The phase variation between wrinkly and smooth colony phenotype affects the virulence of Vibrio parahaemolyticus.

Vibrio parahaemolyticus, the causative agent of seafood-associated gastroenteritis, undergoes wrinkly and smooth colony switching on the plate. The wrinkly spreader grew faster, had stronger motility and biofilm capacity when compared with the smooth one. However, whether the two phenotypes differ in their virulence still needs to be further investigated. In this study, the data showed that the smooth spreader had stronger virulence phenotypes, including the cytotoxicity against HeLa cells, antibacterial activity against E. coli, adhesive capacity toward HeLa cells, and lethality in zebrafish, relative to the wrinkly one. However, the colony morphology variation had no influence on the haemolytic activity. The mRNA levels of major virulence genes including T3SS1, T6SS1, and T6SS2 were significantly enhanced in the smooth colonies relative to those in the wrinkly colonies. Taken together, the presented work highlighted the different virulence profiles of the wrinkly and smooth colony phenotypes.

QsvR and OpaR coordinately repress biofilm formation by Vibrio parahaemolyticus.

Mature biofilm formation by Vibrio parahaemolyticus requires exopolysaccharide (EPS), type IV pili, and capsular polysaccharide (CPS). Production of each is strictly regulated by various control pathways including quorum sensing (QS) and bis-(3'-5')-cyclic di-GMP (c-di-GMP). QsvR, an AraC-type regulator, integrates into the QS regulatory cascade via direct control of the transcription of the master QS regulators, AphA and OpaR. Deletion of qsvR in wild-type or opaR mutant backgrounds altered the biofilm formation by V. parahaemolyticus, suggesting that QsvR may coordinate with OpaR to control biofilm formation. Herein, we demonstrated both QsvR and OpaR repressed biofilm-associated phenotypes, c-di-GMP metabolism, and the formation of V. parahaemolyticus translucent (TR) colonies. QsvR restored the biofilm-associated phenotypic changes caused by opaR mutation, and vice versa. In addition, QsvR and OpaR worked coordinately to regulate the transcription of EPS-associated genes, type IV pili genes, CPS genes and c-di-GMP metabolism-related genes. These results demonstrated how QsvR works with the QS system to regulate biofilm formation by precisely controlling the transcription of multiple biofilm formation-associated genes in V. parahaemolyticus.

Quorum sensing and QsvR tightly control the transcription of vpa0607 encoding an active RNase II-type protein in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a Gram-negative, halophilic bacterium, is a leading cause of acute gastroenteritis in humans. AphA and OpaR are the master quorum sensing (QS) regulators operating at low cell density (LCD) and high cell density (HCD), respectively. QsvR is an AraC-type protein that integrates into the QS system to control gene expression by directly controlling the transcription of aphA and opaR. However, the regulation of QsvR itself remains unclear to date. In this study, we show that vpa0607 and qsvR are transcribed as an operon, vpa0607-qsvR. AphA indirectly activates the transcription of vpa0607 at LCD, whereas OpaR and QsvR directly repress vpa0607 transcription at HCD, leading to the highest expression levels of vpa0607 occurs at LCD. Moreover, VPA0607 acts as an active RNase II-type protein in V. parahaemolyticus and feedback inhibits the expression of QsvR at the post-transcriptional level. Taken together, this work deepens our understanding of the regulation of QsvR and enriches the integration mechanisms of QsvR with the QS system in V. parahaemolyticus.

QsvR represses the transcription of polar flagellum genes in Vibrio parahaemolyticus.

Vibrio parahaemolyticus produces dual flagellar systems, i.e., the sheathed polar flagellum (Pof) and numerous lateral flagella (Laf), both of which are strictly regulated by numerous factors. QsvR is an AraC-type regulator that controls biofilm formation and virulence of V. parahaemolyticus. In the present study, we showed that deletion of qsvR significantly enhanced swimming motility of V. parahaemolyticus, while the swarming motility was not affected by QsvR. QsvR bound to the regulatory DNA regions of flgAMN and flgMN within the Pof gene loci to repress their transcription, whereas it negatively controls the transcription of flgBCDEFGHIJ and flgKL-flaC in an indirect manner. However, over-produced QsvR was also likely to possess the binding activity to the regulatory DNA regions of flgBCDEFGHIJ and flgKL-flaC in a heterologous host. In summary, this work demonstrated that QsvR negatively regulated the swimming motility of V. parahaemolyticus via directly action on the transcription of Pof genes.

AphA directly activates the transcription of polysaccharide biosynthesis gene scvE in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a seafood-borne pathogen, is capable of forming biofilms on surfaces. Exopolysaccharide (EPS) plays crucial roles in holding bacterial cells together and keeping biofilm attached on the surface. The cpsA-K and scvA-O gene clusters are responsible for EPS synthesis in V. parahaemolyticus. AphA, the master quorum sensing (QS) regulator operating at low cell density (LCD), positively regulates transcription of cpsA-K and scvA-O, but lacks the detailed mechanisms. The present data showed that the aphA mutant produced smooth colonies, whereas the wild-type strain produced wrinkled colonies. AphA bound the regulatory DNA region of scvE to activate its transcription, whereas it positively regulated transcription of cpsA and scvA in an indirect manner. The transcriptional level of scvE gradually decreased with increasing cell density, which correlated with the expression level of aphA. Taken together, this work elucidated how AphA regulated the biofilm-associated colony morphology variation in V. parahaemolyticus through its regulatory actions on the expression of EPS genes.

Transcriptomic Analysis of Vibrio parahaemolyticus Underlying the Wrinkly and Smooth Phenotypes.

Vibrio parahaemolyticus, a causative agent of seafood-associated gastroenteritis, undergoes opaque-translucent (OP-TR) colony switching associated with capsular polysaccharide (CPS) production. Here, we showed that V. parahaemolyticus was also able to naturally and reversibly switch between wrinkly and smooth phenotypes. More than 1,000 genes were significantly differentially expressed during colony morphology switching, including the major virulence gene loci and key biofilm-related genes. The genes responsible for type III secretion system 1 (T3SS1), type VI secretion systems (T6SS1 and T6SS2), and flagellar synthesis were downregulated in the wrinkly spreader phenotype, whereas genes located on the pathogenicity island Vp-PAI and those responsible for chitin-regulated pili (ChiRP) and Syp exopolysaccharide synthesis were upregulated. In addition, we showed that the wrinkly spreader grew faster, had greater motility and biofilm capacities, and produced more c-di-GMP than the smooth type. A dozen genes potentially associated with c-di-GMP metabolism were shown to be significantly differentially expressed, which may account for the differences in c-di-GMP levels between the two phenotypes. Most importantly, dozens of putative regulators were significantly differentially expressed, and hundreds of noncoding RNAs were detected during colony morphology switching, indicating that phenotype switching is strictly regulated by a complex molecular regulatory network in V. parahaemolyticus. Taken together, the presented work highlighted the gene expression profiles related to wrinkly-smooth switching, showing that the significantly differentially expressed genes were involved in various biological behaviors, including virulence factor production, biofilm formation, metabolism, adaptation, and colonization. IMPORTANCE We showed that Vibrio parahaemolyticus was able to naturally and reversibly switch between wrinkly and smooth phenotypes and disclosed the gene expression profiles related to wrinkly-smooth switching, showing that the significantly differentially expressed genes between the two colony morphology phenotypes were involved in various biological behaviors, including virulence factor production, biofilm formation, metabolism, adaptation, and colonization.

Characterization of Vibrio parahaemolyticus isolated from stool specimens of diarrhea patients in Nantong, Jiangsu, China during 2018-2020.

Vibrio parahaemolyticus is the leading cause of acute seafood-associated gastroenteritis worldwide. The aim of this study was to investigate the presence of virulence genes, biofilm formation, motor capacities and antimicrobial resistance profile of V. parahaemolyticus isolates isolated from clinical samples in Nantong during 2018-2020. Sixty-six V. parahaemolyticus strains isolated from stool specimens of diarrheal patients were examined. The PCR results showed that there were two tdh+trh+ isolates, four tdh-trh- isolates and sixty tdh+trh- isolates, accounting for 3.0%, 6.1% and 90.9%, respectively. All the tdh carrying isolates manifested the positive reactions for the Kanagawa phenomenon (KP) test. Most of the isolates harbored at least one of the specific DNA markers of 'pandemic group' strains, suggesting that the dominant isolates of V. parahaemolyticus in Nantong might belong to the new O3: K6 or its serovariants. All tdh+ isolates possessed the Vp-PAI genes, but no tdh-trh- isolates carried the T3SS2 genes. All isolates were biofilm producers and had relatively strong motor capacities. In addition, the V. parahaemolyticus isolates were resistant to ampicillin (98.5%), cefuroxime (75.6%), cefepime (66.7%), piperacillin (59.1%) and ampicillin/sulbactam (50.0%), but sensitive to ciprofloxacin (100.0%), levofloxacin (100.0%), trimethoprim-sulfamethoxazole (98.5%), gentamicin (98.5%), amikacin (97%), meropenem (71.2%), and ceftazidime (56.1%). Multidrug-resistant isolates in clinical might be related to the inappropriate use of antimicrobials in aquaculture.

[ToxR represses the synthesis of c-di-GMP in Vibrio parahaemolyticus].

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the ß-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.

Quorum sensing regulates transcription of the pilin gene mshA1 of MSHA pilus in Vibrio parahaemolyticus.

Vibrio parahaemolyticus produces two types of IV pili: mannose-sensitive haemagglutinin type IV pili (MSHA) and chitin-regulated pili (ChiRP). Both of them are required for biofilm formation and the pathogen persistence in hosts. However, there are few reports on the regulation of their expression. In the present study, we showed that the master quorum sensing (QS) regulators AphA and OpaR oppositely regulated the transcription of mshA1 encoding the pilin of MSHA pilus in V. parahaemolyticus. At low cell density (LCD), AphA indirectly repressed mshA1 transcription. In contrast, at high cell density (HCD), OpaR bound to the regulatory DNA region of mshA1 to activate its transcription. Oppositely regulation of mshA1 by AphA and OpaR led to a gradual increase in the expression level of mshA1 from LCD to HCD. Thus, regulation of type IV pili production was one of the mechanisms that V. parahaemolyticus adopted to control biofilm formation.

The Effect of Salinity on Biofilm Formation and c-di-GMP Production in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a moderately halophilic, salt-requiring organism that exhibits optimal growth at approximately 3% salt. Thus, salinity stress is one of the most important stimuli during its lifecycle. The bacterium possesses a strong ability to form biofilms on surfaces, which are thought to be involved in protecting it from adverse environmental conditions. In the present study, salinity-dependent biofilm formation by V. parahaemolyticus was investigated by combining crystal violet staining, colony morphology, intracellular c-di-GMP quantification and quantitative PCR. The results showed that biofilm formation by V. parahaemolyticus was significantly enhanced in low salinity growth conditions and was affected by incubation time. In addition, low salinity reduced intracellular c-di-GMP degradation in V. parahaemolyticus. Transcription of genes encoding ScrABC and ScrG proteins, which are involved in intracellular c-di-GMP metabolism, was inhibited by low salinity growth conditions. Thus, reduced intracellular c-di-GMP degradation in V. parahaemolyticus in low salinity growth conditions may be mediated by repression of scrG and scrABC transcription. Taken together, these results demonstrated for the first time that salinity regulates biofilm formation and c-di-GMP production in V. parahaemolyticus.

OpaR Controls the Metabolism of c-di-GMP in Vibrio parahaemolyticus.

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis worldwide, has a strong ability to form biofilms on surfaces. Quorum sensing (QS) is a process widely used by bacteria to communicate with each other and control gene expression via the secretion and detection of autoinducers. OpaR is the master QS regulator of V. parahaemolyticus operating under high cell density (HCD). OpaR regulation of V. parahaemolyticus biofilm formation has been reported, but the regulatory mechanisms are still not fully understood. bis-(3'-5')-cyclic di-GMP (c-di-GMP) is an omnipresent intracellular second messenger that regulates diverse behaviors of bacteria including activation of biofilm formation. In this work, we showed that OpaR repressed biofilm formation and decreased the intracellular concentration of c-di-GMP in V. parahaemolyticus RIMD2210633. The OpaR box-like sequences were detected within the regulatory DNA regions of scrA, scrG, VP0117, VPA0198, VPA1176, VP0699, and VP2979, encoding a group of GGDEF and/or EAL-type proteins. The results of qPCR, LacZ fusion, EMSA, and DNase I footprinting assays demonstrated that OpaR bound to the upstream DNA regions of scrA, VP0117, VPA0198, VPA1176, and VP0699 to repress their transcription, whereas it positively and directly regulated the transcription of scrG and VP2979. Thus, transcriptional regulation of these genes by OpaR led directly to changes in the intracellular concentration of c-di-GMP. The direct association between QS and c-di-GMP metabolism in V. parahaemolyticus RIMD2210633 would be conducive to precise control of gene transcription and bacterial behaviors such as biofilm formation.

The quorum sensing regulator OpaR is a repressor of polar flagellum genes in Vibrio parahaemolyticus.

Vibrio parahaemolyticus possesses two types of flagella: a single polar flagellum (Pof) for swimming and the peritrichous lateral flagella (Laf) for swarming. Expression of Laf genes has previously been reported to be regulated by the quorum sensing (QS) regulators AphA and OpaR. In the present study, we showed that OpaR, the QS regulator at high cell density (HCD), acted as a negative regulator of swimming motility and the transcription of Pof genes in V. parahaemolyticus. OpaR bound to the promoter-proximal DNA regions of flgAMN, flgMN, and flgBCDEFGHIJ within the Pof gene loci to repress their transcription, whereas it negatively regulates the transcription of flgKL-flaC in an indirect manner. Thus, this work investigated how QS regulated the swimming motility via direct action of its master regulator OpaR on the transcription of Pof genes in V. parahaemolyticus.

Transcriptional regulation of the virulence genes and the biofilm formation associated operons in Vibrio parahaemolyticus.

BACKGROUND: The membrane fusion protein (mfp) gene locus of Vibrio parahaemolyticus consists of two operons, cpsQ-mfpABC and mfpABC, which are both required for biofilm formation. ToxR and CalR are required for the full virulence of V. parahaemolyticus, and their mutual regulation has been demonstrated. Moreover, cell density-dependent expression of toxR was previously observed in V. parahaemolyticus, but details about the related mechanisms remained unclear. QsvR can work with the master quorum sensing (QS) regulators AphA and OpaR to regulate virulence expression and biofilm formation. RESULTS: In the present work, we showed that QsvR bound to the promoter-proximal DNA regions of toxR and calR to repress their transcription as well as occupying the regulatory regions of cpsQ-mfpABC and mfpABC to activate their transcription. Thus, we reconstructed the QsvR-dependent promoter organization of toxR, calR, cpsQ-mfpABC, and mfpABC. CONCLUSION: QsvR directly repressed toxR and calR transcription as well as directly activated cpsQ-mfpABC and mfpABC transcription. The data presented here promotes us to gain deeper knowledge of the regulatory network of the mfp locus in V. parahaemolyticus.