1887

Abstract

The Gram-negative bacterium 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)-1-converting enzyme] and its substrate IL-1 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 -chlorophenylhydrazone (CCCP) dose-dependently inhibited the Mxi-Spa-dependent secretion of IpaB triggered by the dye Congo red and abolished translocation of IpaB into the host-cell cytoplasm of -infected macrophages. CCCP specifically inhibited -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-1, without affecting uptake or escape of 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 .

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2007-09-01
2019-10-18
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vol. , part 9, pp. 2862 - 2876

Reversible inhibition by CCCP of growth in TSB broth. Growth of wild-type in TSB medium was monitored in the absence (circles) or presence (squares, triangles) of 25 μM CCCP. After 4.5 h, all cultures were washed, and CCCP was removed (squares) or added back (triangles). Growth of in broth was abolished by the addition of 25 μM CCCP. The inhibition was completely reversible, as removal of CCCP after 2.5 h restored growth of the bacteria at the initial rate, demonstrating that exposure to CCCP within this time frame does not kill the bacteria. The growth curve shown is representative of three independent experiments. [ PDF] (72 kb)



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