Project description:The foodborne pathogen Listeria monocytogenes experiences osmotic stress in many habitats, including foods and the gastrointestinal tract of the host. While osmotic stress induced changes in expression have been investigated for specific genes, identifying and understanding genome-wide temporal changes in the transcriptome due to salt stress will provide insights into how this pathogen adapts to and survives this stress, leading to development of new intervention strategies. To determine the short-term and long-term responses to salt stress, we exposed exponential phase cells of L. monocytogenes H7858 to 6% NaCl in Brain Heart Infusion (BHI) broth at 7°C and 37°C and extracted RNA after 2.5%, 5%, 10%, and 20% of lag phase and during exponential growth in BHI + 6% NaCl, and evaluated temporal changes in transcript levels with microarrays. The temperature dependent short-term response to salt stress included a significant increase in transcript levels of the alternative sigma factor σB, with maximum expression at 2.5-5% of lag phase at 37°C, and at 20% of lag phase at 7°C. Transcript levels of genes known to be regulated by σB, including inlAB, opuCA, glpFK, and gadBC, were significantly upregulated at the same relative time points as σB As indicated by significantly elevated transcript levels during exponential growth in BHI + 6% NaCl, genes encoding proteins involved in purine and pyrimidine synthesis and amino acid biosynthesis are part of the long-term response to salt stress at 37°C. Transcript levels of virulence genes plcA, mpl, actA, and plcB were also significantly upregulated during exponential growth under salt stress at 37°C. Genes encoding proteins involved in protein degradation and stability and cell membrane modifications are part of the long-term response to salt stress at 7°C. Overall, an initial alteration in the transcriptome occurs, decreasing transcript levels of genes encoding proteins involved in energy conversion and metabolism, while increasing transcript levels of those involved in the stress response controlled by σB, followed by a long-term response, including increased expression of genes encoding compatible solute transporters and general stress proteins, facilitating growth under salt stress.

Project description:Listeria monocytogenes is one of the most heat-resistant non-spore-forming food-borne pathogens and poses a notable risk to food safety, particularly when mild heat treatments are used in food processing and preparation. While general heat stress properties and response mechanisms of L. monocytogenes have been described, accessory mechanisms providing particular L. monocytogenes strains with the advantage of enhanced heat resistance are unknown. Here, we report plasmid-mediated heat resistance of L. monocytogenes for the first time. This resistance is mediated by the ATP-dependent protease ClpL. We tested the survival of two wild-type L. monocytogenes strains-both of serotype 1/2c, sequence type ST9, and high sequence identity-at high temperatures and compared their genome composition in order to identify genetic mechanisms involved in their heat survival phenotype. L. monocytogenes AT3E was more heat resistant (0.0 CFU/ml log10 reduction) than strain AL4E (1.4 CFU/ml log10 reduction) after heating at 55°C for 40 min. A prominent difference in the genome compositions of the two strains was a 58-kb plasmid (pLM58) harbored by the heat-resistant AT3E strain, suggesting plasmid-mediated heat resistance. Indeed, plasmid curing resulted in significantly decreased heat resistance (1.1 CFU/ml log10 reduction) at 55°C. pLM58 harbored a 2,115-bp open reading frame annotated as an ATP-dependent protease (ClpL)-encoding clpL gene. Introducing the clpL gene into a natively heat-sensitive L. monocytogenes strain (1.2 CFU/ml log10 reduction) significantly increased the heat resistance of the recipient strain (0.4 CFU/ml log10 reduction) at 55°C. Plasmid-borne ClpL is thus a potential predictor of elevated heat resistance in L. monocytogenes. IMPORTANCEListeria monocytogenes is a dangerous food pathogen causing the severe illness listeriosis that has a high mortality rate in immunocompromised individuals. Although destroyed by pasteurization, L. monocytogenes is among the most heat-resistant non-spore-forming bacteria. This poses a risk to food safety, as listeriosis is commonly associated with ready-to-eat foods that are consumed without thorough heating. However, L. monocytogenes strains differ in their ability to survive high temperatures, and comprehensive understanding of the genetic mechanisms underlying these differences is still limited. Whole-genome-sequence analysis and phenotypic characterization allowed us to identify a novel plasmid, designated pLM58, and a plasmid-borne ATP-dependent protease (ClpL), which mediated heat resistance in L. monocytogenes. As the first report on plasmid-mediated heat resistance in L. monocytogenes, our study sheds light on the accessory genetic mechanisms rendering certain L. monocytogenes strains particularly capable of surviving high temperatures-with plasmid-borne ClpL being a potential predictor of elevated heat resistance.

Project description:Listeria monocytogenes is well known to survive and grow under several stress conditions, including salt stress, which is important for growth in certain foods as well as for host infection. To characterize the contributions, to salt stress response, of transcriptional regulators important for stress response and virulence (i.e., σ(B) and PrfA), we analyzed three L. monocytogenes parent strains and isogenic mutants (ΔsigB, ΔprfA, and ΔsigBΔprfA), representing different serotypes and lineages, for their ability to grow, at 25°C, in BHI with 1.9 M NaCl. With regard to growth rate, only the lineage IV strain presented a significant difference between the parent strain and both of its respective mutants lacking prfA (ΔprfA and ΔsigBΔprfA). Conversely, the lineage I and II parent strains showed significantly shorter lag phase in comparison to their respective ΔsigB mutant strains. Intestinal epithelial cell invasion assay and hemolytic activity assays showed a significant role for σ(B) in the former and for PrfA in the latter. To explore the mechanism that may contribute to the extended lag phase in the ΔsigB mutant strain and survival and growth of the parent strain upon salt shock, whole genome transcription profiling was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic ΔsigB mutant after 30 min of lag phase growth at 25°C in the presence of 1.9M NaCl (salt shock) without aeration. Microarray data showed significantly higher transcript levels for 173 genes in the parent strain as compared to the ΔsigB strain. Overall, 102 of the 173 σ(B) up-regulated genes had been identified in previous studies, indicating that 71 genes were newly identified as being up-regulated by σ(B) in this study. We hypothesize that, among these genes newly identified as σ(B) up-regulated, four genes (lmo2174, lmo0530, lmo0527 and lmo0529) may play a major role in response to salt stress. Lmo2174 contains domains that facilitate sensing and producing a transduction signal in the form of cyclic di-GMP, which may activate the enzymes Lmo0527, Lmo0529 and Lmo0530, which encode proteins similar to those responsible for synthesis of exopolysaccharides that may protect the cell by changing the cell wall structure during salt stress. Overall, our data showed that σ(B), but not PrfA, contributes to growth under salt stress. Moreover, we show that the σ(B) regulon of a L. monocytogenes lineage I strain challenged with salt shock includes salt stress-specific as well as previously unidentified σ(B) up-regulated genes.

Project description:The foodborne pathogen Listeria monocytogenes experiences osmotic stress in many habitats, including foods and the gastrointestinal tract of the host. While osmotic stress induced changes in expression have been investigated for specific genes, identifying and understanding genome-wide temporal changes in the transcriptome due to salt stress will provide insights into how this pathogen adapts to and survives this stress, leading to development of new intervention strategies. To determine the short-term and long-term responses to salt stress, we exposed exponential phase cells of L. monocytogenes H7858 to 6% NaCl in Brain Heart Infusion (BHI) broth at 7°C and 37°C and extracted RNA after 2.5%, 5%, 10%, and 20% of lag phase and during exponential growth in BHI + 6% NaCl, and evaluated temporal changes in transcript levels with microarrays. The temperature dependent short-term response to salt stress included a significant increase in transcript levels of the alternative sigma factor σB, with maximum expression at 2.5-5% of lag phase at 37°C, and at 20% of lag phase at 7°C. Transcript levels of genes known to be regulated by σB, including inlAB, opuCA, glpFK, and gadBC, were significantly upregulated at the same relative time points as σB As indicated by significantly elevated transcript levels during exponential growth in BHI + 6% NaCl, genes encoding proteins involved in purine and pyrimidine synthesis and amino acid biosynthesis are part of the long-term response to salt stress at 37°C. Transcript levels of virulence genes plcA, mpl, actA, and plcB were also significantly upregulated during exponential growth under salt stress at 37°C. Genes encoding proteins involved in protein degradation and stability and cell membrane modifications are part of the long-term response to salt stress at 7°C. Overall, an initial alteration in the transcriptome occurs, decreasing transcript levels of genes encoding proteins involved in energy conversion and metabolism, while increasing transcript levels of those involved in the stress response controlled by σB, followed by a long-term response, including increased expression of genes encoding compatible solute transporters and general stress proteins, facilitating growth under salt stress. A reference design was used to analyze gene expression differences. Exponential phase cultures at 37C and at 7C were used as a reference for salt stressed samples at each temperature. Salt stressed samples from the same relative time point were compared across temperatures. 4 biological replicates were tested for each comparison.

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Nisin resistance of Listeria monocytogenes is increased by exposure to salt stress and is mediated via LiaR.

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Nisin resistance of Listeria monocytogenes is increased by exposure to salt stress and is mediated via LiaR.

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