INFLUENCE OF AGR ON FIBRINOGEN BINDING IN STAPHYLOCOCCUS-AUREUS NEWMAN

The ability of Staphylococcus aureus to bind fibrinogen is believed to be important in promoting bacterial adherence to both intravascular c atheters and host tissues during infection. We investigated the influe nce of the global regulator agr on the fibrinogen binding capacity and its relationship to expression of coagulase (encoded by coa) and clum ping factor (encoded by clfA) in strain Newman. Strains were obtained by transducing site-specific mutations of clfA, coa, and agr into stra in Newman to obtain single, double, and triple mutants of the respecti ve genes. As expected, the clfA mutant bound less soluble I-125-labele d fibrinogen than the corresponding coa mutant in agr(+) strains; howe ver, with agr mutant strains, the upregulation in fibrinogen binding c apacity correlated mostly with the increased expression and transcript ion of coagulase as shown by Western (immunoblot) and Northern (RNA) b lot analysis. In particular, the coa agr double mutant resulted in a s ignificant reduction in fibrinogen binding compared with that of the a gr mutant. The contribution of clfA to fibrinogen binding in agr-negat ive strains was less than that of coa (32,740 +/- 1,189 versus 18,141 +/- 334 and 38,919 +/- 1,021 cpm for clfA agr, coa agr, and the single agr mutant, respectively). Thus, coagulase is a major binding protein for soluble fibrinogen in the agr-negative background. In in vitro mi crotiter and catheter adherence assays with solid-phase fibrinogen, cl umping factor, but not coagulase, plays a major role in binding to imm obilized fibrinogen. coa transcription was negatively modulated by agr and occurred mainly during the exponential growth phase. In contrast, clfA transcription was agr independent and was strongest during the p ostexponential phase. Although an agr coa clfA triple mutant bound les s soluble fibrinogen than the agr coa double mutant (8,504 +/- 831 ver sus 18,141 +/- 334 cpm), significant residual fibrinogen binding capac ity remained in the triple mutant, thus suggesting an additional fibri nogen binding component. By using direct ligand affinity blotting with I-125-fibrinogen, we could identify coagulase and an additional unide ntified 52-kDa protein as a fibrinogen binding component in cell extra cts. This band was absent in the extract of the coa clfA double mutant .