Sigma B contributes to PrfA-mediated virulence in Listeria monocytogenes.
Ontology highlight
ABSTRACT:
SUBMITTER: Nadon CA
PROVIDER: S-EPMC128067 | biostudies-literature | 2002 Jul
REPOSITORIES: biostudies-literature
Publications
Sigma B contributes to PrfA-mediated virulence in Listeria monocytogenes.
Nadon Celine A CA Bowen Barbara M BM Wiedmann Martin M Boor Kathryn J KJ
Infection and immunity 20020701 7
Transcription of the Listeria monocytogenes positive regulatory factor A protein (PrfA) is initiated from either of two promoters immediately upstream of prfA (prfAp(1) and prfAp(2)) or from the upstream plcA promoter. We demonstrate that prfAp(2) is a functional sigma(B)-dependent promoter and that a sigB deletion mutation affects the virulence phenotype of L. monocytogenes. Thus, the alternative sigma factor sigma(B) contributes to virulence in L. monocytogenes. ...[more]
Similar Datasets
Project description:Listeria monocytogenes sigma(B) and positive regulatory factor A (PrfA) are pleiotropic transcriptional regulators that coregulate a subset of virulence genes. A positive regulatory role for sigma(B) in prfA transcription has been well established; therefore, observations of increased virulence gene expression and hemolytic activity in a DeltasigB strain initially appeared paradoxical. To test the hypothesis that L. monocytogenes sigma(B) contributes to a regulatory network critical for appropriate repression as well as induction of virulence gene expression, genome-wide transcript profiling and follow-up quantitative reverse transcriptase PCR (qRT-PCR), reporter fusion, and phenotypic experiments were conducted using L. monocytogenes prfA*, prfA* DeltasigB, DeltaprfA, and DeltaprfA DeltasigB strains. Genome-wide transcript profiling and qRT-PCR showed that in the presence of active PrfA (PrfA*), sigma(B) is responsible for reduced expression of the PrfA regulon. sigma(B)-dependent modulation of PrfA regulon expression reduced the cytotoxic effects of a PrfA* strain in HepG2 cells, highlighting the functional importance of regulatory interactions between PrfA and sigma(B). The emerging model of the role of sigma(B) in regulating overall PrfA activity includes a switch from transcriptional activation at the P2(prfA) promoter (e.g., in extracellular bacteria when PrfA activity is low) to posttranscriptional downregulation of PrfA regulon expression (e.g., in intracellular bacteria when PrfA activity is high).
| S-EPMC2681731 | biostudies-literature
Project description:Some Listeria monocytogenes internalins are recognized as contributing to invasion of mammalian tissue culture cells. While PrfA is well established as a positive regulator of L. monocytogenes virulence gene expression, the stress-responsive sigma(B) has been recognized only recently as contributing to expression of virulence genes, including some that encode internalins. To measure the relative contributions of PrfA and sigma(B) to internalin gene transcription, we used reverse transcription-PCR to quantify transcript levels for eight internalin genes (inlA, inlB, inlC, inlC2, inlD, inlE, inlF, and inlG) in L. monocytogenes 10403S and in isogenic Delta prfA, Delta sigB, and Delta sigB Delta prfA strains. Strains were grown under defined conditions to produce (i) active PrfA, (ii) active sigma(B) and active PrfA, (iii) inactive PrfA, and (iv) active sigma(B) and inactive PrfA. Under the conditions tested, sigma(B) and PrfA contributed differentially to the expression of the various internalins such that (i) both sigma(B) and PrfA contributed to inlA and inlB transcription, (ii) only PrfA contributed to inlC transcription, (iii) only sigma(B) contributed to inlC2 and inlD transcription, and (iv) neither sigma(B) nor PrfA contributed to inlF and inlG transcription. inlE transcript levels were undetectable. The important role for sigma(B) in regulating expression of L. monocytogenes internalins suggests that exposure of this organism to environmental stress conditions, such as those encountered in the gastrointestinal tract, may activate internalin transcription. Interplay between sigma(B) and PrfA also appears to be critical for regulating transcription of some virulence genes, including inlA, inlB, and prfA.
| S-EPMC1892873 | biostudies-literature
Project description:Listeria monocytogenes is a food-borne facultative intracellular pathogen. It is widespread in the environment and has several distinct life-styles. The key transcriptional activator PrfA positively regulates L. monocytogenes virulence genes to mediate the transition from extracellular, flagellum-propelled cell to intracellular pathogen. Here we report the first evidence that PrfA also has a significant positive impact on extracellular biofilm formation. Mutants lacking prfA were defective in surface-adhered biofilm formation. The DeltaprfA mutant exhibited wild-type flagellar motility, and its biofilm defect occurred after initial surface adhesion. We also observed that mutations that led to the constitutive expression of PrfA-dependent virulence genes had a minimal impact on biofilm formation. Furthermore, biofilm development was enhanced in a mutant encoding a PrfA protein variant unable to fully transition from the extracellular form to the virulent, intracellular activity conformation. These results indicate that PrfA positively regulates biofilm formation and suggest that PrfA has a global role in modulating the life-style of L. monocytogenes. The requirement of PrfA for optimal biofilm formation may provide selective pressure to maintain this critical virulence regulator when L. monocytogenes is outside host cells in the environment.
| S-EPMC2916369 | biostudies-literature
Project description:The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes cellular uptake by reducing the expression of virulence genes. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA-binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds at two separate sites on the protein: one within a hydrophobic pocket or tunnel, located between the C- and N-terminal domains of PrfA, and the second in the vicinity of the DNA-binding helix-turn-helix motif. At both sites the compound interacts with residues important for PrfA activation and helix-turn-helix formation. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.
| S-EPMC4802734 | biostudies-other
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.
| S-EPMC8007333 | biostudies-literature
Project description:While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.
| S-EPMC193959 | biostudies-other
Project description:Listeria monocytogenes isolates belonging to serogroup 4 (subtypes 4a, 4ab, 4b, 4c, 4d, and 4e) exhibit different levels of virulence in mice. Molecular studies indicate that in comparison with the control strain EGD (serotype 1/2a), these strains differ in the expression of the PrfA-regulated virulence genes, including prfA itself. Strains of serotypes 4c, 4d, 4e, and especially 4a show a low level of invasiveness in Caco-2 cells, which correlates in part with the low level of expression of the inlA gene. All serotypes reach the cytoplasm, at the latest, 2 h postinfection and become surrounded by polymerized actin within the next hour, but actin tail formation by serotype 4a, 4c, 4d, and 4e strains is drastically reduced. The actA genes in these serogroup 4 strains are expressed in minimum essential medium and within the phagocytic cell line J774. However, the amounts and (in part) the sizes of the ActA proteins in these strains differ under these conditions. The reduced actin tail formation by serotype 4a, 4c, 4d, and 4e strains may be due to the low level of in vivo expression of ActA. In addition, the loss of one repeat unit in the ActA proteins of serotype 4a and 4e strains may also contribute to the less efficient actin tail formation observed with these strains.
| S-EPMC174330 | biostudies-other
Project description:Upon bacterial entry into the cytosol of infected mammalian host cells, the central virulence regulator PrfA of Listeria monocytogenes becomes activated and induces the expression of numerous factors which contribute to bacterial pathogenesis. The mechanism or signal by which PrfA becomes activated during the course of infection has not yet been determined; however, several amino acid substitutions within PrfA (known as PrfA* mutations) that appear to lock the protein into a constitutively activated state have been identified. In this study, the PrfA activation statuses of several L. monocytogenes mutant strains were subjected to direct isogenic comparison and the mutant with the highest activity, the prfA(L140F) mutant, was identified. The prfA(L140F) strain was subsequently used as a tool to identify gene products secreted as a result of PrfA activation. By use of two-dimensional gel electrophoresis followed by liquid chromatography-electrospray ionization-tandem mass spectroscopy analyses, 15 proteins were identified as up-regulated in the prfA(L140F) secretome, while the secretion of two proteins was found to be reduced. Although some of the proteins identified were known to be subject to direct regulation by PrfA, the majority have not previously been associated with PrfA regulation and their expression or secretion may be influenced indirectly by a PrfA-dependent regulatory pathway. Plasmid insertion inactivation of the genes encoding four novel secreted products indicated that three of the four have significant roles in L. monocytogenes virulence. The use of mutationally activated prfA alleles therefore provides a useful approach towards identifying gene products that contribute to L. monocytogenes pathogenesis.
| S-EPMC2168340 | biostudies-literature
Project description:Listeria monocytogenes is able to efficiently utilize glycerol as a carbon source. In a defined minimal medium, the growth rate (during balanced growth) in the presence of glycerol is similar to that in the presence of glucose or cellobiose. Comparative transcriptome analyses of L. monocytogenes showed high-level transcriptional upregulation of the genes known to be involved in glycerol uptake and metabolism (glpFK and glpD) in the presence of glycerol (compared to that in the presence of glucose and/or cellobiose). Levels of expression of the genes encoding a second putative glycerol uptake facilitator (GlpF(2)) and a second putative glycerol kinase (GlpK(2)) were less enhanced under these conditions. GlpK(1) but not GlpK(2) was essential for glycerol catabolism in L. monocytogenes under extracellular conditions, while the loss of GlpK(1) affected replication in Caco-2 cells less than did the loss of GlpK(2) and GlpD. Additional genes whose transcription levels were higher in the presence of glycerol than in the presence of glucose and cellobiose included those for two dihydroxyacetone (Dha) kinases and many genes that are under carbon catabolite repression control. Transcriptional downregulation in the presence of glycerol (compared to those in the presence glucose and cellobiose) was observed for several genes and operons that are positively regulated by glucose, including genes involved in glycolysis, N metabolism, and the biosynthesis of branched-chain amino acids. The highest level of transcriptional upregulation was observed for all PrfA-dependent genes during early and late logarithmic growth in glycerol. Under these conditions, a low level of HPr-Ser-P and a high level of HPr-His-P were present in the cells, suggesting that all enzyme IIA (EIIA) (or EIIB) components of the phosphotransferase system (PTS) permeases expressed will be phosphorylated. These and other data suggest that the phosphorylation state of PTS permeases correlates with PrfA activity.
| S-EPMC2493282 | biostudies-literature
Project description:Metabolic adaptations are critical to the ability of bacterial pathogens to grow within host cells and are normally preceded by sensing of host-specific metabolic signals, which in turn can influence the pathogen's virulence state. Previously, we reported that the intracellular bacterial pathogen Listeria monocytogenes responds to low availability of branched-chain amino acids (BCAAs) within mammalian cells by up-regulating both BCAA biosynthesis and virulence genes. The induction of virulence genes required the BCAA-responsive transcription regulator, CodY, but the molecular mechanism governing this mode of regulation was unclear. In this report, we demonstrate that CodY directly binds the coding sequence of the L.?monocytogenes master virulence activator gene, prfA, 15 nt downstream of its start codon, and that this binding results in up-regulation of prfA transcription specifically under low concentrations of BCAA. Mutating this site abolished CodY binding and reduced prfA transcription in macrophages, and attenuated bacterial virulence in mice. Notably, the mutated binding site did not alter prfA transcription or PrfA activity under other conditions that are known to activate PrfA, such as during growth in the presence of glucose-1-phosphate. This study highlights the tight crosstalk between L.?monocytogenes metabolism and virulence, while revealing novel features of CodY-mediated regulation.
| S-EPMC4329120 | biostudies-literature