Project description:Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis in humans and calves characterized by diarrhea and polymorphonuclear cell (PMN) influx to the intestinal mucosa. The Salmonella Type III Secretion System encoded at Pathogenicity Island I (SPI-1) translocates the Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into the host epithelial cell cytoplasm. These five effector proteins act in concert to induce fluid secretion and transcription of C-X-C chemokines, which serve to recruit PMNs to the intestine. While the individual molecular interactions of these Salmonella proteins with cultured host cells have been extensively characterized, their combined role in the generation of fluid secretion and inflammation is less well understood. A bovine ligated ileal loop model was used in conjunction with a custom bovine microarray to determine intestinal response to acute S. Typhimurium infection in the calf. Gene expression responses to both wild type S. Typhimurium a delta sipA, sopABDE2 mutant were measured at seven times during the initial 12 hours of infection. Microarray analysis confirmed increased expression of genes encoding proteins previously associated with immune response to Salmonella spp. infection. Gene expression changes were mapped to molecular interaction pathways and changes in expression of mechanistic genes, which are defined as perturbed genes identified by Bayesian genetic network modeling, were strongly involved in the mechanisms of the host immune response. In addition to correctly identifying known effects of wild type S. Typhimurium on host (bovine) gene expression, Bayesian genetic network modeling identified novel effects of S. Typhimurium on several molecular interaction pathways. Novel effects impacted gene regulation in the following pathways: adipocytokine signaling, insulin signaling, complement and coagulation cascades, axon guidance, gap junction, neuroactive ligand-receptor interaction, long-term depression, long-term potentiation, melanogenesis, and natural killer cell mediated cytotoxicity. Known effects were observed in the following pathways: regulation of actin cytoskeleton, apoptosis, cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), MAPK signaling, calcium signaling, Jak-STAT signaling, leukocyte transendothelial migration, adherens junction, tight junction, and ECM-receptor interactions, phosphatidylinositol signaling system, and antigen processing and presentation. Quantitative real-time PCR was used to verify the expression of some of these mechanistic genes.

Project description:Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis with diarrhea and polymorphonuclear cell (PMN) influx into the intestinal mucosa in humans and calves. The Salmonella Type III Secretion System (T3SS) encoded at Pathogenicity Island I translocates Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into epithelial cells and is required for induction of diarrhea. These effector proteins act together to induce intestinal fluid secretion and transcription of C-X-C chemokines, recruiting PMNs to the infection site. While individual molecular interactions of the effectors with cultured host cells have been characterized, their combined role in intestinal fluid secretion and inflammation is less understood. We hypothesized that comparison of the bovine intestinal mucosal response to wild type Salmonella and a SipA, SopABDE2 effector mutant relative to uninfected bovine ileum would reveal heretofore unidentified diarrhea-associated host cellular pathways. To determine the coordinated effects of these virulence factors, a bovine ligated ileal loop model was used to measure responses to wild type S. Typhimurium (WT) and a ΔsipA, sopABDE2 mutant (MUT) across 12 hours of infection using a bovine microarray. Data were analyzed using standard microarray analysis and a dynamic bayesian network modeling approach (DBN). Both analytical methods confirmed increased expression of immune response genes to Salmonella infection and novel gene expression. Gene expression changes mapped to 219 molecular interaction pathways and 1620 gene ontology groups. Bayesian network modeling identified effects of infection on several interrelated signaling pathways including MAPK, Phosphatidylinositol, mTOR, Calcium, Toll-like Receptor, CCR3, Wnt, TGF-β, and Regulation of Actin Cytoskeleton and Apoptosis that were used to model of host-pathogen interactions. Comparison of WT and MUT demonstrated significantly different patterns of host response at early time points of infection (15 minutes, 30 minutes and one hour) within phosphatidylinositol, CCR3, Wnt, and TGF-β signaling pathways and the regulation of actin cytoskeleton pathway.

Project description:Penetration of intestinal epithelial cells by Salmonella enterica serovar Typhimurium requires the expression of invasion genes, found in Salmonella pathogenicity island 1 (SPI1), that encode components of a type III secretion apparatus. These genes are controlled in a complex manner by regulators within SPI1, including HilA and InvF, and those outside SPI1, such as the two-component regulators PhoP/PhoQ and BarA/SirA. We report here that epithelial cell invasion requires the serovar Typhimurium homologue of Escherichia coli csrA, which encodes a regulator that alters the stability of specific mRNA targets. A deletion mutant of csrA was unable to efficiently invade cultured epithelial cells and showed reduced expression of four tested SPI1 genes, hilA, invF, sipC, and prgH. Overexpression of csrA from an induced araBAD promoter also negatively affected the expression of these genes, indicating that CsrA can act as both a positive and a negative regulator of SPI1 genes and suggesting that the bacterium must tightly control the level or activity of CsrA to achieve maximal invasion. We found that CsrA affected hilA, a regulator of the other three genes we tested, probably by controlling one or more genetic elements that regulate hilA. We also found that both the loss and the overexpression of csrA reduced the expression of two regulators of hilA, hilC and hilD, suggesting that csrA exerts its control of hilA through one or both of these regulators. We further found, however, that CsrA could affect the expression of both invF and sipC independent of its effects on hilA. One additional striking phenotype of the csrA mutant, not observed in a comparable E. coli mutant, was its slow growth. Phenotypic revertants that had normal growth rates, while maintaining the csrA mutation, were common. These suppressed strains, however, did not recover the ability to invade cultured cells, indicating that the csrA-mediated loss of invasion cannot be attributed simply to poor growth and that the growth and invasion deficits of the csrA mutant arise from effects of CsrA on different targets.

Project description:Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis in humans and calves characterized by diarrhea and polymorphonuclear cell (PMN) influx to the intestinal mucosa. The Salmonella Type III Secretion System encoded at Pathogenicity Island I (SPI-1) translocates the Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into the host epithelial cell cytoplasm. These five effector proteins act in concert to induce fluid secretion and transcription of C-X-C chemokines, which serve to recruit PMNs to the intestine. While the individual molecular interactions of these Salmonella proteins with cultured host cells have been extensively characterized, their combined role in the generation of fluid secretion and inflammation is less well understood. A bovine ligated ileal loop model was used in conjunction with a custom bovine microarray to determine intestinal response to acute S. Typhimurium infection in the calf. Gene expression responses to both wild type S. Typhimurium a delta sipA, sopABDE2 mutant were measured at seven times during the initial 12 hours of infection. Microarray analysis confirmed increased expression of genes encoding proteins previously associated with immune response to Salmonella spp. infection. Gene expression changes were mapped to molecular interaction pathways and changes in expression of mechanistic genes, which are defined as perturbed genes identified by Bayesian genetic network modeling, were strongly involved in the mechanisms of the host immune response. In addition to correctly identifying known effects of wild type S. Typhimurium on host (bovine) gene expression, Bayesian genetic network modeling identified novel effects of S. Typhimurium on several molecular interaction pathways. Novel effects impacted gene regulation in the following pathways: adipocytokine signaling, insulin signaling, complement and coagulation cascades, axon guidance, gap junction, neuroactive ligand-receptor interaction, long-term depression, long-term potentiation, melanogenesis, and natural killer cell mediated cytotoxicity. Known effects were observed in the following pathways: regulation of actin cytoskeleton, apoptosis, cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), MAPK signaling, calcium signaling, Jak-STAT signaling, leukocyte transendothelial migration, adherens junction, tight junction, and ECM-receptor interactions, phosphatidylinositol signaling system, and antigen processing and presentation. Quantitative real-time PCR was used to verify the expression of some of these mechanistic genes. Microarrays were used to examine the transcriptional profiles of bovine intestinal epithelia infected with wild type Salmonella enterica serotype Typhimurium (control and wild type or delta sipA sopABDE2 mutant infected) across seven time points (15 min, 30 min, 1, 2, 4, 8, and 12 hours). Experiments were performed in quadruplicate (bovine ligated ileal loops surgeries were performed with four calves), generateing a total of 84 arrays.

Dataset Information

Systematic analysis of the regulation of type three secreted effectors in Salmonella enterica serovar Typhimurium.

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Results

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