Regulatory Actions Of ToxR And CalR On Their Own Genes And Type Ⅲ Secretion System 1 (T3SS1) In Vibrio Parahaemolyticus

BackgroundVibrio parahaemolyticus,a Gram-negative halophilic bacterium is the leading cause of seafood-associated diarrhea and gastroenteritis in humans.The consumption of raw or improperly cooked seafood contaminated with virulent V.parahaemolyticus strains results in human infections with clinical symptoms such as diarrhea,vomiting,nausea and abdominal cramping.The pathogenicity of V.parahaemolyticus is as a result of the possession of an arsenal of virulent factors,such as thermostable direct hemolysin（tdh）,TDH related hemolysin（trh）,and two type III secretion systems（T3SS1 and T3SS2）.The type III secretion system 1（T3SS1）is a major virulence determinant expressed by this bacterium,which contributes to V.parahaemolyticus-induced cytotoxicity in most mammalian cell lines and lethality in the mouse infection model.T3SS1 expression is regulated by ToxR and CalR.However,the exact regulation mechanisms have not been elucidated.The clarification of the regulation of the virulence genes expression will enhance our understanding of the pathogenic mechanisms in V.parahaemolyticus.ObjectiveIn the study,we want to investigate the transcriptional regulatory mechanism of ToxR and CalR on their own genes and on T3SS1 in V.parahaemolyticus using molecular biological experiments to elucidate the relative pathogenic mechanism.Methods1.Bacterial strains constructionThe gene deleted mutant strains?calR and?toxR,were constructed using the gene specific PCR site directed mutagenesis method.The recombinant amplicon was inserted between the Pst I and Sph I sites of the suicide plasmid pDS132.The complementary strainsΔcalR/pBAD33-calR andΔtoxR/pBAD33-toxR,were constructed by transforming the mutants with the recombinant plasmid pBAD33-calR and pBAD33-toxR,respectively.2.RNA isolation and quantitative reverse transcription PCR（qRT-PCR）Total RNA was extracted from the WT,ΔcalR,andΔtoxR strains using the TRIzol method and reverse transcribed to cDNAs.qRT-PCR was used to determine the relative mRNA levels of the target genes together with the SYBR Green master mix based on the standard curve of the 16S rRNA（reference gene）expression for each RNA preparation.3.Preparation of 6x His-tagged proteins（CalRhis6 and ToxRhis6）The entire coding region of calR and the truncated toxR were PCR amplified and cloned between the BamH I and Hind III sites of the plasmid pET28a.The recombinant plasmid was transferred into E.coli BL21λDE3 for protein expression at different conditions by induction with 1 mM IPTG.The Ni-column affinity chromatography was used to isolate the proteins.The purity of each protein was confirmed by SDS-PAGE.The protein was stored at-80°C.4.Electrophoretic mobility shift assay（EMSA）The 5′-ends of the promoter DNA region of the calR,toxR,exsB,vp1687,and vp1667 genes were labeled with[γ-³²P]ATP and T4 polynucleotide kinase.EMSA was performed with increasing amounts of CalR_his6 or ToxR_his6 protein and binding products were analyzed in a native 4%（w/v）polyacrylamide gel.The results were detected by autoradiography.5.LacZ fusion andβ-galactosidase assayThe promoter-proximal DNA regions of each target gene was cloned into the pHRP309plasmid between EcoR I and BamH I site and the upstream promoterless lacZ reporter gene.The recombinant plasmids were transferred into WT,ΔtoxR,andΔcalR strains respectively.The V.parahaemolyticus strains transformed with recombinant pHRP309 plasmid were cultivated and then lysed to measure theβ-galactosidase activities in cellular extracts using aβ-Galactosidase Enzyme Assay System.6.Primer extension assayThe 5’end of the sense or antisense primer was labeled with[γ-³²P]and the primer was annealed to the complementary region of the mRNA strand to generate cDNA under the action of the reverse transcription AMV enzyme.The same labeled primer was used for sequencing the corresponding DNA fragments.The primer extension products and sequencing materials were concentrated and analyzed in an 8M urea-6%polyacrylamide denaturing gel electrophoresis and the results were detected with autoradiography.7.DNase I footprinting assayThe promoter-proximal region of each target gene with a single ³²P-labeled end was generated by PCR.The PCR products were purified and DNA binding was performed with increasing amounts of CalR_his6 or ToxR_his6 protein.The reaction was incubated at room temperature for 30 min.Before digestion,10μl of Ca²⁺/Mg²⁺solution was added,followed by incubation for 1 min at room temperature.The optimized RQ1 RNase-Free DNase I（Promega,USA）was then added to the reaction mixture,and the mixture was incubated at room temperature for 40 to 90 s.The reaction was quenched by adding 9μL of stop solution followed by incubation for 1 min at room temperature.The partially digested DNA samples were extracted with phenol/chloroform,precipitated with ethanol,and analyzed in 6%polyacrylamide/8 M urea gel.Protected regions were identified by comparison with the sequence ladders.Radioactive species were detected by autoradiography.8.Murine infection modelOvernight cultures of WT and?toxR strains were washed twice with PBS buffer（pH 7.2）and serially ten-fold diluted with PBS.Selected dilutions were plated on HI plate to determine the colony forming unit（CFU）.For each tested strain,0.1 mL of 10⁸ CFU/mL bacteria suspension was inoculated intraperitoneally into 15 female BALB/c that were 25-28days old.The numbers of mice killed daily were monitored and recorded to calculate the survival rate.9.Cell culture and cytotoxicity assayHeLa cells were maintained in DMEM containing 10%FBS at 37oC and seeded in a 96-well plate and incubated for 16 hours at 37°C,5.0%CO₂ prior to infection.HeLa cells were infected with 10⁶ CFU of bacteria for 3 h at 37°C,5.0%CO₂ at a multiplicity of infection（MOI）of 2.5.After infection,the release of lactate dehydrogenase（LDH）into the medium was quantified with a CytoTox96 kit according to the manufacturer’s instructions.Uninoculated DMEM was added to wells containing the uninfected HeLa cells and the maximal LDH release condition used as controls.The percentage cytotoxicity was then calculated.Results1.ToxR activates the transcription of calR directlyThe results of the qRT-PCR and primer extension indicate that the mRNA level of calR decreased inΔtoxR relative to WT.The results ofβ-galactosidase activity detection in LacZ fusion strains shows that the calR promoter activity significantly decreased inΔtoxR relative to WT.The EMSA result shows that ToxR_his6 was able to bind to the upstream DNA fragment of calR promoter,while the DNase I footprinting assay shows that ToxR_his6protected a single DNA region from 286 to 257 bp upstream of calR against DNase I digestion considered as the ToxR site.2.CalR represses the transcription of toxRThe results of qRT-PCR andβ-galactosidase activities analysis in LacZ fusion shows that the mRNA level of toxR was greatly decreased inΔcalR relative to WT which confirmed the negative correlation between toxR and CalR transcription.The primer extension assay detected a single transcription start site at 101bp upstream of toxR,and its transcriptional activity is under the negative control of CalR.The EMSA results shows that CalR_his6 binds to the upstream DNA fragment of toxR in a dose dependent manner.DNase I footprinting results shows that CalR_his6 protected two different DNA regions upstream of toxR against DNase I digestion that were considered as the CalR sites indicating CalR represses the transcription of toxR in a direct manner.3.Autoregulation of CalRThe primer extension experiment detected a single transcription start site located 156 bp upstream of calR which is under the negative control of CalR.The LacZ fusion result shows that the promoter activity of calR is significantly enhanced inΔcalR than that in WT suggesting a negative correlation between CalR and its own gene transcription.The EMSA and DNase I footprinting assay results shows that CalR_his6 protects a single DNA region from 121 bp to 182 bp upstream of calR against DNase I digestion in a dose dependent manner indicating the direct binding activity and the negative autoregulation of CalR respectively.4.Autoregulation of ToxRThe primer extension assay detected a single transcription start site at 101 bp upstream of toxR which is under the negative control of ToxR.The LacZ fusion assay results shows a significantly enhanced promoter activity inΔtoxR compared to the WT,therefore suggesting a negative correlation between toxR and its own gene transcription.The EMSA analysis shows that ToxR_his6is6 is able to bind to the upstream DNA fragment of its own DNA in a dose dependent manner,but the ToxR_his6 proteins could not bind to the 16S rRNA gene fragment.The DNase I footprinting investigation shows ToxR_his6 protected two different DNA regions upstream of toxR against DNase I digestion,which indicates that ToxR represses the transcription of its own gene in a direct manner.5.ToxR is required for virulence and inhibits the cytotoxic activity against HeLa cellsThe virulence of V.parahaemolyticus ToxR was evaluated using the murine infection model.The mice infected withΔtoxR and PBS（negative control）,led to no death compared with the WT that shows a significantly reduced survival rate of 15%from day 1,indicating ToxR may be required for lethality in mice.Further,the cytotoxicity against HeLa cells infected withΔtoxR/pBAD33 significantly increased than that of WT/pBAD33 orΔtoxR/pBAD33-toxR.In addition,there was no significant change betweenΔtoxR/pBAD33-toxR and WT/pBAD33 confirming that V.parahaemolyticus ToxR may act as a repressor of the cytotoxic activity against HeLa cells.6.CalR inhibition of the cytotoxic activity against HeLa cellsV.parahaemolyticus cytotoxic activity against HeLa cells was evaluated in terms of the release of LDH from cultured cells.The cytotoxicity against HeLa cells infected withΔcalR/pBAD33 significantly increased than that of WT/pBAD33 orΔcalR/pBAD33-calR,while there was no significant change betweenΔcalR/pBAD33-calR and WT/pBAD33confirming that V.parahaemolyticus CalR can act as a repressor of the cytotoxic activity against HeLa cells.7.CalR directly represses the transcription of T3SS1 genesThe three operons vp1700-1688（exsBAD-vscBCD）,vp1667-1655 and vp1687-1686 from the V.parahaemolyticus T3SS1 locus were selected,and the first genes（exsB,vp1667,and vp1687）of each of the operon was subjected to the qRT-PCR,primer extension,LacZ fusion,EMSA and DNase I footprinting experiments.The qRT-PCR results indicate that the mRNA level of each target gene greatly increased inΔcalR relative to WT.The primer extension assay disclosed a single transcription start site for each of the three operons,and their transcription activities are under the negative control of CalR.The LacZ fusion results shows that CalR represses the promoter activity of the above genes.The EMSA experiment shows that CalR_his6 is able to bind to each of the above genes suggesting a negative regulation of each target gene by CalR.The DNase I footprinting shows CalR_his6 protected two different DNA regions upstream of vp1667 against DNase I digestion that are considered as the CalR sites,but in both exsB and vp1687,a single DNA region upstream is protected against DNase I digestion.8.ToxR directly represses the transcription of T3SS1 genesThe T3SS1 related genes exsB,vp1667,and vp1687 were selected for the qRT-PCR,primer extension,LacZ fusion,EMSA and DNase I footprinting experiments.The qRT-PCR results indicated that the mRNA level of each target gene was greatly increased inΔtoxR relative to the WT.The primer extension assay disclosed a single transcription start site for each of the three operons and their transcription activities were under the negative control of ToxR.The LacZ fusion results shows that the promoter activity of each of the three operons inΔtoxR is much higher relative to that of the WT.EMSA results shows that ToxR_his6is6 is able to bind to the target DNA fragments of both vp1687 and vp1667 in a dose dependent manner but could not bind to exsB.The DNase I footprinting shows ToxR_his6 protected a single DNA region from 159 to 112 bp and 109 to 85 bp upstream of vp1687 and vp1667 respectively,against DNase I digestion considered as the toxR sites.Conclusions1.V.parahaemolyticus ToxR,activates the transcription of calR directly;2.CalR directly represses the transcription of toxR by binding to the upstream DNA fragment of toxR in V.parahaemolyticus;3.CalR and ToxR negatively autoregulate their own genes;4.ToxR is essential for lethality in mice and represses the cytotoxic ability of V.parahaemolyticus against HeLa cells;5.CalR may repress the cytotoxic ability of V.parahaemolyticus against HeLa cells by directly regulating the expression of the T3SS1 related genes exsB,vp1667,and vp1687;6.Vibrio parahaemolyticus CalR can directly repress the transcription of the three selected operons of T3SS1;7.ToxR can directly repress the transcription of T3SS1 related genes exsB,vp1667,and vp1687.