Regulation of Listeria monocytogenes internalin-like genes by σB and PrfA as revealed by subgenomic microarray analyses
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PROVIDER: PRJNA99491 | ENA |
REPOSITORIES: ENA
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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: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: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
2008-10-01 | GSE11347 | GEO
Project description:Phosphopeptides were identified in Listeria monocytogesn strain constitutivally expressing PrfA. Also, the phosphoproteins and proteins were identified that are overexpressed/underextressed in response to PrfA.
2014-10-29 | PXD001108 | Pride
Project description:Comparison of Listeria monocytogenes transcripts in different strains (EGD wild-type versus EGD-e wild-type, EGD-e PrfA* versus EGD-e wild-type).
2013-08-01 | E-MTAB-1676 | biostudies-arrayexpress
Project description:Comparison of Listeria monocytogenes transcripts in different strains (EGD wild-type versus EGD-e wild-type, EGD-e PrfA* versus EGD-e wild-type).
2013-08-01 | E-MTAB-1677 | biostudies-arrayexpress
Project description:PrfA activity was studied in L. monocytogenes strain EGD and in an isogenic prfA deletion mutant (EGDΔprfA) carrying multiple copies of the wild-type prfA or the mutant prfA* gene (strains EGDΔprfApPrfA and EGDΔprfApPrfA*) after growth in brain heart infusion (BHI), Luria-Bertani broth (LB) or a defined minimal medium (MM) supplemented either with one of the three PTS-carbohydrates, glucose, mannose and cellobiose, or the non-PTS carbon source glycerol. Low PrfA activity was observed in the wild-type EGD strain in BHI and LB with either of these carbon sources, while PrfA activity was high in minimal medium in presence of glycerol but significantly reduced in presence of cellobiose. The strains expressing the prfA and prfA* gene under the prfA promoters, P1 and P2, produced equally large amounts of PrfA protein and high PrfA activity was observed in strain EGDΔprfApPrfA* under all growth conditions. In contrast, high PrfA activity in strain EGDΔprfApPrfA was only observed when this strain was cultured in BHI but not in LB or MM (in presence of either carbon source). A ptsH mutant (lacking a functional HPr) was able to grow in BHI suggesting that growth of L. monocytogenes in this culture medium is supported by carbon sources whose uptake and metabolism are independent of the PTS pathway. However, this mutant was unable to grow in LB and MM regardless which of the four carbon sources was added, suggesting that uptake of the used carbohydrates and the catabolism of glycerol depend fully on the functional common PTS pathway. Furthermore, the growth rates of L. monocytogenes are strongly reduced in presence of large amounts of PrfA protein when growing MM but less in LB and only slightly in BHI. The expression profiles of the genes encoding PTS permeases were determined in the three strains under various growth conditions. The data suggest that PrfA activity correlates with the expression level and the phosphorylation state of specific PTS permeases. This SuperSeries is composed of the following subset Series: GSE12143: Listeria monocytogenes EGD after growth BHI vs. LB vs. MM GSE12145: Listeria monocytogenes EGDΔprfApPrfA and EGDΔprfApPrfA* compared to the wild type strain EGD GSE12146: Listeria monocytogenes EGD and EGD-e
2008-11-30 | E-GEOD-12151 | biostudies-arrayexpress
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:Glycerol metabolism and PrfA activity in Listeria monocytogenes
| PRJNA106373 | ENA
Project description:The foodborne pathogen Listeria monocytogenes has the ability to develop biofilm in food-processing environment, which becomes a major concern for the food safety. PrfA, a key transcriptional activator that regulates most of the known listerial virulence gene expression, has been shown to promote L. monocytogenes biofilm formation. In this study, the whole genome microarray was used to identify differentially expressed genes associated with the putative interaction between biofilm formation and PrfA in L. monocytogenes. Comparative transcriptome analyses indicated over 21.9% of the L. monocytogenes EGDe genes (627 out of 2857 predicted) were altered in their expression in biofilm cells relative to planktonic cell populations. These genes were classed into different functional categories which cover most of the biochemical functions encountered in bacterial cells, especially involved in ion transport, DNA repair, and cell wall biosynthesis based on significant enrichment of GO terms. Among them, 185 genes were identified to be associated with PrfA and biofilm formation by comparison of the whole gene expression profiles of L. monocytogenes EGDe and its M-NM-^TprfA mutant. The expression tendency of these PrfA-associated and biofilm-specific genes were mainly opposite in M-NM-^TprfA biofilm, and most of them are involved in phage-related function, membrane bioenergetics, and cell wall. Our results indicated that L. monocytogenes biofilm formation is probably controlled by the complex regulation network involved variable genes required for the different biological pathways. This regulatory network is modified in the prfA deletion mutant in order to maintain its stable biofilm lifestyle. Gene expression of planktonic cells and biofilm cells in Listeria monocytogenes EGDe and prfA isogenic deletion strain EGDeM-NM-^TprfA with cultivated in MEM and BHI for 48 hours, were mesasued using Agilent Listeria monocytogenes customized whole-genome microarray 8x15 array. Three replicates.
2013-05-01 | E-GEOD-43052 | biostudies-arrayexpress