Salmonella typhimurium invasion induces apoptosis in infected macrophages.

Abstract

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...

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“…PCRs were performed in a thermal cycler (Hybaid). Programme 1 (used for primer sets 5, 6, 9 and 10) consisted of one cycle of 5 min at 94.5ЊC, 45 s at 55ЊC and 2 min at 72ЊC, followed by 30 or 35 cycles of 30 s at 94.5ЊC, 45 s at 55ЊC and 2 min at 72ЊC, ᮊ Blackwell Science Ltd, Molecular Microbiology, 24, 155-167 Monack et al (1996) then one final cycle of 94.5ЊC for 45 s, 45 s at 55ЊC and 10 min at 72ЊC. Programme 2 (used for the remaining primer sets) consisted of one cycle of 5 min at 94.5ЊC, 45 s at 60ЊC and 2 min at 72ЊC, followed by 30 or 35 cycles of 30 s at 94.5ЊC, 45 s at 60ЊC and 2 min at 72ЊC, then one final cycle of 94.5ЊC for 45 s, 45 s at 60ЊC and 10 min at 72ЊC.…”

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