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

Project description:A long-term-survival (LTS) phase in Listeria monocytogenes was recently discovered. Cells in this phase are coccoid in shape, survive for at least 30 d without any decrease in viable cell numbers, and are very resistant to heat and high pressure. However, how cells of L. monocytogenes transition to this long-term-survival phase is little understood. Therefore, a whole-genome expression analysis was conducted to study the transcription profile of L. monocytogenes as it enters the LTS phase. Transcription profiles at log, stationary and death phases were analyzed since differential gene expressions at these phases may contribute to the eventual transition to the LTS phase. Specifically, cells of L. monocytogenes F2365 at log, stationary, death and LTS phases were obtained by incubating cultures in TSBYE at 35°C for 13 h, 17 h, 24 h and 168-336 h, respectively. Also, to study cells transitioning from the LTS phase back to the log phase, 1 ml of the LTS-phase culture at 336 h was reinoculated into 100 ml of fresh TSBYE with incubation at 35°C for 8 h. Total RNAs of all samples were extracted, reverse transcribed into cDNAs and then hybridized to the L. monocytogenes expression microarray (Roche NimbleGen). During the transition from log phase to stationary phase, differential changes in gene expression involved genes associated with cell envelope, cell division, stress response, energy metabolism, protein synthesis and material transport. During the transition from stationary to death phase, differential changes were observed in genes related to cell envelope, detoxification, pathogenesis, energy metabolism, protein synthesis and material transport. When cultures transitioned from death phase to 168-h LTS phase, significant downregulation of genes associated with amino acid and protein biosynthesis, as well as stress responses, were observed (P < 0.05), while multiple genes related to cell envelope, energy production and material transportation were significantly upregulated (P < 0.05). High similarity of transcription profiles (r = 0.93) within LTS phase was observed when comparing transcriptomes at 168 h and 336 h. RNA quality measurement revealed a high level of degradation of ribosomal RNA during the LTS phase. The transcription profile at 8-h (log-phase) after re-inoculation of LTS cells also resembled that at 13 h (r = 0.94). We hypothesize that the upregulation of some compatible solute transporters during the LTS phase may result in accumulation of these solutes, which may lower intracellular water activity and thus enhance resistance of L. monocytogenes to heat and high pressure. Dormancy may be induced at the LTS phase which is suggested by the downregulation of genes associated with transcription and translation. Once fresh nutrients are provided, LTS cells may quickly exit dormancy and become metabolically active as they transition to the log phase.

Project description:A long-term-survival (LTS) phase in Listeria monocytogenes was recently discovered. Cells in this phase are coccoid in shape, survive for at least 30 d without any decrease in viable cell numbers, and are very resistant to heat and high pressure. However, how cells of L. monocytogenes transition to this long-term-survival phase is little understood. Therefore, a whole-genome expression analysis was conducted to study the transcription profile of L. monocytogenes as it enters the LTS phase. Transcription profiles at log, stationary and death phases were analyzed since differential gene expressions at these phases may contribute to the eventual transition to the LTS phase. Specifically, cells of L. monocytogenes F2365 at log, stationary, death and LTS phases were obtained by incubating cultures in TSBYE at 35°C for 13 h, 17 h, 24 h and 168-336 h, respectively. Also, to study cells transitioning from the LTS phase back to the log phase, 1 ml of the LTS-phase culture at 336 h was reinoculated into 100 ml of fresh TSBYE with incubation at 35°C for 8 h. Total RNAs of all samples were extracted, reverse transcribed into cDNAs and then hybridized to the L. monocytogenes expression microarray (Roche NimbleGen). During the transition from log phase to stationary phase, differential changes in gene expression involved genes associated with cell envelope, cell division, stress response, energy metabolism, protein synthesis and material transport. During the transition from stationary to death phase, differential changes were observed in genes related to cell envelope, detoxification, pathogenesis, energy metabolism, protein synthesis and material transport. When cultures transitioned from death phase to 168-h LTS phase, significant downregulation of genes associated with amino acid and protein biosynthesis, as well as stress responses, were observed (P < 0.05), while multiple genes related to cell envelope, energy production and material transportation were significantly upregulated (P < 0.05). High similarity of transcription profiles (r = 0.93) within LTS phase was observed when comparing transcriptomes at 168 h and 336 h. RNA quality measurement revealed a high level of degradation of ribosomal RNA during the LTS phase. The transcription profile at 8-h (log-phase) after re-inoculation of LTS cells also resembled that at 13 h (r = 0.94). We hypothesize that the upregulation of some compatible solute transporters during the LTS phase may result in accumulation of these solutes, which may lower intracellular water activity and thus enhance resistance of L. monocytogenes to heat and high pressure. Dormancy may be induced at the LTS phase which is suggested by the downregulation of genes associated with transcription and translation. Once fresh nutrients are provided, LTS cells may quickly exit dormancy and become metabolically active as they transition to the log phase. Based on the annotated genome of L. monocytogenes F2365 (GenBank accession# NC_002973) (Donaldson et al., 2009), a gene expression microarray was designed to target 2821 protein-coding genes (including putative protein-coding genes). Each of the 2821 genes was targeted by 12 unique 60-mer oligonucleotide probes. Each unique probe was printed in duplicates on the microarray. The microarrays were in a format of 4 × 72 k (4 microarrays per slide, with each microarray containing 72,000 probes) and provided by Roche NimbleGen (Roche NimbleGen, WI).

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Genome-wide transcriptional profiling of the cell envelope stress response of Listeria monocytogenes

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