Invasive Salmonella typhimurium induces dramatic cytoskeletal changes on the membrane surface of mammalian epithelial cells and RAW264.7 macrophages as part of its entry mechanism. Noninvasive S. typhimurium strains are unable to induce this membrane ruffling. Invasive S. typhimurium strains invade RAW264.7 macrophages in 2 h with 7-to 10-fold higher levels than noninvasive strains. Invasive S. typhimurium and Salmonella typhi, independent of their ability to replicate intracellularly, are cytotoxic to RAW264.7 macrophages and, to a greater degree, to murine bone marrow-derived macrophages. Here, we show that the macrophage cytotoxicity mediated by invasive Salmonella is apoptosis, as shown by nuclear morphology, cytoplasmic vacuolization, and host cell DNA fragmentation. S. typhimurium that enter cells causing ruffles but are mutant for subsequent intracellular replication also initiate host cell apoptosis. Mutant S. typhimurium that are incapable of inducing host cell membrane ruffling fail to induce apoptosis. The activation state of the macrophage plays a significant role in the response of macrophages to Salmonella invasion, perhaps indicating that the signal or receptor for initiating programmed cell death is upregulated in activated macrophages. The ability ofSalmonella to promote apoptosis may be important for the initiation of infection, bacterial survival, and escape of the host immune response.Salmonella typhimurium causes a self-limiting gastroenteritis in humans and typhoid-like systemic disease in mice. S. typhimurium entry into cultured epithelial cells is associated with dramatic host cell membrane ruffling (1, 2) and subsequent intracellular survival. S. typhimurium also invades murine M cells overlying the Peyer's Patch lymphoid follicles with associated membrane ruffling (3). Following invasion, the M cell is destroyed and the bacteria gain access to the subepithelial lymph tissue and the lamina propria, where they encounter macrophages, dendritic cells, lymphocytes, and neutrophils. Many laboratories have investigated the S. typhimuriummacrophage interaction in vitro (4) and found that S. typhimurium replicate in macrophage-like cell lines and survive in spleenic-derived macrophages from susceptible mice strains (5). Recently it was shown that S. typhimurium is cytotoxic to macrophages 14 h subsequent to infection. Noncytotoxic mutants, selected at 48 h postinoculation, were located in ompR, a gene belonging to a family of two-component regulators (6).In this study, we demonstrate that RAW264.7 and murine bone marrow-derived macrophages (BMM) invaded by S. typhimurium show clear manifestations of apoptosis and that mutant S. typhimurium incapable of inducing host cell membrane ruffling fail to induce apoptosis. We conclude that invasion of macrophages by S. typhimurium through a specific pathway associated with membrane ruffling signals the mammalian cell to undergo programmed cell death. MATERIALS AND METHODSBacterial Strains and Growth Conditions. The mousevirulent S. typhimurium st...

The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membraneperforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly + rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly + rBCG (∆ureC hly + rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. ∆ureC hly + rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of ∆ureC hly + rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.

SummaryIn 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS±PAGE. Distinct and characteristic proteins were identi®ed by mass spectrometry and introduced into a dynamic 2-DE database (http:/ /www.mpiibberlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identi®ed. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identi®ed and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.

Caspase-1 (Casp-1) mediates the processing of the proinflammatory cytokines interleukin-1␤ (IL-1␤) and IL-18 to their mature forms. Casp-1-deficient mice succumb more rapidly to Salmonella challenge than do wild-type animals. Both Casp-1 substrates, IL-18 and IL-1␤, are relevant for control of Salmonella enterica serovar Typhimurium. We used IL-18 ؊/؊ and IL-1␤ ؊/؊ mice in addition to administration of recombinant IL-18 to Casp-1 ؊/؊ mice to demonstrate that IL-18 is important for resistance to the systemic infection but not for resistance to the intestinal phase of the infection. This suggests that IL-1␤ is critical for the intestinal phase of the disease. Thus, we show that Casp-1 is essential for host innate immune defense against S. enterica serovar Typhimurium and that Casp-1 substrates are required at distinct times and anatomical sites.