Eur J Immunol. 2014 Mar;44(3):742-51.
The outer membrane protein Ail of Yersinia pestis mediates several virulence functions, including serum resistance. Here, we demonstrate that Ail binds C4b-binding protein (C4BP), the primary fluid-phase regulator of the classical and lectin pathways. Noncovalent binding of C4 and C4b to Ail was also observed. C4BP bound to Ail can act as a cofactor to the serine protease factor I (fI) in the cleavage of fluid-phase C4b. Employing a panel of C4BP alpha-chain mutants, we observed that the absence of complement control protein domain 6 and 8 reduced binding to Ail. Immunoblot analysis of normal human serum (NHS)-treated bacteria revealed minimal C4b alpha’-chain complexes with bacterial outer membrane targets. Addition of the anti-C4BP monoclonal antibody MK104 to NHS restored C4b-alpha’ chain target complexes, suggesting that C4b binds covalently to targets on the Y. pestis surface. C4b bound to Ail noncovalently was also cleaved in a C4BP and fI-dependent manner, leaving the C4c fragment bound to Ail. MK104 also prevented the cleavage of noncovalently bound C4b. Collectively, these data suggest that when C4BP is bound to Ail, fI can cleave and inactivate C4b that has bound covalently to bacterial surface structures as well as C4b bound noncovalently to Ail.
鼠疫耶尔森氏菌的外膜蛋白 Ail 介导多种毒力功能，包括血清抗性。在这里，我们证明 Ail 结合 C4 结合蛋白 (C4BP)，这是经典和凝集素途径的主要液相传感调节剂。还观察到非共价结合的 C4 和 C4b 与 Ail。与 Ail 结合的 C4BP 可以作为丝氨酸蛋白酶因子 I (fI) 在液相传感 C4b 切割中的辅助因子。使用一组 C4BP α-链突变体，我们观察到缺乏补体控制蛋白结构域 6 和 8 减少了与 Ail 的结合。用正常人血清 (NHS) 处理细菌的免疫印迹分析显示，与细菌外膜靶标结合的 C4b alpha’-链复合物很少。向 NHS 添加抗 C4BP 单克隆抗体 MK104 可恢复 C4b-alpha’链靶复合物，表明 C4b 与鼠疫耶尔森氏菌表面的靶标共价结合。以非共价方式与 Ail 结合的 C4b 也以 C4BP 和 fI 依赖性方式被切割，留下与 Ail 结合的 C4c 片段。MK104 还阻止了非共价结合的 C4b 的切割。总之，这些数据表明，当 C4BP 与 Ail 结合时，fI 可以切割和失活已共价结合到细菌表面结构以及与 Ail 非共价结合的 C4b。
