Signal peptides, nucleic acid molecules and methods for treatment of caries

#1. Field of the Invention

#The invention generally relates to compounds and methods that inhibit or disrupt microbial biofilms involved in infections in man and animals and in biofouling of surfaces susceptible to microbial accumulation.

#2. Description of the Related Art

#FIG. 1 shows the schematic layout of the arrangement of the genetic locus encoding the signal peptide precursor (ComC) [SEQ ID NO:4], the histidine kinase (ComD) [SEQ ID NO:2] and the response regulator (ComE) [SEQ ID NO:3]. Note that this arrangement is different from other loci in related streptococci for the following reasons: a). The comC gene [SEQ ID NO:4] is transcribed from its own unique promoter, unlike the genes thus far described in other streptococci that are arranged in an operon-like cluster with the comC/DE genes being transcribed from a single promoter, b) The comC gene [SEQ ID NO:4] is separated by 148 nucleotides from the comD gene [SEQ ID NO:6].

#FIG. 2 shows the nucleic acid molecule that is SEQ ID NO:4. In a preferred embodiment, the figure shows a nucleic acid encoding CSP (competence signal peptide [SEQ ID NO:5]). FIG. 2 also shows histidine kinase [SEQ ID NO:6] sequences and response regulator [SEQ ID NO:7] sequences. FIG. 2A. S. mutans comC gene [SEQ ID NO:4]. Encodes a precursor to a signal peptide [SEQ ID NO:1]. FIG. 2B. S. mutans CSP encoding sequence [SEQ ID NO:5]. Encodes a Competence Signal Peptide [SEQ ID NO:11]. FIG. 2C. S. mutans comD gene [SEQ ID NO:6]. Encodes a response regulator that activates transcription of a number of genes. FIG. 2D. S. mutans comE gene [SEQ ID NO:7].

#FIG. 3. Sequence of the deduced amino acid sequence of the signal peptide [SEQ ID NO:1], histidine kinase [SEQ ID NO:2], and response regulator [SEQ ID NO:3]. FIG. 3A. S. mutans ComC protein (CSP Precursor) [SEQ ID NO:1]. FIG. 3B. S. mutans ComD protein (Histidine Kindase) [SEQ ID NO:2]. FIG. 3C. S. mutans ComE protein (Response Regulator) [SEQ ID NO:3].

#In some Gram-positive bacteria (including Streptococcus mutans), when a specific histidine kinase receptor located in the cell membrane is disrupted, the cells become ineffective at developing a biofilm. The cells growing in this biofilm environment use a small peptide signal molecule to activate the receptor in surrounding cells, thereby communicating the message to form a biofilm. This same signal peptide and histidine kinase are also involved in the induction of genetic competence, the cell's ability to take up and incorporate DNA from its extracellular environment, as well as that of acid tolerance, the cell's ability to survive pH levels as low as pH 3.0. A mechanism that blocks the signal molecule from activating the histidine kinase receptor molecule provides a novel method for controlling microbial biofilms, either alone or in combination with chemical or physical means.

#We have identified a genetic locus in S. mutans consisting of three genes that encode: 1) a peptide precursor [SEQ ID NO:1] that is processed during export into a secreted 21-amino acid peptide (CSP) [SEQ ID NO:11]; 2) a histidine kinase [SEQ ID NO:2] that acts as a cell surface receptor activated by the peptide; 3) a response regulator [SEQ ID NO:3] that activates a number of other genes involved in genetic competence, biofilm formation, and acid tolerance of S. mutans. These properties have been attributed to the bacterium's ability to cause dental caries. Inactivation of any of these three genes or impairment of interaction or activity of any of their encoded proteins will disrupt the bacterium's ability to take up foreign DNA, form biofilms, and tolerate acidic pH.

#Streptococcus mutans is a resident of the biofilm environment of dental plaque, a matrix of bacteria and extracellular material that adheres to the tooth surface. Under appropriate environmental conditions populations of S. mutans and the pH of the surrounding plaque will drop. S. mutans, being among the most acid tolerant organisms residing in dental plaque, will increase its numbers in this acidic environment and eventually become a dominant member of the plaque community. This situation eventually leads to dissolution of the tooth enamel, resulting in the development of dental caries. We control the accumulation and acid tolerance of this bacterium to make it less able to cause caries. We accomplish this by using inhibitors of an extracellular signal peptide that promotes the expression of genes involved in S. mutans biofilm formation and acid tolerance. Compounds are disclosed that inhibit the action of the peptide. These inhibitors can include peptides, antibodies, or other agents that specifically inhibit the activation of the histidine kinase and the family of genes activated as a result of the histidine kinase activation by the signal molecule. Inhibitors include: modified structures of the peptide where amino acids are removed from the N- and/or COOH terminal of the peptide and/or substitutions of internal amino acid residues. We delete, one, two to 5, 6 to 10 and 10 to 15 amino acids from the peptide (for example at either terminal) and measure competitive inhibition of signal peptide binding to histidine kinase (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids are deleted and inhibition measured). Inhibitors also include antibodies raised against the 21-amino acid CSP [SEQ ID NO:11] alone or coupled to a larger molecule to increase immunogenicity. We also test inhibitors described in Barrett et al. Proc. Natl. Acad. Sci. USA 95:5317-5322) and measure competitive inhibition of signal peptide binding to histidine kinase.