OpaR Controls a Network of Downstream Transcription Factors in Vibrio parahaemolyticus BB22
ABSTRACT: 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.
Project description:Vibrio parahaemolyticus is an emerging world-wide human pathogen that is associated with food-borne gastroenteritis when raw or undercooked seafood is consumed. Expression of virulence factors in this organism is modulated by the phenomenon known as quorum sensing, which permits differential gene regulation at low versus high cell density. The master regulator of quorum sensing in V. parahaemolyticus is OpaR. OpaR not only controls virulence factor gene expression, but also the colony and cellular morphology associated with growth on a surface and biofilm formation. Whole transcriptome Next Generation sequencing (RNA-Seq) was utilized to determine the OpaR regulon by comparing strains BB22OP (opaR+, LM5312) and BB22TR (?opaR1, LM5674). This work, using the published V. parahaemolyticus BB22OP genome sequence, confirms and expands upon a previous microarray analysis for these two strains that used an Affymetrix GeneChip designed from the closely related V. parahaemolyticus RIMD2210633 genome sequence. Overall there was excellent correlation between the microarray and RNA-Seq data. Eleven transcription factors under OpaR control were identified by both methods and further confirmed by quantitative reverse transcription PCR (qRT-PCR) analysis. Nine of these transcription factors were demonstrated to be direct OpaR targets via in vitro electrophoretic mobility shift assays with purified hexahistidine-tagged OpaR. Identification of the direct and indirect targets of OpaR, including small RNAs, will enable the construction of a network map of regulatory interactions important for the switch between the nonpathogenic and pathogenic states.
Project description:Proteases play a key role in numerous bacterial physiological events. Microbial proteases are used in the pharmaceutical industry and in biomedical applications. The genus Vibrio comprises protease-producing bacteria. Proteases transform polypeptides into shorter chains for easier utilization. They also function as a virulence factor in pathogens. The mechanism by which protease genes are regulated in Vibrio parahaemolyticus, an emerging world-wide human pathogen, however, still remains unclear. Quorum sensing is the communication system of bacteria. OpaR is the master quorum-sensing regulator in V. parahaemolyticus. In the present study, quantitative reverse transcriptase-polymerase chain reaction and protease gene promoter-fusion reporter assays revealed that OpaR represses seven protease genes-three metalloprotease genes and four serine protease genes-which are involved in environmental survival and bacterial virulence. Furthermore, the electrophoresis mobility shift assay demonstrated that OpaR is bound directly to the promoter region of each of the seven protease genes. DNase I footprinting identified the sequence of these OpaR-binding sites. ChIP-seq analyses revealed 435 and 835 OpaR-binding sites in the late-log and stationary phases, respectively. These OpaR-binding sequences indicated a conserved OpaR-binding motif: TATTGATAAAATTATCAATA. These results advance our understanding of the protease regulation system in V. parahaemolyticus. This study is the first to reveal the OpaR motif within V. parahaemolyticus in vivo, using ChIP-seq, and to provide a database for OpaR direct regulon.
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.
Project description:The type VI secretion system (T6SS) is bacterial protein injection machinery with roles in virulence, symbiosis, interbacterial interaction, antipathogenesis, and environmental stress responses. There are two T6SS loci, T6SS1 and T6SS2, in the two chromosomes of Vibrio parahaemolyticus, respectively. This work disclosed that the master quorum sensing (QS) regulator OpaR repressed the transcription of hcp1 encoding the structural component Hcp1 of T6SS1 in V. parahaemolyticus, indicating that QS had a negative regulatory action on T6SS1. A single ?(54)-dependent promoter was transcribed for hcp1 in V. parahaemolyticus, and its activity was repressed by the OpaR regulator. Since the OpaR protein could not bind to the upstream region of hcp1, OpaR would repress the transcription of hcp1 in an indirect manner.
Project description:<h4>Background</h4>Vibrio parahaemolyticus is a leading cause of infectious diarrhea and enterogastritis via the fecal-oral route. V. harveyi is a pathogen of fishes and invertebrates, and has been used as a model for quorum sensing (QS) studies. LuxR is the master QS regulator (MQSR) of V. harveyi, and LuxR-dependent expression of its own gene, qrr2-4 and aphA have been established in V. harveyi. Molecular regulation of target genes by the V. parahaemolyticus MQSR OpaR is still poorly understood.<h4>Methodology/principal findings</h4>The bioinformatics analysis indicated that V. parahaemolyticus OpaR, V. harveyi LuxR, V. vulnificu SmcR, and V. alginolyticus ValR were extremely conserved, and that these four MQSRs appeared to recognize the same conserved cis-acting signals, which was represented by the consensus constructs manifesting as a position frequency matrix and as a 20 bp box, within their target promoters. The MQSR box-like sequences were found within the upstream DNA regions of opaR, qrr2-4 and aphA in V. parahaemolyticus, and the direct transcriptional regulation of these target genes by OpaR were further confirmed by multiple biochemical experiments including primer extension assay, gel mobility shift assay, and DNase I footprinting analysis. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and OpaR-binding sites were determined for the five target genes of OpaR, which gave a structural map of the OpaR-dependent promoters. Further computational promoter analysis indicated the above regulatory circuits were shared by several other closely related Vibrios but with slight exceptions.<h4>Conclusions/significance</h4>This study gave a comprehensive computational and characterization of the direct transcriptional regulation of five target genes, opaR, qrr2-4 and ahpA, by OpaR in V. parahaemolyticus. These characterized regulatory circuits were conserved in V. harveyi and V. parahaemolyticus.
Project description:To maintain the turgor pressure of the cell under high osmolarity, bacteria accumulate small organic compounds called compatible solutes, either through uptake or biosynthesis. Vibrio parahaemolyticus, a marine halophile and an important human and shellfish pathogen, has to adapt to abiotic stresses such as changing salinity. Vibrio parahaemolyticus contains multiple compatible solute biosynthesis and transporter systems, including the ectABC-asp_ect operon required for de novo ectoine biosynthesis. Ectoine biosynthesis genes are present in many halotolerant bacteria; however, little is known about the mechanism of regulation. We investigated the role of the quorum sensing master regulators OpaR and AphA in ect gene regulation. In an opaR deletion mutant, transcriptional reporter assays demonstrated that ect expression was induced. In an electrophoretic mobility shift assay, we showed that purified OpaR bound to the ect regulatory region indicating direct regulation by OpaR. In an aphA deletion mutant, expression of the ect genes was repressed, and purified AphA bound upstream of the ect genes. These data indicate that AphA is a direct positive regulator. CosR, a Mar-type regulator known to repress ect expression in V. cholerae, was found to repress ect expression in V. parahaemolyticus In addition, we identified a feed-forward loop in which OpaR is a direct activator of cosR, while AphA is an indirect activator of cosR Regulation of the ectoine biosynthesis pathway via this feed-forward loop allows for precise control of ectoine biosynthesis genes throughout the growth cycle to maximize fitness.IMPORTANCE Accumulation of compatible solutes within the cell allows bacteria to maintain intracellular turgor pressure and prevent water efflux. De novo ectoine production is widespread among bacteria, and the ect operon encoding the biosynthetic enzymes is induced by increased salinity. Here, we demonstrate that the quorum sensing regulators AphA and OpaR integrate with the osmotic stress response pathway to control transcription of ectoine biosynthesis genes in V. parahaemolyticus We uncovered a feed-forward loop wherein quorum sensing regulators also control transcription of cosR, which encodes a negative regulator of the ect operon. Moreover, our data suggest that this mechanism may be widespread in Vibrio species.
Project description:The quorum regulatory cascade is poorly characterized in Vibrio parahaemolyticus, in part because swarming and virulence factors--the hallmarks of the organism--are repressed by this scheme of gene control, and quorum sensing seems to be silenced in many isolates. In these studies, we examine a swarming-proficient, virulent strain and identify an altered-function allele of the quorum regulator luxO that is demonstrated to produce a constitutively active mimic of LuxO?P. We find that LuxO* affects the expression of three small regulatory RNAs (Qrrs) and the activity of a translational fusion in opaR, the output regulator. Tests for epistasis showed that luxO* is dominant over luxO and that opaR is dominant over luxO. Thus, information flow through the central elements of the V. parahaemolyticus quorum pathway is proven for the first time. Quorum-sensing output was explored using microarray profiling: the OpaR regulon encompasses ?5.2% of the genome. OpaR represses the surface-sensing and type III secretion system 1 (T3SS1) regulons. One novel discovery is that OpaR strongly and oppositely regulates two type VI secretion systems (T6SS). New functional consequences of OpaR control were demonstrated: OpaR increases the cellular cyclic di-GMP (c-di-GMP) level, positively controls chitin-induced DNA competency, and profoundly blocks cytotoxicity toward host cells. In expanding the previously known quorum effects beyond the induction of the capsule and the repression of swarming to elucidate the global scope of genes in the OpaR regulon, this study yields many clues to distinguishing traits of this Vibrio species; it underscores the profoundly divergent survival strategies of the quorum On/Off phase variants.
Project description:Vibrio parahaemolyticus is an organism well adapted to communal life on surfaces. When grown on a surface or in a viscous layer, the bacterium induces a large gene system and differentiates to swarmer cells capable of movement over and colonization of surfaces. V. parahaemolyticus displays additional phenotypic versatility manifested as variable colony morphology, switching between translucent and opaque colony types. Although not itself luminescent, V. parahaemolyticus produces autoinducer molecules capable of inducing luminescence in Vibrio harveyi. To examine the role of quorum signaling in the lifestyles of V. parahaemolyticus, the functional homolog of the gene encoding the V. harveyi autoinducer-controlled transcriptional regulatory protein LuxR was cloned. Sequence analysis of the clone predicted an open reading frame with a deduced product 96% identical to LuxR. Introduction of the clone carrying the luxR-like locus into V. parahaemolyticus dramatically affected colony morphology, converting a translucent strain to an opaque one. When the coding sequence for the luxR homolog was placed under the control of the Ptac promoter, conversion to the opaque phenotype became inducible by isopropyl-beta-D-thiogalactopyranoside. Allelic disruption of the luxR-like gene on the chromosome of an opaque strain produced a translucent strain proficient in swarming ability. Primer extension mapping demonstrated opaR transcription in opaque but not translucent cell types. It is postulated that this gene, which has been named opaR, encodes a transcription factor controlling cell type. The underlying genetic basis for opaque-translucent variation may be the consequence of a genomic alteration detected in the opaR locus of opaque and translucent strains.
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.
Project description:Vibrio parahaemolyticus is the leading bacterial cause for seafood-related gastroenteritis worldwide. As an intestinal pathogen, V. parahaemolyticus competes with other commensal bacteria for the same pool of nutrients. The major source of nutrition for intestinal bacteria is intestinal mucus. We wanted to determine the expression profile of wild-type V. parahaemolyticus in mouse intestinal mucus and then perform a differential expression analysis in a ∆luxO deletion mutant, in which the high cell density quorum sensing regulator OpaR is constitutively expressed and low cell density regulator AphA is repressed. Overall design: RNA-Seq analysis was performed on RNA isolated from two biological replicates of wild-type Vibrio parahaemolyticus strain RIMD2210633 and three biological replicates the ∆luxO mutant strain, grown in M9 minimal media supplemented with mouse intestinal mucus.