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Microbiology. 2007 Sep;153(Pt 9):2862-76.

Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death.

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Institute of Microbiology, Swiss Federal Institute of Technology (ETH) Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.


The Gram-negative bacterium Shigella flexneri triggers pro-inflammatory apoptotic cell death in macrophages, which is crucial for the onset of an acute inflammatory diarrhoea termed bacillary dysentery. The Mxi-Spa type III secretion system promotes bacterial uptake and escape into the cytoplasm, where, dependent on the translocator/effector protein IpaB, caspase-1 [interleukin (IL)-1beta-converting enzyme] and its substrate IL-1beta are activated. Here, we show that in the course of a macrophage infection, IpaB is secreted intracellularly for more than 1 h post-infection and progressively accumulates in aggregates on the bacterial surface. Concomitantly, the bacterial pool of IpaB is gradually depleted. The protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) dose-dependently inhibited the Mxi-Spa-dependent secretion of IpaB triggered by the dye Congo red in vitro and abolished translocation of IpaB into the host-cell cytoplasm of S. flexneri-infected macrophages. CCCP specifically inhibited S. flexneri-triggered macrophage death in a dose-dependent manner, even if added up to 60 min post-infection. Addition of CCCP 15 min after infection blocked macrophage cell death, the activation of caspase-1 and the maturation of IL-1beta, without affecting uptake or escape of S. flexneri from the phagosome. By contrast, CCCP used at the same concentration had no effect on ATP-induced caspase-1 activation or staurosporine-induced apoptosis. Our results indicate that under the conditions used, CCCP rapidly and specifically blocks bacterial type III secretion, and thus, intracellular type III secretion promotes cytotoxicity of S. flexneri.

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