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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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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.
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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.

Mentions: Gap3 and its homologues in Gram-positive bacteria, Asp3 of S. gordonii, SAG1450 and gbs1519 of S. agalactiae, SP1760 of S. pneumoniae and SE2245 of Staphylococcus epidermidis, were identified from the corresponding genomes and aligned to determine the conserved regions (Fig. 1); a search of protein databases was carried out to predict functional significance (whether they are carbohydrate-biosynthesis related) of the conserved sequences using online bioinformatics programs. A series of in-frame deletion mutants and site-directed mutants of gap3 were constructed to investigate if these conserved or putatively functional regions/residues were required for Gap3 function and involved in Fap1 biogenesis. One region contains the peptide sequence of PDLPIL (residues 62 to 67), which was also found in a signal receiver domain of a bacterial polysaccharide biosynthesis regulator protein, GelA, of Sphingomonas elodea [18]; another region has a peptide sequence of INDEEKKNHIVENR (from residue 144 to 157 of Gap3), which has a putative coiled-coil domain as predicated by an online program [19]. We hypothesized the two regions are functionally relevant to Fap1 glycosylation. Interestingly, deletion of the highly conserved region, Δ62–67, inhibited the production of the 200 kDa mature Fap1 (Fig. 2A, Lane 1), whereas deletion of the predicted coiled-coil domain from amino acid residues 144 to 157 did not affect mature Fap1 production (Fig. 2A, Lane 2), suggesting the amino acid residues from 62 to 67, PDLPIL were important for Gap3 function in Fap1 glycosylation.


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)

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

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

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.
Mentions: Gap3 and its homologues in Gram-positive bacteria, Asp3 of S. gordonii, SAG1450 and gbs1519 of S. agalactiae, SP1760 of S. pneumoniae and SE2245 of Staphylococcus epidermidis, were identified from the corresponding genomes and aligned to determine the conserved regions (Fig. 1); a search of protein databases was carried out to predict functional significance (whether they are carbohydrate-biosynthesis related) of the conserved sequences using online bioinformatics programs. A series of in-frame deletion mutants and site-directed mutants of gap3 were constructed to investigate if these conserved or putatively functional regions/residues were required for Gap3 function and involved in Fap1 biogenesis. One region contains the peptide sequence of PDLPIL (residues 62 to 67), which was also found in a signal receiver domain of a bacterial polysaccharide biosynthesis regulator protein, GelA, of Sphingomonas elodea [18]; another region has a peptide sequence of INDEEKKNHIVENR (from residue 144 to 157 of Gap3), which has a putative coiled-coil domain as predicated by an online program [19]. We hypothesized the two regions are functionally relevant to Fap1 glycosylation. Interestingly, deletion of the highly conserved region, Δ62–67, inhibited the production of the 200 kDa mature Fap1 (Fig. 2A, Lane 1), whereas deletion of the predicted coiled-coil domain from amino acid residues 144 to 157 did not affect mature Fap1 production (Fig. 2A, Lane 2), suggesting the amino acid residues from 62 to 67, PDLPIL were important for Gap3 function in Fap1 glycosylation.

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