Functions Study On T3SS Transposon Gene VopB,vopD And VcrV Of Vibrio Alginolyticus

Vibrio alginolyticus is one of the main pathogenic bacteria that cause bacterial diseases in marine animals,often causing huge economic losses to the aquaculture industry.The type III secretion system(T3SS)of V.alginolyticus is an important pathogenic system.T3 SS is a conservative injection device that can be inserted into the host cell membrane to inject virulent proteins into cells,leading to cell death.In the process of T3 SS injection of virulence protein,the translocation proteins Vop B,Vop D,and Vcr V at the tip of the needle first form pores in the host cell membrane,which is the premise of pathogenesis,however,their role in the pathogenicity of V.alginolyticus remains unclear.In this study,the deletion strains ΔvopB,ΔvopD,ΔvopB-D,and ΔvcrV of V.alginolyticus were constructed by homologous recombination technology,to study the biological characteristics and pathogenicity of each deletion strain.The functions of the transposon genes vopB,vopD,and vcrV of V.alginolyticus were explored through transcriptome,proteome,and metabolome association analysis.The main results are as follows:1.Cloning of transposon genes vopB,vopD,and vcrV and construction of deletion strains ΔvopB,ΔvopD,ΔvopB-D,and ΔvcrV.vopB,vopD,and vcrV genes were cloned and analyzed by bioinformatics.The results showed that the base sequence lengths of each gene were 1 200 bp,1 005 bp,and 1 824 bp,respectively.The amino acid sequences of the three genes were clustered with Vibrio devilish and Vibrio parahaemolyticus.The deletion strains ΔvopB,ΔvopD,ΔvopB-D,and ΔvcrV were successfully constructed by homologous recombination technology.The genetic stability test showed that each deletion strain was genetically stable.2.Biological characteristics of deletion strains ΔvopB,ΔvopD,ΔvopB-D,andΔvcrVThe biological studies of the deletion strains ΔvopB,ΔvopD,ΔvopB-D,and ΔvcrV showed no significant changes in the growth,self-agglutination ability,and extracellular enzyme activity of each deletion strain.Each deletion strain’s swimming and swarming abilities were enhanced,while the virulence,cell adhesion,and antioxidant capacity were weakened.The biofilm formation ability of the deletion strains ΔvopB and ΔvopD was enhanced,while that of ΔvopB-D and ΔvcrV was decreased.The salinity tolerance of deletion strains ΔvopB,ΔvopD,and ΔvopB-D was enhanced,while that of ΔvcrV was decreased.The deletion strains ΔvopB,ΔvopD,andΔvopB-D had enhanced heavy metal tolerance.Except that the glucose of ΔvopB was unchanged and ROS of ΔvcrV decreased,ROS,SOD,CAT,GPX,β-lactamase,peptidoglycan,NADPH,arginine,proline,histidine,glutamine,ATP,and glucose indexes of each deletion strain were all increased.ΔvopB and ΔvopD decreased sensitivity to quinolones and β-lactam cefazolin and cefoperazone.ΔvopB was more sensitive to cefalexin,and ΔvopD was more sensitive to cefuroxime.ΔvopB-D showed enhanced sensitivity to most drugs except penicillins and quinolones.ΔvcrV has enhanced sensitivity to some β-lactam cephalosporins and decreased sensitivity to quinolones.The expression of flagella-related genes fla B,fli G,and drug resistancerelated genes omp U,emr D,vem D,CARB-6,pbp1a/2,and dac A of deletion strainsΔvopB,ΔvopB-D,and ΔvcrV were all up-regulated.The expression of flagella-related genes fla B,fla G,fli G,and drug resistance-related genes omp U and CARB-6 in deletion strain ΔvopD were up-regulated,while fli G,emr D,vem D,and pbp1a/2 were downregulated.These results indicate that vopB,vopD,and vcrV genes are involved in the pathogenicity and multiple physiological functions of V.alginolyticus T3 SS,and have important biological significance.3.Analysis of the immune effect of deletion strains ΔvopB,ΔvopD,ΔvopB-D,andΔvcrV on zebrafishZebrafish were immunized by intramuscular injection of low concentration deletion strains ΔvopB,ΔvopD,ΔvopB-D,and ΔvcrV,and the immune protection rates were 52.9%,70.5%,82.4%,and 64.7%,respectively.Both ΔvopB and ΔvopD immunized groups down-regulated zebrafish liver immune protein genes Ig M,C3 a,inflammatory factor genes TNF-α,TLR5,IL-6R,IL-6,IL-8,IL-1β,C/EPBβ,antioxidant gene Sod1,Cat1,GPx1 a,GSTR,and Lyz genes.In the ΔvopB-D immune group,only Ig M gene expression was up-regulated,while other and other immune and antioxidant-related genes were down-regulated.The expression of IL-1β and C3 a genes was unchanged in ΔvcrV immunized group,and other immune and antioxidant-related genes were down-regulated.The zebrafish liver biochemical and enzyme activity indexes showed that Ig M,GPX,and MDA indexes were all increased in each immunization group.The indexes of IL-6,TNF-α,SOD,GST,and LZM decreased in the ΔvopB immunized group.The TNF-α,SOD,GST,and CAT indexes in the ΔvopD immunization group decreased,while the C3 a and LZM indexes increased.In theΔvopB-D immunization group,the indexes of IL-6 and TNF-α decreased,while the indexes of SOD,GST,and CAT increased.The indexes of IL-6,CAT,and LZM in theΔvcrV immunized group decreased,while the indexes of C3 a,SOD,and GST increased.It indicated that immunizing zebrafish with each deletion strain affected its immune and antioxidant systems.4.Regulation of vopB,vopD,and vcrV on genes related to drug resistance,antioxidation,and motilityTranscriptome,proteome,and metabolome detection of deletion strains ΔvopB,ΔvopD,ΔvopB-D,and ΔvcrV showed that the differential genes of each deletion strain were 2 618,2 053,1 578,and 1 557;the differential proteins were 335,250,283,and545;the differential metabolites were 32,49,77,and 70,respectively.Omics association analysis showed that the deletion of transposon genes vopB,vopD,and vcrV mainly affected the expression of related genes such as β-lactam resistance,bacterial motility,flagellar assembly,antioxidant system,peptidoglycan metabolism,amino acid metabolism,antibiotics synthesis,T2 SS and T6 SS in V.alginolyticus.