purification-of-pathogen-vacuoles-from-legionella-infected-phagocytes

Purification of Pathogen Vacuoles from <em>Legionella</em>-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Ludwig-Maximilians-Universität

Pro-inflammatory macrophage apoptosis is pivotal in the aetiology of bacillary dysentery, an acute inflammatory diarrhoea caused by Shigella spp. S. flexneri triggers its uptake by macrophages, escapes the phagosome and kills the host cell by a cytotoxic pathway, which activates and requires caspase-1 [interleukin (IL)-1beta-converting enzyme] and releases mature IL-1beta. The bacterial type III-secreted translocator/effector protein IpaB triggers cell death and directly binds to caspase-1. Here, we demonstrate that in S. flexneri-infected macrophages, activated caspase-1 is present in the cytoplasm, the nucleus and on vesicular membranes. IpaB partitions with membrane and cytoplasmic fractions and colocalizes with activated caspase-1 on the surface of bacteria, in the macrophage cytoplasm and on vesicular membranes. Macrophages treated with the cholesterol-sequestering compound methyl-beta-cyclodextrin (MCD) were depleted from cholesterol within minutes and were impaired for phagocytosis of S. flexneri. Consequently, cytotoxicity as determined by lactate dehydrogenase release was blocked. Interestingly, if MCD was added 15-30 min post infection, cytotoxicity, activation of caspase-1, and apoptosis were inhibited, while phagocytosis of the bacteria, escape from the phagosome and type III secretion of IpaB was not affected. Inhibition of Shigella cytotoxicity by MCD coincided with a reduced association of IpaB to host cell membranes. Contrarily, the activation of caspase-1 and cytotoxicity triggered by the K+/H+ antiport ionophore nigericin or by ATP was not affected or even increased by MCD. These results indicate that cholesterol is specifically required for caspase-1 activation and apoptosis triggered by Shigella after the escape from phagosomes, and suggest that membrane association of IpaB contributes to the activation of caspase-1.

Cholesterol is required to trigger caspase-1 activation and macrophage apoptosis after phagosomal escape of Shigella.

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.

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

Shigella spp. are gram-negative pathogenic bacteria that evolved from harmless enterobacterial relatives and may cause devastating diarrhea upon ingestion. Research performed over the last 25 years revealed that a type III secretion system (T3SS) encoded on a large plasmid is a key virulence factor of Shigella flexneri. The T3SS determines the interactions of S. flexneri with intestinal cells by consecutively translocating two sets of effector proteins into the target cells. Thus, S. flexneri controls invasion into EC, intra- and intercellular spread, macrophage cell death, as well as host inflammatory responses. Some of the translocated effector proteins show novel biochemical activities by which they intercept host cell signal transduction pathways. An understanding of the molecular mechanisms underlying Shigella pathogenesis will foster the development of a safe and efficient vaccine, which, in parallel with improved hygiene, should curb infections by this widespread pathogen.

Article	Year
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Microbiology (Reading, England)\| PubMed ID: 15632436	2005
Applied and environmental microbiology\| PubMed ID: 16597995	2006
Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 16670325	2006
PLoS pathogens\| PubMed ID: 16710455	2006
Cholesterol is required to trigger caspase-1 activation and macrophage apoptosis after phagosomal escape of Shigella. Cellular microbiology\| PubMed ID: 16925787	2007
Cellular microbiology\| PubMed ID: 16939534	2006
Environmental microbiology\| PubMed ID: 17298357	2007
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Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death. Microbiology (Reading, England)\| PubMed ID: 17768231	2007
Microbiology (Reading, England)\| PubMed ID: 17975091	2007
European journal of immunology\| PubMed ID: 18000955	2007
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Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 21048081	2010
Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 21270399	2011
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Traffic (Copenhagen, Denmark)\| PubMed ID: 22340894	2012
Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 22696443	2012

Hubert Hilbi

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