Abstract
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.
Highlights
Shigella flexneri is a Gram-negative facultative intracellular pathogen that causes bacillary dysentery, a major public-health problem principally in developing countries
Construction of the ompA Mutant Strain HND92 The DompA mutation carried by the E. coli K-12 strain JW0940 was P1 transduced into the S. flexneri 5a wild-type strain M90T (Table 1)
Both Outer membrane protein A (OmpA) of S. flexneri and OmpA expressed by recombinant plasmid pOA behaved as native E. coli OmpA, since both proteins were found to be strictly localized within the membrane fraction and presented the expected heat-variable mobility (Fig. 1 and data not shown)
Summary
Shigella flexneri is a Gram-negative facultative intracellular pathogen that causes bacillary dysentery, a major public-health problem principally in developing countries. Shigella cell-tocell spread requires the expression and polar surface exposition of IcsA (VirG), a 110-kDa autotransporter protein encoded on the Shigella large virulence plasmid. Several host cell proteins have been implicated in protrusion-mediated Shigella cell-to-cell spread, suggesting that a distinct set of actin regulatory factors interacts with motile bacteria after they contact the plasma membrane [18]. Recent experimental evidences indicate that actin nucleation processes, involved in protrusion formation, may be independent of the activity of the Arp2/3 complex [12]. In this context, it has been recently reported that protrusion formation and inter-cellular spreading depend on actin polymerization that requires the activation of the Diaphanous formin Dia [12]. Formins are a family of ubiquitous expressed proteins that, in contrast to the Arp2/3 complex, initiate de novo actin polymerization leading to cross-linking of actin polymers in parallel arrays [12]
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