Cryopyrin (CIAS1, NLRP3) and ASC are components of the inflammasome, a multiprotein complex required for caspase-1 activation and cytokine IL-1beta production. CIAS1 mutations underlie autoinflammation characterized by excessive IL-1beta secretion. Disease-associated cryopyrin also causes a program of necrosis-like cell death in macrophages, the mechanistic details of which are unknown. We find that patient monocytes carrying disease-associated CIAS1 mutations exhibit excessive necrosis-like death by a process dependent on ASC and cathepsin B, resulting in spillage of the proinflammatory mediator HMGB1. Shigella flexneri infection also causes cryopyrin-dependent macrophage necrosis with features similar to the death caused by mutant CIAS1. This necrotic death is independent of caspase-1 and IL-1beta, and thus independent of the inflammasome. Furthermore, necrosis of primary macrophages requires the presence of Shigella virulence genes. While similar proteins mediate pathogen-induced cell death in plants, this report identifies cryopyrin as an important host regulator of programmed pathogen-induced necrosis in animals, a process we term pyronecrosis.

The complete sequence analysis of the 210-kb Shigella flexneri 5a virulence plasmid was determined. Shigella spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the known Shigella virulence determinants are encoded on a large plasmid that is unique to virulent strains of Shigella and enteroinvasive Escherichia coli; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 open reading frames (ORFs) were identified. An astonishing 153 (53%) of these were related to known and putative insertion sequence (IS) elements; no known bacterial plasmid has previously been described with such a high proportion of IS elements. Four new IS elements were identified. Fifty putative proteins show no significant homology to proteins of known function; of these, 18 have a G؉C content of less than 40%, typical of known virulence genes on the plasmid. These 18 constitute potentially unknown virulence genes. Two alleles of shet2 and five alleles of ipaH were also identified on the plasmid. Thus, the plasmid sequence suggests a remarkable history of IS-mediated acquisition of DNA across bacterial species. The complete sequence will permit targeted characterization of potential new Shigella virulence determinants.Shigella spp. continue to be a major health problem worldwide, causing an estimated 1 million deaths and 163 million cases of dysentery annually (29), predominantly in children younger than 5 years of age in developing countries. Shigella spp. cause bacillary dysentery in humans by invading and replicating in epithelial cells of the colon, causing an intense inflammatory reaction, characterized by abscess formation and ulceration, which damages the colonic epithelium.Shigella entry into susceptible host cells requires the coordinated expression of numerous genes that are activated in response to environmental cues (29,68). Upon contact with host cells, the bacteria secrete factors (IpaB, IpaC, and IpaA) that induce large membrane ruffles on the host cell surface associated with cytoskeletal rearrangements that lead to internalization of bacteria into the host cell. Once internalized, Shigella spp. release factors that cause lysis of the phagocytic vacuole, thereby releasing the bacteria into the cell cytoplasm, where the bacterial surface protein VirG (IcsA) assembles actin tails that propel the bacteria through the cell cytoplasm and into adjacent cells. Additional bacterial factors lead to release of proinflammatory cytokines, osmotic leak of the mucosal epithelium (ShET1 and ShET2), and, in macrophages, induction of cell death (IpaB) (20,38,75).Most work on the molecular pathogenesis of Shigella has been carried out in S. flexneri serotypes 2a and 5a. The entire complement of genes critical for invasion of epithelial cells is contained on a large 220-kb plasmid, termed the virulence plasmid or the invasion plasmid, which is present in all pathogenic strains (68). Known to be located on the virulence plasmi...

Page 2874, Acknowledgments, lines 1 and 2: "We thank the members of the University of Wisconsin genomics team for expert technical assistance" should read "We thank the members of the University of Wisconsin genomics team for expert technical assistance, especially Sean Phillips and Nicholas Hermersmann whose contributions were outstanding."

We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organism's distinctive lifestyle that have yet to be explained.