Surface sensing in Vibrio parahaemolyticus triggers a programme of gene expression that promotes colonization and virulence
Ontology highlight
ABSTRACT:
ORGANISM(S): Vibrio parahaemolyticus RIMD 2210633 Vibrio parahaemolyticus
PROVIDER: GSE18763 | GEO | 2010/12/15
SECONDARY ACCESSION(S): PRJNA121609
REPOSITORIES: GEO
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Project description:Vibrio parahaemolyticus senses surfaces via impeded rotation of its polar flagellum. We have exploited this surface-sensing mechanism to trick the organism into thinking it is on a surface when it is growing in liquid. This facilitated studies of global gene expression in a way that avoided many of the complications of surface-to-liquid comparisons, and illuminated ~ 70 genes that respond to surface sensing per se. Almost all are surface-induced (not repressed) and encode swarming motility proteins, virulence factors or sensory enzymes involved with chemoreception and c-di-GMP signalling. Follow-up studies were performed to place the surfaceresponsive genes in a regulatory hierarchy. Mapping the hierarchy revealed two surprises about LafK, a transcriptional activator that until now has been considered to be the master regulator for the lateral flagellar system. First, LafK controls a more diverse set of genes than previously appreciated. Second, some laf genes are not under LafK control, which means LafK is not the master regulator after all. Additional experiments motivated by the transcriptome analyses revealed that growth on a surface lowers c-di-GMP levels and enhances cytotoxicity. Thus, we demonstrate that V. parahaemolyticus can invoke a programme of gene control upon encountering a surface and the specific identities of the surfaceresponsive genes are pertinent to colonization and pathogenesis. The gene expression profiles of swarming and swimming cell types of Vibrio parahaemolyticus were compared using Affymetrix custom microarrays.
2010-12-15 | E-GEOD-18763 | biostudies-arrayexpress
Project description:Vibrio parahaemolyticus scr genes modulate expression of gene sets pertinent to swarming and biofilm formation. They do so by affecting the level of the second messenger c-di-GMP. Here we explore the extent of this regulation by comparing the transcriptomes of a scrABC mutant and its wild-type parental strain. The scope of transcriptional effects modulated by c-di-GMP includes ~100 genes that are positively and negatively regulated. An elevated cellular level of c-di-GMP represses the surface sensing regulon including the genes encoding the lateral flagellar and type three secretion systems while inducing expression of genes encoding cell surface molecules and capsular polysaccharide. Expression of a few transcriptional regulators was also affected, and here we describe the role of one, CpsQ. CpsQ is one of four V. parahaemolyticus homologs in the CsgD/VpsT family of regulators, members of which have been implicated in c-di-GMP signaling. Mutations in cpsQ, like defects in another previously identified capsule regulator, cpsR, suppress the sticky phenotype of scr mutants. By using a combination of mutant and reporter analyses in Vibrio and E. coli, CpsQ is shown to be the direct positive regulator of cpsA transcription and its cpsA-activating ability is found to be responsive to the cellular level of c-di-GMP. Unexpectedly, we find that a low level of this nucleotide diminishes the stability of CpsQ. The molecular interplay in this signaling circuit is further defined by demonstrating that CpsQ is epistatic to CpsS, a negative regulator of capsule. CpsR activates cpsQ, and CpsQ can also regulate its own transcription. Wildtype Vibrio parahaemolyticus (LM5674) and scrABC mutant (LM6567) were grown on rich mediim agar plates and gene expression profiles were compared.
2011-12-31 | E-GEOD-30508 | biostudies-arrayexpress
Project description:Vibrio parahaemolyticus scr genes modulate expression of gene sets pertinent to swarming and biofilm formation. They do so by affecting the level of the second messenger c-di-GMP. Here we explore the extent of this regulation by comparing the transcriptomes of a scrABC mutant and its wild-type parental strain. The scope of transcriptional effects modulated by c-di-GMP includes ~100 genes that are positively and negatively regulated. An elevated cellular level of c-di-GMP represses the surface sensing regulon including the genes encoding the lateral flagellar and type three secretion systems while inducing expression of genes encoding cell surface molecules and capsular polysaccharide. Expression of a few transcriptional regulators was also affected, and here we describe the role of one, CpsQ. CpsQ is one of four V. parahaemolyticus homologs in the CsgD/VpsT family of regulators, members of which have been implicated in c-di-GMP signaling. Mutations in cpsQ, like defects in another previously identified capsule regulator, cpsR, suppress the sticky phenotype of scr mutants. By using a combination of mutant and reporter analyses in Vibrio and E. coli, CpsQ is shown to be the direct positive regulator of cpsA transcription and its cpsA-activating ability is found to be responsive to the cellular level of c-di-GMP. Unexpectedly, we find that a low level of this nucleotide diminishes the stability of CpsQ. The molecular interplay in this signaling circuit is further defined by demonstrating that CpsQ is epistatic to CpsS, a negative regulator of capsule. CpsR activates cpsQ, and CpsQ can also regulate its own transcription.
2011-12-31 | GSE30508 | GEO
Project description:Vibrio parahaemolyticus an emerging pathogen that is a causative agent of foodborne gastroenteritis when raw or undercooked seafood is consumed. Previous microarray data using a Vibrio parahaemolyticus RIMD2210633 chip has shown the master quorum-sensing regulator OpaR controls virulence, type III and type VI secretion systems, and flagellar and capsule production genes. In a parallel study, RNA-Seq was used to comparatively study the transcriptome changes of wild type Vibrio parahaemolyticus BB22 and a ΔopaR strain directly. Differences in mRNA expression were analyzed using next generation Illumina sequencing and bioinformatics techniques to align and count reads. A comparison with the previous microarray data showed good correlation between the shared genes. The RNA-Seq offered an insight into control of genes specific to the Vibrio parahaemolyticus BB22 strain as well as a new look at the sRNAs that are expressed. Eleven transcriptional regulators with greater than 4 fold regulation in the previous microarray study and 2 fold regulation in the RNA-Seq analysis, were chosen to validate the data using qRT-PCR and further characterized with electrophoretic mobility shift assays (EMSAs) to determine if they are direct targets of OpaR. The transcription factors chosen play key roles in virulence, surface motility, cell to cell interactions, and cell surface characteristics. One small RNA was identified in the RNA-Seq data to be quorum-sensing controlled and unidentified by other programs. The RNA-Seq data has aided in understanding and elucidating the hierarchy of quorum-sensing control of OpaR in Vibrio parahaemolyticus. The wild type Vibrio parahaemolyticus BB22 strain LM5312 and an opaR deletion strain LM5674 were analyzed for mRNA expression using RNA-Seq.
2013-12-31 | E-GEOD-53639 | biostudies-arrayexpress
Project description:Vibrio parahaemolyticus an emerging pathogen that is a causative agent of foodborne gastroenteritis when raw or undercooked seafood is consumed. Previous microarray data using a Vibrio parahaemolyticus RIMD2210633 chip has shown the master quorum-sensing regulator OpaR controls virulence, type III and type VI secretion systems, and flagellar and capsule production genes. In a parallel study, RNA-Seq was used to comparatively study the transcriptome changes of wild type Vibrio parahaemolyticus BB22 and a ΔopaR strain directly. Differences in mRNA expression were analyzed using next generation Illumina sequencing and bioinformatics techniques to align and count reads. A comparison with the previous microarray data showed good correlation between the shared genes. The RNA-Seq offered an insight into control of genes specific to the Vibrio parahaemolyticus BB22 strain as well as a new look at the sRNAs that are expressed. Eleven transcriptional regulators with greater than 4 fold regulation in the previous microarray study and 2 fold regulation in the RNA-Seq analysis, were chosen to validate the data using qRT-PCR and further characterized with electrophoretic mobility shift assays (EMSAs) to determine if they are direct targets of OpaR. The transcription factors chosen play key roles in virulence, surface motility, cell to cell interactions, and cell surface characteristics. One small RNA was identified in the RNA-Seq data to be quorum-sensing controlled and unidentified by other programs. The RNA-Seq data has aided in understanding and elucidating the hierarchy of quorum-sensing control of OpaR in Vibrio parahaemolyticus.
2013-12-31 | GSE53639 | GEO
Project description:The quorum regulatory cascade is poorly characterized in Vibrio parahaemolyticus, in part because swarming and pathogenicity - the hallmark traits of the organism - are repressed by this scheme of gene control. As a consequence, many isolates appear silenced for quorum sensing via phase variation. In these studies, we examine a swarm proficient, virulent strain and find an altered function allele of the central quorum regulator luxO. We use this allele, which produces a constitutively active LuxO, to probe the upstream elements of the pathway and demonstrate their functionality for the first time. We find that the state of luxO affects expression of three small regulatory RNAS (Qrrs) and the activity of a translational fusion in opaR, the central output regulator. We use microarray profiling to determine the OpaR regulon, which was found to encompass ~5.2% of the genome. The quorum sensing proficient strain seems adapted for a sessile, community lifestyle; it is competent to uptake DNA, produces much capsular polysaccharide, has a high level of c-di-GMP, and strongly expresses one type six secretion system. Expressing the entire surface sensing regulon and numerous methyl accepting chemotaxis proteins, the quorum-disrupted cell type seems prepared for a mobile lifestyle. It is also cytotoxic to host cells in co-culture and expresses distinct type six as well as type three secretion systems. Thus, the scope and nature of the genes in the OpaR regulon provide many clues to the distinguishing traits of this Vibrio species as well as to the quite divergent survival strategies of the quorum ON/OFF phase variants The gene expression profiles of different strains of Vibrio parahaemolyticus cells grown on rich medium and compared using Affymetrix custom microarrays.
2011-12-01 | E-GEOD-28216 | biostudies-arrayexpress
Project description:Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a unipolar polysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c-di-GMP) lead to surface-contact-independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP-negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c-di-GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive c-di-GMP phosphodiesterase also elevate UPP production and attachment, consistent with c-di-GMP activation of surface-dependent adhesin deployment. Three biological replicates, independent RNA preparations, one dye swap.
2014-08-21 | E-GEOD-60565 | biostudies-arrayexpress
Project description:The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics.The cyclic dinucleotide cyclic-di-guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. Here we provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by cyclic-di- GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, NF-!B and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid-sensing pathways.Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand. Three-condition experiment: macrophages transfected with mono-GMP (negative control), double-stranded DNA (positive control), or cyclic-di-GMP (experimental condition). Biological replicates: two, independently treated, harvested, and hybridized to arrays. One replicate per array, except two technical replicates were performed for one of the positive control samples.
2009-07-21 | E-GEOD-16943 | biostudies-arrayexpress
Project description:Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a unipolar polysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c-di-GMP) lead to surface-contact-independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP-negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c-di-GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive c-di-GMP phosphodiesterase also elevate UPP production and attachment, consistent with c-di-GMP activation of surface-dependent adhesin deployment.
2014-08-21 | GSE60565 | GEO
Project description:The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics.The cyclic dinucleotide cyclic-di-guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. Here we provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by cyclic-di- GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, NF-!B and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid-sensing pathways.Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.
2009-07-22 | GSE16943 | GEO