Functional Characterization Of VPA0961 Regulator In Vibrio Parahaemolyticus

Objective:The VPA0961 mutant strain of Vibrio parahaemolyticus was constructed with homologous recombination and its complement strain with a recombinant plasmid to study the effect of the LysR-type transcriptional regulator VPA0961 on bacterial growth and virulence including the growth curve under different salinity conditions,motility,biofilm formation ability and lethal toxicity in mice,combing with investigation of bacterial phenotype and molecular experiments.The transcriptional regulatory mechanism of T3SS1(vpoN,vpa1687,exsB)VP-PAI(vopB2,tdh2,vtrA),T6SS2(vpa1044)was also performed for further understanding of the pathogenic mechanism of V.parahaemolyticus.Methods:1.Construction of mutant strain:The homologous arm fusion fragment of vpa0961 was amplified by PCR using genomic DNA of V.paraharmolyticus RIMD2210633(WT)as template.The amplified DNA was cloned into the multiple cloning site of the suicide plasmid pDS132.The pDS132 recombinant plasmid was transferred into WT,to prepare a vpa0961 mutant strain of(?vpa0961).2.Construction of the complement strain:The entire coding region sequence of vpa0961 was amplified and directly cloned into the plasmid pBAD33 to obtain a recombinant complement plasmid.The positive recombinant plasmid was then transferred into the mutant strain Avpa0961 to form the vpa0961 complement strain of the mutant(C-Avpa0961).3.Bacterial growth curve:The WT was cultured in HI broth for 13 h,detected the OD600 each hour.The growth curve was plotted with time as the abscissa and OD as the ordinate.4.Swimming:The swimming plate(1%Oxiod trytone,2%NaCl and 0.2%Difco noble agar)was inoculated with 1.5 ?L of the pre-cultured bacteria.The inoculated plates were incubated at 37? for 2 days.The photograph was taken and the diameter of the moss was measured.Each experiment was repeated with at least 4 plates5.Swarming:The bacterial preculture(2 ?L each)was added to the surface of a swarming plate(2.5%Bacto heart infusion,1.5%NaCl and 2.0%Difco noble agar).The culture was allowed to stand at 37? for 2 days,after which photograph was taken and the diameter of the moss was measured.The experiment was repeated with at least 4 replicates6.Transparency experiment:The WT and Avpa0961 was inoculated into 5ml HI broth,shaken at 37? 250 r/min to OD600 of about 1.2,1?L of each bacterial solution inoculated on the same HI plate at 37 ? overnight to observe the difference in phage transparency phenotype between WT and ?vpa09617.Biofilm formation experiment:WT-pBAD33,,?vpa0961-pBAD33,and C-?vpa0961 were cultured in HI medium to log phase and transferred to a 96-well plate,allowed to stand at 30°C for 3 days.The planktonic bacteria were stained with crystal violet after washing off with 30%glacial acetic acid,the colorimetric measurement was carried out at 595 nm after elution with 30%glacial acetic acid8.Hemolytic activity phenotype experiment:WT,?vpa0961 and C-vpa0961 were respectively inoculated to blood agar plate containing human O-type red blood cells,and cultured at 37?.The difference in the size of the transparent hemolysis circle was measured to determine whether VPA0961 regulates the hemolytic activity of V.parahaemolyticus9.Analysis of lethal toxicity in mice:Mice were infected by the intraperitoneal route with WT,?vpa0961,and the PBS was used as the control.The WT strain can cause bacteremia and leads acute death of mice.By plotting survival curves of mice infected with WT,?vpa0961 and PBS,the lethal toxic activity of V parahaemolyticus VPA0961 in mice could be determined10.Cell adhesion assay:Equal amounts of WT-pBAD33,?vpa0961-pBAD33 and C-?vpa0961 strains were added to an equal amount of HeLa cell pre-culture and cultured in an incubator containing 5%CO2 at 37v.After 90 min,the cells were washed three times with PBS and 0.5%of Triton was applied for 10 min.It was then centrifuged for 15 min and the supernatant was discarded.The precipitate was diluted with DMEM and plated to count the number of bacteria adhering to the surface of HeLa cells.By comparing the difference in adhesion rates between the various strains,it was determined whether VPA0961 regulates the adhesion ability of V.parahaemolyticus.11.qRT-PCR analysis of gene expression:Total RNA was extracted from WT and?vpa0961,respectively,and then reverse transcribed into cDNA.Using cDNA as a template and 16S rRNA expression as internal reference,real-time quantitative RT-PCR analysis was performed to study the differential expression of target genes in different strains.Results:1.qRT-PCR analysis results showed that the expression of vpa0961 in V.parahaemolyticus cultured in low salt condition was higher than that in high salt condition.2.Rsults of specific amplification and sequence analysis showed a gene knockout strain of vpa0961(?vpa0961)and the vpa0961 complement strain(C-?vpa0961)was successfully constructed.3.The growth curve detection results showed that the growth of ?vpa0961 in low and high salt condition were similar with the growth of WT.4.The motility analysis revealed that the swimming ability and swarming ability of?vpa0961 were significantly weakened than the WT.5.Transparency experiments showed that there was no significant difference in the transparency between ?vpa0961 and WT colonies.6.The biofilm formation assay showed that the biofilm formation ability of Avpa0961 was lower than the WT.7.Results of hemolytic activity,cell adhesion and the lethal toxicity in mice assays revealed that the hemolytic activity and the ability to adhere to HeLa cells of ?vpa0961 was decreased,while the lethal toxicity in mice of ?vpa0961 was enhanced comparing with the WT.8.qRT-PCR analysis results revealed that VPA0961 positively regulated the expression of flagellar and motile genes including flgM,flab,flaF,lafA,flgB,motY,fliM and fliD.It could enhance the expression of biofilm formation relative gene sypG and T3SS1 genes vpoN,vpal687,exsB and T6SS2 gene vpa1044,but decreased the expression of Vp-PAI genes vopB2,tdh2 and vtrA.9.Results of qRT-PCR analysis showed that there are interaction between VPA0961and Quorum Sensing system central regulators AphA and OpaR.Conclusion:Results of the study show that the expression of the LysR family transcriptional regulator VPA0961 in V.parahaemolyticus was higher in low salt condition.VPA0961 can enhance the bacterial motility,biofilm formation,hemolytic activity and cell adhesion,but decrease the bacterial lethal toxicity in mice.The mechanism of VPA0961 actions may be associated with the up-regulation of the transcription of flagellar genes,T3SS1 and T6SS2 genes,and down-regulation of the VP-PAI genes transcription.VPA0961 may also interact with the Quorum Sensing system central regulators AphA and OpaR.