Streptococcus mutans is recognized as the major etiologic agent of dental caries in humans. Its pathogenicity is enhanced by a number of virulence factors including an adhesin (P1), which promotes colonization in the absence of dietary sucrose and the ability of this species to withstand the effects of low pH caused by the homofermentation of sugars. The present application focuses on each of these virulence traits. The gene encoding the P1 adhesin, spaP, has been cloned and sequenced and the gene product is well characterized. Nothing is known, however, of environmental or genetic regulatory mechanisms controlling its expression. Environmental regulation will be studied by growing a heterodiploid strain containing a chromosomal spaP::lacZ reporter gene fusion construct in a chemostat under varying growth conditions. Both adherent and non-adherent cells will be assayed for gene expression by measuring beta-galactosidase activity. Genetic regulatory loci will be interrupted by using transposon Tn 917 or random chromosomal fragments and screened for loss of adhesin expression. Regulatory genes will be characterized after marker rescue or cloning into E. coli. Also, putative enzymes and transport proteins that aid in the folding and localization of P1 will be isolated by affinity chromatography methods and responsible genes characterized as above. The second thrust of this proposal involves the Ffh-dependent protein translocation system, only recently discovered in S. mutans. An acid-sensitive mutant, known to be interrupted in the ylxM-ffh (sat) operon and unable to assemble optimal amounts of membrane-bound H+/ATPase, will be studied by two-dimensional gel electrophoresis. Major proteins differentially expressed between mutant and wild-type membranes will be recovered, subjected to N-terminal amino acid sequence analysis and appropriate degenerate oligonucleotides synthesized for use in screening the investigator's genomic libraries for responsible genes. By this approach, the investigator hopes to determine the key proteins transported into membranes by the Ffh-pathway. The yeast two-protein hybrid technique will be employed to analyze the binding of S. mutans Ffh-containing fusion proteins to those containing S. mutans ATP-ase subunits or other relevant gene products in vivo in Saccharomyces cerevisiae. Such reactions will be confirmed by affinity chromatography on Ffh- GST columns. Finally, environmental regulation will be studied by growing a heterodiploid strain containing a chromosomal ylxM promoter::lacZ reporter gene fusion construct in a chemostat under varying growth conditions including pH, osmolarity, and nutrient sources.
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