Streptococcus parasanguis, a primary colonizer of the tooth surface, initiates dental plaque formation by adhesion to the tooth surface and acts as a substrate for colonization of other species. Fap1, a fimbriae-associated protein, has been identified as an adhesin of S. parasanguis FW213. Data suggests it is a glycosylated protein and is required for long fimbriae biogenesis and bacterial adhesion. Objectives: to isolate and characterize mutants involved in Fap1 glycosylation. Methods: A transposon mutagenesis system was established and used to generate a mutant library. Mutants were screened by immunoblotting with a monoclonal antibody, F51, which reacts specifically with a glycan epitope of Fap1. Mutants not reacting with F51 were tested for their reactivity to other monoclonal antibodies by ELISA and Western analysis. Results: An efficient and tractable transposon mutagenesis system was established. Over 2300 transposon mutants were screened and 6 were found to be F51 negative. Among the six mutants, one reacted with D10, an antibody specific for a different glycan epitope; two other mutants were negative for both F51 and D10. However, all of the three mutants above reacted with E42, a monoclonal antibody that recognizes the peptide backbone of Fap1. Conclusions: Three mutants were identified which expressed the peptide backbone but did not express all the glycan epitopes of Fap1, suggesting that the mutagenized genes may play a role in the stepwise glycosylation process.

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