Mapping the Functional Domains on Gap3 of Streptococcus parasanguinis

Objectives: Fap1 (fimbriae-associated protein 1) is a major subunit of Streptococcus parasanguinis long fimbriae, and is critical for fimbrial assembly, biofilm formation and bacterial adhesion, which activities are related with dental caries and periodontal disease. The biogenesis and secretion of Fap1 need the products of a gene cluster downstream of fap1 that have been identified to be secY2, gap1-3, secA2, gtf 1 and gtf2. In this study, we aimed to identify the functional domains on Gap3 of S. parasanguinis involved in Gap proteins interaction and Fap1 glycosylation for better understanding the cooperative relationship among fap1 gene locus products. Methods: Electron microscopy, yeast two-hybrid system, Site-directed mutagenesis, GST Pull-Down assay and Western blot were employed to investigate the proteins interaction between Gap proteins and the functional domains of Gap3 involved in proteins interaction and Fap1 glycosylation. Results: Electron micrographs showed both gap1 and gap3 mutants had the same morphological change on cell surface. Combining with the previous finding that both mutants had the same phenotype, it has been suggested that Gap1 and Gap3 might play their roles in Fap1 glycosylation through a way of cooperative interaction. Yeast two-hybrid system was employed to identify the important portions on Gap3 involved in Gap1-Gap3 interaction. Site-directed mutagenesis was used to further locate the functional domains of Gap3 within these regions. Several conserved domains were found to be important in mature Fap1 glycosylation. The Gap1-Gap3 proteins interaction domains were identified using GST Pull-Down assay, with a series of in vitro translation products of Gap3 variants. Western blot results showed that those functional domains are differently involved in the proteins interaction between Gap1 and Gap3. Conclusions: Our finding confirms the important role of the cooperative interaction between Gap proteins in Fap1 glycosylation.