Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1β processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial–host interactions.

Vibrio cholerae is the causal bacterium of the diarrheal disease cholera, and its growth and survival are thought to be curtailed by bacteriovorous predators, e.g., ciliates and flagellates. We explored Caenorhabditis elegans as a test organism after finding that V. cholerae can cause lethal infection of this nematode. By reverse genetics we identified an extracellular protease, the previously uncharacterized PrtV protein, as being necessary for killing. The killing effect is associated with the colonization of bacteria within the Caenorhabditis elegans intestine. We also show that PrtV is essential for V. cholerae in the bacterial survival from grazing by the flagellate Cafeteria roenbergensis and the ciliate Tetrahymena pyriformis. The PrtV protein appears to have an indirect role in the interaction of V. cholerae with mammalian host cells as judged from tests with tight monolayers of human intestinal epithelial cells. Our results demonstrate a key role for PrtV in V. cholerae interaction with grazing predators, and we establish Caenorhabditis elegans as a convenient organism for identification of V. cholerae factors involved in host interactions and environmental persistence.cholera ͉ host interactions ͉ environmental persistence C holera continues to be a major public and individual health problem, especially in those regions of the world where it is endemic. Colwell (1) first hypothesized that coastal waters were an important reservoir of Vibrio cholerae. Huq et al. (2) reported that V. cholerae O1 cells could be observed to be attached to a variety of phytoplankton and zooplankton species. The incidence and severity of epidemics have been linked to salinity, water temperature, turbidity, and plankton blooms (3, 4). Cholera epidemics occur in a regular seasonal pattern. It has been suggested that during interepidemic periods V. cholerae exists in an unexplained ecological association with aquatic organisms (5). During the environmental phase, V. cholerae resides in diverse aquatic environments, often in association with marine plankton (6). The association of V. cholerae with zooplankton has proven to be a key factor in deciphering the global nature of cholera epidemics (7). In such natural bacterioplankton communities V. cholerae and other bacteria are also at the base of the pelagic microbial food web (8). Bacterial growth and survival are subject to constraint by bacteriovorous predators, e.g., protozoa such as ciliates and flagellates (9, 10). Little has been known about mechanisms and adaptations of bacteria to reduce grazing mortality compared with adaptations toward abiotic factors (substrate, temperature, pH, etc.) (11).V. cholerae expresses well characterized factors to establish and cause disease in the mammalian host, including cholera toxin (CT) and toxin-coregulated pili (Tcp). It has been shown that quorum sensing (QS) plays a role in the regulation of virulence in V. cholerae (12). At least three autoinducer signaling circuits function through the action of LuxO, leading to the repression of...

. The most prevalent adhesin was that encoded by the iha gene (91%; 127 of 139 strains), which was distributed in all seropathotypes. toxB and efa1 were present mainly in strains of seropathotypes A and B, which were LEE positive. saa was present only in strains of seropathotypes C, D, and E, which were LEE negative. Two fimbrial genes, lpfA O157/OI-141 and lpfA O157/OI-154 , were strongly associated with seropathotype A. The fimbrial gene lpfA O113 was present in all seropathotypes except for seropathotype A, while sfpA was not present in any of the strains studied. The distribution of STEC adhesins depends mainly on serotypes and not on the source of isolation. Seropathotype A, which is associated with severe disease and frequently is involved in outbreaks, possesses a unique adhesin profile which is not present in the other seropathotypes. The wide distribution of iha in STEC strains suggested that it could be a candidate for vaccine development.

No abstract

A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.