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Physiology and genetic activity of Listeria monocytogenes during multi-hurdle stress induced cell death

ABSTRACT: The physiological basis of the hypothesis that temperature is the primary governing factor of bacterial cell inactivation under otherwise non-growth permissive conditions was investigated. Application of simultaneous low pH (pH 3.5) and low water activity (aw = 0.9; 2.5 M NaCl) conditions, applied to L. monocytogenes strains Scott A and FW03/0035, and increasing incubation temperature from 25°C up to 45°C resulted in increased permeability to ethidium homodimer-1 and corresponded to accelerated declines in esterase activity and ATP basal levels but did not result in autolysis. Triphasic survival curves were readily observable when sufficiently large cell populations were inactivated at 25°C and 35°C; indicative of a mixture of sensitive and resistant sub-populations. Enrichment-based recovery experiments however indicate that the stress conditions eventually lead to complete loss of reproductive capacity, potentially corresponding to an irreversible collapse of pH homeostasis. Transcriptomic analyses were used to further obtain insights into the physiology of the inactivation process occurring at 25°C. RT-PCR, rifampin-enforced decay and microarray experiments revealed transcripts of tufA and other genes become substantially more stable during inactivation during exposure to combined low pH/aw and during non-growth permissive temperature exposure. Gene transcripts were delineated through K-means clustering that appear to be important for initial survival of combined low pH/aw and include an overrepresentation of SigB-activated genes, the response of which fades with increasing time of inactivation exposure. The microarray component of the experiments had the aim of determining: i) Gene expression responses of L. monocytogenes strain Scott A when exposed to a non-growth permissive environment consisting of a broth system acting as a food simulated environment adjusted to low pH (pH 3.5) and low water activity (2.5 M NaCl). ii) To examine the trend in gene expression over time under inactivating (killing) conditions by applying gene set (functional and regulatory) expression trend analysis and K-means cluster analysis. iii) To correlate this data to other physiological and mRNA quantification (real-time-PCR) data.

ORGANISM(S): Listeria monocytogenes

PROVIDER: GSE18796 | GEO | 2009/10/30

SECONDARY ACCESSION(S): PRJNA121203

REPOSITORIES: GEO

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Project description:The physiological basis of the hypothesis that temperature is the primary governing factor of bacterial cell inactivation under otherwise non-growth permissive conditions was investigated. Application of simultaneous low pH (pH 3.5) and low water activity (aw = 0.9; 2.5 M NaCl) conditions, applied to L. monocytogenes strains Scott A and FW03/0035, and increasing incubation temperature from 25Â°C up to 45Â°C resulted in increased permeability to ethidium homodimer-1 and corresponded to accelerated declines in esterase activity and ATP basal levels but did not result in autolysis. Triphasic survival curves were readily observable when sufficiently large cell populations were inactivated at 25Â°C and 35Â°C; indicative of a mixture of sensitive and resistant sub-populations. Enrichment-based recovery experiments however indicate that the stress conditions eventually lead to complete loss of reproductive capacity, potentially corresponding to an irreversible collapse of pH homeostasis. Transcriptomic analyses were used to further obtain insights into the physiology of the inactivation process occurring at 25Â°C. RT-PCR, rifampin-enforced decay and microarray experiments revealed transcripts of tufA and other genes become substantially more stable during inactivation during exposure to combined low pH/aw and during non-growth permissive temperature exposure. Gene transcripts were delineated through K-means clustering that appear to be important for initial survival of combined low pH/aw and include an overrepresentation of SigB-activated genes, the response of which fades with increasing time of inactivation exposure. The microarray component of the experiments had the aim of determining: i) Gene expression responses of L. monocytogenes strain Scott A when exposed to a non-growth permissive environment consisting of a broth system acting as a food simulated environment adjusted to low pH (pH 3.5) and low water activity (2.5 M NaCl). ii) To examine the trend in gene expression over time under inactivating (killing) conditions by applying gene set (functional and regulatory) expression trend analysis and K-means cluster analysis. iii) To correlate this data to other physiological and mRNA quantification (real-time-PCR) data. Experimental design: Two biologically replicated control cultures (each with two technical replicate sper chip) were labelled with Cy3 for each treatment sample for a total of 4 pairs of biological replicates. The treatments consisted of treated (inactivated in brain-heart infusion broth at pH 3.5 and water activity 0.9) L. monocytogenes cells with two biological replicates (each with two technical replicates per chip) labelled with Cy5. The time course (incubation time under inactivating conditions) of treatments was âinitialâ (20 minutes), 24 h, 48 h, and 72 h.

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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Physiology and genetic activity of Listeria monocytogenes during multi-hurdle stress induced cell death

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