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A metabolomics analysis of gallbladder lipid droplet formation due to Salmonella infection

ABSTRACT: To cause disease, Salmonella enterica serovar Typhimurium requires two type-III secretion systems, encoded on Salmonella Pathogenicity Islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver virulence proteins, termed effectors, into the host cell cytosol. While the importance of these effector proteins to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded effector, SseL. Deletion of the sseL gene resulted in bacterial filamentation and elongation and unusual localization of Salmonella within infected epithelial cells. Infection with the ΔsseL strain also caused dramatic changes in lipid metabolism and led to massive accumulation of lipid droplets in infected cells. Some of these changes were investigated through metabolomics of gallbladder tissue. This phenotype was directly attributed to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine resulted in the same phenotype as the deletion mutant. Excessive buildup of lipids due to the absence of a functional sseL gene was also observed in S. Typhimurium-infected livers. These results demonstrate that SseL alters host lipid metabolism in infected epithelial cells by modifying ubiquitination patterns of cellular targets.

ORGANISM(S): Mus musculus

PROVIDER: GSE27604 | GEO | 2011/08/23

SECONDARY ACCESSION(S): PRJNA138279

REPOSITORIES: GEO

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Project description:To cause disease, Salmonella enterica serovar Typhimurium requires two type-III secretion systems, encoded on Salmonella Pathogenicity Islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver virulence proteins, termed effectors, into the host cell cytosol. While the importance of these effector proteins to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded effector, SseL. Deletion of the sseL gene resulted in bacterial filamentation and elongation and unusual localization of Salmonella within infected epithelial cells. Infection with the ?sseL strain also caused dramatic changes in lipid metabolism and led to massive accumulation of lipid droplets in infected cells. Some of these changes were investigated through metabolomics of gallbladder tissue. This phenotype was directly attributed to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine resulted in the same phenotype as the deletion mutant. Excessive buildup of lipids due to the absence of a functional sseL gene was also observed in S. Typhimurium-infected livers. These results demonstrate that SseL alters host lipid metabolism in infected epithelial cells by modifying ubiquitination patterns of cellular targets. Female C57BL/6 mice were infected with the indicated strain of Salmonella enterica serovar Typhimurium by oral gavage. Four gallbladders were collected and pooled per sample group and metabolites extracted using a mixture of methanol and chloroform. Extracts were infused into a 12-T Apex-Qe hybrid quadrupole-FT-ICR mass spectrometer equipped with an Apollo II electrospray ionization source, a quadrupole mass filter and a hexapole collision cell. Raw mass spectrometry data were processed as described elsewhere (Han et al. 2008. Metabolomics. 4:128-140). To identify differences in metabolite composition between different groups of samples, we filtered the list of masses for metabolites which were present on one set of samples but not the other. Additionally, we calculated the ratios between averaged intensities of metabolites from each group of mice. To assign possible metabolite identities, monoisotopic neutral masses of interest were queried against MassTrix (http://masstrix.org). Masses were searched against the Mus musculus database within a mass error of 3 ppm.

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.

Project description:Salmonella Typhimurium is an enteric food-borne pathogen responsible for causing salmonellosis associated with acute diarrhea. The bacterial invasion initially requires flagellar motility to reach the intestinal lumen and to cross the mucus layer of the intestinal epithelia and subsequent adhesion to the host cell, mainly mediated by fimbriae .The key genes involved in the pathogenic process are encoded within highly conserved regions of the bacterial genome called Salmonella Pathogenicity Islands (SPIs). The key regulator HilA, which is positively regulated by HilC and HilD, is located in SPI-1;however, other virulence-related regulators, such as RtsA, are encoded outside this island . During the invasion, proteins encoded by the prgHIJK, spaMNOPQRS, and invABCEFGH SPI-1 operons constitute a Type-3 Secretion System. Effector proteins,such as SipA and SipC (encoded in SPI-1) and SigD/SopB (encoded in SPI-5), translocate to the host cytosol through this system causing intracellular changes and inducing the immune response . The survival and replication of intracellular Salmonella inside Salmonella-containing vacuoles (SCVs) is mainly mediated by the genes located in SPI-2. The immune response is activated through the recognition of pathogen-associated molecular patterns (PAMPs) by specific receptors expressed on the surface and/or inside different cell types. The most important PAMPs are flagellin (monomers comprising the flagella filaments), particularly the FliC subunit, which is highly expressed on the bacterial surface, and lipopolysaccharide (LPS).Oxygen availability is limited in the inflamed gut whereas other compounds, such as hydrogen sulfide (H2S) and H2 are abundantly produced by the colonic bacteria. In addition, nutrients are also limited inside the SCV, while reactive oxygen as wellas nitrogen species may be found . In anaerobic conditions, Salmonella is able to survive by using alternative energy sources, such as nitrate or fumarate, through the use of specific enzymes: nitrate and nitrite reductases, fumarate reductase , DMSO reductase and respiratory hydrogenases.In a previous study, we compared transcriptomes of a clinical isolate of S. Typhimurium (strain 50-wt) and its derivative-multidrug-resistant mutant (strain 50-64) using microarrays . In addition to showing antimicrobial resistance, strain was also less invasive. In the present study, we focused on the genes of unknown function that showed impaired expression. We further selected those genes with a putative role in the acquired resistance phenotype or in the observed repressed virulence observed. Here, 4 putative membrane proteins and the yedF of unknown function were investigated to evaluate their involvement inthese two phenotypes.

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A metabolomics analysis of gallbladder lipid droplet formation due to Salmonella infection

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