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

<div><p>Outer membrane protein A (OmpA) is a multifaceted predominant outer membrane protein of <em>Escherichia coli</em> and other <em>Enterobacteriaceae</em> whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of <em>Shigella flexneri</em> has been investigated. An <em>ompA</em> mutant of wild-type <em>S. flexneri</em> 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 <em>ompA</em> mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the <em>ompA</em> 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 <em>ompA</em> 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 <em>ompA</em> 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 <em>ompA</em> mutation might exert pleiotropic effects on other <em>S. flexneri</em> genes, complementation of the <em>ompA</em> mutation with a recombinant plasmid carrying the <em>S. flexneri ompA</em> gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent <em>ompA</em> mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the findings presented in this study.</p>