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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Differential fluorescence induction technology was used to identify promoters of Streptococcus pneumoniae genes that are expressed during lung infection of the mouse. Among the promoter clones that were identified multiple times was the psa promoter, which drives expression of the psaBCA operon. These genes have been identified previously and shown to encode a manganese permease system as well as play a role in the virulence of this organism. Mutations in psaB, psaC or psaA result in growth limitation in low manganese. The expression of the psa operon was examined in vivo and the virulence of deletion mutants of psaB, psaC, psaA and psaBCA was assessed in four different animal models of infection. The psa promoter was induced more than ten-fold in vivo using an intraperitoneal chamber implant model. The psaB, psaC and psaA mutants were completely attenuated in systemic, respiratory tract and otitis media infections. In addition, these mutants were unable to grow in an implanted peritoneal chamber, but growth was restored by the addition of manganese to the chambers.

SummaryDifferential fluorescence induction (DFI) in Streptococcus pneumoniae was used as a method for the discovery of genes activated in specific growth environments. Competence stimulatory peptide (CSP) was used as the model inducing system to identify differentially expressed genes. To identify CSP-induced promoters, a plasmid library was constructed by inserting random pieces of S. pneumoniae chromosomal DNA upstream of the promoterless gfpmut2 gene in an Escherichia coli/S. pneumoniae shuttle vector. S. pneumoniae carrying the library were induced with CSP and enriched for green fluorescent protein (GFP)-expressing bacteria using fluorescenceactivated cell sorting. A total of 886 fluorescent clones was screened, and 12 differentially activated promoter elements were identified. Sequence analysis of these clones revealed that three were associated with novel competence loci, one of which we show is essential for DNA uptake, and six are known CSP-inducible promoters. We also explored whether competence proteins have a role in virulence and found that mutations in three CSP-inducible genes resulted in attenuated virulence phenotypes in either of two murine infection models. These results demonstrate the utility of DFI as a method for identifying differentially expressed genes in S. pneumoniae and the potential utility of applying DFI to other Gram-positive bacteria.

virR is the central regulatory locus required for coordinate temperature-regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI-AccI DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique HpaI restriction site resulted in a loss of VirR+ activity. The virR ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone-like protein, H-NS. Based on the predicted amino acid sequence of E. coli H-NS, only a single conservative base-pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNA(Tyr). These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.