Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death.
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.
Schroeder
Gunnar N
GN
Institute of Microbiology, Swiss Federal Institute of Technology (ETH) Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.
Jann
Naja J
NJ
Institute of Microbiology, Swiss Federal Institute of Technology (ETH) Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.
Hilbi
Hubert
H
Institute of Microbiology, Swiss Federal Institute of Technology (ETH) Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.
D000818:Animals; D017209:Apoptosis; D001426:Bacterial Proteins / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D020170:Caspase 1 / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000382:microbiology*; D004789:Enzyme Activation; D053583:Interleukin-1beta / Q000378:metabolism; D008264:Macrophages / Q000382:microbiology / Q000502:physiology*; D017737:Macrophages, Peritoneal / Q000382:microbiology / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D012763:Shigella flexneri / Q000187:drug effects / Q000472:pathogenicity*
D001426:Bacterial Proteins; D053583:Interleukin-1beta; C087447:ipaB protein, Shigella; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D020170:Caspase 1