Isolation and characterization of Fap1, a fimbriae‐associated adhesin ofStreptococcus parasanguisFW213

Abstract

SummaryAn adhesin of Streptococcus parasanguis FW213, a primary colonizer of the tooth surface, has been purified from the culture medium by immunoaffinity chromatography. The purified protein has a molecular mass of 200 kDa and stains positively for carbohydrate. The amino-terminal sequence indicated that this protein represented a unique streptococcal surface protein. Immunogold labelling of the bacterium indicated that this protein was associated with fimbriae and designated Fap1 (fimbriae-associated protein). A polymerase chain reaction (PCR) product based on the amino terminus of Fap1 was used to probe an FW213 genomic library. A 9 kb fragment containing the fap1 gene was isolated and 2.5 kb have been sequenced. Generation of fap1 mutants by a single cross-over (Campbell insertion) or a non-polar allelic exchange abolished the expression of Fap1. The inactivation of fap1 resulted in a dramatic reduction in the expression of the long peritrichous fimbriae and adhesion to saliva-coated hydroxylapatite (SHA). Northern blots probed with an internal gene fragment of fap1 hybridized to a 9 kb transcript, which suggests that fap1 is transcribed as a polycistronic message. These data demonstrate that Fap1 is a unique streptococcal adhesin that is involved in the assembly of S. parasanguis FW213 fimbriae and adhesion to SHA.

Molecular Strategies for Fimbrial Expression and Assembly

“…A greater than 150-Da fimbrial component is associated with the adhesion of FW213 to SHA (Elder and Fives-Taylor, 1986). Manipulation of fimbriae of S. parasanguis alters the adhesion capability of this bacterium to SHA (Fachon-Kalweit et al, 1985;Wu et al, 1998). Fimbriated Streptococcus sanguis co-aggregates consistently well with A. naeslundii and F nucleatum, whereas non-fimbriated strains do not (Hogg et al, 1981;Handley et al, 1985).…”

“…Like other known streptococcal fimbriae, the purified fimbriae of S. parasanguis FW213 are resistant to dissociation into smaller subunits by a variety of chemical treatments (Elder and Fives-Taylor, 1986). Like other sanguis streptococcus fimbriae-associated proteins, Fapl is also glycosylated (Fachon-Kalweit et al, 1985;Wu et al, 1998). Because these fimbriae are difficult to anaylze by biochemical means, an understanding of streptococcal fimbrial assembly is limited.…”

“…(4) Fap 1 is a major structural subunit of S. parasanguis fimbriae In search of a major fimbrial subunit of S. parasanguis, we identified and isolated Fapl (Wu et al, 1998 (Wu and Fives-Taylor, 1999 (Wu, 1999).…”

“…Discovery of a common feature in the predicted Fapl protein is of interest, since the cell-wall sorting signal is required for the anchoring of proteins to cell surfaces (Schneewind et al, 1993). Fap I is likely to be anchored to the cell surface to execute its function(s), since it is a major adhesin that promotes the attachment of FW213 to SHA, and is the subunit of fimbriae (Elder and FivesTaylor, 1986;Wu et al, 1998). A mutation of the cell-wall sorting signal region of FapI abrogates Fapl anchorage and fimbrial presentation and significantly reduces the adhesion ability of S. parasanguis FW213.…”

“…Secretion and anchoring of some proteins is accomplished by a signal sequence at the N-terminus and a cell-wall sorting signal at the C-terminus (Navarre and Schneewind, 1999). The few known putative fimbrial subunits, FimA, FimP, and Fapl, from oral Gram-positive bacteria, all have signal sequences and cell-wall sorting signals (Yeung and Ragsdale, 1997;Wu et al, 1998;Yeung et al, 1998;Wu and Fives-Taylor, 1999 …”