Characterization of the SigB-PrfA regulatory network in Listeria monocytogenes
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ABSTRACT:
ORGANISM(S): Listeria innocua Listeria monocytogenes
PROVIDER: GSE11347 | GEO | 2008/10/01
SECONDARY ACCESSION(S): PRJNA106563
REPOSITORIES: GEO
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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 The experimental design included 4 mutant strains of L. monocytogenes 10403S (PrfA*, delta prfA, delta prfA delta sigB, and PrfA* delta sigB), of which cDNA generated from 4 biological replicates were hybridized in all possible pairwise comparisons. Data were analyzed using a one way ANOVA in R/MAANOVA to determine significant differences in gene expression among the different strains. A two way ANOVA implemented in R/MAANOVA determined significant differences in gene expression due to the presence or absence of SigB and PrfA.
2010-05-19 | E-GEOD-11347 | biostudies-arrayexpress
Project description:Purpose: The objective of the present study was to refine the PrfA regulatory network using RNA-seq to characterize the L. monocytogenes transcriptome in a wt, ∆prfA, and prfA* under PrfA-inducing conditions. Methods: Sequence reads were aligned to the L. monocytogenes 10403S genome using the BWA-MEM algorithm, version 0.7.3a. The data for coverage per base on the sense and antisense strands were analyzed separately using SAMtools. Differential expression of genes in each strain comparison (wt/∆prfA, wt/prfA*, and prfA*/∆prfA) were analyzed using the baySeq package for R version 2.2.0. Transcripts were considered differentially expressed if the FDR (False Discovery Rate) was ≤ 0.05, the FC (Fold Change) was ≥ 2.0 for up-regulated transcripts and ≤ 0.5 for down-regulated transcripts. Results: Our data indicate that we (i) confirmed transcription of known PrfA-dependent genes and (ii) identified a novel PrfA box and its putative ������A-dependent promoter region. Conclusions: Combined with many studies that have investigated the PrfA regulon and virulence in L. monocytogenes, we have shown tha PrfA directly regulates a small subset of genes; however, using RNA-seq and bioinformatics we were able to identify a novel PrfA-dependent gene and its putative PrfA box. This finding highlights the importance of using advanced approaches to analyze RNA-seq data, which can potentially reveal previously unidentified genes regulated under specific conditions.
2020-10-06 | GSE146507 | GEO
Project description:Listeria monocytogenes is well known to have the ability to survive and grow under a variety of stress conditions. The ability to survive and grow under osmotic stress conditions in particular appears to be important for both growth in certain foods and food associated environments as well as for host infection. To characterize the contributions of transcriptional regulators important for stress response and virulence (i.e., Sigma B - σB and PrfA), we initially analyzed three L. monocytogenes parent strains and isogenic mutants ( delta sigB, delta prfA, and delta sigB delta prfA), representing different serotypes and lineages, for their ability to grow in BHI with 11% NaCl (1.9M) at 25°C. No significant differences were observed in terms of growth between the parent strains and their respective mutants lacking prfA (i.e. delta prfA, and delta sigB delta prfA mutant strains). While for all strains, the delta sigB mutant showed a prolonged lag phase as compared to the parent strains, maximum growth rates were only reduced for the delta sigB mutant of lineage I and IV strains. Interestingly, for the serotype 1/2b strain, the delta sigB mutant reached a higher maximum cell density than the parent strain or the delta prfA mutant. Caco-2 intestinal epithelial cells invasion assays and hemolytic activity assays showed a significant role for σB in the former and for PrfA in the latter. To initially explore the mechanism that may contribute to the extended lag phase in the delta sigB mutant, microarray was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic delta sigB mutant in lag phase at 25°C in the presence of 11% NaCl. Microarray data showed significant lower and higher transcript levels for 135 and 173 genes, respectively, in the parent strain as compared to the delta sigB strains. Overall, 51 of the 173 σB up-regulated genes had previously been found among the 249 σB-dependent genes identified in a microarray study conducted in stationary phase cells, indicating that up to 122 genes may be transcribed in a σB-dependent manner during lag phase under salt stress. Notable genes that showed higher transcript levels in the parent strain include inlD (encoding an internalin protein that may be involved in virulence), sigH (encoding an alternative σ factor), glpK (encoding a glycerol kinase) and resD (encoding a two-component response regulator ResD); while, genes that showed lower transcript levels in the parent strain include dnaK (encoding a dihydroxyacetone kinase), groES (encoding a class I heat-shock protein GroES), grpE (encoding a co-chaperone GrpE) and fri (encoding a non-heme iron-binding ferritin). These data showed that although PrfA does not contribute to growth under osmotic stress at 25°C, σB does contribute to survival of L. monocytogenes under high salt conditions. Moreover, the σB-dependent transcriptome of L. monocytogenes lag phase cells under salt stress was characterized and includes previously identified as well as novel σB-dependent genes, including a number of stress response and virulence-associated genes.
2013-07-20 | GSE49056 | GEO
Project description:The Listeria monocytogenes genome contains more than 20 genes that encode cell surface-associated proteins termed internalins, which are characterized by the presence of multiple leucine-rich repeats. Subsets of internalin genes have been reported previously as transcriptionally regulated by the stress responsive sigma factor B (inlA, inlB, inlC2 and inlD) and the pleiotropic transcriptional activator PrfA (inlA, inlB, inlC). To investigate contributions of σB and PrfA to internalin gene regulation, we designed a sub-genomic microarray containing two probes for each of the 24 internalin-like genes present in the L. monocytogenes 10403S genome. Competitive microarray hybridization was performed on RNA extracted from (i) the 10403S parent strain and an isogenic ΔsigB strain; (ii) 10403S and an isogenic ΔprfA strain; (iii) a 10403S derivative that expresses the constitutively active PrfA* (G155S) and the ΔprfA strain; and (iv) 10403S and an isogenic ΔsigBΔprfA strain. σB- and PrfA-dependent transcription of selected genes was further confirmed by quantitative reverse transcriptase PCR. Statistical analyses of microarray data demonstrated that use of two probes on a microarray for a given target gene yielded more reliable transcriptional profiling information than use of a single probe. The suitability of the PrfA* strain for examining PrfA-dependent gene expression was established quantitatively by the observation that the plcA and prfA transcript levels generated by the PrfA* strain were similar to those obtained from intracellular L. monocytogenes and significantly higher than those in a wildtype strain grown in BHI broth. Among the 24 internalin-like genes examined, 4 and 6 were positively regulated by PrfA and σB, respectively, including inlA and inlB, which were positively regulated by both PrfA and σB. In summary, our findings clearly establish broad roles for both PrfA and σB in regulating L. monocytogenes internalin gene expression Keywords: Listeria monocytogenes, Internalins, sigB, PrfA, Microarrays
2007-04-28 | GSE6421 | GEO
Project description:Listeria monocytogenes is well known to have the ability to survive and grow under a variety of stress conditions. The ability to survive and grow under osmotic stress conditions in particular appears to be important for both growth in certain foods and food associated environments as well as for host infection. To characterize the contributions of transcriptional regulators important for stress response and virulence (i.e., Sigma B - M-OM-^CB and PrfA), we initially analyzed three L. monocytogenes parent strains and isogenic mutants ( delta sigB, delta prfA, and delta sigB delta prfA), representing different serotypes and lineages, for their ability to grow in BHI with 11% NaCl (1.9M) at 25M-BM-0C. No significant differences were observed in terms of growth between the parent strains and their respective mutants lacking prfA (i.e. delta prfA, and delta sigB delta prfA mutant strains). While for all strains, the delta sigB mutant showed a prolonged lag phase as compared to the parent strains, maximum growth rates were only reduced for the delta sigB mutant of lineage I and IV strains. Interestingly, for the serotype 1/2b strain, the delta sigB mutant reached a higher maximum cell density than the parent strain or the delta prfA mutant. Caco-2 intestinal epithelial cells invasion assays and hemolytic activity assays showed a significant role for M-OM-^CB in the former and for PrfA in the latter. To initially explore the mechanism that may contribute to the extended lag phase in the delta sigB mutant, microarray was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic delta sigB mutant in lag phase at 25M-BM-0C in the presence of 11% NaCl. Microarray data showed significant lower and higher transcript levels for 135 and 173 genes, respectively, in the parent strain as compared to the delta sigB strains. Overall, 51 of the 173 M-OM-^CB up-regulated genes had previously been found among the 249 M-OM-^CB-dependent genes identified in a microarray study conducted in stationary phase cells, indicating that up to 122 genes may be transcribed in a M-OM-^CB-dependent manner during lag phase under salt stress. Notable genes that showed higher transcript levels in the parent strain include inlD (encoding an internalin protein that may be involved in virulence), sigH (encoding an alternative M-OM-^C factor), glpK (encoding a glycerol kinase) and resD (encoding a two-component response regulator ResD); while, genes that showed lower transcript levels in the parent strain include dnaK (encoding a dihydroxyacetone kinase), groES (encoding a class I heat-shock protein GroES), grpE (encoding a co-chaperone GrpE) and fri (encoding a non-heme iron-binding ferritin). These data showed that although PrfA does not contribute to growth under osmotic stress at 25M-BM-0C, M-OM-^CB does contribute to survival of L. monocytogenes under high salt conditions. Moreover, the M-OM-^CB-dependent transcriptome of L. monocytogenes lag phase cells under salt stress was characterized and includes previously identified as well as novel M-OM-^CB-dependent genes, including a number of stress response and virulence-associated genes. Independent RNA isolations were performed for one wildtype (lineage I) and M-NM-^TsigB strains from cells grown to lag 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.
2013-07-20 | E-GEOD-49056 | biostudies-arrayexpress
Project description:Listeria monocytogenes is a food-borne pathogen which causes listeriosis. It is an intracellular parasite invading the epithelial cells where it escapes from the vacuole into the host cytoplasm to replicate, using actin-based motility to move within and between cells. The intracellular life cycle is well documented whereas the time spent in the lumen of the intestine is poorly understood. The aim of this study was to investigate the mechanism by which L. monocytogenes adapts to the environment of the small intestine prior to invasion. Specifically, to determine if the PrfA regulon, that encodes the virulence factors of L. monocytogenes, is switched on by signals within the intestinal lumen. L. monocytogenes were grown under aerobic or microaerobic conditions with glucose or glycerol as carbon source.
2023-11-24 | E-MTAB-12856 | biostudies-arrayexpress
Project description:Analysis of L. monocytogenes ptsH, hprK and ccpA mutants defective in carbon catabolite repression (CCR) control revealed significant alterations in the expression of PrfA-dependent genes. The hprK mutant showed high up-regulation of PrfA-dependent virulence genes upon growth in glucose-containing media whereas expression of these genes was even slightly down-regulated in the ccpA mutant when compared to the wild-type strain. The ptsH mutant could only grow in a rich culture medium and here the PrfA-dependent genes were up-regulated as in the hprK mutant. As expected, HPr-Ser-P was not produced in the hprK and ptsH mutants and synthesized at a similar level in the ccpA mutant as in the wild-type strain. However, no direct correlation was found between the level of HPr-Ser-P or HPr-His-P and PrfA activity when L. monocytogenes was grown in minimal medium with different PTS carbohydrates. Comparison of the transcript profiles of the hprK and ccpA mutants with that of the wild-type strain indicates that the up-regulation of the PrfA-dependent virulence genes in the hprK mutant correlates with the down-regulation of genes known to be controlled by the efficiency of PTS-mediated glucose transport. Furthermore, growth in the presence of the non-PTS substrate glycerol results in high PrfA activity. These data suggest that not component(s) of the CCR or the common PTS pathway but rather of subsequent steps seem to be involved in the modulation of PrfA activity Keywords: PTS components PrfA Listeria monocytogenes
2007-02-01 | GSE6028 | GEO
Project description:Listeria monocytogenes (Lm) strains expressing high levels of the virulence regulator PrfA (mutant PrfA* or wild-type PrfA) show strong growth inhibition in minimal media when supplemented with glucose but not when supplemented with glucose-6-phosphate compared to the isogenic strains expressing low levels of PrfA. A significantly reduced rate of glucose uptake is observed in the PrfA* over-expressing strain growing in LB supplemented with glucose. Comparative transcriptome analyses were performed with RNA isolated from a prfA mutant and the isogenic strain carrying multiple prfA or prfA* copies on a plasmid. The analysis reveals in addition to high transcriptional up-regulation of the known PrfA-regulated virulence genes (group I), less pronounced up-regulation in the expression of several phage- and metabolic genes (group II) and strong down-regulation of several genes involved mainly in carbon- and nitrogen- metabolism in the PrfA* over-expressing strain (group III). Among the latter genes are in particular the nrgAB-, gltAB-, glnRA- (involved in nitrogen metabolism), ilvB- operons (involved in biosynthesis of the branched chain amino acids) and genes for some ABC transporters. Most of the down-regulated genes have been shown previously in Bacillus subtilis to belong to a class of genes whose expression is negatively affected by impaired glucose uptake. The results lead to the conclusion that excess PrfA(*) interferes with component(s) essential for PTS-mediated glucose transport. Keywords: Metabolism and Virulence gene expression
2006-05-01 | GSE4414 | GEO
Project description:The Listeria monocytogenes genome contains more than 20 genes that encode cell surface-associated proteins termed internalins, which are characterized by the presence of multiple leucine-rich repeats. Subsets of internalin genes have been reported previously as transcriptionally regulated by the stress responsive sigma factor B (inlA, inlB, inlC2 and inlD) and the pleiotropic transcriptional activator PrfA (inlA, inlB, inlC). To investigate contributions of Ï?B and PrfA to internalin gene regulation, we designed a sub-genomic microarray containing two probes for each of the 24 internalin-like genes present in the L. monocytogenes 10403S genome. Competitive microarray hybridization was performed on RNA extracted from (i) the 10403S parent strain and an isogenic Î?sigB strain; (ii) 10403S and an isogenic Î?prfA strain; (iii) a 10403S derivative that expresses the constitutively active PrfA* (G155S) and the Î?prfA strain; and (iv) 10403S and an isogenic Î?sigBÎ?prfA strain. Ï?B- and PrfA-dependent transcription of selected genes was further confirmed by quantitative reverse transcriptase PCR. Statistical analyses of microarray data demonstrated that use of two probes on a microarray for a given target gene yielded more reliable transcriptional profiling information than use of a single probe. The suitability of the PrfA* strain for examining PrfA-dependent gene expression was established quantitatively by the observation that the plcA and prfA transcript levels generated by the PrfA* strain were similar to those obtained from intracellular L. monocytogenes and significantly higher than those in a wildtype strain grown in BHI broth. Among the 24 internalin-like genes examined, 4 and 6 were positively regulated by PrfA and Ï?B, respectively, including inlA and inlB, which were positively regulated by both PrfA and Ï?B. In summary, our findings clearly establish broad roles for both PrfA and Ï?B in regulating L. monocytogenes internalin gene expression Keywords: Listeria monocytogenes, Internalins, sigB, PrfA, Microarrays For all experiments, each strain was grown in BHI at 37oC with shaking (200 rpm) to OD600=0.4, then diluted 1:100 into fresh BHI and grown to OD600=0.4. Cells were then exposed to conditions designed to induce PrfA or sB activity (0.2% charcoal or 0.3 M NaCl, respectively). Specifically, to collect RNA for identification of PrfA-dependent genes, 0.2% charcoal (â??BHI-charcoalâ??) was added to early-log phase (OD600=0.4) 10403S and DprfA cells, which were subsequently incubated with shaking for 120 min at 37°C. To collect RNA for identification of PrfA-and sB-dependent genes, early-log phase 10403S and DprfADsigB cells were incubated with shaking for 120 min at 37°C in BHI with 0.2% charcoal. NaCl was added (0.3 M final concentration) for the final 10 min (â??BHI-charcoal+NaClâ??). To collect RNA for identification of sB-dependent genes, early-log phase 10403S and DsigB cells were incubated for 120 min in BHI with NaCl added (0.3 M final concentration) for the final 10 min (â??BHI+NaClâ??). To collect RNA for identification of PrfA- dependent genes in the prfA* strain, early-log phase prfA* and DprfA cells were subsequently incubated for 120 min in BHI. After these exposures, RNA was stabilized by the addition of two volumes of RNAprotectâ?¢ (Qiagen, CA, USA). Bacterial cells were then harvested by centrifugation and stored at â??80oC for no longer than 24 h before RNA isolation. For each growth condition described above, RNA isolations were performed on three different days to provide independent biological replicates. Each set of RNA samples was used to perform competitive hybridization microarray experiments.
2010-06-11 | E-GEOD-6421 | biostudies-arrayexpress
Project description:Cultivation based analyses were used to determine the resistance of a set of 140 Listeria monocytogenes strains to weak organic acids. Strains of lineage I tended to exhibit greater overall resistant to four different organic acids compared to lineage II strains. Resistant strains also possessed higher survival levels following challenge to pH 2.4 compared to sensitive strains. Transcriptomic analyses were performed to determine genetic responses relevant to growth at mildly acidic conditions (pH 5.0) and to the presence of sodium diacetate. Lineage I and II strain representatives, clinical strain ATCC 19115 and food isolate FW04/0023, were found to exhibit similar transcriptomic changes when habituated to pH 5.0 in brain heart infusion broth (BHIB) relative to growth at pH 7.2 though considerably more divergent responses were detected when 21 mM sodium diacetate was present. Homogeneity in acid habituation-related gene expression was reflected in relatively homogenous SigB, PrfA, HrCA and CodY regulon expression responses. In the presence of sodium diacetate, SigB and PrfA regulon genes found to be upregulated in sigB and prfA null mutants were overall repressed but SigB dependent-gene activation was not evident. L. monocytogenes strains responses to sodium diacetate were observed in different expression trends amongst various functionally-related gene sets and in particular the expression of the PrfA, Atp, Kdp, Cob, Pdu, Eut and Dlt operons; iron transporter and heat shock-related proteins. The results suggest there is diversity in the specific responses to weak organic acids and subsequent cytosolic acidification amongst L. monocytogenes strains though the acid tolerance response itself manifests as a more conserved set of expression responses.
2008-05-29 | GSE11570 | GEO