Mishra Meenu, Ressler Adam, Schlesinger Larry S, Wozniak Daniel J
Department of Microbial Infection and Immunity, The Ohio State University, Columbus, Ohio, USA Center for Microbial Interface Biology, The Ohio State University, Columbus, Ohio, USA.
Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA.
Infect Immun. 2015 Aug;83(8):3006-14. doi: 10.1128/IAI.00081-15. Epub 2015 May 11.
Pseudomonas aeruginosa is a versatile opportunistic pathogen that can cause devastating persistent infections. Complement is a highly conserved pathway of the innate immune system, and its role in the first line of defense against pathogens is widely appreciated. One of the earliest events in the complement cascade is the conversion of C3 to C3a and C3b, the latter typically binds to one or more acceptor molecules on the pathogen surface. We previously demonstrated that complement C3b binding acceptors exist on the P. aeruginosa surface. In the current study, we utilized either C3 polyclonal or C3b monoclonal antibodies in a far-Western technique followed by mass spectroscopy to identify the C3b acceptor molecule(s) on the P. aeruginosa surface. Our data provide evidence that OprF (an outer membrane porin, highly conserved in the Pseudomonadaceae) binds C3b. An oprF-deficient P. aeruginosa strain exhibits reduced C3 deposition compared to the wild type. We observed reduced internalization of oprF-deficient bacteria by neutrophils after opsonization compared with wild-type P. aeruginosa. Heterologous expression of OprF significantly enhanced C3b binding and increased serum-mediated bactericidal effects in complement-susceptible Escherichia coli. Furthermore, the predicted secondary structure of the C-terminal, surface-exposed region of OprF has high structural identity to the OmpA domain of several other Gram-negative bacteria, one of which is known to bind C3b. Therefore, these findings provide new insights into the biology of complement interactions with P. aeruginosa and other Gram-negative bacteria.
铜绿假单胞菌是一种具有多种致病能力的机会致病菌，可引发破坏性的持续性感染。补体是固有免疫系统中一条高度保守的途径，其在抵御病原体的第一道防线中的作用已得到广泛认可。补体级联反应中最早发生的事件之一是C3转化为C3a和C3b，后者通常会与病原体表面的一个或多个受体分子结合。我们之前证明铜绿假单胞菌表面存在补体C3b结合受体。在本研究中，我们采用远缘Western技术，随后进行质谱分析，利用C3多克隆抗体或C3b单克隆抗体来鉴定铜绿假单胞菌表面的C3b受体分子。我们的数据表明，OprF（一种外膜孔蛋白，在假单胞菌科中高度保守）能结合C3b。与野生型相比，oprF基因缺失的铜绿假单胞菌菌株的C3沉积减少。我们观察到，与野生型铜绿假单胞菌相比，调理后oprF基因缺失的细菌被中性粒细胞内化的能力降低。OprF的异源表达显著增强了C3b结合，并增加了血清介导的对补体敏感的大肠杆菌的杀菌作用。此外，OprF C端表面暴露区域的预测二级结构与其他几种革兰氏阴性菌的OmpA结构域具有高度的结构相似性，其中一种已知能结合C3b。因此，这些发现为补体与铜绿假单胞菌及其他革兰氏阴性菌相互作用的生物学机制提供了新的见解。
