Project description:Listeria monocytogenes strain F2365 was the first strain representative of serotype 4b (lineage I) to be sequenced in 2004, suggesting it could become the model organism for this serotype, which is associated with most human outbreaks of listeriosis worldwide to date. F2365 itself is an outbreak strain involved in the Mexican-style soft cheese outbreak in California in 1985. In this study we show through phenotypic and transcriptomic analysis that L. monocytogenes strain F2365 has reduced ability to respond to stress due to the absence of a functional σB-dependent stress response system. F2365 shows no B-dependent ability to survive acid or oxidative stress nor B-dependent ability to infect Caco-2 epithelial cell in vitro or guinea pigs in vivo. Therefore, there is substantial evidence that F2365 is an atypical strain and is not a suitable representative of outbreak-associated serotype 4b strains.
Project description:As the foodborne pathogen Listeria monocytogenes has the ability to grow at refrigeration temperatures, whole-genome microarray experiments were performed using L. monocytogenes strain 10403S to define the cold stress regulon and to identify genes differentially expressed during growth at 4°C and 37°C. Microarray analysis using a stringent cutoff (adjusted p<0.001; fold-change >2.0) revealed 105 and 170 genes that showed higher transcript levels in logarithmic- and stationary-phase cells, respectively, at 4°C (compared to cells at 37°C). A total of 74 and 102 genes showed lower transcript levels in logarithmic- and stationary-phase cells grown at 4°C, respectively. Genes upregulated at 4°C during both stationary- and log-phase included those encoding a two-component response regulator (lmo0287), a cold shock protein (cspL), and two RNA helicases (lmo0866 and lmo1722), whereas genes encoding selected virulence factors and heat shock proteins were downregulated at 4°C. Selected genes that were upregulated at 4°C during both stationary- and log-phase were confirmed by quantitative reverse transcriptase PCR. Our data show (i) a large number of L. monocytogenes genes are differentially expressed at 4 and 37°C with a larger number of genes showing higher transcript level at 4°C than genes showing lower transcript levels at 4°C; (ii) L. monocytogenes genes upregulated at 4°C include a number of genes and operons with previously reported or plausible roles in cold adaptation; and (iii) L. monocytogenes genes downregulated at 4°C include a number of virulence and virulence-associated genes as well as some heat shock genes. Keywords: Listeria monocytogenes, cold regulon, temperature
Project description:Listeria monocytogenes strain F2365 was the first strain representative of serotype 4b (lineage I) to be sequenced in 2004, suggesting it could become the model organism for this serotype, which is associated with most human outbreaks of listeriosis worldwide to date. F2365 itself is an outbreak strain involved in the Mexican-style soft cheese outbreak in California in 1985. In this study we show through phenotypic and transcriptomic analysis that L. monocytogenes strain F2365 has reduced ability to respond to stress due to the absence of a functional M-OM-^CB-dependent stress response system. F2365 shows no M-oM-^AM-3B-dependent ability to survive acid or oxidative stress nor M-oM-^AM-3B-dependent ability to infect Caco-2 epithelial cell in vitro or guinea pigs in vivo. Therefore, there is substantial evidence that F2365 is an atypical strain and is not a suitable representative of outbreak-associated serotype 4b strains. Independent RNA isolations were performed for F2365 and M-NM-^TsigB strains from cells grown to early stationary phase. Three biological replicates were used in competitive whole-genome microarray experiments. For each set of hybridizations, RNA from a L. monocytogenes wildtype strain was hybridized with RNA from its isogenic M-NM-^TsigB null mutant.
Project description:Listeria monocytogenes SigB and PrfA are pleiotropic regulators of stress response and virulence gene expression, which have been shown to co-regulate genes in L. monocytogenes. We performed whole genome transcriptional profiling in the presence of PrfA* and active SigB, to identify the overlaps between the PrfA virulence regulon and the SigB stress response regulon. In L. monocytogenes, the PrfA* allele contributes to the activation of virulence genes to a level comparable to that of intracellular growing L. monocytogenes. Our results showed that the core PrfA regulon consists of 12 genes previously described as PrfA regulated. Furthermore, we found that the role of SigB during virulence gene regulation changes, dependent on the presence or absence of PrfA*. In the absence of PrfA*, SigB activated the transcription of virulence genes such as inlA and inlB. In the presence of PrfA*, SigB negatively influenced the transcription of genes in the PrfA core regulon. The observed effect of SigB on the transcript level of PrfA regulated genes was shown to reduce the cytotoxic effect of the PrfA* allele in HepG-2 cells. Our results indicate that the SigB-PrfA regulatory network is important for the adjustment of virulence gene transcription to ensure L. monocytogenes success as an intracellular pathogen. Keywords: comparison of gene expression of regulatory mutants
Project description:Investigation of whole genome gene expression level changes in Listeria monocytogenes LO28 delta-lhrC1-5 mutant, compared to the wild type strain. The lhrC1-5 genes encode the regulatory sRNAs LhrC1-5. The microarray studied the gene expression of unstressed cells and cells exposed to cefuroxime for 30 min. The lhrC1-5 mutant employed in this study is further described in Sievers et al. (2014) A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB. Nucleic Acids Res. 42:9383-98.
Project description:In several gram-positive bacterial genera including Bacillus, Staphylococcus, and Listeria, sigma B (σB) has been identified as a stress-responsive alternative sigma factor responsible for initiating transcription of genes (the σB regulon) involved in response to stress-inducing environmental conditions. In L. monocytogenes, a foodborne pathogen of considerable threat to public health and the food industry, σB is involved in regulation of stress response and virulence gene expression. We have defined the σB regulon in L. monocytogenes during early stationary phase and under salt stress (0.3M NaCl) conditions using whole-genome microarrays, identifying 168 genes that generated ≥2.0-fold higher signals in the parental strain 10403S than in an isogenic sigB null mutant (ΔsigB), categorized into nine functional groups including stress-response genes (12), virulence genes (5), and genes related to transport (26) and metabolism (45). To gain a broader biological perspective of the σB stress response system, we applied these microarrays to Listeria innocua under the same environmental conditions. Our studies revealed 64 candidates in the L. innocua σB regulon with ≥2.0-fold higher signals in the parent than in a ΔsigB mutant; 49 of the 64 genes overlap with the L. monocytogenes σB regulon, indicating extensive overlap in σB-controlled genes between the two species. Further transcriptional analysis using TaqMan quantitative real time RT-PCR was performed for selected genes that displayed contrasting fold changes among the four microarray data sets (two stress conditions per species). We report novel members of the L. monocytogenes σB regulon, as well as the initial definition of the L. innocua σB regulon. Our comparative studies of the σB stress response systems in L. monocytogenes and L. innocua revealed features of the σB regulon that are conserved and unique to the two species. Keywords: Listeria monocytogenes, Listeria innocua, SigB regulon, salt stress, stationary phase
Project description:The foodborne pathogen Listeria monocytogenes (Lm) can cause invasive infection in susceptible animals and humans. To survive and proliferate within hosts, this facultative intracellular pathogen needs to tightly coordinate the expression of a complex regulatory network, including virulence factors. Here, we identify and characterize MouR a novel virulence regulator of Lm. Through RNA-seq transcriptomic analysis, we characterized the MouR regulatory network and demonstrated how MouR positively controls the expression of the Agr system (agrBDCA) of Lm. Resolving MouR 3D structure revealed a dimeric DNA-binding transcription factor belonging to the VanR class of the GntR superfamily of regulatory proteins. We showed that by direct binding to the agr promoter region, MouR ultimately modulates chitinase activity and biofilm formation. Importantly, we demonstrated by in vitro cell invasion assays and in vivo mice infections the crucial role of MouR for Lm full virulence.
Project description:The foodborne pathogen Listeria monocytogenes uses a number of transcriptional regulators, including the negative regulator CtsR, to control gene expression under different environmental conditions and in response to stress. Gene expression patterns of DctsR log phase cells were compared to both wt and ictsR-mcsA log phase cells grown with 0.5mM IPTG to identify CtsR-dependent genes.We identified 62 CtsR-dependent genes that showed significant expression ratios (adj. P < 0.05), with ≥ 1.5-fold differential expression either between ΔctsR and wt or between ΔctsR and ictsR-mcsA. Keywords: Listeria monocytogenes, CtsR regulon, log phase
Project description:The foodborne pathogen Listeria monocytogenes uses a number of transcriptional regulators, including the negative regulator HrcA, to control gene expression under different environmental conditions and in response to stress. Gene expression patterns of DhrcA stationary phase cells were compared to wt to identify hrcA-dependent genes. We identified 61 HrcA-dependent genes that showed significant expression ratios (adj. P < 0.05), with ≥ 1.5-fold differential expression between ΔhrcA and wt. Combined with microarray analysis, Hidden Markov Model searches show HrcA directly repress at least 8 genes. Keywords: Listeria monocytogenes, HrcA regulon, stationary phase