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

Peng Z, Fives-Taylor P, Ruiz T, Zhou M, Sun B, Chen Q, Wu H - BMC Microbiol. (2008)

Bottom Line: A gene encoding a glycosylation-associated protein, Gap3, was found to be important for Fap1 glycosylation, long fimbrial formation and Fap1-mediated biofilm formation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatric Dentistry, University of Alabama at Birmingham School of Dentistry, Birmingham, AL 35294, USA. zxpeng@uab.edu

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.

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Related in: MedlinePlus

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.
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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.

Mentions: Fap1 is important for S. parasanguinis adhesion [8]. The above data showed that Gap3 site-directed mutation affected Fap1 glycosylation and biogenesis. An adhesion experiment was performed to determine if the partially glycosylated form of Fap1 could still function in adhesion of the Gap3 mutants (Fig. 5). Like fap1 and gap3 mutants, the L64R, P65R and L67T mutants failed to adhere to SHA, suggesting the partially glycosylated form was unable to function in bacterial adhesion. I66N had a modest reduction in bacterial adhesion, indicating the I66 mutation only had a minor effect on adhesion.


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

Peng Z, Fives-Taylor P, Ruiz T, Zhou M, Sun B, Chen Q, Wu H - BMC Microbiol. (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335109&req=5

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.
Mentions: Fap1 is important for S. parasanguinis adhesion [8]. The above data showed that Gap3 site-directed mutation affected Fap1 glycosylation and biogenesis. An adhesion experiment was performed to determine if the partially glycosylated form of Fap1 could still function in adhesion of the Gap3 mutants (Fig. 5). Like fap1 and gap3 mutants, the L64R, P65R and L67T mutants failed to adhere to SHA, suggesting the partially glycosylated form was unable to function in bacterial adhesion. I66N had a modest reduction in bacterial adhesion, indicating the I66 mutation only had a minor effect on adhesion.

Bottom Line: A gene encoding a glycosylation-associated protein, Gap3, was found to be important for Fap1 glycosylation, long fimbrial formation and Fap1-mediated biofilm formation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatric Dentistry, University of Alabama at Birmingham School of Dentistry, Birmingham, AL 35294, USA. zxpeng@uab.edu

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.

Show MeSH
Related in: MedlinePlus