dramatically different phenotype when compared to siblings of said cells grown in liquid media. In some, this altered physiological state is due to gene activation initiated by contact with the surface (Finlay & Falkow, Microbiol Molec Rev, 61: 136-169) or perceived cell density by these cells. (Quorum sensing) has been shown to result from signal molecules produced by bacteria (Davies et al. Appl Environ Microbiol, 61: 860-867). Biofilms can also function as “genotype reservoirs” that allow the persistence, transmission and selection of genetic elements that confer resistance to antimicrobial compounds.
Caries and periodontal diseases are the most common chronic infectious diseases that affect humans and are always accompanied by plaque that forms as a biofilm on the tooth surface. Thus, the goal of prevention of caries and periodontal disease is plaque control.

Plaque is caused by the continuous adherence of diverse bacteria and depends on both the species involved and the surface composition (Kawashima et al. 2003, Oral Microbiol Immunol, 18: 220-225). Oral streptococci and actinomycetes are the first to appear on the tooth surface. Streptococcus (Streptococcus) accounts for almost 20% of salivary bacteria, including Streptococcus species, such as Streptococcus mutans, Streptococcus sobrinus, St. sanguis ), Streptococcus gordonii, St. oralis and St. mitis. Two “mutans” of Streptococcus, S. mutans and S. sobrinus are directly involved in the initiation of caries (Devulapalle et al. 2004, Carbohydr Res 339: 029-1034). S. mutans, along with its role as an opportunistic pathogen, has evolved to rely on the biofilm lifestyle for oral survival and persistence, thus forming a biofilm pathogenic Streptococcus (RA Burne, J Dent Res, 1998, 77: 445-452).
Many streptococci use population sensing systems to regulate several physiological processes. These include exogenous DNA uptake capacity, acid tolerance performance, biofilm formation ability, and ability to become pathogenic. These collective sensing systems are primarily composed of small receptive stimulating peptides (CSPs) that are detected by neighboring cells via histidine kinase / response regulator pairs. It has been shown that analogs of population sensing peptides (analogs of CSP) can competitively inhibit S. mutans biofilm formation (Cvitkovitch et al. US Patent Application 20020081302, 2002).
Any known type of antibiotic against S. mutans cannot be satisfactorily controlled. There is a need to identify new ways to control S. mutans-induced caries.

SUMMARY OF THE INVENTION According to certain embodiments of the invention, compounds are provided that competitively inhibit the binding of CSP (SEQ ID NO: 30) to S. mutans histidine kinase (SEQ ID NO: 4). . In certain embodiments, the compound is a peptide or antibody. In some embodiments, the compound is a derivative of SEQ ID NO: 2, a fragment of SEQ ID NO: 2, or a derivative of a fragment of SEQ ID NO: 2.
According to certain embodiments of the present invention, there is provided a method for producing the compounds described above. In another embodiment of the present invention, there is provided a method of using a compound as described above for inhibiting growth of S. mutans, suppressing caries, or improving dental health.
In one aspect of the invention there is provided an isolated polypeptide comprising a fragment of SEQ ID NO: 2 or SEQ ID NO: 30 and capable of inhibiting the genetic capacity of S. mutans.
In one aspect of the invention, there is provided an isolated polypeptide that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 30 and capable of inhibiting the genetic capacity of S. mutans.
In certain embodiments of the invention, 1-5 amino acids have been removed from the COOH terminus of SEQ ID NO: 2 or SEQ ID NO: 30.
In one aspect of the invention, an isolated polypeptide comprising a fragment of SEQ ID NO: 2 or SEQ ID NO: 30 and capable of inhibiting S. mutans biofilm formation is provided.
In one aspect of the invention, there is provided 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.
In certain embodiments of the invention, 1-5 amino acids of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 30 are modified to include up to 1 amino acid substitution each for 10 amino acids.

In one aspect of the invention, a composition for reducing biofilm formation of S. mutans comprising a peptide derivative of CSP is provided.
In one aspect of the invention, a composition for reducing the transformation efficiency of S. mutans comprising a peptide derivative of CSP is provided.
In one embodiment of the invention, the peptide analog has a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47. It is.
In one embodiment of the invention, the peptide analog is a peptide having the amino acid sequence of SEQ ID NO: 44.
In one aspect of the invention, a cell genetically engineered to produce any of the compositions of the invention set forth above is provided.
In one embodiment of the invention, the cell is a bacterium.
In one embodiment of the invention, the cell is S. mutans.
In one aspect of the invention there is provided the use of a cell of the invention for the purpose of producing any of the compositions of the invention indicated above.
In one aspect of the invention, a composition comprising a cell of the invention and a pharmaceutically acceptable adjuvant is provided.
In one aspect of the invention, there is provided a method of inhibiting the growth of S. mutans comprising administering a composition according to any of claims 9 to 12 or 17.
In one aspect of the invention, there is provided a method for inhibiting caries comprising administering any of the compositions of the invention set forth above.
In one aspect of the invention, there is provided a method for improving dental health comprising administering any of the compositions of the invention set forth above.
In one aspect of the invention, an isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47, Provided.

In one aspect of the invention, peptide analogs of S. mutans receptivity stimulating peptide (CSP) are provided that inhibit biofilm formation.
In one aspect of the invention, a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47 and orally acceptable. A composition that inhibits biofilm formation is provided.
In one aspect of the invention, a pharmaceutical composition is provided comprising at least one CSP inhibitor and a pharmaceutically acceptable carrier.
In one aspect of the invention, there is provided 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 of the invention.
In one aspect of the invention, there is provided a method for preventing plaque formation comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention.
In one aspect of the invention, there is provided a method of treating or preventing a condition caused by bacteria associated with dental plaque comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention.
One aspect of the invention provides the use of a CSP inhibitor for the manufacture of a medicament for treating or preventing an infection caused by a biofilm-forming bacterium.
In one embodiment of the invention, the CSP inhibitor is a peptide analog of S. mutans receptivity stimulating peptide (CSP) that inhibits biofilm formation.
In another embodiment of the invention, the CSP inhibitor comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47. It is polypeptide which has.
In one embodiment of the invention, the CSP inhibitor is a polypeptide having the amino acid sequence of SEQ ID NO: 44.

In one embodiment of the invention, the CSP inhibitor is an antibody or fragment thereof specific for CSP.
In one embodiment of the invention, the CSP inhibitor is an antisense oligonucleotide that inhibits CSP expression or transcription.
In one embodiment of the invention, the CSP inhibitor is an antisense oligonucleotide that inhibits export of CSP peptides.
In one aspect of the invention, there is provided a composition that inhibits biofilm formation of Streptococcus species, comprising: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: At least one peptide having an amino acid sequence selected from the group consisting of: 47.
In one embodiment of the invention, the composition further comprises one or more ingredients selected from the group consisting of surfactants, preservatives and antibiotics.
In one embodiment of the invention the composition comprises 1 μg / mL to 100 μg / mL of the peptide.
In one aspect of the invention, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47, for the manufacture of a medicament that inhibits biofilm formation in Streptococcus species. There is provided the use of at least one peptide having an amino acid sequence selected from the group consisting of:
In one aspect of the invention, from SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47, intended to prevent or inhibit Streptococcus species biofilm formation. Use of at least one peptide having an amino acid sequence selected from the group consisting of is provided.
In one aspect of the invention, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 37, intended to treat or prevent symptoms caused by Streptococcus species associated with dental plaque, The use of at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 47 is provided.

In one aspect of the invention, the therapeutic of at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47. A method of treating or preventing Streptococcus spp. Infection in a patient in need thereof, comprising administering an effective amount.
In one aspect of the invention, the therapeutic of at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47. Methods are provided for inhibiting or preventing Streptococcus sp. Biofilm formation in a patient in need thereof, comprising administering an effective amount.
In one aspect of the invention, the therapeutic of at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47. Methods are provided for treating or preventing symptoms caused by plaque-associated Streptococcus species in a patient in need thereof, including administering an effective amount.
In one embodiment of the invention, the symptoms caused by Streptococcus species associated with plaque are selected from the group consisting of caries, gingivitis and endocarditis.
In one embodiment of the invention, the peptide has the amino acid sequence of SEQ ID NO: 44.
In one embodiment of the present invention, the Streptococcus species is Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus ococcus ococcus Strococcus ococcus mitis), Streptococcus salivarius, Streptococcus cristatus, Streptococcus pneumoniae, S. pyogenes, and Streptococcus agaloccus agaloccus Is done.
In one embodiment of the invention, the Streptococcus species is Streptococcus pneumoniae.
These and other embodiments, features and advantages of the present invention will become apparent with reference to the following detailed description and drawings.

DETAILED DESCRIPTION OF THE INVENTION In some gram positive bacteria (including Streptococcus mutans), when certain histidine kinase receptors present in the cell membrane are destroyed, the cells are unable to grow biofilms. Cells that proliferate in this biofilm environment use small peptide signal molecules to activate peripheral cell receptors, thereby exchanging information for biofilm formation. This same signal peptide and histidine kinase are also required to induce genetic tolerance (cell ability to take up DNA from the extracellular environment) as well as acid tolerance ability (cell ability to survive at pH levels as low as pH 3.0). It is. The mechanism of blocking activation of the signal molecule histidine kinase receptor provides a novel way to control microbial biofilms alone or in combination with chemical or physical means.
We have identified an S. mutans locus consisting of three genes that encode the following: 1) Peptide precursor (sequence) processed into 21 amino acid secreted peptide (CSP) (SEQ ID NO: 30) during export Number: 2); 2) Histidine kinase that functions as a cell surface receptor activated by the peptide (SEQ ID NO: 4); 3) Focused on the genetic acceptability, biofilm formation and acid resistance of S. mutans Response regulator (SEQ ID NO: 6) that activates many other genes that are involved These characteristics are believed to be the result of the ability of bacteria to cause caries. If the inactivation of any of these three genes, or the interaction or activity of any of their encoded proteins, is impaired, the ability of the bacteria to take up foreign DNA, biofilm formation and acid pH resistance is destroyed. I will.

  Streptococcus mutans is a resident bacterium in the biofilm environment of plaque, which is a bacterial matrix and extracellular material attached to the tooth surface. Under appropriate environmental conditions, the pH of the S. mutans population and surrounding plaque will drop. S. mutans, the most acid-resistant microorganism that settles in plaque, increases in number in this acidic environment and eventually becomes the dominant member of the plaque community. This situation ultimately leads to tooth enamel dissolution and advances caries. We control the accumulation and acid resistance of this bacterium, making it difficult for the bacterium to cause caries. We achieve this goal by using inhibitors of extracellular signal peptides that promote the expression of genes required for S. mutans biofilm formation and acid resistance. Compounds that inhibit the action of the peptides are disclosed. These inhibitors may include peptides, antibodies, or other substances that specifically inhibit the gene family that is activated as a result of activation of histidine kinase and activation of histidine kinase by the signal molecule. . Inhibitors include modified structures of wild-type mature CSP peptides in which amino acids have been removed from the N- and / or COOH termini and / or internal amino acid residues have been replaced. We deleted one, two to five, six to ten, and ten to fifteen amino acids from the wild-type mature CSP peptide (eg from either end) and competitive binding of the signal peptide to histidine kinase Inhibition is measured (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acids are deleted and inhibition is measured). Inhibitors also include antibodies made against the 21-amino acid CSP (SEQ ID NO: 30) alone or in combination with larger molecules to increase immunogenicity. We also test inhibitors described in the literature (Barrett et al. Proc Natl Acad Sci USA, 95: 5317-5322) and measure competitive inhibition of binding of signal peptides to histidine kinase.

In addition to identifying the genes encoding this signaling / sensing system, we identified and chemically synthesized a 21-amino acid peptide (SEQ ID NO: 14) that promotes S. mutans biofilm formation and acid resistance. . By searching literature and databases, genes similar to this signal-receptor system are present in most Gram-positive bacteria, and therefore one inhibitor or a family of related inhibitors is effective in inhibiting biofilm formation in a large group of bacteria. It became clear to get.
The treatment or prevention of caries involves the addition of a compound that inhibits the promoting action of the 21-amino acid peptide (SEQ ID NO: 30) on S. mutans biofilm formation and acid resistance. This is accomplished by adding these compounds to the biofilm and / or incorporating these inhibitors into materials that control growth on the surface. These include delivery by topical application alone or with other compounds (including toothpastes, mouth washes, foods or food additives).
Streptococcus mutans is also involved in the development of endocarditis. Inhibitors of biofilm formation and therefore aggregation are equally useful for treating these bacterial infections.
We also have other plaque bacterial species, such as Actinomyces spp. And other Streptococcus (eg, Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis and Streptococcus oralis ) Was identified as a compound that inhibits biofilm formation. Streptococcus is about 20% of salivary bacteria. This compound comprises the S. mutans wild type mature CSP, wherein amino acids have been removed from the N- and / or C-terminus of the S. mutans wild type mature CSP peptide and / or have internal amino acid residue substitutions. Includes modified structures of peptides.

Identification and characterization of receptor-stimulating peptides (CSP), histidine kinase (HK) and response regulator (RR)
Receptivity stimulating peptides : CSPs isolated from S. mutans are provided according to certain embodiments of the invention. Furthermore, provided in accordance with certain embodiments of the present invention is a recombinant isolation produced by a cell comprising a nucleic acid molecule (SEQ ID NO: 1) encoding CSP operably linked to a promoter. CSP (SEQ ID NO: 2). Further provided according to certain embodiments of the present invention is an isolated nucleic acid molecule (SEQ ID NO: 1) encoding CSP. The peptide we use for the experiment is preferably chemically synthesized (SEQ ID NO: 14).
A CSP-encoding nucleic acid molecule (SEQ ID NO: 1) and a molecule that has sequence identity with or hybridizes with the CSP coding sequence and further encodes a peptide having CSP activity (a preferred percentage of sequence identity is And vectors containing said molecules are provided according to various embodiments of the present invention. In certain embodiments of the invention, there are provided peptides having CSP (SEQ ID NO: 2) or sequence identity (preferred percentages are described below) or having CSP activity. The nucleic acid molecules and peptides disclosed herein can be obtained from S. mutans, and furthermore, they can be isolated from natural sources, synthetic or recombinant. CSP (SEQ ID NO: 2) or peptides having sequence identity and having CSP activity (prepared by the methods described in this application) are also provided according to the present invention.

Histidine kinase : According to certain embodiments of the present invention, HK (SEQ ID NO: 4) isolated from S. mutans is disclosed. Also disclosed is a recombinant isolated HK polypeptide produced by a cell comprising a nucleic acid molecule encoding HK (SEQ ID NO: 3) operably linked to a promoter. In yet another embodiment of the invention, an isolated nucleic acid molecule encoding an HK polypeptide (SEQ ID NO: 4) is disclosed.
HK-encoding nucleic acid molecules and molecules that have sequence identity with or hybridize with the HK-encoding sequence (SEQ ID NO: 3) and further encode proteins with HK activity (a preferred percentage of sequence identity is described below. And vectors containing said molecules are disclosed as part of the present invention. In accordance with some embodiments of the invention, polypeptides having HK (SEQ ID NO: 4) or sequence identity (preferred percentages are described below) or HK activity are disclosed. The nucleic acid molecules and peptides disclosed herein can be obtained from S. mutans, and furthermore they can be isolated from natural sources, synthetic or recombinant. Furthermore, provided according to certain embodiments of the invention are HK (SEQ ID NO: 4) or peptides having sequence identity and HK activity (prepared by the methods described in this application). is there.

Response modulators : According to certain embodiments of the invention, RR (SEQ ID NO: 6) isolated from S. mutans is disclosed. A recombinant isolated RR polypeptide (SEQ ID NO: 6) produced by a cell containing a nucleic acid molecule (SEQ ID NO: 5) encoding RR operably linked to a promoter is also present in certain other types of the invention. Provided according to an embodiment. Yet another embodiment of the present invention includes an isolated nucleic acid molecule encoding an RR polypeptide.
Certain embodiments of the invention include an RR-encoding nucleic acid molecule and a polypeptide having sequence identity with or hybridizing to the RR-encoding sequence (SEQ ID NO: 5) and further having RR activity. And vectors containing these molecules (a preferred percentage of sequence identity is described below). Some embodiments of the invention also include polypeptides having RR (SEQ ID NO: 6) or sequence identity (preferred percentages are described below) or having RR activity. The nucleic acid molecules and polypeptides of the present invention can be obtained from S. mutans, and they can be isolated from natural sources, synthetic or recombinant. Certain embodiments of the present invention include RR (SEQ ID NO: 6) or peptides having sequence identity and having RR activity (prepared by the methods described in this application).
The comA and comB nucleotide sequences (SEQ ID NO: 25 and SEQ ID NO: 27) and amino acid sequences (SEQ ID NO: 26 and SEQ ID NO: 28) are also characteristic of certain embodiments of the present invention. comA and comB are components of the CSP exporter. Considerations regarding variants, sequence identity, etc. for CSP, HK, RR apply to both the complete sequences shown in the figure and the bracketed parts (coding regions) of those sequences. The peptides and polypeptides may be natural, recombinantly produced, or synthetic.

Functionally Equivalent Nucleic Acid Molecules Certain embodiments of the present invention include nucleic acid molecules that are functional equivalents of all or part of the CSP sequence of SEQ ID NO: 1. (In this application, a nucleic acid molecule may also be referred to as a DNA sequence or a nucleotide sequence. These terms all have the same meaning as the nucleic acid molecule). Functionally equivalent nucleic acid molecules are DNA and RNA encoding a peptide having the same or similar CSP activity as the CSP peptide shown in SEQ ID NO: 2 (eg, genomic DNA, complementary DNA, synthetic DNA, and messenger) RNA molecules). A functionally equivalent nucleic acid molecule can encode a peptide comprising a region of the CSP peptide (SEQ ID NO: 2), or more preferably a region having sequence identity with the complete CSP peptide. Identity is calculated according to methods known in the art. Most preferred is the ClustalW program described below (preferably using default parameters) (JD Thompson et al. Nucleic Acid Res 22: 4673-4680). For example, if a nucleic acid molecule (assuming “sequence A”) is 90% identical to a portion of the nucleic acid molecule of SEQ ID NO: 1, then sequence A is preferably the reference portion of the nucleic acid molecule of SEQ ID NO: 1. (However, sequence A can contain up to 10 point mutations (eg, substitution with other nucleotides) for each 100 nucleotides of the reference portion of the nucleic acid molecule of SEQ ID NO: 1). The mutations described in this application preferably do not destroy the reading frame of the coding sequence. Nucleic acid molecules that are functionally equivalent to a CSP sequence can occur in a variety of forms, as described below.
The nucleic acid molecule can encode a conservative amino acid change in the CSP peptide (SEQ ID NO: 2). Certain embodiments of the invention include functionally equivalent nucleic acid molecules that encode conservative amino acid changes within the CSP amino acid sequence and produce silent amino acid changes.

The nucleic acid molecule can encode non-conservative amino acid substitutions, additions or deletions in the CSP peptide. Some embodiments of the invention include functionally equivalent nucleic acids that produce non-conservative amino acid changes within the CSP amino acid sequence of SEQ ID NO: 2. Functionally equivalent nucleic acid molecules have non-conservative amino acid substitutions (preferably chemically similar amino acid substitutions), additions or deletions, but the same or similar CSP as the CSP peptide shown in SEQ ID NO: 2. Peptides that also retain activity, DNA and RNA encoding peptides and proteins are included. The DNA or RNA can encode a fragment or variant of CSP. Fragments are useful as immunogens and in immunogenic compositions (US Pat. No. 5,837,472). The CSP or CSP-like activity of such fragments and variants is identified by the assay described below. Fragments and variants of CSPs encompassed by the present invention are not identical to the sequence of SEQ ID NO: 1, preferably a naturally occurring CSP nucleic acid molecule, or a region of the sequence (eg coding sequence), or the nucleic acid It should have at least about 40%, 60%, 80% or 95% sequence identity with one of the conserved domains of the molecule. Sequence identity is preferably measured with the ClustalW program (preferably using default parameters) (JD Thompson et al. Nucleic Acid Res 22: 4673-4680).
A nucleic acid molecule functionally equivalent to the CSP nucleic acid molecule of SEQ ID NO: 1 will be apparent from the following description. For example, the sequence shown in SEQ ID NO: 1 can be altered in its full length by natural or artificial mutations (eg, partial nucleotide insertions or deletions), and thus the full length of the coding sequence in SEQ ID NO: 1. Assuming 100%, functionally equivalent nucleic acid molecules preferably have a length of about 60-120% of the coding sequence, more preferably about 80-110%. Fragments can be less than 60%.
A portion such that several codons encode different amino acids within the sequence of the nucleic acid molecule, but the resulting peptide retains a CSP activity that is the same as or similar to the activity of a naturally occurring CSP peptide (SEQ ID NO: 2). Nucleic acid molecules that contain natural or artificial mutations (typically less than 80%, preferably less than 60%, more preferably less than 40%) of the total length are functionally equivalent nucleic acid molecules. The mutant DNA generated in such a manner is preferably at least about 40%, preferably at least about 60%, at least about 80%, more preferably at least about 90% with the amino acid sequence of the CSP peptide of SEQ ID NO: 2. It should encode a peptide with 95% sequence identity. Preferably, sequence identity is assessed by the ClustalW program.

Since the genetic code is degenerate, the nucleic acid sequence of SEQ ID NO: 1 is not the only sequence that can encode a peptide having CSP activity. The present invention includes a nucleic acid molecule having essentially the same genetic information as the nucleic acid molecule shown in SEQ ID NO: 1. Nucleic acid molecules (including RNA) that contain one or more nucleic acid variations compared to the sequences described in this application and that result in the production of the peptide shown in SEQ ID NO: 2 are within the scope of the various embodiments of the present invention. include.
Other functionally equivalent forms of CSP-encoding nucleic acids can be isolated using DNA-DNA or DNA-RNA hybridization techniques. Accordingly, certain embodiments of the present invention also include a nucleic acid molecule that hybridizes to one or more of the sequences of SEQ ID NO: 1 or a complementary sequence thereof, and a CSP produced by the sequence of SEQ ID NO: 1 or a variant thereof. Nucleic acid molecules encoding the expression of peptides, peptides and proteins that exhibit the same or similar activity as that of the peptide are included. Such a nucleic acid molecule preferably hybridizes with the sequence of SEQ ID NO: 1 under conditions of moderate to high stringency (see Sambrook et al. Molecular Cloning: A Laboratory Manual, ( Latest edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). A high stringency wash contains a low salt concentration (preferably about 0.2% SSC) and a low stringency wash contains a high salt concentration (preferably about 2% SSC). A temperature of about 37 ° C. to 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 a nucleic acid molecule encoding a CSP activator peptide (preferably a mammalian peptide). The method comprises contacting a sample comprising a nucleic acid molecule comprising all or part of SEQ ID NO: 1 (preferably comprising at least about 15 or 20 nucleotides of SEQ ID NO: 1) under moderate or high stringency hybridization conditions. Identifying a nucleic acid molecule that hybridizes with a nucleic acid molecule comprising all or part of SEQ ID NO: 1. A similar method is described in US Pat. No. 5,851,788, which is hereby incorporated by reference in its entirety.

Certain embodiments of the present invention also identify all or part of a nucleic acid molecule that hybridizes to all or part of SEQ ID NO: 1, eg, as a probe or to identify antagonists or inhibitors of peptides purified by said nucleic acid. Methods used in the assay for (described below). Some embodiments of the present invention include methods of using nucleic acid molecules having sequence identity with CSP nucleic acid molecules (as described below) in a similar manner.
Certain embodiments of the invention also include a nucleic acid molecule detection kit. Said kit is preferably a nucleic acid molecule and detection reagent as disclosed herein encoding CSP (SEQ ID NO: 2) or a peptide having CSP activity, which is attached in a suitable container means or on a surface (eg Detectable label). Other variations of the kit will be apparent from the description and patent teachings (eg, US Pat. Nos. 5,837,472 and 5,801,233, which are incorporated herein by reference).
The nucleic acid molecule described above has a function substantially equivalent to that of the CSP nucleic acid molecule of the present invention (SEQ ID NO: 1) if the peptide (SEQ ID NO: 2) produced by the nucleic acid molecule has CSP activity. It is thought to have. A peptide has CSP activity if it can stimulate the genetic capacity and acid resistance of S. mutans. Activation of HK (SEQ ID NO: 4) / RR (SEQ ID NO: 6) is shown when a peptide can stimulate uptake and integration of foreign DNA. We describe below how the activity of these peptide-mediated processes can be measured by determining plasmid uptake efficiency (which is a measure of genetic acceptability). The ability to transport and take up foreign DNA requires activation of HK (SEQ ID NO: 4) / RR (SEQ ID NO: 6) and subsequent genes activated by a signal cascade initiated by the signal peptide. The measured value of erythromycin resistance conferred by cells exposed to the peptide and erythromycin resistance conferring plasmid DNA is an indicator of its functional level. Conversely, if an inhibitory factor can interfere with the action of the peptide, the competence assay can reduce erythromycin resistance as described in the following assays (genetic acceptability assay and biofilm transformation assay). This will be indicated by a corresponding decrease in the number of cells acquired. Activation of HK (SEQ ID NO: 4) / RR (SEQ ID NO: 6) will also be shown if a peptide can stimulate an acid tolerance response. We describe below how we can measure the activity of these peptide-mediated processes by determining cell viability under acidic pH conditions. Since the ability to withstand exposure to acidic pH depends on the activation of HK / RR and the subsequent gene activated by the signal peptide, the measure of survival of S. mutans under low pH conditions is It is an indicator of the functional level of the signal peptide. Conversely, if an inhibitor is able to interfere with the signal peptide sensing system, cells grown under acidic pH conditions by an acid adaptation assay as described in the assay below (acid adaptation assay). That would be indicated by a corresponding decrease in the survival rate.

Production of CSPs in Eukaryotic and Prokaryotic Cells The nucleic acid molecules disclosed herein can be obtained from cDNA libraries. The nucleotide molecule can also be obtained from other sources known in the art (eg, tag analysis of expressed sequences or in vitro synthesis). The DNA described in this application (including variants that are functional equivalents) can be introduced and expressed in a variety of eukaryotic and prokaryotic cells. The recombinant nucleic acid molecule of CSP contains appropriate transcriptional or translational regulatory elements operably linked. Suitable regulatory elements are derived from a variety of sources and can be easily selected by those skilled in the art (J. Sambrook, EE Fritsch & T. Maniatis, (latest edition), Molecular Cloning: A Laboratory Manual. Cold Spring). Harbor Laboratory Press. New York; Ausubel et al. (Latest edition), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). For example, if it is desired to up-regulate nucleic acid molecule expression, a sense sequence and an appropriate promoter could be inserted into the vector. The promoter may be inducible, constitutive, environmental or developmental, or cell specific or tissue specific. Transcription is enhanced using promoters known in the art for expression. CMV and SV40 promoters are commonly used to express the desired peptide in cells. Other promoters known in the art can also be used (many suitable promoters and vectors are described in the patent applications and patents cited in this application).
If it is desired to down-regulate the expression of the nucleic acid, an antisense sequence and a suitable promoter could be inserted into the vehicle. Nucleic acid molecules can be isolated from natural sources (in sense or antisense orientation), synthesized, or mutated in natural or synthetic sequences, or a combination of the foregoing.

Examples of regulatory elements include transcriptional promoters and enhancers or RNA polymerase binding sequences, ribosome binding sequences (including translation initiation signals). Furthermore, depending on the vector used, other genetic elements (eg, selectable markers) can be incorporated into the recombinant molecule. Other regulatory regions that can be used include enhancer domains and termination regions. The regulatory element can be derived from bacteria, fungi, viruses, or birds. Similarly, the regulatory elements can be obtained from animals, plants, yeast, insects or other sources, including synthetically produced elements and mutant elements.
In addition to the use of the expression vectors described above, the peptide can be expressed by inserting the recombinant nucleic acid molecule into a known expression system derived from bacteria, viruses, yeasts, mammals, insects, fungi or birds. it can. Depending on the cell type, the recombinant molecule may be a technique such as Agrobacterium tumefaciens mediated transformation, particle bombardment mediated transformation, direct uptake, microinjection, co-precipitation, transfection and electroporation. Can be introduced into cells. Retrovirus vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, DNA virus vectors and liposomes can be used. The appropriate construct is inserted into the expression vector. The vector can also include a marker for selection of transformed cells. The construct can be inserted at a site created by a restriction enzyme.
In one embodiment of the invention, a nucleic acid molecule of the invention inserted into an expression vector is transfected into a cell to produce a cell that expresses a peptide encoded by the nucleic acid molecule.
Another embodiment of the present invention transfects cells with a nucleic acid molecule disclosed herein inserted into an expression vector to produce cells that express the CSP peptide (SEQ ID NO: 2) or other peptides of the present invention. Regarding the method. In accordance with certain embodiments of the present invention, methods for expressing the disclosed peptides in cells are provided. A preferred method comprises culturing a cell comprising a recombinant DNA vector comprising a nucleic acid molecule encoding CSP (SEQ ID NO: 1) (or another nucleic acid molecule of the invention) so that the peptide is expressed in a culture medium. Would include that. Preferably, the method further comprises recovering the peptide from the cell or cell culture medium.

Certain embodiments of the probe present invention, the oligonucleotide probe prepared from other nucleic acid molecules disclosed CSP nucleic acid molecule or herein were cloned according to the present application (see Materials and Methods) Including. The probe will be 15 to 20 nucleotides in length. A preferred probe is at least 15 nucleotides of the CSP of SEQ ID NO: 1. Certain embodiments of the invention also include at least 15 contiguous nucleotides of SEQ ID NO: 1. The probe is useful for identifying a nucleic acid encoding a peptide functionally equivalent to CSP as well as the CSP peptide. The oligonucleotide probe can hybridize with the sequence shown in SEQ ID NO: 1 under stringent hybridization conditions. Nucleic acid molecules encoding the peptides disclosed herein can be isolated from other organisms by screening the library with labeled probes under moderate to highly stringent hybridization conditions. The activity of the peptide encoded by the nucleic acid molecule can be assessed by cloning and expression of the DNA. After isolating the expression product, the peptide is assayed for CSP activity as described in this application.
Functionally equivalent CSP nucleic acid molecules from other cells or equivalent CSP-encoding cDNA or synthetic DNA can also be isolated by amplification using the polymerase chain reaction (PCR) method. An oligonucleotide primer (for example, a degenerate primer) based on SEQ ID NO: 1 is prepared, and PCR and reverse transcriptase are used (ES Kawasaki, 1990, In Innis et al., Eds., PCR Protocols, Academic Press, San Diego) , Chapter 3, p.21), functionally equivalent DNA can be amplified from genomic libraries or cDNA libraries of other organisms. The oligonucleotide library can also be used as a probe to screen a cDNA library.

Functionally equivalent peptides, peptides and proteins The present invention includes not only peptides encoded by the sequences disclosed herein, but also functionally equivalent peptides, peptides and proteins that exhibit the same or similar CSP peptide activity. Including.
We designed and synthesized analogs based on the natural sequence of S. mutans CSP, and further assayed their ability to interfere with capacity development, acid tolerance response and biofilm formation.
Based on the natural CSP amino acid sequence of S. mutans, 17 peptide analog panels were designed and synthesized with modified length and hydrophobicity of the peptide analogs. The first set of peptide analogues lacks the first, second, third, fourth or fifth residues from the N- and C-terminus of the mature S. mutans CSP sequence (SEQ ID NO: 30). Generated by letting go. The second set included peptide analogs in which charged internal residues were replaced with neutral (valine) or hydrophobic (alanine) residues. The peptide analogs synthesized and tested in this experiment are listed in Table 1.

Peptide analog H1 transforms all 17 peptides designed and synthesized based on the sequence of natural S. mutans CSP capable of inhibiting genetic acceptability with S. mutans wild type UA159 strain They were first screened for their ability to interfere. Among them, the analog H1 (SEQ ID NO: 39) significantly (18-fold) transformed efficiency compared to the natural (no added exogenous CSP) transformation efficiency of S. mutans UA159 strain. ) Reduced (Table 2 and Figure 2). These results indicate that H1 inhibits natural genetic transformation of S. mutans. The competent regulon identified and characterized in our laboratory showed that transformation of S. mutans is a comD-dependent process. To test the hypothesis that the peptide analog H1 can compete with the native CSP produced by S. mutans for occupation of the ComD histidine kinase receptor, we inherited with the S. mutans comD null mutant. The ability of H1 to induce sexual acceptability was tested. As expected, the results indicated that the action of the peptide analog H1 was achieved through the comD receptor and therefore a ComD-dependent process (FIG. 3).
In contrast, the peptide analogs IH-1 (SEQ ID NO: 31), IH-2 (SEQ ID NO: 32), B1 (SEQ ID NO: 33), and C1 (SEQ ID NO: 34) are expressed in the wild type S. mu. It did not show a significant effect on transformation efficiency compared to Tans CSP (Table 2). These results suggested that these peptide analogs retained native CSP activity despite sequence modifications. However, peptide analogs D1 (SEQ ID NO: 35), E1 (SEQ ID NO: 36), F1 (SEQ ID NO: 37), G1 (SEQ ID NO: 38), A2 (SEQ ID NO: 40), B2 (SEQ ID NO: 41), C2 (SEQ ID NO: 42), D2 (SEQ ID NO: 43), E2 (SEQ ID NO: 44), F2 (SEQ ID NO: 45), G2 (SEQ ID NO: 46), or B3 (SEQ ID NO: 47) The transformation efficiency of S. mutans UA159 in the presence of) is reduced compared to wild-type S. mutans CSP (5 μg CSP). This suggests that these peptide analogs show similar aspects to CSP in terms of stimulatory competence but do not have the same affinity for the comD receptor as native wild-type S. mutans CSP. It suggests that there is.

Table 1: Modified forms of mature CSP peptides

^a効果なし：CSPとの比較で顕著な相違なし Table 2: Effect of 5 μg / mL peptide analogue on the capacity of S. mutans wild-type UA159

^aNo effect: No significant difference compared to CSP

Table 3: Effect of 5 μg / mL peptide analogue on growth, acid tolerance and biofilm formation of S. mutans wild-type UA159 at pH 7.5

Multiple peptide analogues affect cell growth of S. mutans in acidic media The peptide analogues are resistant to S. mutans acid resistance mechanisms (cells that are resistant to the acidic attack typically encountered in plaque). S. mutans UA159 cells were grown in THYE medium at pH 7.5 and pH 5.5 in the presence of various concentrations of peptide analogs. . The results presented in Table 3 showed that the peptide analogs F1, F2, and G2 reduce cell proliferation at pH 7.5 and pH 5.5. The peptide analogs H1, A2, B2, C2, D2, E2, and B3 do not affect the growth of S. mutans cells at pH 7.5. Furthermore, when the same peptide analog was tested at pH 5.5, the results showed that there was a significant reduction in cell proliferation. Interestingly, the peptide analog H1, which is centrally involved in the inhibition of genetic acceptability, can also inhibit the growth of S. mutans cells in acidic media (Figure 4), while neutral There was no effect on growth at pH (Figure 5).

S. mutans peptide analogues inhibit Streptococcus biofilm formation
S. mutans comC null mutants that cannot produce a CSP signal peptide form a biofilm that lacks wild-type structure. Furthermore, exogenous addition of synthetic CSP restores the wild-type phenotype to comC-deficient mutants (Li et al. 2002). Therefore, CSP seems to be responsible for an essential part of S. mutans biofilm formation. Thus, the analogs were tested for their ability to inhibit S. mutans biofilm formation. In the first experiment (the results are presented in Table 3), the S. mutans peptide analogs F1 (SEQ ID NO: 37), G1 (SEQ ID NO: 38), E2 (SEQ ID NO: 44), F2 ( SEQ ID NO: 45), G2 (SEQ ID NO: 46), and B3 (SEQ ID NO: 47) are 24.4% to 38.9% compared to S. mutans biofilm grown in the presence of wild-type CSP. Significantly reduced biomass in range, suggesting that these peptide analogs could interfere with signal pathways that regulate biofilm formation by S. mutans.
In the second experiment, B2 (SEQ ID NO: 41), B3 (SEQ ID NO: 47), C2 (SEQ ID NO: 42), E2 (SEQ ID NO: 44), F1 (SEQ ID NO: 37), and H1 (sequence) The anti-biofilm activity of No. 39) was examined. The anti-biofilm activity against S. mutans of the peptide analogs converted to percentage inhibition varied between 0 and 80% (FIG. 18). Peptide analog E2 (SEQ ID NO: 44) showed the best anti-biofilm activity (80% inhibition) among the six synthetic CSP analogs tested. Peptide analog E2 could be a potent S. mutans QS inhibitor. This is because this peptide analog causes a marked decrease in biofilm formation and inhibits cell growth at pH 5.5 without affecting cell growth at neutral pH.
In yet another experiment, S. mutans peptide analogs were further tested for their ability to inhibit biofilm formation by other types of bacteria, particularly other plaque-associated bacteria. Peptide analogs B3 (SEQ ID NO: 47), C2 (SEQ ID NO: 42), E2 (SEQ ID NO: 44) and F1 (SEQ ID NO: 37) are plaque-associated Streptococcus (S. sobrinus, S. sanguis, S It has been found to significantly reduce biofilm formation of non-plaque associated Streptococcus (eg, Streptococcus pneumoniae) (including Gordonii, S. Oralis, S. Mitis) and non-plaque.

S. mutans-derived E2 (SEQ ID NO: 44) peptide is grown at a concentration as low as 5 μg / mL and is It showed an inhibitory effect on both film formation. The percent inhibition of biofilm formation in these microorganisms varied from 40 to 75% (Figures 19, 20, 21, 22, 23, 24 and 25). Furthermore, the anti-biofilm activity of the E2 (SEQ ID NO: 44) peptide was tested against a mixed culture of the above Streptococcus species. The peptide also showed a significant inhibitory effect on mixed film biofilm formation (data not shown).
When a certain peptide has CSP activity, it is considered that the peptide possesses a function substantially equivalent to the function of the CSP peptide (SEQ ID NO: 2). CSP activity means that the peptide can confer genetic competence to S. mutans, and when exogenous genetic material is added to the cells, as described in the genetic acceptability assay below. It is measured by the increased ability to take up and express the substance. CSP activity also means that the peptide can confer acid resistance to S. mutans and, as described in the acid adaptation assay below, by increasing cell survival under acidic pH conditions when added to the cells. Measured. Functionally equivalent peptides, peptides and proteins include peptides, peptides and proteins that have the same or similar protein activity as CSP when assayed. That is, they can stimulate genetic acceptability and low pH tolerance (ability to withstand acid attack at pH 3.5-pH 3.0 for up to 3 hours) for S. mutans. If a peptide can increase the frequency of uptake and expression of foreign DNA as described in the assay for genetic acceptability below, then the peptide can be converted into an acid as described in the assay for acid adaptation. If the resistance response can be promoted, the peptide has CSP activity.
Identity refers to the similarity of two peptides or proteins that have been aligned to give the highest degree of matching. Identity is calculated according to methods known in the art, such as the ClustalW program. For example, if a peptide (assuming “sequence A”) has 90% identity to a portion of the peptide of SEQ ID NO: 30, sequence A is identical to the reference portion of the peptide of SEQ ID NO: 30. However, sequence A can include up to one point mutation (eg, substitution with another amino acid) for each 10 amino acids of the reference portion of the peptide of SEQ ID NO: 30. Peptides, peptides and proteins functionally equivalent to CSP peptides can exist in a variety of forms as described below.

Peptides that are biologically functionally equivalent to the CSP peptide include amino acid sequences that contain amino acid changes in the CSP sequence (SEQ ID NO: 2). A functionally equivalent peptide is at least about 40% sequence identity, preferably at least about 60%, at least about 75%, at least about 80 to the native CSP peptide (SEQ ID NO: 2) or corresponding region. %, At least about 90%, at least about 95% sequence identity. Preferably, sequence identity is determined by the ClustalW program. Most preferably 1, 2, 3, 4, 5, 5-10, 10-15 amino acids are modified.
Variants of CSP peptides can also be created by splicing. A combination of techniques known in the art can be used to substitute, delete or add amino acids. For example, a hydrophobic residue (eg, methionine) can be replaced with another hydrophobic residue (eg, alanine). Alanine residues can be substituted with more hydrophobic residues such as leucine, valine or isoleucine. Tyrosine can be substituted with an aromatic residue (eg, phenylalanine). An acidic, negatively charged amino acid (eg, aspartic acid) can replace glutamic acid. A positively charged amino acid (eg lysine) can also be replaced by another positively charged amino acid (eg arginine). Modifications to the peptides disclosed herein also convert, for example, a hydrogen group to another group (eg, a hydroxy or amino group) by treating such a peptide with an agent that chemically modifies the side chain group. Can be achieved.
Peptides having one or more D-amino acids are included in certain embodiments of the invention. Also included are peptides in which one or more amino acids are acetylated at the N-terminus. Peptides having the same or similar biological activity as the corresponding peptides disclosed herein, but having a more favorable activity than said peptides in terms of properties such as solubility, stability and / or susceptibility to hydrolysis and proteolysis Those skilled in the art will recognize that a variety of techniques can be used to construct mimetics (ie, modified peptides or peptides or proteins). See, for example: Morgan & Gainor, Ann Rep Med Chem, 1989, 24: 243-252.

Certain embodiments of the invention also include hybrid nucleic acid molecules and peptides. For example, a nucleic acid molecule derived from a nucleic acid molecule disclosed herein is combined with another nucleic acid molecule to produce a nucleic acid molecule that expresses the fusion peptide. One or more other domains of the CSP described in this application can also be used to generate fusion peptides. For example, nucleotide domains derived from the molecule of interest can be linked to all or part of a nucleic acid molecule (or molecule having sequence identity) that encodes the CSP peptides described in this application. Fusion nucleic acid molecules and peptides can also be chemically synthesized or generated using other known techniques. Certain embodiments of the invention include a nucleic acid molecule encoding a fusion peptide, or a recombinant vector comprising said nucleic acid molecule.
The variant preferably retains 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 or techniques known in the art.
Variants that are generated by a combination of the techniques described above but retain the same or similar CSP activity as a naturally occurring CSP (SEQ ID NO: 2) are also included in certain embodiments of the invention ( For example, a combination of amino acid addition and substitution).
Variants of CSP produced by the techniques described above that competitively inhibit CSP activity are also included in certain embodiments of the invention (eg, combinations of amino acid additions and substitutions).
Variants of CSP produced by the techniques described above that reduce bacterial transformation efficiency are also encompassed by the present invention (eg, a combination of amino acid additions and substitutions).
Variants of CSP produced by the techniques described above that reduce biofilm formation are also included in certain embodiments of the invention (eg, combinations of amino acid additions and substitutions).
Variants of CSPs encompassed by the present invention are preferably at least about 60%, 75%, 80%, 90% relative to the naturally occurring peptide, or the corresponding region or portion of the peptide, or the corresponding region. % Or 95% sequence identity. Sequence identity is preferably measured by ClustalW.

Histidine kinases and response regulators Certain embodiments of the invention also include histidine kinases, response regulators of the invention, and sequences having identity to comA and comB. The preferred percentage of identity (nucleic acid molecules and polypeptides) is the same as the percentage described for CSP.
Similarly, histidine kinase (SEQ ID NO: 3 and SEQ ID NO: 4), response regulator (SEQ ID NO: 5 and SEQ ID NO: 6), comA (SEQ ID NO: 25 and SEQ ID NO: 26), or comB (SEQ ID NO: Probes and antibodies against: 27 and SEQ ID NO: 28) can also be prepared using the descriptions in this application and techniques known in the art. The description for the preparation of CSP variants and variants is also 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 sequence No. 26) and comB (SEQ ID NO: 27 and SEQ ID NO: 28). Certain embodiments of the invention also include fragments of HK having HK activity, fragments of RR having RR activity (SEQ ID NO: 5 and SEQ ID NO: 6), and comA having activity (SEQ ID NO: 25 and sequence). No. 26) or comB (SEQ ID NO: 27 and SEQ ID NO: 28).

Design of competitive inhibitors of CSP peptides
The activity of the CSP peptide (SEQ ID NO: 2) can be altered by performing position-directed mutagenesis. We have characterized the binding domains and other important amino acid residues of peptides that are candidates for mutation, insertion and / or deletion. Sequence variants can be synthesized. USE (Unique site elimination) mutagenesis kit (Pharmacia Biotech) or other commercially available mutations in CSP nucleic acid molecules (SEQ ID NO: 1) or DNA plasmids or expression vectors containing nucleic acid molecules with sequence identity It can be used in these experiments using introduction kits or PCR. Peptide analogs of S. mutans CSP peptides can be prepared by deleting and / or replacing amino acids at the C ′ or N ′ terminus of the CSP peptide 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 for identifying CSP inhibitors. All of these modifications of the CSP DNA sequence presented in this application (SEQ ID NO: 1) and peptides produced by these modified sequences are encompassed by the present invention.
Peptide analogs of S. mutans CSP peptides prepared by deleting and / or substituting amino acids of the CSP peptide can be screened for biofilm formation inhibition. Screening assays are described below.

Pharmaceutical composition
CSP inhibitors are also useful when combined with a carrier in pharmaceutical compositions. The composition is useful when administered in a method of medical treatment or prevention of disease, abnormalities or abnormal physical conditions caused by S. mutans. Certain embodiments of the present invention may also be used in disease, abnormal or abnormal body conditions characterized by excessive levels or activity of excess S. mutans or CSP peptide (SEQ ID NO: 2), such as carriers and CSPs. A method of medical treatment by administering a pharmaceutical composition comprising an inhibitor. Caries are an example of a disease and can be treated or prevented by CSP (SEQ ID NO: 2) acting as an antagonist. The composition also includes diseases, abnormalities or abnormalities caused by other bacteria that cause plaque, including but not limited to Actinomyces spp and other Streptococci spp Useful when administered in a medical treatment or prevention method for a physical condition.
The pharmaceutical composition is used in medical treatment methods, for example for foods, food additives, toothpaste gels, toothpastes, mouth washes, dental floss, denture cleansers, denture adhesives, chewing gums, candies, biscuits, soft drinks or sports drinks. It can be administered to humans or animals by such methods. The CSP inhibitors of the present invention can be conjugated to lipids or carbohydrates. This enhances the ability of the inhibitor to adhere to the tooth by extending the attachment time or enhancing its affinity, or both. The can also be combined with polymers or dental floss, for example of dental work (eg crowns, orthodontic appliances, fillers). The pharmaceutical composition can be administered to humans or animals. The dose to be administered depends on the condition of the individual, the drug indication, the physical and chemical stability of the drug, the toxicity of the desired action and the route of administration chosen (Robert Rakel, ed., Conn's Current Therapy (1995, WB Saunders Company, USA)). The pharmaceutical composition is used to treat diseases (eg caries, periodontal disease and endocarditis) caused by streptococcal infection. In a preferred embodiment, the pharmaceutical composition is used to treat diseases caused by Actinomyces species and Streptococcus species. In a further preferred embodiment, the pharmaceutical composition is used to produce S. mutans, S. sobrinus, S. oralis, S. sanguis, S. mytis S. gordonii, Streptococcus pneumoniae, S. pyogenes, and S. agalactiee. Infection by Streptococcus caused by (but not limited to) is treated.

The pharmaceutical composition of the present invention can be produced using a CSP inhibitor that is an antisense oligonucleotide. For example, CSP activity could block antisense mRNA that inhibits CSP expression or transcription. Alternatively, the antisense oligonucleotide may inhibit the activity of an exporter that secretes CSP from cells. We have an array of these exporters. There are two copies of the gene (comAB) required for export (SEQ ID NO: 25 and SEQ ID NO: 27). Antisense preparation techniques are well known in the art.
In certain embodiments, the CSP inhibitor is an antisense oligonucleotide that inhibits CSP expression or transcription. Preferably, said antisense oligonucleotide is an oligonucleotide complementary to at least 10 consecutive nucleotides of an oligonucleotide encoding CSP (the oligonucleotide encoding CSP has the nucleic acid sequence of SEQ ID NO: 1) ).
In another embodiment, the CSP inhibitor is an antisense oligonucleotide that inhibits export of CSP peptides. Preferably, the antisense oligonucleotide is an oligonucleotide complementary to at least 10 consecutive nucleotides of an oligonucleotide encoding a CSP exporter (the oligonucleotide encoding the CSP exporter is SEQ ID NO: 25 Or 27 nucleic acid sequences).
A nucleic acid molecule (SEQ ID NO: 1) (an antisense inhibitor of CSP) and a competitive inhibitor of CSP (SEQ ID NO: 2), or a peptide analog of S. mutans CSP, is provided in an in vivo delivery vehicle (eg, a liposome) Can be introduced into cells. They can also be introduced into these cells using physical techniques (eg microinjection and electroporation) or chemical methods (eg co-precipitation) or using liposomes. In some cases, it is desirable to utilize liposomes that are derived from the bacteria in question.

The pharmaceutical composition of the present invention can be produced using an antibody or fragment thereof that selectively inhibits CSP activity. A more detailed discussion of the preparation of CSP specific antibodies is given below.
In a preferred embodiment, the pharmaceutical composition of the present invention can be manufactured using one or more CSP peptide analogs capable of inhibiting plaque-associated bacterial biofilm formation. The inhibitory CSP peptide analog may be a naturally occurring mutated CSP peptide obtained from a bacterium of the genus Streptococcus whose ability to form a biofilm is inhibited. The inhibitory CSP peptide analog may be a synthetic peptide prepared using methods known in the art. In a more preferred embodiment, the inhibitory 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 F1 (SEQ ID NO: 37). In a further preferred embodiment, the pharmaceutical composition is manufactured using the E2 (SEQ ID NO: 44) peptide.
A pharmaceutical composition comprising a CSP peptide analog or a pharmaceutically acceptable salt thereof, in particular B3 (SEQ ID NO: 47), C2 (SEQ ID NO: 42), E2 (SEQ ID NO: 44) and F1 (SEQ ID NO: 37) Is particularly useful as a method of treatment or prevention of diseases, abnormalities or abnormal body conditions caused by Streptococcus infections. Such pharmaceutical compositions may be used to treat infections caused by one or more of the oral streptococcal bacteria, such as S. mutans, S. sobrinus, S. oralis, S. sanguis, S. mitis, S. gordonii. Is particularly useful. The pharmaceutical compositions are also useful for the treatment and prevention of other types of Streptococcus infections, such as S. pneumoniae, S. pyogenes and S. agaractie.

For example, “pharmaceutically acceptable” in the description such as “pharmaceutically acceptable carrier” or “pharmaceutically acceptable salt” refers herein to a biologically or otherwise undesirable substance. Means not. That is, such substances do not cause any undesired biological effects or interact with the patient in a deleterious manner with any of the other ingredients in the composition in which the substance is contained. It can be incorporated into the pharmaceutical composition to be administered.
As used herein, “carrier” or “vehicle” refers to a common pharmaceutically acceptable carrier material suitable for administration of a drug, and is a non-toxic pharmaceutical composition known in the art. Or any substance that does not interact in a deleterious manner with other components of the drug delivery system.
The pharmaceutical composition can be prepared by known methods for the preparation of a pharmaceutically acceptable composition that can be administered to a patient, and an effective amount of the nucleic acid molecule or peptide is a pharmaceutically acceptable vehicle. And put together as a mixture. 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 buffers, solubilizers, dispersants, emulsifiers, viscosity modifiers, fragrances, lactose fillers, antioxidants, preservatives or dyes. There are preferred excipients that stabilize the peptide for parenteral and other administration. Such excipients include serum albumin, glutamic acid or aspartic acid, phospholipids and fatty acids.
On this basis, the pharmaceutical composition is an active, combined with one or more pharmaceutically acceptable vehicles or diluents, contained in a buffer solution that has an appropriate pH and isotonic with physiological fluids. A compound or substance (eg, a CSP inhibitor) could be included. The pharmaceutical carrier will depend on the route of administration used. Methods for combining active molecules with a vehicle or combining them with a diluent are well known to those skilled in the art. The composition also includes additives, such as antioxidants, buffers, bactericides, bactericidal antibiotics, and solutes that make the formulation isotonic in the intended recipient; dispersants and swelling agents. Aqueous and non-aqueous sterile suspensions may be included. The composition could include a targeting agent for transporting the active compound to a specific site.

The pharmaceutical composition of the present invention can be produced in a manner known per se, for example, by usual mixing, dissolution, granulation, sugar-coating production, homogenization, emulsification, encapsulation, entrapment or lyophilization treatment. .
A pharmaceutical composition for use in accordance with the present invention thus comprises one or more pharmaceutically acceptable ones comprising excipients and adjuvants (which facilitate the processing of the active compound into pharmaceutically usable preparations). It can be formulated in a conventional manner using a carrier. Proper formulation is dependent upon the route of administration chosen.
The pharmaceutical compositions of the invention can be administered by any suitable route known in the art. If the infection is local, the pharmaceutical composition can be administered locally to the infected area. If the infection is systemic, the pharmaceutical composition can be administered orally, intravenously, or parenterally.
For injection, the substances of the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline.
For oral administration, the compounds can be readily formulated by combining the active compound with pharmaceutically acceptable carriers well known in the art. Such carriers may be formulated for oral intake by the patient to be treated, such as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. enable. Pharmaceutical preparations for oral use can be obtained by grinding the resulting mixture with solid excipients and, if desired, adding appropriate adjuvants to obtain tablets or dragees after adding the granule mixture Can be obtained by processing. Suitable excipients are in particular fillers such as sugars (including lactose, sucrose, mannitol or sorbitol) or cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum arabic, methylcellulose , Hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

In accordance with another aspect of the present invention, a method is provided for producing a cell that has been genetically modified to produce a CSP derivative that inhibits transformation efficiency. Another feature involves administering to the patient genetically modified S. mutans to produce a CSP derivative that inhibits transformation efficiency. Methods for producing and administering genetically engineered cells are known in the art. See for example WO02 / 44230.
In yet another aspect of the present invention, a genetically modified transfection that produces a CSP derivative that inhibits transformation efficiency in the manufacture of a medicament for administration to a mammalian patient for the purpose of reducing caries. Use of live S. mutans is provided.
In accordance with yet another aspect of the present invention, there is provided a method for producing cells genetically modified to produce CSP derivatives that inhibit biofilm formation. Another feature includes administering to the patient cells that have been genetically modified to produce CSP derivatives that inhibit biofilm formation.
In yet another aspect of the present invention, genetically produced to produce CSP derivatives that inhibit biofilm formation in the manufacture of a medicament for administration to a mammalian patient for improving oral health or reducing caries. Use of artificially modified live transfected cells is provided.
The pharmaceutical compositions of the present invention can be administered by any suitable route known in the art. If the infection is local, the pharmaceutical composition can be administered locally to the infected area. If the infection is systemic, the pharmaceutical composition can be administered orally, intravenously, or parenterally.
In some cases, one or more pharmaceutical compositions of the present invention can be administered for the treatment of infections caused by more than one type of bacteria. For example, a pharmaceutical composition comprising an antisense oligonucleotide can be administered together with a pharmaceutical composition comprising a CSP peptide analog. In another case, a pharmaceutical composition comprising an antibody can be administered with a composition comprising a CSP peptide analog. Alternatively, the pharmaceutical composition can be prepared using one or more types of CSP inhibitors to obtain a unit dosage form.

In some cases, a pharmaceutical composition of the invention can be administered with a known antimicrobial agent (eg, antibiotic). In some cases, pharmaceutical compositions that inhibit biofilm formation are also useful to make bacterial cells more sensitive to antibiotics.
As used herein, the term “antibiotic” refers to any compound known to those of skill in the art that inhibits bacterial growth or kills bacteria. The term “antibiotic” includes, but is not limited to: beta-lactam (penicillin and cephalosporin), vancomycin, bacitracin, macrolide (erythromycin), lincosamide (clindamycin), chloram Phenicol, tetracycline, aminoglycoside (gentamicin), amphotericin, cefazolin, clindamycin, mupirocin, sulfonamide and trimethoprim, rifampicin, metronidazole, quinolone, novobiocin, polymyxin, gramicidin, or any salt or variant thereof. The antibiotic used depends on the type of bacterial infection.
The therapeutically effective dosage of the pharmaceutical composition of the present invention depends on the CSP inhibitor, the type and severity of the infection, and whether the pharmaceutical composition further comprises additional components (eg, antibiotics). The In general, a therapeutically effective dose is the lowest amount sufficient to control biofilm formation and is not toxic to the human being and animal being treated. Methods for determining effective dose and toxicity are known in the art.

In an additional feature of the anti-biofilm composition and its use , the present invention provides a composition useful for inhibiting and destroying biofilms of Streptococcus species. The composition comprises at least one peptide analog of S. mutans CSP that inhibits or destroys biofilm formation of Streptococcus species. Peptide analogs useful in the practice of the present invention include: F1 (SEQ ID NO: 37), H1 (SEQ ID NO: 39), B2 (SEQ ID NO: 41), C2 (SEQ ID NO: 42), E2 (SEQ ID NO: 44) and B3 (SEQ ID NO: 47). In a preferred embodiment of the invention, the composition is prepared using the E2 (SEQ ID NO: 44) peptide.
The compositions of the present invention are effective in inhibiting and destroying Streptococcus biofilms utilizing a quorum sensing system. The composition is effective for both oral and non-oral Streptococcus species. Examples of oral Streptococcus infections that can be controlled using the compositions of the present invention include (but are not limited to) Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus mitis. ). Examples of infections of non-oral Streptococcus species that can be regulated using the compositions of the present invention include (but are not limited to) Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae.
In certain embodiments of the invention, the composition may be formulated as a pharmaceutical composition suitable for administration to a human or animal patient infected with one or more Streptococcus species. The pharmaceutical composition comprises one or more F1 (SEQ ID NO: 37), H1 (SEQ ID NO: 39), B2 (SEQ ID NO: 41), C2 (SEQ ID NO: 42), E2 (SEQ ID NO: 44) and A B3 (SEQ ID NO: 47) peptide analog, or a pharmaceutically acceptable salt as described above, and a pharmaceutically acceptable carrier can be included. In a preferred embodiment of the invention, the pharmaceutical composition comprises an E2 (SEQ ID NO: 44) peptide analog.
The peptide analog can be conjugated to a lipid or carbohydrate. Thereby, the ability of the peptide analogue to adhere to an infected surface (eg, a tooth) is enhanced by extending the attachment time or increasing the affinity, or both.

In some cases, pharmaceutical compositions of the invention can be administered with known antimicrobial agents (eg, antibiotics, examples of which are listed above). The antibiotic used depends on the type of bacterial infection. For example, in some cases, the biofilm inhibitory pharmaceutical compositions of the present invention are also useful for making bacterial cells more sensitive to antibiotics. Alternatively, the pharmaceutical composition can be prepared with one or more active ingredients (eg, antibiotics) in addition to the CSP peptide analog to obtain a unit dosage form.
In addition to the usual oral pharmaceutical formulations such as tablets, capsules and syrups, the composition of the present invention can be used for foods, food additives, toothpaste gels, toothpastes, mouthwashes, dental floss, denture cleansers, denture adhesives. It can be administered to humans and animals in the form of chewing gum, candy, biscuits, soft drinks or sports drinks.
In addition, the antimicrobial composition can include additional components including, but not limited to, surfactants, chelators, antibodies, preservatives, and antibiotics (see, but not limited to, the examples listed above).
In a further aspect, the present invention provides a method for treating or preventing Streptococcus infection in a patient in need thereof. The methods include F1 (SEQ ID NO: 37), H1 (SEQ ID NO: 39), B2 (SEQ ID NO: 41), C2 (SEQ ID NO: 42), E2 (SEQ ID NO: 44) and B3 (SEQ ID NO: 47). Administering a therapeutically effective amount of at least one peptide having an amino acid sequence selected from the group consisting of: In a preferred embodiment, the method comprises administering a therapeutically effective amount of an E2 (SEQ ID NO: 44) peptide. The peptide analog can be administered in any form of the pharmaceutical composition encompassed by the present invention. In some cases, the method of treating or preventing Streptococcus spp. Infection further comprises administration of a therapeutically effective amount of another active agent, such as an antibiotic (see examples above). The antibiotic can be administered at the same time as the peptide analog. If it is desired to enhance the entry of the antibiotic into the biofilm, it is preferable to first administer the peptide analog to destroy the biofilm.

A “therapeutically effective amount” or “therapeutically effective dose” of a pharmaceutical composition of the present invention is a peptide analog, the type and severity of the infection, and the pharmaceutical composition is a further active ingredient ( For example, it will depend on whether antibiotics are included. In general, a therapeutically effective dose is the lowest amount sufficient to control biofilm formation and / or eliminate infection and is not toxic to the human or animal being treated. Methods for determining effective dose and toxicity are known in the art.
The method includes treatment of infections caused by both oral and non-oral Streptococcus species. In the case of oral Streptococcus species (including, for example, Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus mitis), the pharmaceutical composition is useful for plaque reduction and control. The composition is also useful for the treatment and prevention of symptoms caused by dental plaque, including caries, periodontal disease (eg gingivitis) and endocarditis. In such cases, it is generally desirable to administer the pharmaceutical composition locally to the infected area. For example, the pharmaceutical composition may be administered in the form of food, food additives, toothpaste gel, toothpaste, mouthwash, dental floss, denture wash, denture adhesive, chewing gum, candy, biscuit, soft drink or sports drink. Can do.
In the case of infections caused by non-oral Streptococcus species such as, but not limited to, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae, the pharmaceutical composition depends on the nature and severity of the infection. Administration can be local or systemic.

The exact dosage of any pharmaceutical composition of the present invention will depend on a number of factors that will be apparent to those skilled in the art and in view of the description herein. These factors include in particular: the substance of the compound to be administered, the formulation, the route of administration used, the gender, age and weight of the patient, and the severity of the condition being treated. Methods for determining dosage and toxicity are well known in the art, and testing is generally initiated in animals and subsequently performed in humans if no significant toxicity is observed in the animals. For example, in cases where the pharmaceutical composition is used for infection, dosage suitability can be assessed by monitoring the severity of the infection using conventional methods known in the art. If at least 3 to 14 days after treatment, the provided dose does not reduce the bacterial level to normal or acceptable levels, the dose can be increased. Patients must be monitored for drug side effects and signs of toxicity.
In a preferred embodiment of the invention, the therapeutically effective amount of said peptide analog will be from 1 μg / mL to 1 mg / mL per day. In a further preferred embodiment, the therapeutically effective amount of said peptide analog will be from 1 μg / mL to 100 μg / mL. In a more preferred embodiment, the peptide analog administered is E2 (SEQ ID NO: 44) peptide in an amount of 1 μg / mL to 100 μg / mL. In cases involving the co-administration of yet another active ingredient (eg, antibiotic), the amount of the peptide analog can be reduced. Alternatively, it may be appropriate to reduce the normal dose of the antibiotic.

The above-described CSP inhibitors used in the manufacture of antimicrobial compositions pharmaceutical compositions may also be used in the manufacture of antimicrobial compositions useful for inhibiting biofilm formation on various surfaces, such as disinfectants. it can.
The antimicrobial composition that inhibits biofilm formation can comprise any of the peptides, antisenses and antibody CSP inhibitors described above. In a preferred embodiment of the invention, the CSP inhibitor used in the manufacture of the antimicrobial composition is a peptide analog of S. mutans CSP that inhibits biofilm formation. More preferably, the CSP inhibitor is one or more of the following S. mutans CSP peptide analogs: B3 (SEQ ID NO: 47), C2 (SEQ ID NO: 42), E2 (SEQ ID NO: 44) And F1 (SEQ ID NO: 37). In a further preferred embodiment, the antimicrobial composition is produced using the E2 (SEQ ID NO: 44) peptide.
The antimicrobial composition can further comprise additional components including, but not limited to, surfactants, preservatives, and antibiotics (see, but not limited to, the examples listed above).
The CSP inhibitor is one or more S. mutans CSP peptide analogs B3 (SEQ ID NO: 47), C2 (SEQ ID NO: 42), E2 (SEQ ID NO: 44) and F1 (SEQ ID NO: 37) In some cases, the amount of the CSP inhibitor is preferably 1 μg / mL to 1 mg / mL. In a preferred embodiment, the CSP inhibitor is an E2 (SEQ ID NO: 44) peptide, and the amount of the E2 (SEQ ID NO: 44) peptide is preferably 1 μg / mL to 100 μg / mL.

vaccine
Antibodies raised against CSP (SEQ ID NO: 2 or SEQ ID NO: 30) will provide protection against caries. Antibodies can be produced as described below. Alternatively, the disclosed peptide (SEQ ID NO: 2 or SEQ ID NO: 30) or a fragment thereof can be used with a carrier to produce a vaccine. The peptide or fragment can also be conjugated to another molecule to increase its antigenicity. The antibody can also be conjugated to a peptide (LJ Brady et al. “Monoclonal Antibody-Mediated Modulation of the Humoral Immune Response against Mucosally Applied Streptococcus mutans” (in press)). To enhance the immune response, the peptide can be conjugated to KLH, ovalbumin, or thyroglobulin prior to immunization. The vaccine will trigger the mammalian immune system to produce antibodies. Certain embodiments of the present invention comprise a vaccine composition and a method of vaccinating a mammal (preferably a human) against caries, said method comprising administering an effective amount of the vaccine composition to the mammal. Achieved by: Techniques for making and using vaccines are known in the art. To produce a vaccine, the peptide, a fragment of the peptide, can be mixed with other antigens (with different immunogenicity), vehicles, or excipients. Examples of peptide vaccines are found in US Pat. Nos. 5,679,352, 5,194,254, and 4,950,480. Techniques for producing vaccines containing site-directed mutagenesis are described in U.S. Pat.Nos. 5,714,372, 5,543,302, 5,433,945, 5,358,868, 5,332,583, 5,244,657, 5,221,618, 5,147,643, 5,085,862 and 5,073,494. . The vaccine can be administered by known techniques, such as topical or parenteral administration. With the introduction of new technologies, significant changes have occurred in vaccine immunology. Cell-free purified fraction without side effects, non-pathogenic but immunogenic variants, recombinant technology, conjugate vaccine, combination vaccine (to limit the number of injections). Vaccine delivery systems can deliver multiple doses of vaccine at a single point of contact. Genetically engineered oral vaccines are useful to confer better and long lasting immunity. Oral vaccines are useful. The nose is also useful as a route for immunization. Vaccines can be composed solely of DNA and can induce humoral and cellular immune responses. Recombinant live vaccines are also useful. A powerful adjuvant is added for the effectiveness of the vaccine. Furthermore, mouse monoclonal antibodies can be “humanized” by genetic manipulation and further expressed efficiently in plants. These recombinant antibodies have opened an era of highly specific and safe therapeutic intervention. The advantage of pre-made antibodies administered in appropriate amounts induced against a specific target compared to vaccines is the certainty of efficacy in individual recipients, but the quality of the immune response and The amount varies from individual to individual. For example, nasal immunity is described by C. Jespersgaad et al. (“Protective Immunity against Streptococcus mutans Infection in Mice after Intranasal Immunization with the Glucan-Binding Region of S. mutans Glucosyltransferase”, Infection and Immunity, December 1999, p.6543-6549, vol. 67, No. 12). Vaccine compositions can include solid or liquid formulations such as gels, syrups, inhalants, tablets, toothpastes, mouth washes or chewing gum.
For vaccine applications, cholera toxin can be utilized by conjugating a peptide and the B-subunit of the toxin (ie, binding to CTB) to stimulate the production of secreted antibodies.

Screening inhibitors for inhibitors of CSP The inhibitor is preferably directed to CSP (SEQ ID NO: 2 or SEQ ID NO: 30) to prevent S. mutans capacity, low pH tolerance and biofilm formation.
A method for identifying a compound that decreases the interaction between CSP (SEQ ID NO: 2 or SEQ ID NO: 30) and HK (SEQ ID NO: 4) can include: (i) CSP (SEQ ID NO: 2 or SEQ ID NO: 30) is contacted with (ii) HK (SEQ ID NO: 4), CSP binding fragment of HK (SEQ ID NO: 4) or one of the aforementioned derivatives in the presence of the compound; and (b) ( determining whether the interaction between i) and (ii) has been reduced, whereby the compound is bound between CSP (SEQ ID NO: 2 or SEQ ID NO: 30) and HK (SEQ ID NO: 4) Indicates to reduce the interaction. A CSP inhibitor (a compound for the treatment or prevention of caries) inhibits the interaction between (i) and (ii). For example, a synthetic peptide library can be screened. Furthermore, non-peptide small organic molecules could be screened.
In one embodiment, the invention includes an assay for determining whether a test compound can act as an agonist or antagonist of CSP (SEQ ID NO: 2) or a peptide having a functional activity of CSP, said assay comprising Culturing cells comprising DNA capable of expressing CSP (SEQ ID NO: 1) or a peptide having CSP activity, said culturing carried out in the presence of at least one compound to determine the ability to modulate CSP activity Followed by monitoring the cells for increased or decreased levels of CSP (SEQ ID NO: 2 or SEQ ID NO: 30) or CSP activity. Other assays (as well as variations of the above assay) will be apparent from the description of the invention and the techniques disclosed in the following patent documents: US Pat. The entirety of which is included herein)). For example, the level of the test compound can be fixed or varied.

Antibody preparation
CSP (SEQ ID NO: 2 or SEQ ID NO: 30) peptides are also useful as antigens for the preparation of antibodies that can be used for purification or detection of other CSP-like peptides. The antibody can also block binding of CSP (SEQ ID NO: 2) to HK (SEQ ID NO: 4). The antibody preferably targets the entire CSP (SEQ ID NO: 2) sequence. CSP (SEQ ID NO: 2 or SEQ ID NO: 30) peptides can be conjugated to other compounds to enhance immunogenicity.
We created a polyclonal antibody against the unique sequence CSP (SEQ ID NO: 2 or SEQ ID NO: 30). Monoclonal and polyclonal antibodies are prepared according to the description in this application and techniques known in the art. For the preparation and use of monoclonal antibodies, see U.S. Pat. Which is incorporated herein by reference in its entirety. Examples of the preparation of polyclonal antibodies are disclosed in US Pat. Nos. 5,512,282, 4,828,985, 5,225,331 and 5,124,147, which are hereby incorporated by reference in their entirety. Antibodies that recognize CSP (SEQ ID NO: 2 and SEQ ID NO: 30) can be used for screening organisms or tissues that contain a CSP peptide (SEQ ID NO: 2) or a CSP-like peptide. The antibodies are also useful for immunopurifying CSP or CSP-like peptides from crude extracts.
Using an antibody (preferably the antibody described above), CSP (SEQ ID NO: 2) or similar peptide, for example, under conditions that allow the formation of an immune complex between the antibody recognized by the antibody and the antibody Detecting by contacting a biological sample with said antibody and detecting the presence or absence of an immunological complex, thereby detecting the presence of CSP (SEQ ID NO: 2) or similar peptide in said sample Can do. Certain embodiments of the invention also preferably comprise a composition comprising: an antibody, a medium suitable for the formation of an immunological complex of said antibody and a peptide recognized by said antibody, and immunological A reagent that can detect the complex and confirm the presence of CSP (SEQ ID NO: 2) or a similar peptide. Certain embodiments of the present invention also include a kit for detecting in vitro the presence or absence of CSP (SEQ ID NO: 2) or similar peptide in a biological sample, said kit preferably comprising an antibody, A medium suitable for the formation of an immunological complex between the antibody and a peptide recognized by the antibody, and a CSP (SEQ ID NO: 2) or similar in a biological sample by detecting the immunological complex Reagents that can confirm the presence of the peptide are included. Further information regarding the use of antibodies is provided, for example, in US Pat. Nos. 5,695,931 and 5,837,472, which are hereby incorporated by reference in their entirety.

Genetic acceptability assay
The ability of the peptide to activate HK (SEQ ID NO: 4) and RR (SEQ ID NO: 6) and subsequent genes that are required for conferring properties for genetic acceptability, acid resistance and biofilm formation is signal It can be determined by measuring the uptake and expression efficiency of S. mutans DNA (preferably plasmid DNA) when exposed to peptides and / or inhibitors. Genetic receptivity is assayed using two methods based on the protocol described by Perry et al. (Infect Immun, 41: 722-727) and Lindler & Macrina (J Bacteriol, 166: 658-665) . The method comprises adding DNA and CSP (SEQ ID NO: 1) (preferably plasmid DNA) to an S. mutans culture (or a culture of a bacterium expressing CSP (SEQ ID NO: 1) or a variant thereof). Need. Subsequently, the transformation rate is determined. S. mutans is preferably grown in THYE + 5% horse serum (THYE-HS). After 2 hours of incubation, 1 μg / mL plasmid DNA or 10 μg / mL chromosomal DNA is added to the culture. To assay for induction of acceptability, a synthetic competence stimulating peptide (SCSP) (SEQ ID NO: 14) was subsequently added to the culture and SCSP was added to each sample at a final concentration of 500 ng / mL. Incubate for 30 minutes. After a 30 minute incubation, an equal volume of DNA is added to each well (1 μg / mL plasmid or 10 μg / mL chromosomal DNA) and incubation is continued for an additional 2 hours. Immediately cell dilutions were spread on THYE agar plates with appropriate antibiotics. Transformation frequency was expressed as the number of transformants (antibiotic resistant cells) per number of surviving recipients. This frequency is determined by comparing the number of cells that can grow in the presence of antibiotics against the total number of cells present (ie, the number of cells that grow in the absence of antibiotics). Larger values indicate a higher rate of transformation and thus reflect the stimulatory effect of the peptide. As a result, the addition of a molecule that can act as an inhibitor results in a reduction of the transformant / recipient ratio, indicating that the inhibitor is effective in blocking the activity of CSP (SEQ ID NO: 2) . CSP (SEQ ID NO: 1 or SEQ ID NO: 2) deficient cells can also be used in these assay variants. A compound that inhibits CSP (SEQ ID NO: 2) or a variant thereof can be identified by adding a test compound to the mixture and determining whether the transformation rate is reduced by the addition of the test compound.
The activity of the system is also a marker protein expressed from a fusion with a promoter controlled by a signal cascade initiated by CSP (SEQ ID NO: 2) / HK (SEQ ID NO: 4) / RR (SEQ ID NO: 6). For example, it can be measured by an in vitro assay based on measurement of the expression of green fluorescent protein (GFP). Some such promoters are present immediately 5 'to the S. mutans comX gene. S. mutans grown in microtiter wells were exposed to CSP (SEQ ID NO: 2) and / or inhibitor, measured the fluorescence level of comX :: GFP strain, and stimulated with CSP (SEQ ID NO: 2) ( And vice versa a quantitative measurement of inhibitor activity). Inhibiting CSP (SEQ ID NO: 2) or a variant thereof by adding a test compound to the mixture and determining whether quantitative measurement of CSP (SEQ ID NO: 2) stimulation is reduced by the addition of the test compound Can be identified.

Acid Tolerance Assay The ability of CSP (SEQ ID NO: 2) to promote acid tolerance tolerance is determined by measuring cell viability of S. mutans when exposed to acidic pH. In one example, S. mutans was first grown in batch culture and the method previously described in so-called standard log phase and stationary phase cells (Svensaeter et al. Oral Microbiol Immunol, 12: 266-73). The acid tolerance response is assayed by using the modified method. Transfer one volume of overnight culture to 9 volumes of fresh TYG medium (pH 7.5) (1:10) and obtain log mid-phase cells by incubating at 37 ° C. with 5% CO ₂ for 2 hours. These cells are then collected by centrifugation at 8,000 g for 10 minutes and resuspended in 2 mL of fresh TYG (pH 5.5) at various cell concentrations as determined by OD ₆₀₀ . Cells are acid-adapted by incubating with 5% CO ₂ at 37 ° C. for 2 hours at pH 5.5. Subsequently, the adapted log phase cells are exposed to a killing pH. The killing pH is predetermined by incubating non-adapted log metaphase cells in TYG medium with a pH value of 6.0 to 2.0. Stationary phase cells are prepared by suspending log late cells in TY (tryptone-yeast extract) medium without glucose. The culture is incubated at 37 ° C. for 2 hours to allow the cells to fully enter stationary phase. Induction of acid adaptation in stationary phase cells follows a procedure similar to the induction of acid adaptation in log phase cells. Adaptation of log phase cells and stationary phase cells to acidic pH is determined by measuring the ability of bacterial cells to survive 3 hours at the killing pH. Acid killing is initiated by suspending cells in the same volume of fresh TYG (pH 3.5), and a small amount of cell suspension is immediately taken from each sample to determine the total number of viable cells at 0 hours. The cells are then incubated for 3 hours at 37 ° C. with 5% CO ₂ , a small sample is taken and the viability determined by counting viable cells. The addition of a molecule that can act as an inhibitor results in a reduction in acid tolerance of S. mutans, correspondingly reducing cell survival, said inhibitor being CSP (SEQ ID NO: 2) It is effective in the inhibition activity. CSP (SEQ ID NO: 1 or SEQ ID NO: 2) deficient cells can also be used in these assays where the acid adaptation deficient phenotype of the comC (SEQ ID NO: 1 or SEQ ID NO: 2) deficient cells can be complemented by the addition of a signal peptide. Can be used for deformation. A compound that inhibits CSP (SEQ ID NO: 1 or SEQ ID NO: 2) or a variant thereof by adding a test compound to the mixture and determining whether cell viability is reduced by the addition of the test compound Can be identified.

Cells transformed with the nucleic acid molecules disclosed herein (histidine kinase (SEQ ID NO: 3), CSP (SEQ ID NO: 1) or response regulator (SEQ ID NO: 5)) are useful as research tools. For example, cells that do not express histidine kinase (SEQ ID NO: 3), CSP (SEQ ID NO: 1) or response regulator (SEQ ID NO: 5) (or cell lines such as immortalized or primary cell cultures) Obtaining and inserting a histidine kinase (SEQ ID NO: 3), CSP (SEQ ID NO: 1) or response regulator (SEQ ID NO: 5) nucleic acid molecule into the cell, and determining the expression level and activity. Alternatively, histidine kinase (SEQ ID NO: 3), CSP (SEQ ID NO: 3), CSP (SEQ ID NO: 1) or response regulator (SEQ ID NO: 5) in cells expressing nucleic acid molecules, histidine kinase (SEQ ID NO: 3), CSP (sequence Number: 1) or response regulator (SEQ ID NO: No. 5) The nucleic acid molecule can be overexpressed, in another example various types of histidine kinase (SEQ ID NO: 3), CSP (SEQ ID NO: 1) or response regulator (SEQ ID NO: 5) Cells of multiple experimental groups can be transformed with vectors containing nucleic acid molecules to determine the level of polypeptide and peptide produced, their function, and the phenotype of the cell. Useful for in vitro analysis of the activity or structure of histidine kinase (SEQ ID NO: 4), CSP (SEQ ID NO: 2) or response regulator (SEQ ID NO: 6). The peptides can be used for microscopic or X-ray crystallographic studies.
Nucleic acid molecules and polypeptides of histidine kinase (SEQ ID NO: 3 and SEQ ID NO: 4), CSP (SEQ ID NO: 1 and SEQ ID NO: 2) or response regulator (SEQ ID NO: 5 and SEQ ID NO: 6) are also It is useful in assays for the identification and development of compounds that directly inhibit and / or enhance the function of a polypeptide or peptide. For example, they determine whether a test compound can act as an antagonist to histidine kinase (SEQ ID NO: 4), CSP (SEQ ID NO: 2) or response regulator (SEQ ID NO: 6) by: Are useful in assays that: (a) have histidine kinase (SEQ ID NO: 4), CSP (SEQ ID NO: 1) or response regulator (SEQ ID NO: 5) (or have histidine kinase, CSP or regulator response factor activity) Cells containing nucleic acid molecules expressing said fragment or variant), wherein said culture is histidine kinase (SEQ ID NO: 4), CSP (SEQ ID NO: 2) or response regulator (SEQ ID NO: 6) Carried out in the presence of increasing concentrations of at least one test compound to determine its ability to inhibit, and (b) the inhibition level as a function of the concentration of the test compound. Le monitored by cell, thereby histidine kinase (SEQ ID NO: 4), CSP (SEQ ID NO: 2) or response regulator (SEQ ID NO: 6) illustrates the ability of the test compound that inhibits the activity of.
A suitable assay can be applied, for example, from US Pat. No. 5,851,788.

Example
Materials and methods
Cell growth conditions :
Cells are grown in various dilutions of Todd Hewitt yeast extract medium with and without 5% horse serum and 0.01% porcine stomach mucin.
Biofilm growth cell transformation protocol :
Biofilms are polystyrene microtiter plates to provide a quick and convenient way to assay biofilm formation and thus the activity of the peptide (SEQ ID NO: 2) / receptor (SEQ ID NO: 6) / kinase (SEQ ID NO: 4) system. Grow on. Biofilm formation was performed by inoculating 20 μL of the cell suspension into each well containing 2 mL of biofilm medium (4-fold diluted Todd-Huwit yeast extract medium supplemented with porcine gastric mucin at a final concentration of 0.01%) and anaerobic. Start by incubating overnight at 37 ° C under sex conditions. After 20 hours of incubation, the liquid medium is removed and 2 mL of pre-warmed fresh THYW + 5% horse serum is added. The culture is incubated for 30 minutes and each well is supplemented with synthetic receptor capacity stimulating peptide (SCSP) and various concentrations of inhibitors at a final concentration of 200 ng / mL and incubation is continued. After 30 minutes, plasmid DNA (1 mg / mL) or chromosomal DNA (10 mg / mL) is added to each well and the culture is incubated for an additional 2 hours. Subsequently, the floating cells are removed and the wells are washed once with PBS buffer. Biofilm cells are collected in 2 mL of fresh culture medium by washing the wells with gentle sonication and a pipette. Centrifuge the sample at 12,000 xg for 5 minutes. Both biofilm and suspension cells are resuspended in 200 μL fresh media and immediately spread on THYE agar + appropriate antibiotics. Transformation frequency is determined after 48 hours of incubation.

Genome database analysis :
A homologue of the Streptococcus pneumoniae comD (SEQ ID NO: 3) / E (SEQ ID NO: 5) gene encoding the histidine kinase (SEQ ID NO: 4) / response regulator (SEQ ID NO: 6) system was identified. Primers were designed using this sequence to amplify regions from numerous S. mutans isolates. An open reading frame consisting of 138 nucleotides was present in the opposite direction 148 nucleotides 5 'proximal from the end of the comD homologue (Figure 1). This ORF encodes a 46 amino acid long peptide (SEQ ID NO: 2) and was found to be a precursor of 21 amino acid CSP (SEQ ID NO: 30).
PCR amplification and nucleotide sequencing :
The comCDE gene (SEQ ID NO: 21) was amplified by PCR from the genomes of several S. mutans isolates using primers designed based on the genome database sequence, and their nucleotide sequence was determined. The deduced amino acid sequence is compared between isolates by sequence alignment to confirm identity.
Gene inactivation :
The gene is inactivated by incorporating internal homologous fragments into the suicide vector pVA8912. Individual genes (comC (SEQ ID NO: 1), comD (SEQ ID NO: 3), comE (SEQ ID NO: 5)) inactivate mutants with deletions, biofilm-forming ability, acidic pH (pH2 -4) Compare their phenotypes with the parent strain NG8 for performance and DNA transport and uptake capacity. ComD (SEQ ID NO: 3) and comE (SEQ ID NO: 5) knockout mutants were constructed by insertion-overlapping mutagenesis, while comC (SEQ ID NO: 1) knockout mutants were comC (SEQ ID NO: 1). ) Created by allelic exchange through insertion of erythromycin resistance determinants at the locus (Li et al. 2001). All mutants were therefore resistant to erythromycin. Wild type strains are routinely subcultured on Todd Hewitt Yeast Extract (THYE) agar plates (BBL ™; Becton Dickinson, Cockeysville, MD), while mutants are THYE agar + 10 μg / Maintained on mL erythromycin. Minimal medium (DMM) was prepared and biofilms were grown by modifying a previously described method (Loo et al. 2000). The medium contains 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 ₄ .7H ₂ O, 0.2% (wt / vol) casamino acid. In addition, filter sterilized vitamins (0.04 mM nicotinic acid, 0.1 mM pyridoxine HCl, 0.01 mM pantothenic acid, 1 μM riboflavin, 0.3 μM thiamine HCl and 0.05 μM 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 were supplemented.

Creation of comD deletion mutants :
The S. mutans UA159 comD null mutant was constructed by a PCR-based deletion procedure involving restriction-ligation and allelic exchange as previously described (Lau et al. 2002). Primers used for the construction and confirmation of S. mutans comD deletion mutants were P1-HK13 (5'-CACAACAACTTATTGACGCTATCCC-3 '), P2-HK13 (5'-GGCGCGCCAACTGGCAACAGGCAGCAGACC-3'), P3-HK13 (5 '-GGCCGGCCTCAAAACGATGCTGTCAAGGG-3'), P4-HK13 (5'-AGATTATCATTGGCGGAAGCG-3 '), Erm-19 (5'-GGCGCGCCCCGGGCCCAAAATTTGTTTGAT-3') and Erm-20 (5'-GGCCGGCCAGTCGGCAGCGACTCATAGAAT-3 ').
Synthesis of synthetic peptides :
The sequence of the processed peptide was deduced by determining the cleavage site that should be present beside the gly-gly amino acid residues (positions 24 and 25) (FIG. 4). A peptide corresponding to the amino acid sequence of residues 26-46 (inclusive) was synthesized.
Synthesis of peptide analogs :
The sequence of peptide analogues used in this experiment is listed in Table 4. This peptide was synthesized by methods known in the art.

Table 4: Synthetic CSP analogs

A receptor stimulating peptide (CSP) analog was synthesized based on the sequence of mature 21 amino acid CSP (SGSLSTFFRLFNRSFTQALGK). CSP peptide analogs (F1 (SEQ ID NO: 37), H1 (SEQ ID NO: 39), B2 (SEQ ID NO: 41), C2 (SEQ ID NO: 42), E2 (SEQ ID NO: 44) and B3 (SEQ ID NO: 47)) was synthesized at the following facilities: Advanced Protein Technology Centre, Peptide Synthesis Facility of Hospital for Sick Children (Toronto, ON) and Mimotopes (San Diego). F1 and H1 analogs were created by deleting the 2nd and 4th residues from the C 'terminus, respectively, but in B2 and C2, the charged residues remain neutral (alanine) or hydrophobic (valine) residues. Substituted with group. In the E2 analog, the second arginine (from the C ′ end) was replaced with neutral alanine. B3 analogs were created by replacing the third residue from N ′ with threonine and then deleting the first, second and third residues from the C ′ end.
The peptide was dissolved at 1 mg / mL in sterile deionized distilled water. For all insoluble peptides, 10% (vol / vol) acetic acid, 20% (vol / vol) acetonitrile or 100% (vol / vol) dimethylformamide (DMF) was subsequently added. The peptide was stored at -20 ° C until use.

Recovery of the defect phenotype by the addition of CSP :
To determine whether a synthetic peptide (SEQ ID NO: 14) can restore the defective phenotype of a comC (SEQ ID NO: 2) mutant, a chemically synthesized 21 amino acid receptor-stimulating peptide (CSP) ) (SEQ ID NO: 14) (Li et al. 2001) was used in complementation experiments. The peptide was freshly dissolved in sterile distilled water at a concentration of 1 mg / mL. This CSP solution was then added to the culture at a final concentration of 2 μg / mL 2 hours after inoculation with bacterial cells.
Growth rate :
The strains were grown in THYE medium to assay the growth curves of the parent and mutant strains using a bioscreen microbiology reader incorporating multi-well disposable microtiter plates (Biscreen C, Helsinki, Finland). The bioscreen reader is equipped with a Biolink software program that allows automatic recording and display of growth curves and growth rate calculations. Growth of the strain was initiated by inoculating 5 μL of cell suspension into each well containing 200 μL of fresh THYE medium. The cell suspension was preconditioned to the same optical density at OD ₆₀₀ before inoculation. The plate was then placed in a bioscreen system. The system was set to automatically read the optical density every 15 minutes while shaking. Optical density readings are automatically recorded and converted to growth curves. Each assay was performed in quadruplicate.

Bacterial strain and growth state :
Seven strains of S. mutans were used in this experiment (strains include: BM71, GB14, H7, JH1005, LT11, NG8 and UAB159). All strains were cultivated from lyophilized ampoules and routinely maintained on Todd-Huwit yeast extract (THYE) plates. The medium was supplemented with either erythromycin (Em) (10 μg / mL) or kanamycin (Km) (500 μg / mL) for selection of antibiotic resistant colonies after transformation.
S. mutans strain wild-type UA159 and its comD null mutant were routinely used on a 0.3% (wt / vol) yeast extract supplemented Todd-Huwit (THYE) agar plate at 37 ° C in an atmosphere containing 5% CO ₂ Allowed to grow. For biofilm experiments, S. mutans strains were grown in semi-limited minimal medium (SDM) supplemented with 5 mM glucose as previously described (Li et al. 2002). The replicable plasmid pDL289 (Buckley et al. 1995) was used as donor DNA for genetic transformation experiments. Plasmid DNA was prepared from E. coli by using a commercially available plasmid preparation kit (Qiagen). When needed, antibiotics were added as follows: 10 μg / mL erythromycin or 500 μg / mL kanamycin for S. mutans and 50 μg / mL kanamycin for E. coli.
Streptococcus species (including S. sobrinus, S. sanguis, S. gordonii, S. oralis, S. mititus, Streptococcus pneumoniae) were also used to experiment with the inhibitory effects of synthetic peptide analogs. They were grown at pH 5.5 or 7.5 in Todd-Huwit broth (THYE) containing 0.3% yeast extract. They were routinely passaged on THYE agar plates and incubated in a 37 ° C. anaerobic chamber (5% CO ₂ ). In liquid medium, the culture was incubated in a closed screw cap tube without stirring in a 37 ° C. anaerobic chamber (5% CO ₂ ).

Assay of S. mutans biofilms formed on polystyrene microtiter plates (a) :
Biofilms are grown on polystyrene microtiter plates to provide a quick and convenient method for assaying genetic transformation. A 4-fold diluted THYE medium supplemented with porcine gastric mucin at a final concentration of 0.01% was used as biofilm medium (BM). Biofilm formation was initiated by inoculating 20 μL of cell suspension into each well containing 2 mL of BM and provided with 4 wells (2 for transformation assay and 2 for biofilm quantification) . After incubating the culture at 37 ° C. for 20 hours under anaerobic conditions, the liquid medium was removed for viable cell counting. The biofilm cells were collected in 2 mL of PBS by washing the wells once with 10 mM PBS buffer (pH 7.2) and gently sonicating for 5 seconds. Both biofilm and suspension cells were immediately spread on THYE plates using a spiral system (Spiral Plater, Model D, Cincinnati, OH) and incubated at 37 ° C. under anaerobic conditions. Biofilm formation was quantified by viable cell count after 48 hours of incubation.
Assay of S. mutans biofilm formed on polystyrene microtiter plate (b) :
Biofilms were grown in 96 well polystyrene microtiter plates. Biofilm growth is performed overnight in 300 μL SDM-glucose containing peptide analogs of varying concentrations (0, 0.1, 0.5, 2 and 5 μg / mL) in individual wells of a 96-well microtiter plate. Start by inoculating 10 μL of UA159. Wells without cells were used as blank controls. Subsequently, the microtiter plate was incubated without stirring at 37 ° C. under 5% CO _{2 for} 16 hours. After incubation, the floating cells were carefully removed and the plates were air dried overnight. Subsequently, the plate was stained with 0.01% (wt / vol) safranin for 10 minutes, washed with sterile distilled water and air-dried. Biofilm was quantified by measuring the absorbance of the stained biofilm at 490 nm using a microplate reader (Model 3550; Bio-Rad Laboratories, Richmond, Calif.).

Assays of S. mutans, S. sobrinus, S. sanguis, S. gordonii, S. oralis, S. mitis and S. pneumoniae biofilms formed on polystyrene microtiter plates :
96 well microtiter plates to determine the anti-biofilm activity of synthetic E2 peptides against Streptococcus species (including S. mutans, S. sobrinus, S. sanguis, S. gordonii, S. oralis and S. mitis) Biofilm growth on top, 10 μL of overnight Streptococcus species, 300 μL of semi-limited minimal medium (58 mM K ₂ HPO) containing E2 peptide (0 and 5 μg / mL) in individual wells of a 96-well microtiter plate ₄ , 15 mM KH ₂ PO ₄ , 10 mM (NH ₄ ) ₂ SO ₄ , 35 mM NaCl and 2 mM MgSO ₄ .7H ₂ O). The semi-limited minimal medium consists of filter sterilized vitamins (0.04 mM nicotinic acid, 0.1 mM pyridoxine HCl, 0.1 mM pantothenic acid, 1 μM ribofragment bottle, 0.3 μM thiamine HCl and 0.05 μM D-biotin), amino acids (4 mM L- Supplemented with glutamic acid, 1 mM L-arginine HCl, 1.3 mM L-cysteine HCl, 0.1 mM L-tryptophan), 0.2% (wt / wt) casamino acid and 20 mM glucose. Wells without cells were used as blank controls. Subsequently, the microtiter plate was incubated in an anaerobic chamber (5% CO ₂ ) without stirring for 24 hours at 37 ° C. After incubation, the proliferation was measured at 600 nm with a microplate reader. Suspended cells were carefully removed and the plates were air dried overnight. Subsequently, the plate was stained with 0.4% crystal violet for 10 minutes, washed with sterile distilled water, and air-dried for 15 minutes. The biofilm was quantified by measuring the absorbance of the stained biofilm at 630 nm using a microplate reader.

S. mutans receptivity assay :
To determine whether peptide analogs have any effect on the development of genetic competence, S. mutans UA159 wild type cells were assayed for genetic transformation. An overnight culture of S. mutans UA159 is diluted with pre-warmed THEY broth (1:20) and brought to 37 ° C in an atmosphere containing 5% CO ₂ until the optical density (OD) at 600 nm reaches approximately 0.1. And incubated. The culture was divided into 6 aliquots containing 1 μg / mL plasmid pDL289 and various concentrations (0, 0.1, 0.5, 2 and 5 μg / mL) of peptide analogs. The culture was incubated at 37 ° C. for 2.5 hours under air containing 5% CO _{2 and} gently sonicated for 10 seconds to disperse the Streptococcus chains and spread on kanamycin-containing THYE plates. Plates were incubated at 37 ° C. for 48 hours under air containing 5% CO ₂ . All recipient cells were counted by spreading serial dilutions on a THYE plate without antibiotics. Transformation efficiency was expressed as a percentage of kanamycin resistant transformants relative to the total number of recipient cells.
S. Mutans acid resistance assay :
The effect of peptides on acid tolerance was assessed by determining growth in THYE at pH 7.5 and pH 5.5. An overnight culture of S. mutans wild-type UA159 cells is diluted with pre-warmed THEY broth (1:20) and incubated at 37 ° C in air containing 5% CO ₂ until the OD ₆₀₀ reaches approximately 0.4. did. Make 20-fold dilutions with 400 μL of pH7.5 THYE or pH5.5 THYE broth containing peptide analogs at various concentrations (0, 0.1, 0.5, 2, and 5 μg / mL), 100-well Bioscreen C plates Triplicate was added to each individual well. Wells without cells were used as blank controls. Cells were continuously grown using a Bioscreen microbiology reader (Labsystems, Helsinki, Finland) and cell growth was measured at 37 ° C. for 16 hours. Measurements were performed every 20 minutes with shaking to prevent cell aggregation.

“Steady state” biofilm assays :
Biofilms were also grown on chemostat biofilm fermenters to identify and optimize conditions for genetic acceptability of biofilm growing cells. The biofilm fermenter was modified at the Mechanical Engineering and Glass Bloeing Shops, University of Tronto, based on a similar system previously described (Li and Bowden, 1994). The container was made of glass with a working volume of 400 mL. The container lid is constructed of stainless steel with 10 sampling ports (diameter 0.5 cm, sinking area in liquid medium approximately 4.0 cm ² ) allowing aseptic insertion and retrieval of glass rods Provide an abiotic surface for biofilm accumulation. The temperature in the chemostat vessel was maintained at 37 ° C. ± 0.1 by a temperature controller (Model R-600F, Cole Parmer Instrument Corp. Vernon Hill, IL). The culture pH was controlled by addition of 1M KOH or 1M HCl by a pH control unit (Digital pHMeter / Controller, Model 501-3400, Barnant Corp. Barrington, IL). The vessel was placed on a magnetic stirrer (Fisher Scientific), and the culture was stirred at 200 rpm with a polypropylene-coated magnetic stir bar (3 cm in length). Continuous culture was performed by pumping fresh 4-fold diluted THYE broth (final concentration 0.01% porcine gastric mucin (type III, Sigma) supplement) into the container (400 mL) at the desired dilution. Daily maintenance of the chemostat included optical density readings, viable cell counts, and liquid medium pH measurements. When the culture reached “steady state” (at least 10 average generation time), a glass rod was aseptically inserted into the chemostat for initiation of biofilm formation. Subsequently, biofilms at various times were removed from the culture for biotransformation using genetic transformation and viable cell counting.

Scanning electron microscopy (SEM) :
To examine the thickness of the spatial distribution and biofilm by scanning electron microscopy, the various times biofilms taken out scraping the bottom of the microtiter wells, followed the washed once with KPO ₄ of 10 mM, Fix overnight with 3.7% formaldehyde (2 mL) in 10 mM KPO ₄ buffer. Subsequently, the samples were dehydrated in a series of alcohol baths (30%, 50%, 70%, 95% and 100%), critical point dried with liquid CO ₂ , mounted and sputter coated with gold. Subsequently, the samples were examined using a scanning electron microscope (Model S-2500, Hitachi Instruments, San Jose, CA).

Transformation protocol :
Biofilm cells using two methods modified on the basis of protocols described in the literature (Perry et al. Infect Immun, 41: 722-727; and Linder amd Macrina, J Bacteriol, 166: 658-665) The natural transformation of was assayed. To the biofilm formed on the polystyrene microtiter plate, 2 mL of freshly heated THYE + 5% horse serum (THYE-HS) pre-warmed immediately after removing the BM medium was added, and incubation was continued at 37 ° C. After a 2 hour incubation, a final concentration of 1 μg / mL plasmid DNA or 10 μg / mL chromosomal DNA was added to each well. The culture was further incubated for 2 hours and cells were harvested for plating. To assay for induction of capacity by a synthetic capacity-stimulating peptide (SCSP) (SEQ ID NO: 14), the culture was incubated for 30 minutes and SCSP (SEQ ID NO: 14) was added to each well at a final concentration of 500 ng / mL. Added to. After 30 minutes incubation, an equal volume of DNA was added to each well (1 μg / mL plasmid DNA or 10 μg / mL chromosomal DNA) and the incubation continued for another 2 hours. Subsequently, the liquid medium was removed from each well and the wells were washed once with PBS buffer. Biofilm cells were collected in 2 mL PBS buffer by gentle sonication or by washing the wells with a pipette. The sample was centrifuged at 12,000 × g for 5 minutes. Both biofilm and suspension cells were resuspended in 200 μL fresh medium and spread immediately on THYE agar plates + appropriate antibiotics. For biofilms grown on a chemostat, the glass rods containing the biofilm cells were removed, placed in 2 mL of freshly warmed THYE-HS medium and incubated for 30 minutes. Subsequently, transformation was initiated using the same method as described above. Suspension cells were also removed and the transformation frequency was compared. After completion of the transformation process, both biofilm and suspension cells were spread on THYE agar + appropriate antibiotics. Transformation frequency was determined after 48 hours of incubation. The frequency of transformation was expressed as the number of transformed cells / μg DNA / viable recipient cells upon addition of DNA.

Donor DNA :
In this experiment, both plasmid DNA and chromosomal DNA were used as donor DNA to assay genetic transformation. The plasmid DNA contained an integration plasmid, pVAGTFA (which retains the erythromycin resistance (Em ^r ) determinant) and a fragment of the S. mutans gtfA gene. A replicable plasmid, pDL289 (which carries the kanamycin resistance gene (Km ^r )) was also used. Chromosomal DNA that holds em ^r gene were prepared from recombinant S. mutans strain carrying a copy of pVAGTFA integrated into a chromosome.

Although the present invention has been described in detail above with specific reference to the preferred embodiments, those skilled in the art will recognize that modifications can be made without departing from the scope of the invention. For example, if the application refers to peptides, it is clear that polypeptides can often be used. Similarly, when genes are described in this application, it is clear that nucleic acid molecules or gene fragments can often be used.
All publications (including GenBank registrations), patents and patent applications are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually displayed. Included in the book.

Example 1 CSP Peptide Analogues Performed In Vitro Assays to Perform Streptococcus mutans Biofilm Formation as described in the Materials and Methods section to inhibit Streptococcus mutans biofilm formation. The effects of synthetic CSP analogs (F1, H1, B2, C2, E2, and B3; Table 4) were determined. The analog was synthesized as described in Materials and Methods. The anti-biofilm activity of analogs against S. mutans varied from 0 to 80% in terms of percent inhibition (FIG. 18). Since the E2 peptide showed the best anti-biofilm activity (80% inhibition) among the 6 synthetic CSP analogs tested, the peptide was selected for further in vitro experiments.
Example 2 E2 CSP Peptide Analogues are In Vitro Assays as described in Materials and Methods to Inhibit Biofilm Formation in S. Sobrinus, S. Sanguis, S. Gordoni, S. Oralis and S. Mytis To determine whether E2 analogs of CSP can inhibit biofilm formation in Streptococcus species such as S. sobrinus, S. sanguis, S. gordonii, S. oralis and S. mitis . The E2 peptide showed an inhibitory effect on both growth and biofilm formation in all five bacteria tested at concentrations as low as 5 μg / mL. The percent inhibition of biofilm formation in these bacteria varied between 40 and 75% (Figures 19, 20, 21, 22, and 23). Furthermore, the anti-biofilm activity of the E2 peptide was tested against the mixed culture of the above Streptococcus species. The E2 peptide also showed a significant inhibitory effect on biofilm formation in mixed cultures (data not shown).
Example 3 E2 CSP Peptide Analogs Inhibit Streptococcus pneumoniae Biofilm Formation This Example demonstrates the effect of S. mutans CSP analogs on the CSP-mediated population sensing signaling system of non-oral Streptococcus pathogens. In vitro assays were performed as described in the Materials and Methods section to determine whether E2 analogs of CSP could inhibit S. pneumoniae biofilm formation. At concentrations as low as 5 μg / mL, E2 peptide showed significant anti-biofilm activity against S. pneumoniae (25% inhibition, FIG. 24).

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Schematic diagram showing the arrangement of loci encoding signal peptide precursor (ComC) (SEQ ID NO: 1), histidine kinase (ComD) (SEQ ID NO: 3), and response regulator (ComE) (SEQ ID NO: 5) It is. This arrangement is different from other related Streptococcus. This is because, unlike the other genes described so far, which are arranged as an operon-like cluster with the comC / DE gene transcribed from a single promoter, the comC gene (SEQ ID NO: 1) has its own unique This is because it is transcribed from the promoter and the comC gene (SEQ ID NO: 1) is 148 nucleotides away from the comD gene (SEQ ID NO: 3). Nucleic acid molecule SEQ ID NO: 1, 3, 5 and 29 are shown. Figure 2A: S. mutans comC gene (SEQ ID NO: 1). Encodes precursor of signal peptide (SEQ ID NO: 2). Figure 2B: S. mutans CSP coding sequence (SEQ ID NO: 29). Encodes a receptor stimulating peptide (SEQ ID NO: 30). FIG. 2C: S. mutans comD gene (SEQ ID NO: 3). Figure 2D: S. mutans comE gene (SEQ ID NO: 5). It encodes a response regulator (SEQ ID NO: 6) that activates the transcription of a number of genes. The sequence of the deduced amino acid sequences of the signal peptide (SEQ ID NO: 2), histidine kinase (SEQ ID NO: 4) and response regulator (SEQ ID NO: 6) is shown. FIG. 3A: S. mutans ComC protein (CSP precursor) (SEQ ID NO: 2). Figure 3B: S. mutans ComD protein (histidine kinase) (SEQ ID NO: 4). FIG. 3C: S. mutans ComE protein (response regulator) (SEQ ID NO: 6). The deduced amino acid sequences of the signal peptide precursors of various strains and their predicted cleavage sites are shown. The original peptide is expressed as a 46 amino acid peptide and is cleaved behind the glycine-glycine residue to produce an active signal peptide. Fig. 2 shows a synthetic signal peptide (SEQ ID NO: 14) that is effective for induction of acceptability, biofilm formation and acid tolerance in Streptococcus mutans. 2 shows the natural activity of a signal / receptor system that functions in vitro in a model biofilm as determined by the ability of various S. mutans strains to accept donor plasmid DNA conferring erythromycin resistance. It is a table | surface which shows the influence of the synthetic peptide with respect to the genetic acceptability of a S. mutans cell. FIG. 3 shows the induction of Streptococcus mutans genetic transformation by a synthetic receptivity stimulating peptide (SCSP). A list of primers used in amplification by polymerase chain reaction (PCR) of a target gene or an internal region of a target gene for subsequent sequencing or inactivation. The ComCDE locus region (SEQ ID NO: 21) is shown. ComC (first highlight region; nucleotides 101 to 241), ComD (second highlight region; nucleotides 383 to 1708) and ComE (third highlight region; nucleotides 1705 to 2457) are highlighted. The comX DNA sequence (SEQ ID NO: 22), protein sequence (SEQ ID NO: 23), and comX locus region (SEQ ID NO: 24) containing 100 bp upstream and downstream (promoter upstream). FIG. 10A: S. mutans comX gene (SEQ ID NO: 22). FIG. 10B: S. mutans ComX protein (SEQ ID NO: 23). FIG. 10C: S. mutans comX locus region (SEQ ID NO: 24). The comA and comB nucleotide sequences (SEQ ID NO: 25) and (SEQ ID NO: 27), as well as the amino acid sequences (SEQ ID NO: 26) and (SEQ ID NO: 28) are shown. ComA and ComB are components of the CSP exporter. FIG. 11A: S. mutans comA gene (SEQ ID NO: 25). FIG. 11B: S. mutans ComA protein (SEQ ID NO: 26). FIG. 11C: S. mutans comB gene (SEQ ID NO: 27). FIG. 11D: S. mutans ComB protein (SEQ ID NO: 28). Figure 2 shows the effect of synthetic peptides on acid tolerance tolerance in S. mutans comC deficient cells. Addition of a synthetic signal peptide (CSP) (SEQ ID NO: 14) to the comC mutant culture restored the ability of the mutant to survive low pH challenge when compared to the parental strain NG8. It is a schematic diagram of the collective sensing circuit of S. mutans. Figure 2 shows the effect of various concentrations of H1 on genetic transformation of S. mutans wild type UA159. Results are expressed as the mean ± SE of 3 separate experiments. Figure 5 shows the effect of various concentrations of H1 on genetic transformation of S. mutans comD null mutants. FIG. 6 shows the effect of various concentrations (μg / mL) of CSP and H1 on cell growth of S. mutans wild type UA159 in THYE (pH 5.5). Average OD ₆₀₀ value ± SE. Results represent the average of three separate experiments. FIG. 6 shows the effect of various concentrations (μg / mL) of CSP and H1 on cell growth of S. mutans wild type UA159 in THYE (pH 7.5). Average OD ₆₀₀ value ± SE. Results represent the average of three separate experiments. Figure 6 shows the effect of synthetic CSP analogs (B2, B3, C2, E2, F1 and H1 peptides) on Streptococcus mutans biofilm formation. Figure 3 shows the effect of E2 peptide on Streptococcus sobrinus biofilm formation. Figure 3 shows the effect of E2 peptide on Streptococcus oralis biofilm formation. Figure 2 shows the effect of E2 peptide on Streptococcus sanguis biofilm formation. Figure 2 shows the effect of E2 peptide on Streptococcus mitis biofilm formation. Figure 2 shows the effect of E2 peptide on Streptococcus gordonii biofilm formation. Figure 2 shows the effect of E2 peptide on Streptococcus pneumoniae biofilm formation.

Claims (71)

  An isolated polypeptide comprising a fragment of SEQ ID NO: 2 or SEQ ID NO: 30, and further capable of inhibiting the genetic acceptability of S. mutans.   An isolated polypeptide that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 30 and that can further inhibit the genetic acceptability of S. mutans.   The isolated polypeptide according to claim 1 or 2, wherein 1-5 amino acids have been removed from the COOH terminus of SEQ ID NO: 2 or SEQ ID NO: 30.   An isolated polypeptide comprising a fragment of SEQ ID NO: 2 or SEQ ID NO: 30 and further 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 that can further inhibit S. mutans biofilm formation.   The isolated polypeptide of claim 5 or 6, wherein 1-5 amino acids of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 30 have been modified to contain up to one.   A composition for reducing biofilm formation of S. mutans, comprising a peptide derivative of CSP.   A composition comprising a peptide derivative of CSP, which reduces the transformation efficiency of S. mutans.   9. The peptide analogue is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 47. Composition.   10. The composition of claim 9, wherein the peptide analog is a peptide having the amino acid sequence of SEQ ID NO: 44.   A cell genetically engineered to produce a composition according to any one of claims 7 to 10.   The cell according to claim 11, wherein the cell is a bacterium.   The cell according to claim 12, which is an S. mutans cell.   Use of a cell according to any one of claims 11 to 13 for the purpose of producing a composition according to any one of claims 7 to 10.   A composition comprising the cell of any one of claims 11 to 13 and a pharmaceutically acceptable adjuvant.   A method for inhibiting the growth of S. mutans comprising administering the composition according to any one of claims 7 to 10 or 15.   A method for inhibiting caries, comprising administering the composition according to any one of claims 7 to 10 or 15.   A method for improving dental health comprising administering a composition according to any one of claims 7 to 10 or 15.   An isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47.   A peptide analog of S. mutans-receptive stimulating peptide (CSP) that inhibits biofilm formation.   21. A peptide analog according to claim 20, which is naturally occurring.   21. The peptide analog of claim 20, which is a synthetic product.   23. The peptide analog of claim 22, wherein the synthetic peptide is produced by deletion and / or substitution of amino acids at the C 'or N' terminus of S. mutans CSP.   The peptide of claim 21, wherein the S. mutans CSP has the amino acid sequence of SEQ ID NO: 30.   21. The peptide of claim 20, wherein the peptide analog inhibits a biofilm formed by plaque associated bacteria.   21. The peptide according to claim 20, wherein the plaque-associated bacterium is Streptococci or Actinomyces.   The Streptococcus bacterium is selected from the group consisting of S. mutans, S. sorbinus, S. sanguis, S. gordonni, S. oralis and S. mitis 27. The peptide analog of claim 26.   Peptide analogues inhibit biofilms formed by Streptococcus bacteria selected from the group consisting of S. pneumoniae, S. pyogenes, and S. agalactiae The peptide according to claim 20.   A pharmaceutical composition comprising at least one CSP inhibitor and a pharmaceutically acceptable carrier.   30. The pharmaceutical composition of claim 29, wherein the CSP inhibitor is a peptide analog of S. mutans receptivity stimulating peptide (CSP) that inhibits biofilm formation.   The CSP inhibitor is a polypeptide sequence having an amino acid selected from the group consisting of the polypeptides of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47. Item 31. A pharmaceutical composition according to item 29 or 30.   32. The pharmaceutical composition according to claim 31, wherein the amino acid sequence is SEQ ID NO: 44.   30. The pharmaceutical composition according to claim 29, wherein the CSP inhibitor is an antibody specific to CSP or a fragment thereof.   34. The pharmaceutical composition of claim 33, wherein the antibody is specific for S. mutans CSP.   30. The pharmaceutical composition of claim 29, wherein the CSP inhibitor is an antisense oligonucleotide that inhibits CSP expression and transcription.   36. The pharmaceutical composition of claim 35, wherein the antisense oligonucleotide is an oligonucleotide complementary to at least 10 consecutive nucleotides of a CSP-encoded oligonucleotide, and wherein the CSP-coded oligonucleotide has a nucleic acid sequence of SEQ ID NO: 1. .   30. The pharmaceutical composition of claim 29, wherein the CSP inhibitor is an antisense oligonucleotide that inhibits export of the CSP peptide.   The antisense oligonucleotide is an oligonucleotide complementary to at least 10 contiguous nucleotides of a CSP exporter-encoding oligonucleotide, wherein the CSP exporter-encoding oligonucleotide has a nucleic acid sequence of SEQ ID NO: 25 or 27. 37. A pharmaceutical composition according to 37.   39. A method for treating or preventing a bacterial infection caused by a biofilm-forming bacterium comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 29 to 38.   39. A method for preventing plaque formation comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 29 to 38.   40. A method of treating or preventing a condition caused by plaque associated bacteria comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 29 to 38.   42. The method of claim 41, wherein the symptom is selected from the group consisting of caries, periodontal disease, gingivitis and endocarditis.   Use of a CSP inhibitor for the manufacture of a medicament or an infection inhibitor for the treatment of infections caused by biofilm-forming bacteria.   44. The use of claim 43, wherein the CSP inhibitor is a peptide analog of S. mutans receptivity stimulating peptide (CSP) that inhibits biofilm formation.   The CSP inhibitor is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47. 44. Use according to 44.   46. The use according to claim 45, wherein the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 44.   44. Use according to claim 43, wherein the CSP inhibitor is an antibody specific to CSP or a fragment thereof.   48. Use according to claim 47, wherein the antibody is specific for S. mutans CSP.   44. The use of claim 43, wherein the CSP inhibitor is an antisense oligonucleotide that inhibits CSP expression or transcription.   50. The use of claim 49, wherein the antisense oligonucleotide is an oligonucleotide that is complementary to at least 10 consecutive nucleotides of a CSP-encoding oligonucleotide, wherein the CSP-encoding oligonucleotide has a nucleic acid sequence of SEQ ID NO: 1.   44. Use according to claim 43, wherein the CSP inhibitor is an antisense oligonucleotide that inhibits export of the CSP peptide.   52. Use according to claim 51, wherein the antisense oligonucleotide is an oligonucleotide complementary to at least 10 consecutive nucleotides of a CSP exporter oligonucleotide, said oligonucleotide having the nucleic acid sequence of SEQ ID NO: 25 or 27.   A composition that inhibits biofilm formation of Streptococcus spp, comprising: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, and Comprising at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 47, and wherein the Streptococcus species is Streptococcus sobrinus, S. sanguis, Streptococcus gordonii (S gordonii), S. oralis, Streptococcus mitis, S. salivarius, Streptococcus cristatus, S. pneumoniae, S. pyogenes and Streptococcus aga It is selected from the group consisting of frontispiece (S. agalactiae), the composition.   54. The composition of claim 53, wherein the peptide has the amino acid sequence of SEQ ID NO: 44.   55. The composition of claim 53 or 54, further comprising one or more ingredients selected from the group consisting of surfactants, preservatives, and antibiotics.   56. A composition according to any one of claims 53 to 55, comprising 1 [mu] g / mL to 100 [mu] g / mL of said peptide.   57. A composition according to any one of claims 53 to 56, comprising 1 [mu] g / mL to 10 [mu] g / mL peptide.   58. The composition of any one of claims 53 to 57, comprising 1 [mu] g / mL to 5 [mu] g / mL peptide.   The group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47 for the manufacture of a medicament that inhibits biofilm formation of Streptococcus species Use of at least one peptide having an amino acid sequence selected from Said use, selected from the group consisting of Tuus, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae.   The group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 47, intended to prevent or inhibit Streptococcus species biofilm formation Use of at least one peptide having an amino acid sequence selected from Said use, selected from the group consisting of Tuus, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae.   SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: for the purpose of treating or preventing symptoms caused by Streptococcus species associated with plaque The use of at least one peptide having an amino acid sequence selected from the group consisting of 47: Said use, selected from the group consisting of Sarivarius, Streptococcus critus.   62. Use according to claim 61, wherein the symptoms are selected from the group consisting of caries, gingivitis and endocarditis.   62. Use according to any one of claims 59 to 61, wherein the Streptococcus species is Streptococcus pneumoniae.   64. Use according to any one of claims 59 to 63, wherein the peptide has the amino acid sequence of SEQ ID NO: 44.   A method of treating or preventing Streptococcus spp. Infection in a patient in need thereof, comprising: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, and Administering the therapeutically effective amount of at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 47.   A method of inhibiting or preventing the formation of a Streptococcus species biofilm in a patient in need thereof, comprising SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, and Administering the therapeutically effective amount of at least one peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 47.   Streptococcus sp. 67. A method according to claim 65 or 66.   A method of treating or preventing symptoms caused by Streptococcus species associated with dental plaque in a patient in need thereof, comprising: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42. The method comprising administering a therapeutically effective amount of at least one peptide having an amino acid sequence selected from the group consisting of 42, SEQ ID NO: 44, and SEQ ID NO: 47.   69. The method of claim 68, wherein the symptom is selected from the group consisting of caries, periodontal disease, gingivitis and endocarditis.   69. The method of claim 68, wherein the Streptococcus species is selected from the group consisting of Streptococcus sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, and Streptococcus cristatus.   71. The method of any one of claims 65 to 70, wherein the peptide has the amino acid sequence of SEQ ID NO: 44.

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