Wolz C, McDevitt D, Foster T J, Cheung A L
The Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, New York 10021, USA.
Infect Immun. 1996 Aug;64(8):3142-7. doi: 10.1128/iai.64.8.3142-3147.1996.
The ability of Staphylococcus aureus to bind fibrinogen is believed to be important in promoting bacterial adherence to both intravascular catheters and host tissues during infection. We investigated the influence of the global regulator agr on the fibrinogen binding capacity and its relationship to the expression of coagulase (encoded by coa) and clumping factor (encoded by clfA) in strain Newman. Strains were obtained by transducing site-specific mutations of clfA, coa, and agr into strain Newman to obtain single, double, and triple mutants of the respective genes. As expected, the clfA mutant bound less soluble 125I-labeled fibrinogen than the corresponding coa mutant in agr+ strains; however, with agr mutant strains, the upregulation in fibrinogen binding capacity correlated mostly with the increased expression and transcription of coagulase as shown by Western (immunoblot) and Northern (RNA) blot analysis. In particular, the coa agr double mutant resulted in a significant reduction in fibrinogen binding compared with that of the agr mutant. The contribution of clfA to fibrinogen binding in agr-negative 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 microtiter and catheter adherence assays with solid-phase fibrinogen, clumping factor, but not coagulase, plays a major role in binding to immobilized 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 postexponential phase. Although an agr coa clfA triple mutant bound less soluble fibrinogen than the agr coa double mutant (8,504 +/- 831 versus 18,141 +/- 334 cpm), significant residual fibrinogen binding capacity remained in the triple mutant, thus suggesting an additional fibrinogen binding component. By using direct ligand affinity blotting with 125I-fibrinogen, we could identify coagulase and an additional unidentified 52-kDa protein as a fibrinogen binding component in cell extracts. This band was absent in the extract of the coa clfA double mutant.
金黄色葡萄球菌结合纤维蛋白原的能力被认为在感染期间促进细菌黏附于血管内导管和宿主组织方面很重要。我们研究了全局调节因子agr对纤维蛋白原结合能力的影响及其与纽曼菌株中凝固酶（由coa编码）和凝聚因子（由clfA编码）表达的关系。通过将clfA、coa和agr的位点特异性突变转导至纽曼菌株中获得单突变体、双突变体和三突变体。正如预期的那样，在agr+菌株中，clfA突变体比相应的coa突变体结合的可溶性125I标记纤维蛋白原更少；然而，对于agr突变菌株，纤维蛋白原结合能力的上调主要与凝固酶表达和转录的增加相关，如蛋白质免疫印迹法（Western blot）和RNA印迹法（Northern blot）所示。特别是，与agr突变体相比，coa agr双突变体导致纤维蛋白原结合显著减少。在agr阴性菌株中，clfA对纤维蛋白原结合的贡献小于coa（clfA agr、coa agr和单agr突变体分别为32,740±1,189、18,141±334和38,919±1,021 cpm）。因此，在agr阴性背景下，凝固酶是可溶性纤维蛋白原的主要结合蛋白。在使用固相纤维蛋白原的体外微量滴定和导管黏附试验中，凝聚因子而非凝固酶在与固定化纤维蛋白原的结合中起主要作用。coa转录受agr负调控，主要发生在指数生长期。相比之下，clfA转录不依赖于agr，在指数生长期后最强。尽管agr coa clfA三突变体比agr coa双突变体结合的可溶性纤维蛋白原更少（8,504±831与18,141±334 cpm），但三突变体中仍保留有显著的残余纤维蛋白原结合能力，因此表明存在另一种纤维蛋白原结合成分。通过使用125I-纤维蛋白原直接配体亲和印迹法，我们可以在细胞提取物中鉴定出凝固酶和另一种未鉴定的52 kDa蛋白作为纤维蛋白原结合成分。该条带在coa clfA双突变体提取物中不存在。
