Role of gap3 in Fap1 glycosylation, stability, in vitro adhesion, and fimbrial and biofilm formation of Streptococcus parasanguinis.
OBJECTIVE
Streptococcus parasanguinis is a primary colonizer of the tooth surface. Its adhesion is mediated by the long fimbriae, which are composed of multiple subunits of a serine-rich glycoprotein, Fap1. Previous studies revealed that a chromosomal region located downstream of fap1 is involved in the secretion and glycosylation of Fap1. In this study, we investigated the role of a glycosylation-associated gene, gap3, in Fap1 biogenesis.


METHODS
A gap3 non-polar mutant was constructed by insertional inactivation. The phenotype of the mutant and the subcellular distribution of its products were investigated. The binding ability of the mutant was tested with saliva-coated hydroxyapatite (SHA). Electron microscopy was used to observe the morphological changes on the mutant cell surface. Confocal microscopy was utilized to determine biofilm formation ability.


RESULTS
The gap3 mutant produced a partially glycosylated Fap1 precursor, that was less stable than mature Fap1. The Fap1 precursor was distributed in all subcellular fractions including the cell surface and culture medium although in decreased amounts. These data suggest a role for Gap3 in Fap1 glycosylation as well as a link between glycosylation and secretion of Fap1. The gap3 mutant had reduced binding to saliva-coated hydroxyapatite. Electron microscopy revealed that the gap3 mutant had lost its long fimbriae. Biofilm formation was also inhibited by the gap3 mutation. Fewer gap3 mutant cells adhered to the biofilm surface and microcolony formation was decreased.


CONCLUSIONS
Gap3 is required for the complete glycosylation and secretion of Fap1, which is important for fimbrial assembly, bacterial adhesion, and in vitro biofilm formation.
Peng
Z
Z
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, USA.
Wu
H
H
Ruiz
T
T
Chen
Q
Q
Zhou
M
M
Sun
B
B
Fives-Taylor
P
P
D001422:Bacterial Adhesion / Q000235:genetics*; D018441:Biofilms / Q000254:growth & development*; D002462:Cell Membrane / Q000648:ultrastructure; D002473:Cell Wall / Q000648:ultrastructure; D003593:Cytoplasm / Q000648:ultrastructure; D017886:Durapatite; D036781:Fimbriae Proteins / Q000032:analysis / Q000235:genetics*; D010861:Fimbriae, Bacterial / Q000235:genetics* / Q000648:ultrastructure; D020868:Gene Silencing; D005822:Genetic Vectors; D006031:Glycosylation; D016695:Glycosyltransferases / Q000032:analysis / Q000235:genetics*; D006801:Humans; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D010641:Phenotype; D010957:Plasmids; D011498:Protein Precursors / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012463:Saliva / Q000502:physiology; D013291:Streptococcus / Q000235:genetics / Q000502:physiology*
D011498:Protein Precursors; C403708:fap1 protein, Streptococcus; D036781:Fimbriae Proteins; D017886:Durapatite; D016695:Glycosyltransferases