Streptococcus mutans resides in the biofilm of dental plaque where it produces acid from dietary carbohydrate to cause caries. S. mutans uses a quorum sensing signaling system in biofilms to activate genetic competence, acid tolerance, and influence biofilm architecture. This proposal addresses mechanisms by which cell-cell and environmental signals activate this 'biofilm phenotype'. S. mutans quorum sensing system is encoded by the comCDE genes that encode a competence-stimulating peptide (CSP) precursor, a histidine kinase and a response regulator. We expect to answer 1)Which phenotypic changes does CSP invoke through its various putative receptors and regulators and how do these changes facilitate optimized growth/survival in a biofilm environment? 2) How are these pathways regulated? 3) Can we inhibit these pathways to attenuate the biofilm phenotype of S. mutans? DMA microarray analysis will be used to identify genes and proteins that are activated by CSP. A number of mutants defective in various components of the system will be used to elucidate these complex signaling networks. Genes encoding products with altered expression in a biofilm context will be cloned and mutated using a novel allelic exchange technique; the mutants'genetic competence and biofilm forming abilities will be tested. Cell segregation and the 3D architecture of the mutant biofilms will be assessed by SEM and Confocal Scanning Laser Microscopy (CSLM). Reporter gene fusions (gfp, lacZ and luc) constructed in selected genes will be used to measure gene expression temporally in response to CSP using fluorimetric and luminetric analysis and CSLM to give insight into how the CSP signaling cascade functions to influence biofilm phenotype in real time. CSP analogs will be tested for their inhibition of the signal transduction process. The regulatory processes involved at modulating this network will be investigated using DNA-protein binding assays, protein-protein interactions and phosphorylation assays. These experiments will give insight into the signaling mechanisms used by S. mutans and potentially other biofilm-forming pathogens for optimal survival in biofilms and will hopefully lead to the design of means to control them.
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