Characterization of monoclonal antibodies specific for adhesion: isolation of an adhesin of Streptococcus sanguis FW213

Abstract

Monoclonal antibodies reactive to an adhesive strain of Streptococcus sanguis (FW213) and nonreactive to a nonadhesive mutant (JL7) were derived from the fusion of myeloma line X63Ag8.653 and spleen cells from BALB/c mice immunized with live S. sanguis cells. Five cell lines, belonging to subclasses of immunoglobulin G, produced monoclonal antibodies specifically directed against the adhesive strain. All five antibodies also failed to react with five additional, independently isolated, nonadhesive mutants. A spontaneous mutant of FW213 (VT508) that no longer reacted with monoclonal antibody F51 (MAbF51) was isolated by serial agglutination with the antibody. Langmuir adsorption isotherms of VT508 indicated that this mutant also had altered ability to adhere to saliva-coated hydroxyapatite further confirming the specificity of MAbF51 for adhesion. Electron microscopy revealed that VT508 had lost the peritrichous fimbriae associated with the adhesion of FW213. MAbF51 was used to purify the adhesin from lysozyme cell extracts by using an affinity column of MAbF51 linked to Sephacryl S1000. Purity was suggested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis (CIEP). The adhesin had a molecular weight greater than 150,000 and was not denatured in sodium dodecyl sulfate reducing gels. Two peaks of near electrophoretic mobility were detected in CIEP when the purified material was run against polyclonal antibody to the whole cell. Tandem CIEP analysis and immunoprecipitation provided evidence that the two peaks represented the same antigen in two different forms.

Identification of dipeptide repeats and a cell wall sorting signal in the fimbriae‐associated adhesin, Fap1, of Streptococcus parasanguis

“…Discovery of this common feature in the predicted Fap1 protein is of interest, as this cell wall sorting signal is required for the anchoring of proteins to cell surfaces (Schneewind et al, 1993;Homonylo-McGavin and Lee, 1996). Fap1 is likely to be anchored on the cell surface to execute its function(s), as it is a major adhesin that promotes the attachment of FW213 to SHA and is the subunit of fimbriae (Elder and Fives-Taylor, 1986;Wu et al, 1998). A mutation in the cell wall sorting signal region abrogated Fap1 anchorage, fimbriae formation and significantly reduced the adhesion ability of S. parasanguis FW213.…”

“…Almost nothing is known about fimbriae biogenesis in Gram-positive bacteria. Gram-positive fimbriae have been exceedingly difficult to dissociate (Elder and Fives-Taylor, 1986;Yeung et al, 1987). Recently, A. naeslundii T14V type 1 fimbriae and type 2 fimbriae have been studied.…”

“…This protein is located at the tip of fimbriae (Fenno et al, 1995) and is involved in adherence (Oligino and Fives-Taylor, 1993). Fap1, a 200 kDa fimbriae-associated protein, has been purified by immunoaffinity chromatography based on its reactivity with a fimbriae-specific monoclonal antibody (mAb) F51 (Elder and Fives-Taylor, 1986;Wu et al, 1998). This F51 antibody reacts specifically with Fap1 and inhibits the adhesion of FW213 to SHA.…”

“…This F51 antibody reacts specifically with Fap1 and inhibits the adhesion of FW213 to SHA. A spontaneous mutant of S. parasanguis, which lacks F51 binding activity, does not adhere and has lost its long fimbriae (Elder and Fives-Taylor, 1986). The fap1 locus coding for this protein has been cloned and partially sequenced.…”

A conserved domain of previously unknown function in Gap1 mediates protein–protein interaction and is required for biogenesis of a serine‐rich streptococcal adhesin

“…To determine the role of Gap1 in secretion when the Fap1 is not glycosylated, we constructed a gtf1 gap1 double mutant. A spontaneous non-adherent mutant VT508 has been isolated previously (Elder and Fives-Taylor, 1986) and characterized to a gtf1 mutant in this study. VT508 has a mutation at Gtf1 coding amino acid 203 ( Fig.…”