Bacteria employ several mechanisms, and most notably secretion systems, to translocate effectors from the cytoplasm to the extracellular environment or the cell surface. Pseudomonas aeruginosa widely employs secretion machineries such as the Type III Secretion System to support virulence and cytotoxicity. However, recently identified P. aeruginosa strains that do not express the Type III Secretion System have been shown to express ExlA, an exolysin translocated through a two-partner secretion system, and are the causative agents of severe lung hemorrhage. Sequence predictions of ExlA indicate filamentous hemagglutinin (FHA-2) domains as the prevalent features, followed by a C-terminal domain with no known homologs. In this work, we have addressed the mechanism employed by ExlA to target membrane bilayers by using NMR, small-angle X-ray scattering, atomic force microscopy, and cellular infection techniques. We show that the C-terminal domain of ExlA displays a “molten globule-like” fold that punctures small holes into membranes composed of negatively charged lipids, while other domains could play a lesser role in target recognition. In addition, epithelial cells infected with P. aeruginosa strains expressing different ExlA variants allow localization of the toxin to lipid rafts. ExlA homologs have been identified in numerous bacterial strains, indicating that lipid bilayer destruction is an effective strategy employed by bacteria to establish interactions with multiple hosts.

细菌利用多种机制，最著名的是分泌系统，将效应子从细胞质转移到细胞外环境或细胞表面。铜绿假单胞菌广泛使用诸如III型分泌系统之类的分泌机制来支持毒力和细胞毒性。但是，最近发现的铜绿假单胞菌已经表明，不表达III型分泌系统的菌株可表达ExlA（一种通过两伙伴分泌系统转移的外溶素），是严重的肺出血的病因。ExlA的序列预测表明丝状血凝素（FHA-2）结构域为主要特征，其后为C端结构域，其同源性未知。在这项工作中，我们已经解决了ExlA通过使用NMR，小角度X射线散射，原子力显微镜和细胞感染技术靶向膜双层的机制。我们显示，ExlA的C末端结构域显示出“熔融小球状”折叠，将小孔刺入由带负电荷的脂质组成的膜中，而其他结构域在目标识别中的作用较小。另外，上皮细胞感染了表达不同ExlA变体的铜绿假单胞菌菌株使毒素定位于脂质筏。已经在许多细菌菌株中鉴定出ExlA同源物，表明脂质双层破坏是细菌用来建立与多个宿主的相互作用的有效策略。