Outer Membrane Protein A (OmpA): A New Player in <em>Shigella flexneri</em> Protrusion Formation and Inter-Cellular Spreading

Outer membrane protein A (OmpA) is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92) and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the findings presented in this study.外膜蛋白A（Outer membrane protein A，OmpA）是大肠杆菌（Escherichia coli）及其他肠杆菌科（Enterobacteriaceae）细菌中一种多功能的主要外膜蛋白，其在多种细菌性感染发病机制中的作用近年来已得到学界广泛认可。本研究针对外膜蛋白A在弗氏志贺菌（Shigella flexneri）毒力中的作用展开探究，构建了野生型弗氏志贺菌5a株M90T的ompA突变株（命名为HND92），实验显示该突变株的细胞间扩散能力严重受损，无法在HeLa细胞单层上形成噬斑（plaque）。缺失外膜蛋白A会显著降低IcsA的表达水平，但细胞结合型以及释放型的经IcsP切割的成熟蛋白氨基端95千道尔顿片段的表达水平则无明显差异。尽管如此，ompA突变株的整个细菌表面均暴露有IcsA蛋白。令人意外的是，ompA突变株可形成正常的纤维状肌动蛋白（F-actin）彗星尾，这表明细菌侧表面异常暴露的IcsA并未影响肌动蛋白成核蛋白的正常激活，提示外膜蛋白A的缺失可能暴露了细菌侧表面的成熟或细胞结合型IcsA。此外，ompA突变株仍可侵袭并在HeLa细胞单层内增殖，但被内化的细菌会被困在宿主细胞胞质中。本研究发现，ompA突变株形成的突起数量显著少于野生型菌株，这表明该缺陷可能是其无法形成噬斑的原因。尽管本研究无法完全排除ompA突变可能对弗氏志贺菌其他基因产生多效性影响，但通过携带弗氏志贺菌ompA基因的重组质粒对ompA突变株进行互补实验，结果清晰表明：具有功能的外膜蛋白A对于IcsA的正常暴露、噬斑形成以及突起生成既是必需的，也是充分的。此外，本研究还构建了另一株独立的ompA突变株，实验发现两株突变株的毒力表型完全一致，这些结果进一步支持了本研究的实验发现。