Recently, Salmonella spp. were shown to induce apoptosis in infected macrophages. The mechanism responsible for this process is unknown. In this report, we establish that the Inv-Spa type III secretion apparatus target invasin SipB is necessary and sufficient for the induction of apoptosis. Purified SipB microinjected into macrophages led to cell death. Binding studies show that SipB associates with the proapoptotic protease caspase-1. This interaction results in the activation of caspase-1, as seen in its proteolytic maturation and the processing of its substrate interleukin-1␤. Caspase-1 activity is essential for the cytotoxicity. Functional inhibition of caspase-1 activity by acetyl-Tyr-Val-Ala-Aspchloromethyl ketone blocks macrophage cytotoxicity, and macrophages lacking caspase-1 are not susceptible to Salmonella-induced apoptosis. Taken together, the data demonstrate that SipB functions as an analog of the Shigella invasin IpaB.

We report here that the Shigella invasion plasmid antigen (Ipa)B, which is sufficient to induce apoptosis in macrophages, binds to caspase (Casp)-1, but not to Casp-2 or Casp-3. Casp-1 is activated and its specific substrate interleukin-1␤ is cleaved shortly after Shigella infection. Macrophages isolated from Casp-1 knock-out mice are not susceptible to Shigella-induced apoptosis, although they respond normally to other apoptotic stimuli. Shigella kills macrophages from casp-3, casp-11, and p53 knock-out mice as well as macrophages overexpressing Bcl-2. We propose that Shigella induces apoptosis by directly activating Casp-1 through IpaB, bypassing signal transduction events and caspases upstream of Casp-1. Taken together these data indicate that Shigella-induced apoptosis is distinct from other forms of apoptosis and seems uniquely dependent on Casp-1.

Salmonella typhimurium invades host macrophages and induces apoptosis and the release of mature proinflammatory cytokines. SipB, a protein translocated by Salmonella into the cytoplasm of macrophages, is required for activation of Caspase-1 (Casp-1, an interleukin [IL]-1β–converting enzyme), which is a member of a family of cysteine proteases that induce apoptosis in mammalian cells. Casp-1 is unique among caspases because it also directly cleaves the proinflammatory cytokines IL-1β and IL-18 to produce bioactive cytokines. We show here that mice lacking Casp-1 (casp-1 −/ − mice) had an oral S. typhimurium 50% lethal dose (LD50) that was 1,000-fold higher than that of wild-type mice. Salmonella breached the M cell barrier of casp-1 −/ − mice efficiently; however, there was a decrease in the number of apoptotic cells, intracellular bacteria, and the recruitment of polymorphonuclear lymphocytes in the Peyer's patches (PP) as compared with wild-type mice. Furthermore, Salmonella did not disseminate systemically in the majority of casp-1 −/ − mice, as demonstrated by significantly less colonization in the PP, mesenteric lymph nodes, and spleens of casp-1 −/ − mice after an oral dose of S. typhimurium that was 100-fold higher than the LD50. The increased resistance in casp-1 −/ − animals appears specific for Salmonella infection since these mice were susceptible to colonization by another enteric pathogen, Yersinia pseudotuberculosis, which normally invades the PP. These results show that Casp-1, which is both proapoptotic and proinflammatory, is essential for S. typhimurium to efficiently colonize the cecum and PP and subsequently cause systemic typhoid-like disease in mice.

We have developed a system that recreates in vitro the generation of post-Golgi vesicles from an isolated Golgi fraction prepared from vesicular stomatitis virus- or influenza virus-infected Madin-Darby canine kidney or HepG2 cells. In this system, vesicle generation is temperature- and ATP-dependent and requires a supply of cytosolic proteins, including an N-ethylmaleimide-sensitive factor distinct from NSF. Cytosolic proteins obtained from yeast were as effective as mammalian cytosolic proteins in supporting vesicle formation and had the same requirements. The vesicles produced (50-80 nm in diameter) are depleted of the trans Golgi marker sialyltransferase, contain the viral glycoprotein molecules with their cytoplasmic tails exposed, and do not show an easily recognizable protein coat. Vesicle generation was inhibited by brefeldin A, which indicates that it requires the activation of an Arf-like GTP-binding protein that promotes assembly of a vesicle coat. Vesicles formed in the presence of the nonhydrolyzable GTP analogue guanosine 5'-3-O-(thio)triphosphate retained a nonclathrin protein coat resembling that of COP-coated vesicles, and sedimented more rapidly in a sucrose gradient than the uncoated ones generated in its absence. This indicates that GTP hydrolysis is not required for vesicle generation but that it is for vesicle uncoating. The activity of a Golgi-associated protein kinase C (PKC) was found to be necessary for the release of post-Golgi vesicles, as indicated by the capacity of a variety of inhibitors and antibodies to PKC to suppress it, as well as by the stimulatory effect of the PKC activator 12-O-tetradecanoylphorbol-13-acetate.

Infection by the gram-negative bacterium Shigella flexneri results in dysentery, an acute inflammatory disease of the colon. Essential events in the pathogenesis of Shigella infections include bacterial invasion of epithelial cells, escape from the phagosome, and induction of apoptosis in macrophages. The Shigella virulence factor invasion plasmid antigen B (IpaB) is required for all of these processes. Induction of apoptosis is dependent on IpaB binding to the cysteine protease caspase-1 (Casp-1). The activation of this enzyme triggers both apoptosis and release of the proinflammatory cytokine interleukin-1␤. Several IpaB mutants were generated to correlate function with protein subdomains. We determined that the N-terminal portion of IpaB is necessary for stable expression of IpaB. A putative amphipathic ␣-helical domain preserves the structure of IpaB. We found 10 consecutive residues within the amino terminus of the hydrophobic region that play a critical role in invasion, phagosomal escape, and cytotoxicity. An IpaB mutant carrying a mutation in this region binds to Casp-1 yet is not cytotoxic, even following direct delivery to the macrophage cytoplasm. These results indicate that the association between IpaB and Casp-1 is only a step in the activation of macrophage apoptosis.Infections with enterobacteria of the genus Shigella cause dysentery, a severe bloody diarrhea. Dysentery is characterized by an acute inflammation of the colon with mucosal erosion (11, 23). Development of shigellosis requires bacterial penetration across the intestinal epithelial barrier via M cells. Upon reaching the underlying lymphoid follicles, the bacteria are engulfed by resident macrophages (30,38). Once inside a macrophage, Shigella escapes from the phagosome into the cytoplasm and kills this cell by inducing apoptosis (42). The dying macrophage releases mature interleukin-1␤ (IL-1␤) (40) and IL-18 (32), two cytokines important in the initiation of inflammation (34). Shigella also invades epithelial cells through pathogen-directed endocytosis (23). Invasion of enterocytes and bacterial cell-to-cell spread enhance tissue damage (20,33).The Shigella invasion plasmid antigens B (IpaB), IpaC, and IpaD are required for epithelial cell entry and phagosome escape (14, 23). However, IpaB alone is sufficient to activate macrophage apoptosis (5). The Ipa proteins interact with host cells upon being secreted by a type III secretion apparatus (26). In the macrophage cytoplasm, IpaB binds to caspase-1 (Casp-1; also called IL-1␤ converting enzyme [ICE]), a proapoptotic and proinflammatory cysteine protease that cleaves IL-1␤ and IL-18 to their biologically active forms (6, 37). The activation of Casp-1 leads to macrophage apoptosis by an as yet ill-defined pathway (15, 40). Casp-1-deficient macrophages' resistance to Shigella-induced cell death demonstrates that Casp-1 is essential for this process (16).IpaB contains a hydrophobic region (amino acids [aa] 310 to 430) that contains two putative membrane-spanning domains (aa 313 to 346 and 4...

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