Fap1 is a serine-rich streptococcal adhesin and essential for fimbrial biogenesis and biofilm formation of Streptococcus parasanguis. Fap1-like proteins are found in many streptococci and staphylococci and implicated in bacterial pathogenesis. Fap1 contains two serine-rich repeat regions (RI and RII) that are modified by O-glycosylation. A seven-gene genomic island mediating Fap1 biogenesis has been identified and localized adjacent to the fap1 gene. Objectives: To determine poorly understood molecular mechanisms governing initiation of Fap1 glycosylation. Methods: A recombinant Fap1 (Fap1ΔRII) that is devoid of the repeat region II was used as a model to investigate monosaccharide composition of Fap1 by glycosyl composition analyses, and to determine oligasaccharides attached to the Fap1 peptide by glycosyl linkage, lectin and Western blot analyses. An in vivo Fap1 glycosylation system was established in E. coli to study the initial glycosylation step catalyzed by two glycosyltransferases: Gtf1 and Gtf2. Results: Fap1ΔRII has a monosaccharide composition profile similar to the native Fap1 and is modified by glucose and N-acetyl glucosamine residues. Inactivation of two glycosyltransferases in both S. parasanguis and E. coli abolished Fap1 glycosylation. Gtf1 and Gtf2 catalyze the transfer of GlcNAc to Fap1ΔRII. Furthermore, Gtf1 and 2 homologues of Streptococcus sanguis and Streptococcus pneumoniae are also able to glycosylate Fap1ΔRII. Conclusions: Our work shows the requirement of two glycosyltransferases Gtf1 and Gtf2 for Fap1ΔRII glycosylation, this mechanism is conserved across Gram-positive bacteria that express serine-rich glycoproteins. This work was supported by NIH K22 DE014726, R21 DE016891 and P41 RR018502 grants.

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