Dental plaque is the oral biofilm responsible for the etiology of Dental caries and periodontal disease. A number of salivary proteins have been shown to interact with bacteria in plaque, and such interactions likely play critical roles in plaque formation. One such interaction is that between amylase, the most abundant enzyme in saliva, and the amylase-binding streptococci (ABS), which are numerous in plaque. Based on our recent findings generated during the previous funding period, this once-amended competitive renewal application seeks to continue our studies of the biochemical, physiological, cariological and ecological consequences of amylase-binding to ABS. We found that while amylase binding-deficient mutants adhere less well to amylase-coated surfaces and demonstrate defective biofilm formation in vitro, they colonize rat teeth better than wild type strains, out-compete their parental strains in rats fed sucrose/starch diet. These surprising findings led to the realization that AbpA inhibits sucrose-dependent colonization determinants such as, but perhaps not limited to, GtfG of S. gordonii. Thus, our Aims for the next funding period are to: 1) investigate potential interactions of genes and proteins involved in amylase binding (abpA and abpB) and genes involved in glucan synthesis (rgg and gtfG). We will evaluate the transcription of abpA, abpB and gtfG in mutant strains to determine if Abp modulates gtfG expression (or vice versa) at the transcriptional level. 2) compare wildtype and mutant strains in standard in vitro adhesion and biofilm models. 3) assess the physical interaction between Gtf and amylase-binding proteins using proteomic approaches such as Western blot-ligand binding assays, Maldi-Tof (Matrix-assisted, Laser-Desorption-Ionization/Time of Flight) mass spectrometry, or phage display. 4) determine if amylase-binding mutations in Gtf-deficient or -proficient S. gordonii modulate S. gordonii oral colonization and cariogenicity in rats. 5) determine if S. gordonii-amylase interactions modulate S. mutans colonization competition and cariogenicity and if strains of S. mutans made to express AbpA show altered colonization and/or cariogenic abilities. Integration of in vitro with in vivo studies is crucial for mechanistic understanding of Dental plaque formation.
