Nallapareddy S R, Qin X, Weinstock G M, Höök M, Murray B E
Division of Infectious Diseases, Department of Internal Medicine, Houston, Texas 77030, USA.
Infect Immun. 2000 Sep;68(9):5218-24. doi: 10.1128/IAI.68.9.5218-5224.2000.
Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalis isolates, after growth at 46 degrees C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an ace disruption mutant from E. faecalis strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46 degrees C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single approximately 105-kDa band of mutanolysin extracts from OG1RF grown at 46 degrees C, while no band was detected in extracts from OG1RF grown at 37 degrees C, nor from the OG1RF ace mutant grown at 37 or 46 degrees C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 microg/ml, and also inhibited the 46 degrees C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalis OG1RF to collagen type IV and laminin in addition to collagen type I.
黏附素介导的与细胞外基质（ECM）蛋白的结合被认为是许多细菌感染致病过程中的关键步骤。我们之前报道过，大多数粪肠球菌分离株在46℃生长后会有条件地黏附于ECM蛋白I型和IV型胶原蛋白以及层粘连蛋白；鉴定出一个粪肠球菌特异性基因ace，其编码的蛋白具有细菌黏附素的特征；并认为Ace参与了与I型胶原蛋白的结合。在本研究中，我们构建了一株来自粪肠球菌OG1RF菌株的ace缺失突变体，与46℃生长后的亲本OG1RF菌株相比，该突变体对I型和IV型胶原蛋白以及层粘连蛋白的黏附显著降低。针对OG1RF来源的重组Ace A结构域产生的多克隆免疫血清与46℃生长的OG1RF的变溶菌素提取物中的一条约105 kDa的单条带发生反应，而在37℃生长的OG1RF提取物中未检测到条带，在37℃或46℃生长的OG1RF ace突变体提取物中也未检测到条带。从抗Ace A免疫血清中纯化的IgG在低至1μg/ml的浓度下就能抑制46℃生长的粪肠球菌OG1RF对固定化IV型胶原蛋白、层粘连蛋白以及I型胶原蛋白的黏附，并且还能抑制所测试的两种临床分离株在46℃诱导的黏附。我们还在远缘Western印迹中展示了IV型胶原蛋白与OG1RF变溶菌素提取物中的Ace的体外相互作用。在酶联免疫吸附测定中证实了重组Ace A与固定化的I型和IV型胶原蛋白以及层粘连蛋白的结合，并且显示出浓度依赖性。这些结果表明，Ace A除了介导粪肠球菌OG1RF与I型胶原蛋白的结合外，还介导其与IV型胶原蛋白和层粘连蛋白的条件性结合。
