Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Pig SISP jejunal segments: Lactobacillus plantarum and msa/srt mutant strains vs control, perfusion 4 or 8 hours

ABSTRACT: Mannose-specific interactions of Lactobacillus plantarum 299v with jejunal epithelium were investigated using an in situ pig small intestinal segment perfusion (SISP) model. L. plantarum 299v wildtype strain was compared to two isogenic mutant strains either lacking the gene encoding for the mannose-specific adhesin (msa) or sortase (srtA; responsible for anchoring of cell surface proteins like Msa to the cell wall). Salmonella typhimurium served as a positive control for gene expression analysis. Scrapings from jejunal segments were collected after perfusion with bacterial suspensions or PBS (control) for 4 or 8 hours, and host gene expression was assessed using a home-made cDNA porcine microarray. Keywords: host-microbe interaction, Lactobacillus plantarum, mannose-specific adhesion A Small Intestinal Segment Perfusion (SISP) test was performed using 4 pigs. 10 segments were prepared in the jejunum of each pig and perfused with Lactobacillus plantarum 299v wildtype, Lactobacillus plantarum 299v msa mutant strain, Lactobacillus plantarum 299v srtA mutant strain, Salmonella typhimurium or PBS (control) for 4 or 8 hours. Pooled samples from each treatment at each timepoint were used for microarray analysis. 8 comparisons were done: L. plantarum wildtype vs control (4 hours), L. plantarum wildtype vs control (8 hours), L. plantarum msa mutant vs control (4 hours), L. plantarum msa mutant vs control (8 hours), L. plantarum srt mutant vs control (4 hours), L. plantarum srt mutant vs control (8 hours), S. typhimurium vs control (8 hours), samples taken at the beginning of the experiment vs control (8 hours). Dye-swaps were performed for each comparison.

ORGANISM(S): Sus scrofa

SUBMITTER: Jan van der Meulen

PROVIDER: E-GEOD-9209 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Project description:Background: Lactobacillus plantarum is found in a variety of fermented foods and as such, consumed for centuries. Some strains are natural inhabitants of the human gastro-intestinal tract and like other Lactobacillus species, L. plantarum has been extensively studied for its immunomodulatory properties and its putative health-promoting effects (probiotic). Being the first line of host defense intestinal epithelial cells (IEC) are key players in the recognition and initiation of responses to gut microorganisms. Results: Using high-density oligonucleotide microarrays we examined the gene expression profiles of differentiated Caco-2 cells exposed to various doses of L. plantarum. In addition, the effects were correlated to monolayer permeability studies and measurement of lactic acid production. A transcriptional dose-dependent IEC response to L. plantarum was found. Incubation of Caco-2 with a low bacterial dose induced a specific response, not due to cytotoxicity or production of lactic acid, including modulation of cell cycle and cell signaling functions. Exposure of Caco-2 cells to larger amounts of bacteria, accompanied by the production of lactic acid and glucose depletion, provoked increased permeability and supposed non-specific defense responses. Conclusions: These results suggest that IEC are able to sense and react to the presence of gut bacteria. This study provides the first description of global transcriptional response of human IEC to a commensal lactic acid bacterium, and it shows the importance of choosing physiological bacterial doses to prevent the observation of non-specific host reactions. Caco-2 cells were exposed for 10h to Lactobacillus. Fourteen samples are analyzed: 4 control Caco-2, 4 Caco-2 exposed to a low dose (10) of Lactobacillus, 4 Caco-2 exposed to a medium dose (100) of Lactobacillus, 2 Caco-2 exposed to a high dose (1000) of Lactobacillus. All 14 RNA samples are labeled with Cy5 and hybridized to a common reference (undifferentiated Caco-2, untreated) RNA labeled with Cy3

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Pig SISP jejunal segments: Lactobacillus plantarum and msa/srt mutant strains vs control, perfusion 4 or 8 hours

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