Characterization of a Glycosylation Pathway for an Oral Streptococcal Adhesin

Streptococcus parasanguis, a primary colonizer of teeth, plays an important role in the formation of dental plaque. Adhesion of S. parasanguis is mediated by peritrichous fimbriae. Fap1, a fimbriae-associated 200-kDa glycoprotein, is essential for bacterial fimbriae and biofilm formation. Fap1 possesses two serine-rich repeat regions that comprise 80% of the entire Fap1 sequences. The glycan moiety of Fap1 appears to be O-linked within the serine-rich region. Objective: To determine the Fap1 glycosylation pathway. Methods: A serine-rich sequence specific antibody and seven characterized antibodies were used to examine Fap1 expression. Screening of a S. parasanguis genomic library identified genes involved in Fap1 glycosylation. Those genes were inactivated by non-polar allelic replacement mutagenesis. Results: Two previously undetected high molecular weight forms (360-kDa and 470-kDa) of Fap1 intermediates were identified by immunoblot analysis. A gene locus consisting of seven genes immediately downstream of fap1 was isolated and characterized. Two genes shared homology with genes involved in bacterial secretion, one exhibited similarity to glucosyltransferase gene, and four genes did not have any significant homology with known genes in the databases. Inactivation of one gene coding for the secretion protein SecY2 resulted in partial glycosylation of Fap1 and accumulation of 470-kDa Fap1 intermediate, whereas mutation of a glucosyltransferase gene led to complete defect in Fap1 glycosylation and thus accumulation of 360-kDa Fap1 protein. Conclusions: These data suggest that the locus we identified is involved in Fap1 glycosylation. A processing pathway for Fap1 glycosylation is proposed wherein the 360-kDa polypeptide form of Fap1 is glycosylated to produce the partially glycosylated 470-kDa Fap1 intermediate by glucosyltransferase and the fully glycosylated 200 kDa Fap1 protein is formed upon action of SecY2. This work was supported by the NIH R37-DE1100 grant.