Transcriptional regulation of Salmonella enterica serovar Typhimurium genes by bile.

DNA microarrays and two-dimensional (2-D) gel electrophoresis were utilized to analyze the global effect of bile on transcription and protein synthesis in Salmonella enterica serovar Typhimurium. Two bile-regulated proteins, YciF and PagC, were identified by 2-D gel electrophoresis and mass spectrometry fingerprinting. The operon yciGFE-katN demonstrated increased transcriptional activity in the presence of bile. While this operon has previously been shown to be RpoS-regulated, data from this study suggested that yciGFE-katN is regulated by bile independent of RpoS. The PhoP-PhoQ-regulated PagC is decreased in the presence of bile. Characterization of the untranslated leader of pagC demonstrated that a 97-bp region is necessary for the bile-mediated repression of this promoter. Analysis of data from the DNA microarray revealed an effect of bile on important global mechanistic pathways in S. enterica serovar Typhimurium. Genes involved in type III secretion-mediated invasion of epithelial cells demonstrated an overall repression of transcription in the presence of bile, corroborating previously reported data from this laboratory [Infect. Immun. 68 (2000) 6763]. In addition, bile-mediated transcriptional repression of genes involved in flagellar biosynthesis and motility was observed. These data further demonstrate that bile is an important environmental signal sensed by Salmonella spp. and that bile plays a role in regulating bacterial gene expression in multiple virulence-associated pathways.

Bile-salt-mediated induction of antimicrobial and bile resistance in Salmonella typhimurium.

By DNA microarray, the Salmonella typhimurium marRAB operon was identified as being bile-activated. Transcriptional assays confirm that marRAB is activated in the presence of bile and that this response is concentration-dependent. The bile salt deoxycholate is alone able to activate transcription, while there was no response in the presence of other bile salts tested or a non-ionic detergent. Deoxycholate is able to interact with MarR and interfere with its ability to bind to the mar operator. In addition, incubation of salmonellae in the presence of sublethal concentrations of bile is able to enhance resistance to chloramphenicol and bile, by means of both mar-dependent and mar-independent pathways. To further characterize putative marRAB-regulated genes that may be important for the resistance phenotype, acrAB, which encodes an efflux pump, was analysed. In S. typhimurium, acrAB is required for bile resistance, but while transcription of acrAB is activated by bile, this activation is independent of marRAB, as well as Rob, RpoS or PhoP-PhoQ. These data suggest that bile interacts with salmonellae to increase resistance to bile and other antimicrobials and that this can occur by marRAB- and acrAB-dependent pathways that function independently with respect to bile activation.

Salmonella pathogenicity island 2-dependent macrophage death is mediated in part by the host cysteine protease caspase-1.

Salmonella typhimurium invades host macrophages and can either induce a rapid cell death or establish an intracellular niche within the phagocytic vacuole. Rapid cell death requires the Salmonella pathogenicity island (SPI)1 and the host protein caspase-1, a member of the pro-apoptotic caspase family of proteases. Salmonella that do not cause this rapid cell death and instead reside in the phagocytic vacuole can trigger macrophage death at a later time point. We show here that the human pathogen Salmonella typhi also triggers both rapid, caspase-1-dependent and delayed cell death in human monocytes. The delayed cell death has previously been shown with S. typhimurium to be dependent on SPI2-encoded genes and ompR. Using caspase-1(-/-) bone marrow-derived macrophages and isogenic S. typhimurium mutant strains, we show that a large portion of the delayed, SPI2-dependent death is mediated by caspase-1. The two known substrates of activated caspase-1 are the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and IL-18, which are cleaved to produce bioactive cytokines. We show here that IL-1beta is released during both SPI1- and SPI2-dependent macrophage killing. Using IL-1beta(-/-) bone marrow-derived macrophages and a neutralizing anti-IL-18 antibody, we show that neither IL-1beta nor IL-18 is required for rapid or delayed macrophage death. Thus, both rapid, SPI1-mediated killing and delayed, SPI2-mediated killing require caspase-1 and result in the secretion of IL-1beta, which promotes inflammation and may facilitate the spread of Salmonella beyond the gastrointestinal tract in systemic disease.

Helicobacter pylori genetic diversity within the gastric niche of a single human host.

Isolates of the gastric pathogen Helicobacter pylori harvested from different individuals are highly polymorphic. Strain variation also has been observed within a single host. To more fully ascertain the extent of H. pylori genetic diversity within the ecological niche of its natural host, we harvested additional isolates of the sequenced H. pylori strain J99 from its human source patient after a 6-year interval. Randomly amplified polymorphic DNA PCR and DNA sequencing of four unlinked loci indicated that these isolates were closely related to the original strain. In contrast, microarray analysis revealed differences in genetic content among all of the isolates that were not detected by randomly amplified polymorphic DNA PCR or sequence analysis. Several ORFs from loci scattered throughout the chromosome in the archival strain did not hybridize with DNA from the recent strains, including multiple ORFs within the J99 plasticity zone. In addition, DNA from the recent isolates hybridized with probes for ORFs specific for the other fully sequenced H. pylori strain 26695, including a putative traG homolog. Among the additional J99 isolates, patterns of genetic diversity were distinct both when compared with each other and to the original prototype isolate. These results indicate that within an apparently homogeneous population, as determined by macroscale comparison and nucleotide sequence analysis, remarkable genetic differences exist among single-colony isolates of H. pylori. Direct evidence that H. pylori has the capacity to lose and possibly acquire exogenous DNA is consistent with a model of continuous microevolution within its cognate host.

Dynamic nature of host-pathogen interactions in Mycobacterium marinum granulomas.

Mycobacterium marinum causes long-term subclinical granulomatous infection in immunocompetent leopard frogs (Rana pipiens). These granulomas, organized collections of activated macrophages, share many morphological features with persistent human tuberculous infection. We examined organs of frogs with chronic M. marinum infection using transmission electron microscopy in conjunction with immunohistochemistry and acid phosphatase cytochemistry to better define the bacterium-host interplay during persistent infection. Bacteria were always found within macrophage phagosomes. These phagosomes were often fused to lysosomes, in sharp contrast to those formed during in vitro infection of J774 macrophage-like cells by M. marinum. The infected macrophages in frog granulomas showed various levels of activation, as evidenced by morphological changes, including epithelioid transformation, recent phagocytic events, phagolysosomal fusion, and disintegration of bacteria. Our results demonstrate that even long-term granulomas are dynamic environments with regard to the level of host cell activation and bacterial turnover and suggest a continuum between constantly replicating bacteria and phagocytic killing that maintains relatively constant bacterial numbers despite an established immune response. Infection with a mutant bacterial strain with a reduced capacity for intracellular replication shifted the balance, leading to a greatly reduced bacterial burden and inflammatory foci that differed from typical granulomas.

Mimicry of a G protein mutation by pertussis toxin expression in transgenic Caenorhabditis elegans.

Pathogens produce virulence factors that interact directly with host molecules, but in many cases the host targets are unknown. The genetic and molecular identification of these orphan targets is often not feasible with mammalian experimental models. However, a substantial number of known targets are molecules and pathways that are conserved among eukaryotes, and therefore the use of nonmammalian model hosts to identify orphan targets may prove useful. To demonstrate the feasibility of this approach, we transformed the nematode Caenorhabditis elegans with a gene encoding the catalytic subunit of pertussis toxin (PTX), which in mammals inactivates G(o/i)alpha proteins. Expression of PTX in C. elegans produced phenotypes almost identical to those of a null mutation in the nematode gene encoding G(o/i)alpha. Furthermore, PTX suppressed the phenotype of a constitutively active form of nematode G(o/i)alpha protein. These results indicate that PTX is functional in nematodes and acts specifically on the C. elegans homologue of the mammalian target.

The meaning and impact of the human genome sequence for microbiology.

The characterization of life is immeasurably enhanced by determination of complete genome sequences. For organisms that engage in intimate interactions with others, the genome sequence from one participant, and associated tools, provide unique insight into its partner. We discuss how the human genome sequence will further our understanding of microbial pathogens and commensals, and vice versa. We also propose criteria for implicating a host gene in microbial pathogenesis, and urge consideration of a'second human genome project'.

New approaches for validation of lethal phenotypes and genetic reversion in Helicobacter pylori.

BACKGROUND: Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to develop such tools for H. pylori. MATERIALS AND METHODS: We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would permit the gene's disruption. For genes that yielded mutants with selectable phenotypes, a strategy was developed for reversion whereby an intact copy of the gene is introduced to the organism by transformation with PCR products. Following this procedure, revertants were selected by phenotypic tests then tested for genetic reversion. RESULTS: After failure to attain transformants upon attempted mutation of genes HP0166 and HP1365, we inserted a second copy of each gene within the H. pylori chromosome. In each case the second copy relieved the block of transformation. Mutation of genes HP0703 and HP1021 gave non-motile and small-colony phenotypes, respectively. Following transformation with PCR products containing intact copies of the genes, both phenotype and genotype had reverted following phenotypic selections. CONCLUSIONS: The methods used in this study provide new approaches for confirming suspected genotype/phenotype associations and should be widely applicable in the study of H. pylori.

Host microarray analysis reveals a role for the Salmonella response regulator phoP in human macrophage cell death.

Bacterial pathogens manipulate host cells to promote pathogen survival and dissemination. We used a 22,571 human cDNA microarray to identify host pathways that are affected by the Salmonella enterica subspecies typhimurium phoP gene, a transcription factor required for virulence, by comparing the expression profiles of human monocytic tissue culture cells infected with either the wild-type bacteria or a phoPTn10 mutant strain. Both wild-type and phoPTn10 bacteria induced a common set of genes, many of which are proinflammatory. Differentially expressed genes included those that affect host cell death, suggesting that the phoP regulatory system controls bacterial genes that alter macrophage survival. Subsequent experiments showed that the phoPTn10 mutant strain is defective for killing both cultured and primary human macrophages but is able to replicate intracellularly. These experiments indicate that phoP plays a role in Salmonella-induced human macrophage cell death.

Identification of attenuated Yersinia pseudotuberculosis strains and characterization of an orogastric infection in BALB/c mice on day 5 postinfection by signature-tagged mutagenesis.

Yersinia pseudotuberculosis localizes to the distal ileum, cecum, and proximal colon of the gastrointestinal tract after oral infection. Using signature-tagged mutagenesis, we isolated 13 Y. pseudotuberculosis mutants that failed to survive in the cecum of mice after orogastric inoculation. Twelve of these mutants were also attenuated for replication in the spleen after intraperitoneal infection, whereas one strain, mutated the gene encoding invasin, replicated as well as wild-type bacteria in the spleen. Several mutations were in operons encoding components of the type III secretion system, including components involved in translocating Yop proteins into host cells. This indicates that one or more Yops may be necessary for survival in the gastrointestinal tract. Three mutants were defective in O-antigen biosynthesis; these mutants were also unable to invade epithelial cells as efficiently as wild-type Y. pseudotuberculosis. Several other mutations were in genes that had not previously been associated with growth in a host, including cls, ksgA, and sufl. In addition, using Y. pseudotuberculosis strains marked with signature tags, we counted the number of different bacterial clones that were present in the cecum, mesenteric lymph nodes, and spleen 5 days postinfection. We find barriers in the host animal that limit the number of bacteria that succeed in reaching and/or replicating in the mesenteric lymph nodes and spleen after breaching the gut mucosa.

Bile-induced 'pili' in Campylobacter jejuni are bacteria-independent artifacts of the culture medium.

In 1996, it was reported that the enteric pathogen Campylobacter jejuni produces pilus-like appendages in response to bile salts such as deoxycholate (DOC), and that the formation of these appendages requires the putative peptidase PspA. Pili were known to be important virulence determinants in other pathogenic bacteria but had never before been observed for C. jejuni. We report here that these appendages are not pili, but are instead a bacteria-independent morphological artifact of the growth medium. Furthermore, the pspA gene is not required for their formation. Broth cultures containing a threshold concentration of DOC inoculated with no bacteria produced identical abundant, fibrous, pilus-like structures as those cultures that had been inoculated with C. jejuni. These fibres were also found in growth media from DOC-containing pspA:CmR mutant cultures. These results are consistent with the absence of candidate pilin monomers in protein gel analyses as well as the dearth of pilin-like genes and pilus formation gene clusters in the C. jejuni genome.

Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses.

Helicobacter pylori enhances the risk for ulcer disease and gastric cancer, yet only a minority of H. pylori-colonized individuals develop disease. We examined the ability of two H. pylori isolates to induce differential host responses in vivo or in vitro, and then used an H. pylori whole genome microarray to identify bacterial determinants related to pathogenesis. Gastric ulcer strain B128 induced more severe gastritis, proliferation, and apoptosis in gerbil mucosa than did duodenal ulcer strain G1.1, and gastric ulceration and atrophy occurred only in B128+ gerbils. In vitro, gerbil-passaged B128 derivatives significantly increased IL-8 secretion and apoptosis compared with G1.1 strains. DNA hybridization to the microarray identified several strain-specific differences in gene composition including a large deletion of the cag pathogenicity island in strain G1.1. Partial and complete disruption of the cag island in strain B128 attenuated induction of IL-8 in vitro and significantly decreased gastric inflammation in vivo. These results indicate that the ability of H. pylori to regulate epithelial cell responses related to inflammation depends on the presence of an intact cag pathogenicity island. Use of an H pylori whole genome microarray is an effective method to identify differences in gene content between H. pylori strains that induce distinct pathological outcomes in a rodent model of H. pylori infection.

Vacuolating cytotoxin of Helicobacter pylori plays a role during colonization in a mouse model of infection.

Helicobacter pylori, the causative agent of gastritis and ulcer disease in humans, secretes a toxin called VacA (vacuolating cytotoxin) into culture supernatants. VacA was initially characterized and purified on the basis of its ability to induce the formation of intracellular vacuoles in tissue culture cells. H. pylori strains possessing different alleles of vacA differ in their ability to express active toxin. Those strains expressing higher toxin levels are correlated with more severe gastric disease. However, the specific role(s) played by VacA during the course of infection and disease is not clear. We have used a mouse model of H. pylori infection to begin to address this role. A null mutation of vacA compromises H. pylori in its ability to initially establish infection. If an infection by a vacA mutant is established, the bacterial load and degree of inflammation are similar to those associated with an isogenic wild-type strain. Thus, in this infection model, vacA plays a role in the initial colonization of the host, suggesting that strains of H. pylori expressing active alleles of vacA may be better adapted for host-to-host transmission.

Identification of Legionella pneumophila genes important for infection of amoebas by signature-tagged mutagenesis.

Legionella pneumophila is a facultative intracellular gram-negative rod that causes pneumonia in humans. Free-living amoebas are thought to serve as a reservoir for Legionella infections. Signature-tagged mutagenesis was employed to identify Legionella pneumophila genes necessary for survival in the amoeba Acanthamoeba castellanii. Six mutant strains were defective in assays of invasion and intracellular growth. Four mutants also exhibited invasion and replication defects in Hartmannella vermiformis, an amoeba linked to hospital outbreaks of Legionella pneumonia. The six mutants also were tested in macrophages derived from peripheral blood mononuclear cells. Two mutants had intracellular replication defects, and two different strains entered cells less efficiently. Two transposon insertions were in known L. pneumophila genes, lspK and aroB. The other four were in novel genes. One gene has similarity to a cytochrome c-type biogenesis protein of Pseudomonas fluorescens. Another has similarity to a transcriptional activator regulating flagellar biosynthesis in Vibrio cholera. The third is similar to traA of Rhizobium sp. strain NGR234, which is involved in conjugal transfer of DNA. The fourth has no homology. By using survival in amoeba as a selection, we have isolated mutant strains with a range of phenotypes; and we have potentially identified new L. pneumophila virulence genes.

Salmonella-induced macrophage death: the role of caspase-1 in death and inflammation.

Salmonella typhimurium invades host macrophages and can induce either an almost immediate cell death or establish an intracellular niche within the phagocytic vacuole. Rapid cell death depends on the Salmonella pathogenicity island SPI1 and the host protein caspase-1, a member of the pro-apoptotic caspase family of proteases. Caspase-1-dependent cell death leads to the activation of the potent pro-inflammatory cytokines interleukin (IL)-1beta and IL-18 to produce bioactive cytokines. Animal studies indicate that the activation of these cytokines is necessary for efficient colonization of the mouse gastrointestinal tract. Salmonella that reside in the phagocytic vacuole do not cause this early cell death and can trigger a macrophage death at a much later time point. This late-phase cell death is dependent on SPI2-encoded genes and ompR.

A whole-genome microarray reveals genetic diversity among Helicobacter pylori strains.

Helicobacter pylori colonizes the stomach of half of the world's population, causing a wide spectrum of disease ranging from asymptomatic gastritis to ulcers to gastric cancer. Although the basis for these diverse clinical outcomes is not understood, more severe disease is associated with strains harboring a pathogenicity island. To characterize the genetic diversity of more and less virulent strains, we examined the genomic content of 15 H. pylori clinical isolates by using a whole genome H. pylori DNA microarray. We found that a full 22% of H. pylori genes are dispensable in one or more strains, thus defining a minimal functional core of 1281 H. pylori genes. While the core genes encode most metabolic and cellular processes, the strain-specific genes include genes unique to H. pylori, restriction modification genes, transposases, and genes encoding cell surface proteins, which may aid the bacteria under specific circumstances during their long-term infection of genetically diverse hosts. We observed distinct patterns of the strain-specific gene distribution along the chromosome, which may result from different mechanisms of gene acquisition and loss. Among the strain-specific genes, we have found a class of candidate virulence genes identified by their coinheritance with the pathogenicity island.

mig-14 is a horizontally acquired, host-induced gene required for salmonella enterica lethal infection in the murine model of typhoid fever.

We have characterized a host-induced virulence gene, mig-14, that is required for fatal infection in the mouse model of enteric fever. mig-14 is present in all Salmonella enterica subspecies I serovars and maps to a region of the chromosome that appears to have been acquired by horizontal transmission. A mig-14 mutant replicated in host tissues early after infection but was later cleared from the spleens and livers of infected animals. Bacterial clearance by the host occurred concomitantly with an increase in gamma interferon levels and recruitment of macrophages, but few neutrophils, to the infection foci. We hypothesize that the mig-14 gene product may repress immune system functions by interfering with normal cytokine expression in response to bacterial infections.

Apoptosis as a common bacterial virulence strategy.

The comparison of common strategies used by bacterial pathogens to overcome host defenses provides us with the opportunity to analyze the biology of pathogenicity, as well as point out the unique interactions between a particular pathogen and its host. Here we compare and contrast apoptosis induced by three enteric pathogens, Salmonella, Shigella, and Yersinia. We point out that all three enteric pathogens induce apoptosis in macrophages in vitro, but the proposed mechanisms are quite different. Yersinia induces apoptosis by inhibiting the translocation of the transcriptional activator, NF-kappaB, into the nucleus, which results in the suppression of TNFalpha production; whereas Salmonella- and Shigella-induced apoptosis depend on the activation of caspase-1 (casp-1). The result of casp-1 activation is to induce apoptosis as well as to process the proinflammatory cytokines, pro-IL-1beta and pro-IL18 into their mature bioactive forms. Thus, in contrast to Yersinia, Salmonella and Shigella-induced apoptosis results in a proinflammatory cascade.

Salmonella exploits caspase-1 to colonize Peyer's patches in a murine typhoid model.

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]-1beta-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-1beta 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 (LD(50)) 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 LD(50). 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.