Identification of critical residues in Gap3 of Streptococcus parasanguinis involved in Fap1 glycosylation, fimbrial formation and in vitro adhesion

Abstract

Background: Streptococcus parasanguinis is a primary colonizer of human tooth surfaces and plays an important role in dental plaque formation. Bacterial adhesion and biofilm formation are mediated by long peritrichous fimbriae that are composed of a 200 kDa serine rich glycoprotein named Fap1 (fimbriae-associated protein). Glycosylation and biogenesis of Fap1 are modulated by a gene cluster downstream of the fap1 locus. A gene encoding a glycosylation-associated protein, Gap3, was found to be important for Fap1 glycosylation, long fimbrial formation and Fap1-mediated biofilm formation.

Results: Deletion and site-directed mutagenesis were employed to dissect the regions within Gap3 that were important for its function in Fap1 glycosylation and biogenesis. A deletion of 6 consecutive amino acids, PDLPIL, eliminated the production of the mature 200 kDa Fap1 protein and gave rise instead to a 470 kDa Fap1 intermediate that was only partially glycosylated. Site-directed mutagenesis of the 6 amino acids revealed that only three of these amino acids were required. Mutants in these amino acids (L64R, P65R and L67T) produced the premature 470 kDa Fap1 intermediate. Mutants in the remaining amino acids produced the mature form of Fap1. Cell surface expression of the Fap1 precursor among L64R, P65R and L67T mutants was reduced to levels consistent with that of a gap3 insertional mutant. Electron micrographs showed that these 3 mutants lost their long peritrichous fimbriae. Furthermore, their in vitro adhesion ability to saliva-coated hydroxylapatite (SHA) was inhibited.

Conclusion: Our data suggest that 3 highly conserved, hydrophobic residues L64, P65 and L67 in Gap3 are essential for Gap3 function and are important for complete glycosylation of Fap1, fimbrial formation and bacterial adhesion.

Figure 1

Alignment of Gap3 and its homologues. The black box represents identical or highly conserved residues. The gray box represents similar or less conserved residues. The red arrowed lines indicate the putatively important regions. The red numbers represent the residues chosen for site-directed mutagenesis. Gap3: glycosylation associated protein-3 of S. parasanguinis. Asp3: accessory secretory protein-3 of S. gordonii. SAG1450: hypothetical protein of S. agalactiae. SP1760: hypothetical protein of S. pneumoniae. SE2245: hypothetical protein of Staphylococcus epidermidis. gbs1519: hypothetical protein gbs1519 of S. agalactiae.

Figure 2

Western blot analysis of expression of Fap1 and Gap3 variants. Gap3 deletion mutants, Δ62–67 mutant (Lane 1), Δ144–157 mutant (Lane 2), wild type strain (Lane 3), gap3 allelic replacement mutant VT1619 (Lane 4), gap3 complemented strain VT1732 (Lane 5) and a control strain (VT1619 transformed with empty vector pVT1666) (Lane 6) were subjected to Western blot analysis with the use of MAb D10 (A). Expression of Fap1 by Gap3 site-directed mutants, V33R, F35H, N37I, P38R, S42L, N54I, R59L, P62G, D63V, L64R, P65R, I66N, L67T and L75S (Lanes 1–14) and wild type strain (Lane 15) probed with MAb D10 (B) and MAb F51 (C). Expression of Gap3-GFP by gap3 site-directed mutants, Gap3 L64R (Lane 1), P65R (Lane 2), I66N (Lane3), L67T (Lane4), Gap3 complemented strain VT1732 (Lane 5), control (VT1619 with empty vector pVT1666) (Lane 6) and gap3 mutant VT1619 (Lane 7) probed with anti-GFP MAb (C). Gap3-GFP fusion proteins migrate at 48 kDa; GFP protein migrates at 27 kDa.

Figure 3

Cell surface expression of Fap1 determined by BactELISA. MAbs F51 and E42 were used in BactELISA analysis to determine Fap1 expression in wild type FW213, fap1 mutant VT1393, gap3 mutant VT1619, gap3 complemented strain gap3-/gap3+ and Gap3 site-directed mutants, L64R, P65R, I66N and L67T. Data were obtained from three experiments in triplicates and are presented as means ± standard deviation.

Figure 4

Electron micrographs of S. parasanguinis bacteria. S. parasanguinis bacteria, L64R (A), P65R (B), I66N (C), L67T (D) mutant variants and wild type FW213 (E) were placed on grids, and negatively stained with 2% phosphotungstic acid pH7.0 and visualized by electron microscopy. White arrows point to the short fimbriae. Black arrows point to the long fimbriae. Scale bar = 100 nm.

Figure 5

Adhesion of S. parasanguinis to saliva-coated hydroxylapatite. Wild type of S. parasanguinis FW213, Gap3 site-directed mutants (L64R, P65R, I66N and L67T), fap1 mutant VT1393, gap3 mutant VT1619 and gap3 complemented strain gap3-/gap3+ were labeled with [³H] thymidine respectively. Labeled cells were incubated with SHA. The amounts of radioactivity associated with beads and supernatants were determined in a Wallac 1400 liquid scintillation counter and calculated to determine adhesion percentage. The data were obtained from three independent experiments in three replicates and are presented as means ± standard deviation.