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Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane.

ABSTRACT: Mutations in the lipopolysaccharide (LPS) of Shigella spp. result in attenuation of the bacteria in both in vitro and in vivo models of virulence, although the precise block in pathogenesis is not known. We isolated defined mutations in two genes, galU and rfe, which directly affect synthesis of the LPS of S. flexneri 2a, in order to determine more precisely the step in virulence at which LPS mutants are blocked. The galU and rfe mutants invaded HeLa cells but failed to generate the membrane protrusions (fireworks) characteristic of intracellular motility displayed by wild-type shigellae. Furthermore, the galU mutant was unable to form plaques on a confluent monolayer of eucaryotic cells and the rfe mutant generated only tiny plaques. These observations indicated that the mutants were blocked in their ability to spread from cell to cell. Western immunoblot analysis of expression of IcsA, the protein essential for intracellular motility and intercellular spread, demonstrated that both mutants synthesized IcsA, although they secreted less of the protein to the extracellular medium than did the wild-type parent. More strikingly, the LPS mutants showed aberrant surface localization of IcsA. Unlike the unipolar localization of IcsA seen in the wild-type parent, the galU mutant expressed the protein in a circumferential fashion. The rfe mutant had an intermediate phenotype in that it displayed some localization of IcsA at one pole while also showing diffuse localization around the bacterium. Given the known structures of the LPS of wild-type S. flexneri 2a, the rfe mutant, and the galU mutant, we hypothesize that the core and O-antigen components of LPS are critical elements in the correct unipolar localization of IcsA. These observations indicate a more precise role for LPS in Shigella pathogenesis.

SUBMITTER: Sandlin RC

PROVIDER: S-EPMC172982 | biostudies-other | 1995 Jan

REPOSITORIES: biostudies-other

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Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane.

Sandlin R C RC Lampel K A KA Keasler S P SP Goldberg M B MB Stolzer A L AL Maurelli A T AT

Infection and immunity 19950101 1

Mutations in the lipopolysaccharide (LPS) of Shigella spp. result in attenuation of the bacteria in both in vitro and in vivo models of virulence, although the precise block in pathogenesis is not known. We isolated defined mutations in two genes, galU and rfe, which directly affect synthesis of the LPS of S. flexneri 2a, in order to determine more precisely the step in virulence at which LPS mutants are blocked. The galU and rfe mutants invaded HeLa cells but failed to generate the membrane pro ...[more]

PMID: 7528731

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Project description: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.

Dataset Information

Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane.

Publications

Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane.

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