Unknown,Transcriptomics,Genomics,Proteomics

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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 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.

ORGANISM(S): Listeria monocytogenes

SUBMITTER: John Bowman

PROVIDER: E-GEOD-18796 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A screening process was used to determine the aciduric capacities of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at pH 5.0 in the presence 4 different organic acids. Food and clinical isolates tended to be more resistant but strain genetic groupings were found to be only weakly linked to sodium diacetate (SDA)-resistance. Representative and comparatively aciduric food isolates FW04/0023 and FW04/0025 were found to accumulate reduced levels of acetate anion and K+ ion during growth in the presence of SDA, compared to more acid sensitive reference strains EGD (ATCC BAA-739) and ATCC 19111. The aciduric nature of FW04/0023 and FW04/0025 was also reflected by their comparatively high tolerance to pH 2.4 acid challenge; a property boosted by the presence of SDA, and growth-phase dependent acid tolerance. Transcriptomic analyses revealed increased levels of SDA did not activate or promulgate the response of any of the known acid protective mechanisms, except for acetoin biosynthesis. Elevated levels of unprotonated SDA (20 mM SDA at pH 5.0) was found to have broad effects on gene expression that could be differentiated from effects caused by mildly acidic conditions (pH 5.0) and substantial strain variation was also found. Collated SDA mainly effect genes associated with virulence, cell wall processes, DNA repair, and cofactor and lipid biosynthesis. Strain FW04/0025 was more responsive to elevated unprotonated SDA increasing the expression of 222 genes (>2-fold change, p<0.05), compared to 112 genes for strain EGD. Key differences between the strains in relation to SDA-enhanced transcript abundance in FW04/0023 were primarily associated with the cell wall, oxidative stress management, intermediary metabolism, and several hypothetical proteins. This complement of different responses could potentially alter phenotypes resulting in different cell envelope properties and metabolic properties that effect accumulation of acetate anion. The data demonstrates that amongst different L. monocytogenes strains acetate has differing effects that may ultimately influence growth efficiency under stressful conditions relevant to acidic foods and the gastrointestinal environment. The microarray component of the experiments had the aim of determining: i) To examine the affect of elevated levels of unprotonated sodium diacetate (21 mM sodium diacetate added to cultures at pH 5.0, resulting in ~7.7 mM unprotonated acetate) compared to mild acid stress at pH 5.0 in which lower levels of acetate accumulates through natural end-product formation. ii) To examine the gene expression responses of two L. monocytogenes strains that had different intrinsic acid resistances, including an organic acid resistant strain FW04/0025 and a more sensitive reference strain EGD on which genome the microarray oligonucleotide set is based. Two to four biologically replicated control cultures were labelled with Cy3 for each treatment sample with two sets of experimental conditions investigated for two different L. monocytogenes strains . Condition 1) â Mildly acidic stress: Control culture (Cy3-labelled RNA) - strains were grown to early- mid exponential growth phase at 25Â°C (in a shaking water bath) in brain-heart infusion broth at pH 7.3. Test cultures (Cy5-labelled RNA) â strains grown to early- mid exponential growth at 25Â°C phase in BHI broth adjusted to pH 5.0 (in a shaking water bath). Condition 2) â Organic acid (sodium diacetate) stress: Control culture (Cy3-labelled RNA) - strains were grown to exponential growth phase at 25Â°C (in a shaking water bath) in BHI broth at pH 5.0. Test cultures (Cy5-labelled RNA) â strains grown to exponential growth at 25Â°C phase in BHI broth adjusted to pH 5.0 and amended with 21 mM (0.3% w/v)sodium diacetate (in a shaking water bath).

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 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.

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

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