Fibroblast transcription pattern in Salmonella infected Vs uninfected cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE71727 | GEO | 2016/08/04
SECONDARY ACCESSION(S): PRJNA291925
REPOSITORIES: GEO
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Project description:Human fibroblasts were infected with GFP-expressing Salmonella enterica serovar Typmimurium and further separated by Fluorescence Activated Cell Sorting. RNA from infected and uninfected cells sub-populations were used for genome-wide expression studies 3 independent experiments enabled us to obtain 3 infected and 3 uninfected sub-populations.
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Project description:Human fibroblasts were infected with GFP-expressing Salmonella enterica serovar Typmimurium and further separated by Fluorescence Activated Cell Sorting. RNA from infected and uninfected cells sub-populations were used for genome-wide expression studies 3 independent experiments enabled us to obtain 3 infected and 3 uninfected sub-populations.
2016-08-04 | E-GEOD-71727 | ArrayExpress
Project description:The early interaction of Salmonella enterica serovar typhimurium DT104 with intact small intestinal mucosa was studied in a Small Intestinal Segment Perfusion (SISP) model. Intestinal segments were infected with or without Salmonella. Scrapings from jejunal segments were collected after perfusion for 0, 2, 4, or 8 hours. Details of the SISP experiment are described in: Niewold TA, Veldhuizen EJ, van der Meulen J, Haagsman HP, de Wit AA, Smits MA, Tersteeg MH, Hulst MM. Using the Operon 13K pig oligonucleotide array differences in host gene expression were recorded between infected and uninfected segments within a single pig (isogenic comparisons), and between identical treated segments collected from 3 individual SISP pigs, all responding markedly different to infection with Salmonella (inter-animal comparisons). A Small Intestinal Segment Perfusion (SISP) test was performed with 3 pigs (pig no. 2, 3, and 4) (cross-bred YorkshireM-CM-^W(Large WhiteM-CM-^WLandrace)). Two adjacent segments prepared in the mid-jejunum of each pig were perfused for 1 hour with Salmonella enterica serovar typhimurium DT104 suspended in peptone solution (10E-09 CFU/ml) or with peptone solution alone (mock infected segment) respectively. Subsequently, segments were perfused with peptone solution alone for a maximum period of 8 hours. At 2, 4, and 8 hours a part of the infected segment was dissected to obtain mucosal scrappings. The same was done at 0 and 8 hours for the uninfected (mock) control segment. RNA isolated from scrappings was used for microarray comparisons using the Operon 13K pig oligonucleotide array. 9 comparisons were done. For each of the 3 SISP pigs, expression in the 8 hours perfused infected segment, perfused for 8 hours, was compared to expression in its adjacent mock infected segment (3 isogenic comparisons, 8 hpi.). Expression in the infected segment of each SISP pig, dissected after 2 or 4 hours of perfusion, was compared to expression in an infected segment dissected from another SISP pig (2 versus 3, 2 versus 4, and 3 versus 4 / 3 comparisons at 2 hpi., and 3 comparisons at 4 hpi.). Dye-swaps were performed for each comparison. jejunum pig, host-microbe interaction, Salmonella enterica serovar typhimurium DT104.
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Project description:Three strains of Salmonella enterica serotype Enteritidis were compared to Salmonella enterica serotype Heidelberg, Salmonella enterica serotype Newport, and Salmonella enterica serovar Typhimurium for growth in the presence of 240 antibiotics arranged within a commercial high-throughput phenotype microarray. The results show that antibiotic resistances were different for subpopulations of serotype Enteritidis separated only by genetic drift.
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Project description:The interplay between pathogens and hosts has been studied for decades using targeted approaches such as the analysis of mutants and host immunological responses. Although much has been learned from such studies, they focus on individual pathways and fail to reveal the global effects of infection on the host. To alleviate this issue, high-throughput methods such as transcriptomics and proteomics have been used to study host-pathogen interactions. Recently, metabolomics was established as a new method to study changes in the biochemical composition of host tissues. We report a metabolomics study of Salmonella enterica serovar Typhimurium infection. We used Fourier Transform Ion Cyclotron Resonance Mass Spectrometry with Direct Infusion to reveal that dozens of host metabolic pathways are affected by Salmonella in a murine infection model. In particular, multiple host hormone pathways are disrupted. Our results identify unappreciated effects of infection on host metabolism and shed light on mechanisms used by Salmonella to cause disease, and by the host to counter infection. Female C57BL/6 mice were infected with Salmonella enterica serovar Typhimurium SL1344 cells by oral gavage. Feces and livers were collected and metabolites extracted using acetonitrile. For experiments with feces, samples were collected from 4 mice before and after infection. For liver experiments, 11 uninfected and 11 infected mice were used. Samples were combined into 3 groups of 3-4 mice each, resulting in the analysis of 3 group samples of uninfected and 3 of infected mice. 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 [PMID 19081807]). To identify differences in metabolite composition between uninfected and infected 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 uninfected and infected 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. Data were analyzed by unpaired t tests with 95% confidence intervals.
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Project description:Gene expression in murine dendritic cells (DCs) infected with green fluorescent protein-expressing Salmonella enterica serovar Typhimurium BRD509 was studied by mRNA differential display. Infected DCs were sorted from uninfected cells by flow cytometry. The mRNA expression patterns of infected and uninfected cells revealed a number of differentially expressed transcripts, which included the macrophage-derived chemokine (MDC). Up-regulation of MDC transcription in infected DCs was confirmed by Northern blotting, and the kinetics of MDC expression was examined by real-time reverse transcription-PCR, with which 31- and 150-fold increases were detected at 2 and 6 h postinfection, respectively. The increased release by DCs of MDC into culture media was detected by an enzyme-linked immunosorbent assay. The biological activity of MDC was investigated in in vitro and in vivo assays. In vitro, supernatants from S. enterica serovar Typhimurium-infected DCs were chemoattractive to T cells, and neutralization of MDC in these supernatants inhibited T-cell migration. Passive transfer of anti-MDC antibody to mice infected with BRD509 revealed that neither growth of the bacterium nor resistance of the mice to reinfection was affected and that in vivo inhibition of MDC did not affect T-cell responses, as measured by the gamma interferon ELISPOT method 3 days after challenge infection.
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Project description:The eukaryotic transcriptional regulator Nuclear Factor kappa B (NF-?B) plays a central role in the defense to pathogens. Despite this, few studies have analyzed NF-?B activity in single cells during infection. Here, we investigated at the single cell level how NF-?B nuclear localization - a proxy for NF-?B activity - oscillates in infected and uninfected fibroblasts co-existing in cultures exposed to Salmonella enterica serovar Typhimurium. Fibroblasts were used due to the capacity of S. Typhimurium to persist in this cell type. Real-time dynamics of NF-?B was examined in microfluidics, which prevents cytokine accumulation. In this condition, infected (ST+) cells translocate NF-?B to the nucleus at higher rate than the uninfected (ST-) cells. Surprisingly, in non-flow (static) culture conditions, ST- fibroblasts exhibited higher NF-?B nuclear translocation than the ST+ population, with these latter cells turning refractory to external stimuli such as TNF-? or a second infection. Sorting of ST+ and ST- cell populations confirmed enhanced expression of NF-?B target genes such as IL1B, NFKBIA, TNFAIP3, and TRAF1 in uninfected (ST-) fibroblasts. These observations proved that S. Typhimurium dampens the NF-?B response in the infected fibroblast. Higher expression of SOCS3, encoding a "suppressor of cytokine signaling," was also observed in the ST+ population. Intracellular S. Typhimurium subverts NF-?B activity using protein effectors translocated by the secretion systems encoded by pathogenicity islands 1 (T1) and 2 (T2). T1 is required for regulating expression of SOCS3 and all NF-?B target genes analyzed whereas T2 displayed no role in the control of SOCS3 and IL1B expression. Collectively, these data demonstrate that S. Typhimurium attenuates NF-?B signaling in fibroblasts, an effect only perceptible when ST+ and ST- populations are analyzed separately. This tune-down in a central host defense might be instrumental for S. Typhimurium to establish intracellular persistent infections.
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Project description:The gut microbiota confers resistance to pathogens of the intestinal ecosystem, yet the dynamics of pathogen-microbiome interactions and the metabolites involved in this process remain largely unknown. Here, we use gnotobiotic mice infected with the virulent pathogen Salmonella enterica serovar Typhimurium or the opportunistic pathogen Candida albicans in combination with metagenomics and discovery metabolomics to identify changes in the community and metabolome during infection. To isolate the role of the microbiota in response to pathogens, we compared mice monocolonized with the pathogen, uninfected mice "humanized" with a synthetic human microbiome, or infected humanized mice. In Salmonella-infected mice, by 3 days into infection, microbiome community structure and function changed substantially, with a rise in Enterobacteriaceae strains and a reduction in biosynthetic gene cluster potential. In contrast, Candida-infected mice had few microbiome changes. The LC-MS metabolomic fingerprint of the cecum differed between mice monocolonized with either pathogen and humanized infected mice. Specifically, we identified an increase in glutathione disulfide, glutathione cysteine disulfide, inosine 5'-monophosphate, and hydroxybutyrylcarnitine in mice infected with Salmonella in contrast to uninfected mice and mice monocolonized with Salmonella These metabolites potentially play a role in pathogen-induced oxidative stress. These results provide insight into how the microbiota community members interact with each other and with pathogens on a metabolic level.IMPORTANCE The gut microbiota is increasingly recognized for playing a critical role in human health and disease, especially in conferring resistance to both virulent pathogens such as Salmonella, which infects 1.2 million people in the United States every year (E. Scallan, R. M. Hoekstra, F. J. Angulo, R. V. Tauxe, et al., Emerg Infect Dis 17:7-15, 2011, https://doi.org/10.3201/eid1701.P11101), and opportunistic pathogens like Candida, which causes an estimated 46,000 cases of invasive candidiasis each year in the United States (Centers for Disease Control and Prevention, Antibiotic Resistance Threats in the United States, 2013, 2013). Using a gnotobiotic mouse model, we investigate potential changes in gut microbial community structure and function during infection using metagenomics and metabolomics. We observe that changes in the community and in biosynthetic gene cluster potential occur within 3 days for the virulent Salmonella enterica serovar Typhimurium, but there are minimal changes with a poorly colonizing Candida albicans In addition, the metabolome shifts depending on infection status, including changes in glutathione metabolites in response to Salmonella infection, potentially in response to host oxidative stress.
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Project description:The objective of this research was to determine whether variation in the presence of fimbrial protein SefD would impact efficacy of bacterins as measured by recovery of Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) from the spleens of hens. Two bacterins were prepared that varied in SefD content. Also, two adjuvants were tested, namely, water-in-oil and aluminum hydroxide gel (alum). Control groups for both adjuvant preparations included infected nonvaccinated hens and uninfected nonvaccinated hens. At 21 days postinfection, Salmonella Enteritidis was recovered from 69.7%, 53.1%, and 86.0% from the spleens of all hens vaccinated with bacterins lacking SefD, bacterins that included SefD, and infected nonvaccinated control hens, respectively. No Salmonella was recovered from uninfected nonvaccinates. Results from individual trials showed that both bacterins reduced positive spleens, but that the one with SefD was more efficacious. Alum adjuvant had fewer side effects on hens and egg production as compared to water-in-oil. However, adjuvant did not change the relative recovery of Salmonella Enteritidis from spleens. These results suggest that SefD is a promising target antigen for improving the efficacy of immunotherapy in hens, and is intended to reduce Salmonella Enteritidis in the food supply.
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Project description:Transcriptomic analysis of Salmonella enterica serovar Typhimurium wild-type inside fibroblast cell
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