Biogenesis of Oral Streptococcal Adhesin Requires Interactions Among Glycosylation-Associated Proteins

Objectives: Many oral diseases are caused by bacterial species that often cannot colonize unless a layer of initial colonizers has developed first. These initial colonizers often include the sanguis streptococci, which comprise a large proportion of oral bacterial species. Therefore, the initial binding of sanguis streptococci to the oral cavity has significant implications for oral health and disease. Our research involves one species in particular- Streptococcus parasanguinis- which adheres to the tooth surface by long peritrichous fimbriae. These fimbriae contain a surface glycoprotein, Fap1, which is crucial for bacterial adhesion, colonization, and biofilm formation. An eleven gene cluster is required for Fap1 biogenesis and glycosylation; however, the exact mechanism of Fap1 biogenesis and glycosylation remains a mystery. Methods: To investigate the interactions among three glycosylation-associated proteins- Gap1, Gap2, Gap3- that are encoded by three of the eleven genes involved in Fap1 biogenesis, we created independent gene knock-out mutants and gain of function strains that overexpress each protein individually or a combination of the three proteins. We then analyzed protein expression and stability through stability assays and Western Blot analysis. Results: The three glycosylation-associated proteins form a protein complex. Moreover, Gap3 is unstable without Gap1 and Gap2 alters the modification of Gap3, indicated by a shift in migration of Gap3 on SDS-PAGE gels. Conclusions: The three glycosylation-associated proteins interact with each other and have specific effects on the other two, including affecting protein stability and molecular weight. We are in the process of dissecting the mechanism of how Gap2 affects Gap3. This study will help us understand the molecular mechanism of how sanguis streptococci adhere to the oral cavity. Furthermore, because these three proteins are highly conserved among pathogenic bacteria, understanding this new mechanism may aid in designing novel therapeutics against this previously unknown pathway in pathogenic species.