A Genetic Locus Mediates Fap1 Glycosylation and Biofilm Formation

Fap1 is a Streptococcus parasanguinis glycoprotein and required for biofilm formation. Glycosyltransferase Gtf1 and Gtf2 interact with each other and catalyze the first step of Fap1 glycosylation. Gtf3 and GalT2, located in a glycosyltransferase gly-gtf3-galT1-galT2 locus, are also involved in Fap1 glycosylation and biofilm formation. Gtf3 mediates the second glycosylation step. However, nothing was known about the roles of gly and galT1 in Fap1 glycosylation and biofilm formation. In addition, the exact GalT2 function in Fap1 glycosylation is unknown. Objectives: To dissect functional contribution of gly, galT1 and galT2 to Fap1 glycosylation and biofilm formation. Methods: The gly and galT1 mutants were constructed by insertional mutagenesis. Biofilm formation was monitored using fluorescence microscopy. An E. coli glycosylation system was established to dissect Fap1 glycosylation steps. In this system, plasmid pHSG576 harboring a recombinant Fap1 substrate, Fap1DRII, was co-transformed with two compatible plasmids. The first plasmid carries gtf1-gtf2, and the second plasmid carries gtf3, galT1, galT2 and gly. Fap1 production and glycosylation was determined by Western blot using Fap1-specific antibodies and by carbohydrate staining respectively. Results: The gly mutant still expressed mature Fap1 albeit the expression level was reduced significantly, suggesting Gly may participate in the last step of Fap1 glycosylation. The galT1 mutant produced a Fap1 precursor that cannot be detected by Fap1 glycan-specific antibodies F51 and D10, indicating GalT1 contributes to Fap1 glycosylation. Both gly and galT1 mutants decreased biofilm formation. Furthermore, Fap1DRII glycosylated by Gtf1-Gtf2-Gtf3 was further modified in the presence of GalT1, GalT2, GalT1-GalT2, Gly-GalT1, Gly-GalT2 or Gly-GalT1-GalT2, but not by Gly. These studies suggest GalT1-GalT2 catalyze the subsequent glycosylation step mediated by Gtf1-Gtf2-Gtf3, whereas Gly catalyzes the last step of Fap1 glycosylation. Conclusions: The glycosyltransferase gly-gtf3-galT1-galT2 locus is involved in Fap1 glycosylation and important for biofilm formation. This study was supported by NIH/NIDCR R01DE011000 and R01DE017954.