The curli regulator CsgD mediates stationary phase counter-silencing of csgBA in Salmonella Typhimurium.

Integration of horizontally acquired genes into transcriptional networks is essential for the regulated expression of virulence in bacterial pathogens. In Salmonella enterica, expression of such genes is repressed by the nucleoid-associated protein H-NS, which recognizes and binds to AT-rich DNA. H-NS-mediated silencing must be countered by other DNA-binding proteins to allow expression under appropriate conditions. Some genes that can be transcribed by RNA polymerase (RNAP) associated with the alternative sigma factor σS or the housekeeping sigma factor σ70 in vitro appear to be preferentially transcribed by σS in the presence of H-NS, suggesting that σS may act as a counter-silencer. To determine whether σS directly counters H-NS-mediated silencing and whether co-regulation by H-NS accounts for the σS selectivity of certain promoters, we examined the csgBA operon, which is required for curli fimbriae expression and is known to be regulated by both H-NS and σS . Using genetics and in vitro biochemical analyses, we found that σS is not directly required for csgBA transcription, but rather up-regulates csgBA via an indirect upstream mechanism. Instead, the biofilm master regulator CsgD directly counter-silences the csgBA promoter by altering the DNA-protein complex structure to disrupt H-NS-mediated silencing in addition to directing the binding of RNAP.

Charge separation in donor-acceptor spiro compounds at metal and metal oxide surfaces investigated by surface photovoltage.

Molecules with donor (diphenylamine) and acceptor moieties (dicyano or cyanoacrylic acid moieties) were linked by fluorene or spirobisfluorene cores and the chain length has been changed by introducing a thiophene group between fluorene and diphenylamine. Four different kinds of fluorene and spirobisfluorene compounds were adsorbed from highly diluted solutions at ultra-thin nanoporous TiO2 (np-TiO2), Au and ITO surfaces. Charge separation has been investigated by surface photovoltage spectroscopy in the fixed capacitor and Kelvin probe arrangements in vacuum. Striking differences between the interaction of linking (dicyano or cyanoacrylic moieties) and different substrates were observed. Intra-molecular charge separation and electron injection have been distinguished and the directed adsorption of spiro compounds was deduced.

Salmonella enterica causes more severe inflammatory disease in C57/BL6 Nramp1G169 mice than Sv129S6 mice.

Salmonella enterica serovar Typhimurium (S. Typhimurium) causes systemic inflammatory disease in mice by colonizing cells of the mononuclear leukocyte lineage. Mouse strains resistant to S. Typhimurium, including Sv129S6, have an intact Nramp1 (Slc11a1) allele and survive acute infection, whereas C57/BL6 mice, homozygous for a mutant Nramp1 allele, Nramp1(G169D) , develop lethal infections. Restoration of Nramp1 (C57/BL6 Nramp1(G169) ) reestablishes resistance to S. Typhimurium; mice survive at least 3 to 4 weeks postinfection. Since many transgenic mouse strains are on a C57/BL6 genetic background, C57/BL6 Nramp1(G169) mice provide a model to examine host genetic determinants of resistance to infection. To further evaluate host immune response to S. Typhimurium, we performed comparative analyses of Sv129S6 and C57/BL6 Nramp1(G169) mice 3 weeks following oral S. Typhimurium infection. C57/BL6 Nramp1(G169) mice developed more severe inflammatory disease with splenic bacterial counts 1000-fold higher than Sv129S6 mice and relatively greater splenomegaly and blood neutrophil and monocyte counts. Infected C57/BL6 Nramp1(G169) mice developed higher proinflammatory serum cytokine and chemokine responses (interferon-γ, tumor necrosis factor-α, interleukin [IL]-1ß, and IL-2 and monocyte chemotactic protein-1 and chemokine [C-X-C motif] ligand 1, respectively) and marked decreases in anti-inflammatory serum cytokine concentrations (IL-10, IL-4) compared with Sv129S6 mice postinfection. Splenic dendritic cells and macrophages in infected compared with control mice increased to a greater extent in C57/BL6 Nramp1(G169) mice than in Sv129S6 mice. Overall, data show that despite the Nramp1 gene present in both strains, C57/BL6 Nramp1(G169) mice develop more severe, Th1-skewed, acute inflammatory responses to S. Typhimurium infection compared with Sv129S6 mice. Both strains are suitable model systems for studying inflammation in the context of adaptive immunity.

Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro.

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox(-/)-, iNOS(-/)-, and doubly immunodeficient phox(-/)-iNOS(-/)- mice. The respiratory burst and NO radical (NO.) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase-dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O(2).(-)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S. typhimurium. Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon gamma appears to augment antibacterial activity predominantly by enhancing NO. production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo.

The roles of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) in host resistance to virulent Salmonella typhimurium were investigated in gp91phox(-/)-, iNOS(-/)-, and congenic wild-type mice. Although both gp91phox(-/)- and iNOS(-/)- mice demonstrated increased susceptibility to infection with S. typhimurium compared with wild-type mice, the kinetics of bacterial replication were dramatically different in the gp91phox(-/)- and iNOS(-/)- mouse strains. Greater bacterial numbers were present in the spleens and livers of gp91phox(-/)- mice compared with C57BL/6 controls as early as day 1 of infection, and all of the gp91phox(-/)- mice succumbed to infection within 5 d. In contrast, an increased bacterial burden was detected within reticuloendothelial organs of iNOS(-/)- mice only beyond the first week of infection. Influx of inflammatory CD11b(+) cells, granuloma formation, and serum interferon gamma levels were unimpaired in iNOS(-/)- mice, but the iNOS-deficient granulomas were unable to limit bacterial replication. The NADPH phagocye oxidase and iNOS are both required for host resistance to wild-type Salmonella, but appear to operate principally at different stages of infection.

Homocysteine antagonism of nitric oxide-related cytostasis in Salmonella typhimurium.

Nitric oxide (NO) is associated with broad-spectrum antimicrobial activity of particular importance in infections caused by intracellular pathogens. An insertion mutation in the metL gene of Salmonella typhimurium conferred specific hypersusceptibility to S-nitrosothiol NO-donor compounds and attenuated virulence of the organism in mice. The metL gene product catalyzes two proximal metabolic steps required for homocysteine biosynthesis. S-Nitrosothiol resistance was restored by exogenous homocysteine or introduction of the metL gene on a plasmid. Measurement of expression of the homocysteine-sensitive metH gene indicated that S-nitrosothiols may directly deplete intracellular homocysteine. Homocysteine may act as an endogenous NO antagonist in diverse processes including infection, atherosclerosis, and neurologic disease.

Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase.

A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has been found to be required for virulence and survival within macrophages. Here, SPI2 was shown to allow Salmonella typhimurium to avoid NADPH oxidase-dependent killing by macrophages. The ability of SPI2-mutant bacteria to survive in macrophages and to cause lethal infection in mice was restored by abrogation of the NADPH oxidase-dependent respiratory burst. Ultrastructural and immunofluorescence microscopy demonstrated efficient localization of the NADPH oxidase in the proximity of vacuoles containing SPI2-mutant but not wild-type bacteria, suggesting that SPI2 interferes with trafficking of oxidase-containing vesicles to the phagosome.

DNA repair is more important than catalase for Salmonella virulence in mice.

Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites such as hydrogen peroxide (H2O2), which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxify reactive oxygen species, and DNA repair systems which repair damage resulting from oxidative stress. To determine the relative importance of these two potentially protective defense mechanisms against oxidative stress encountered by Salmonella during infection of the host, a Salmonella typhimurium double mutant unable to produce either the HPI or HPII catalase was constructed, and compared with an isogenic recA mutant deficient in DNA repair. The recA mutant was hypersusceptible to H2O2 at low cell densities in vitro, while the catalase mutant was more susceptible to high H2O2 concentrations at high cell densities. The catalase mutant was found to be resistant to macrophages and retained full murine virulence, in contrast to the recA mutant which previously was shown to be macrophage-sensitive and attenuated in mice. These observations suggest that Salmonella is subjected to low concentrations of H2O2 while at relatively low cell density during infection, conditions requiring an intact DNA repair system but not functional catalase activity.

Cellular routes of invasion by enteropathogens.

The cellular pathways of infection utilized by pathogenic enteric bacteria have important implications for their clinical manifestations. Yersinia reaches Peyer's patches via M cells and uses plasmid-encoded factors to resist phagocytic cells. Shigella also translocates via M cells and incapacitates phagocytes, but subsequently re-enters the epithelium basolaterally to elicit an acute inflammatory response. Salmonella has recently been shown to both colonize Peyer's patches via M cells and independently disseminate to extraintestinal sites via CD18-expressing phagocytes. M cell-mediated entry can lead to gastroenteritis and mucosal antibody production, while systemic dissemination can result in septicemia and elicitation of systemic immune responses.

Oxygen-dependent anti-Salmonella activity of macrophages.

Numerous observations have established a crucial role for phagocytic cells in host resistance to Salmonella. Activated macrophages rely on a complex array of oxygen-dependent antimicrobial molecules to inhibit or kill intracellular Salmonella. An initial oxidative bactericidal phase, which is dependent on the respiratory burst phagocyte oxidase (phox) is succeeded by a prolonged nitrosative bacteriostatic phase, which is dependent on inducible nitric oxide synthase (iNOS). The sequential contribution of phox and iNOS to anti-Salmonella innate immunity has been demonstrated both in vitro and in vivo. The temporal progression from the predominant production of reactive oxygen species to the production of nitrogen oxides could optimize the initial reduction in microbial burden while minimizing the immunopathological consequences of the host inflammatory response.

Growth-phase regulation of plasmid virulence genes in Salmonella.

Virulence genes in the genus Salmonella are regulated by growth phase and by environmental signals, which allows a sequential program of expression during infection. Conditions that promote the expression of loci required in systemic infection, including the plasmid-encoded spv genes, are the opposite of the factors that induce genes involved in the invasion of epithelial cells in the gastrointestinal tract.

Cloning and sequencing of the gene encoding the RpoS (KatF) sigma factor from Salmonella typhimurium 14028s.

The gene encoding the alternative sigma factor RpoS in Salmonella typhimurium was cloned by its ability to complement acid susceptibility in rpoS mutant Escherichia coli. Sequence determination and comparison with rpoS from E. coli demonstrates a high degree of conservation, although significant differences are found within the extragenic regulatory regions.

Mutations in the gene encoding the replication-initiation protein of plasmid RK2 produce elevated copy numbers of RK2 derivatives in Escherichia coli and distantly related bacteria.

Mini-replicons of the broad-host-range plasmid RK2 with increased copy number (cn) due to mutations in the gene encoding the essential replication initiation protein TrfA are described. The cn of these derivatives have been determined in Escherichia coli, Pseudomonas aeruginosa and Agrobacterium tumefaciens and were found to be elevated in all three bacterial hosts. One of the cn mutations was introduced into the intact 60-kb RK2 plasmid by homologous recombination in vivo, resulting in an approximately twofold cn increase. The expression of trfA from this mutant RK2 plasmid did not respond to the cn change as predicted by a simple transcription rate-limitation, replication control model. Implications for the model of RK2 replication control and the potential use of mutant RK2 mini-replicons as high-copy broad-host-range gene cloning vectors are discussed.

Salmonella evasion of the NADPH phagocyte oxidase.

The bacteria-phagocyte interaction is of central importance in Salmonella pathogenesis. Immediately following phagocytosis, the NADPH phagocyte oxidase complex assembles in vesicles and produces highly toxic reactive oxygen species that play a major role in initial Salmonella killing by phagocytes. However, Salmonella has evolved a number of strategies to reduce the efficacy of oxygen-dependent phagocyte antimicrobial systems. Some of these strategies, such as superoxide dismutases, hydroperoxidases, oxidoreductases, scavengers and repair systems are common to most aerobic bacteria. In addition, Salmonella has acquired, by horizontal gene transfer, a type III secretory system encoded by Salmonella pathogenicity island 2 that interferes with the trafficking of vesicles containing functional NADPH phagocyte oxidase to the phagosome, thereby enhancing the survival of Salmonella within macrophages.

Human infection with Salmonella dublin.

Twenty-seven cases of human infection with Salmonella dublin were identified over a 12-year period at the University of California at San Diego-affiliated hospitals. Important epidemiologic risk factors were the ingestion of unpasteurized dairy products or treatment with nutritional therapy that included raw calf-liver extracts. Nearly all patients had underlying chronic diseases. Like Salmonella choleraesuis, S. dublin infections were associated with a high incidence of bacteremia (91%), metastatic sites of infection (30%), and mortality (26%) relative to other non-typhoidal Salmonellae. This pattern of disease expression may be related to a plasmid-encoded virulence factor common to both of these organisms.

Legionella sainthelensi serogroup 2 isolated from patients with pneumonia.

Three Legionella-like organisms isolated from patients with pneumonia are shown to belong to the species Legionella sainthelensi by DNA hybridization studies and to a new serogroup, serogroup 2, by serological studies (ATCC 49322). L. sainthelensi serogroup 2 and L. santicrucis are indistinguishable by slide agglutination, but are separable on the basis of their cell wall fatty acid profiles.

Diverse presentation of aberrant origin of the right subclavian artery: two case reports.

Aberrant origin of the right subclavian artery occurs in up to 1% of the population and can result in a wide range of symptoms. In this report, two cases of this anomaly are presented. In the first case, a patient developed fatal group A streptococcal aortitis. In the second case, the patient complained of chronic cough and intermittent dyspnea. The embryologic genesis of this abnormality is discussed and the current literature is summarized. Although relatively uncommon, it is important to consider this vascular anomaly in the differential diagnosis of patients with dysphagia, dyspnea, chest pain, fever, or mediastinal widening evidenced on chest roentgenography.

Plasmid-mediated virulence genes in non-typhoid Salmonella serovars.

Specific non-typhoid Salmonella serovars carry large virulence plasmids that promote sustained extra-intestinal infections. These plasmids all share a highly conserved 8-kb region containing the spv operon, consisting of the regulatory spvR locus and the four structural spvABCD genes. The SpvR protein belongs to the LysR/MetR family of transcriptional activators, and induces spvABCD expression in the stationary phase in response to nutrient limitation. spv expression also depends on the chromosomal stationary phase sigma factor RpoS (KatF), and is markedly induced when salmonellae enter eukaryotic cells. Additional plasmid genes encode complement resistance including the rck locus which is homologous to ail from Yersinia. Rck blocks formation of the complement membrane attack complex on the bacterial surface. Several loci involved in plasmid replication and stable maintenance have also been identified.

Invasive disease due to extended spectrum beta-lactamase-producing Klebsiella pneumoniae in a neonatal unit: the possible role of cockroaches.

We recently experienced an outbreak of nosocomial disease due to extended spectrum beta-lactamase-producing Klebsiella pneumoniae in a neonatal unit infested with cockroaches. Organisms isolated from cockroaches were indistinguishable by pulsed-field gel electrophoresis from those colonizing infants or causing clinical disease using. Cockroach elimination together with standard infection-control measures resulted in control of the outbreak. We suggest cockroaches are possible vectors of pathogenic bacteria in the hospital environment.