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Shigella flexneri Inhibits Staurosporine-Induced Apoptosis in Epithelial Cells

Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799

Received 27 November 2006/ Returned for modification 26 December 2006/ Accepted 21 February 2007

Shigella flexneri is a facultative intracellular organism that causes bacillary dysentery. The Shigella IpaB protein activates caspase 1 in macrophages, which eventually leads to apoptosis. In contrast, epithelial cells infected with Shigella undergo a stress response but do not die. Therefore, the objective of this study was to determine if Shigella has the ability to inhibit apoptosis in epithelial cells. A modified gentamicin protection assay was used to investigate if HeLa cells infected with S. flexneri are able to resist the induction of apoptosis following treatment with 4 µM of staurosporine. Nuclear staining and immunofluorescence revealed that infected cells remained healthy while uninfected cells appeared apoptotic. Only uninfected cells had detectable levels of activated caspase 3 upon immunofluorescence, and this was verified by Western blot analysis. Despite interfering with caspase 3 activation, Shigella-infected cells treated with staurosporine did have cytochrome c release and caspase 9 activation, indicating that Shigella protects epithelial cells from apoptosis by inhibiting caspase 3 activation. Analysis of S. flexneri mutants showed that invasion and a functional type III secretion system were required to block apoptosis. In addition, a mutant with a deletion in mxiE, which encodes a transcriptional activator for genes induced intracellularly, failed to inhibit apoptosis. Therefore, protection of epithelial cells from apoptosis by S. flexneri is regulated by one or more of the bacterial genes under the control of mxiE. We believe that S. flexneri, like other pathogens, inhibits apoptosis in epithelial cells but causes apoptosis in macrophages to ensure survival inside the host.

Published ahead of print on 5 March 2007.

Editor: D. L. Burns

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