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Characterization of the transcriptomes of genetically diverse Listeria monocytogenes exposed to hyperosmotic and low temperature conditions

ABSTRACT: A screening process was used to determine hyperosmotic as well as cold stress tolerance of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at sub lethal levels of NaCl and 4°C. Analysis of gene expression in cells adapted to cold and hyperosmotic stress revealed significant strain specificity in terms of individual gene expression profiles though general patterns of expression within functional gene subsets were largely similar. . Strain ATCC19115 revealed 150 significantly up- and 146 down-regulated genes in cells adapted to both hyperosmotic and cold stress, 82 up- and 56 down-regulated genes were matching in response to both stresses in strain 70-1700, whereas ScottA, a relatively stress-tolerant strain, showed up-regulation of 77 genes whilst 106 genes were significantly down-regulated. Among these homologously expressed genes only 22 were found to be significantly up-regulated and only 7 were down-regulated in all three strains adapted to hyperosmotic stress and cold temperature, suggesting a dominating strain specific response. Overall adaptation to hyperosmotic and low temperature growth conditions in three L. monocytogenes strains were found to have many parallels, especially when examined using a statistically-based ontological approach. Evidence was presented for strong activation of genes associated with protein synthesis, in particular those coding proteins of the ribosome and involved directly in transcription. Genes associated with DNA maintenance; modification to cell envelope, and cell division were also up-regulated. On the other hand strong suppression of genes associated with carbohydrate metabolism and transport as well as flagella assembly was evident in stress adapted cells most likely due to energy preservation via CodY regulon. This suggests that adaptation to these adverse conditions engages similar mechanisms to cope with the induced stressful environments. The microarray component of the experiments had the aim of determining: i) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic salt resistances following adaptation to hyperosmotic stress. ii) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic cold tolerances following adaptation to cold stress. iii) To evaluate common pathways of adaptation to hyperosmotic and cold stresses

ORGANISM(S): Listeria monocytogenes

PROVIDER: GSE46612 | GEO | 2013/05/04

SECONDARY ACCESSION(S): PRJNA201229

REPOSITORIES: GEO

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Project description:A screening process was used to determine hyperosmotic as well as cold stress tolerance of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at sub lethal levels of NaCl and 4M-BM-0C. Analysis of gene expression in cells adapted to cold and hyperosmotic stress revealed significant strain specificity in terms of individual gene expression profiles though general patterns of expression within functional gene subsets were largely similar. . Strain ATCC19115 revealed 150 significantly up- and 146 down-regulated genes in cells adapted to both hyperosmotic and cold stress, 82 up- and 56 down-regulated genes were matching in response to both stresses in strain 70-1700, whereas ScottA, a relatively stress-tolerant strain, showed up-regulation of 77 genes whilst 106 genes were significantly down-regulated. Among these homologously expressed genes only 22 were found to be significantly up-regulated and only 7 were down-regulated in all three strains adapted to hyperosmotic stress and cold temperature, suggesting a dominating strain specific response. Overall adaptation to hyperosmotic and low temperature growth conditions in three L. monocytogenes strains were found to have many parallels, especially when examined using a statistically-based ontological approach. Evidence was presented for strong activation of genes associated with protein synthesis, in particular those coding proteins of the ribosome and involved directly in transcription. Genes associated with DNA maintenance; modification to cell envelope, and cell division were also up-regulated. On the other hand strong suppression of genes associated with carbohydrate metabolism and transport as well as flagella assembly was evident in stress adapted cells most likely due to energy preservation via CodY regulon. This suggests that adaptation to these adverse conditions engages similar mechanisms to cope with the induced stressful environments. The microarray component of the experiments had the aim of determining: i) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic salt resistances following adaptation to hyperosmotic stress. ii) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic cold tolerances following adaptation to cold stress. iii) To evaluate common pathways of adaptation to hyperosmotic and cold stresses Control cultures (four biological replicates; exception strain ATCC19115 for the 10%NaCl stress which had three biological replicates) were labeled with Cy3 (exception strain ATCC19115 for the 10%NaCl-3 dyeswapped, where the control was labeled with Cy5 and sample with Cy3) for each treatment sample for three different L. monocytogenes strains. Sample cultures (four biological replicates; exception strain ATCC19115 for the 10%NaCl which had three biological replicates) were labeled with Cy5 (exception strain ATCC19115 for the 10%NaCl-3 dyeswapped, where the control was labeled with Cy5 and sample with Cy3) for each treatment sample for three different L. monocytogenes strains. Condition one M-bM-^@M-^Ssub-lethal hyperosmotic stress: Control culture (Cy3-labelled RNA, except ATCC19115 for the 10%NaCl-3 dyeswapped , where the control was labeled with Cy5 and sample with Cy3) - strains were grown to mid exponential growth phase at 25M-BM-0C (in a shaking water bath) in brain-heart infusion broth. Test cultures (Cy5-labelled RNA, except ATCC19115 for the 10%NaCl-3 dyeswapped , where the control was labeled with Cy5 and sample with Cy3) M-bM-^@M-^S strains grown to mid exponential growth at 25M-BM-0C phase (in a shaking water bath) in BHI broth supplemented with 8% additional NaCl (strain 701700), 10% additional NaCl (strain ATCC19115) or 12% additional NaCl (ScottA). Condition two M-bM-^@M-^S cold stress: Control culture (Cy3-labelled RNA) - strains were grown to mid exponential growth phase at 25M-BM-0C (in a shaking water bath) in brain-heart infusion broth. Test cultures (Cy5-labelled RNA) M-bM-^@M-^S strains grown to exponential growth phase at 4M-BM-0C in BHI broth.

Project description:Stress leads to important adaptive physiological responses including release of catecholamines from the adrenal medulla. Repeated or prolonged stress increases the propensity for many disorders including cardiovascular disease and neuropsychiatric disorders. The susceptibility to stress is determined by a number of factors including prior experience with stress. It has been found that while there is adaptation to prolonged cold stress; it leads to sensitization when cold stressed animals are exposed to a different (novel) stressor. Our study aims to determine the mechanism of this adaptation and sensitization.,We will determine the gene expression patterns in rat adrenal medulla in response to several times of cold stress. We will also determine the changes in gene expression with single immobilization stress in adrenal medulla of rat with and without preexposure to cold stress to investigate the mechanism of sensitization in cold adapted animals. ,We hypothesize that during the course of cold stress there are alterations in gene expression of several neurotransmitter related genes and transcription factors. In cold adapted animals, there is increased susceptibility to stress due to sustained changes in expression of several transcription factors, such as FRA-2 which lead to an exaggerated response to the novel stressor. ,Rats were exposed to cold stress (4oC) for 1, 7 or 28 days. A parallel group was kept at room temperature (unstressed). Unstressed or rats pre-exposed to 28 days cold were immobilized for 2 hrs and killed by decapitation. The adrenal medullae, separately from right and left side, have been dissected and individually frozen at -70oC. Our recent immunoblots with the tissue from one side revealed that there is increase in tyrosine hydroxylase and c-fos with the short term cold stress and in dopamine beta hydroxylase and Fra-2 even with the prolonged cold stress. The response of several transcription factors to immobilization stress was enhanced in the cold adapted animals. RNA will be prepared with RNAqueous-Micro Isolation kit from Ambion (alternatively we can ship the tissues). We will pool samples from 4 rats per treatment to control for variability.

Project description:Listeria monocytogenes is a gram-positive, food-borne pathogen responsible for invasive infections with high overall mortality. Early host defenses encountered by L. monocytogenes following ingestion include low pH of the stomach and bile present in the small intestinal lumen. We hypothesized that “epidemic” strains are better able to withstand exposure to low pH and bile encountered in the gastrointestinal tract as compared to most “environmental” strains. Furthermore, we hypothesized that epidemic and environmental strains would have distinct transcriptional programs upon exposure to these conditions. Our treatments included 1 hr exposure to acid (pH 5.5 and 3.5) and bile (0.3%) stress. Strains were pre-exposed to pH 5.5 (1 hr) before being treated with pH 3.5. We used a collection of 12 previously characterized epidemic and environmental strains and each strainXtreatment combination included 3 biological replicates for each microarray experiment. All microarray experiments were two color competitive hybridizations that paired experimental conditions with the same strain at neutral pH for acid stress and pH 5.5 for bile stress. Transcriptomes of environmental strains exposed to acid and bile stress showed remarkably greater number of genes with differences of ≥2-fold expression levels as compared to epidemic strains (5 and 7, respectively). Environmental strains were characterized by up-regulation of several stress related genes and down-regulation of several cell envelope biosynthesis and virulence related genes, suggesting that complex regulatory networks orchestrate the cellular changes in the environmental strains to overcome stressful environments. The transcriptome of epidemic strains, in contrast, showed muted responses to these stress conditions implying their pre-adaptability to acid and bile stress encountered during natural infection that may enable epidemic strains to survive and become “primed” for subsequent colonization and infection in the lower gastrointestinal tract. Keywords: stress response, comparative transcriptomics, acid-adaptation, differential virulence, acid-stress response, bile-stress response

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Characterization of the transcriptomes of genetically diverse Listeria monocytogenes exposed to hyperosmotic and low temperature conditions

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