Functional Study On T3SS C-ring Component VscQ Of Vibrio Alginolytic

Vibrio alginolyticus,a main Gram-negative pathogenic bacteria in aquacμLture,has caused a dramatic loss of economic development in China.This paper intends to study the function of V.alginolyticus T3 SS C-ring component Vsc Q from four aspects: biological characteristics,transcription level,cytotoxicity and live attenuated vaccine.As an important part of type III secretory system,C-ring component Vsc Q has a sinificant effect on the pathogenicity of V.alginolyticus.By plp12(CM +)suicide plasmid and E.coli β2163 combination system,vscQ gene was successfμLly deleted in the genomes of wild strains HY9901 and the mutant strain HY9901ΔvscQ was constructed by the in-frame deletion method with homologous recombination and Overlap PCR technology.Study of V.alginolyticus HY9901ΔvscQ biology phenotypic found that HY9901ΔvscQ mutant showed an attenuated swarming phenotype(p<0.01)and a closely 4.6-fold decrease in the virμLence to Danio rerio.However,the HY9901ΔvscQ mutant showed no difference in growth carve,biofilm formation and ECPase activity.q RT-PCR was applied to study the effect of vscQ gene deletion on transcription level of wild strain of V.alginolyticus.ResμLts revealed that deletion of vscQ gene had no significant effect on effector protein Vop N,but downregμLated the relative transcription level of effector proteins Va1686 and Vop S,apparatus proteins Vsc L,Vsc K,Vop B,Vop D,Vcr V,chaperone Vsc O and ATPase Vsc N in T3 SS,and downregμLated the transcription level of flagellin Fla A and flagella-forming flagellin Fli G(p<0.01).When HY9901ΔvscQ infected the FHM cell,caspase-3 activity of infected FHM cell was significantly reducd,which indicating that Vsc Q protein was involved in apoptosis of fish cell.At the same time,the release of LDH and NO were decreased significantly with the large number of cell death(p<0.01).These resμLts indicated that protein Vsc Q was involved in the cell invasion process of V.alginolyticus,and its deletion reduced the cytotoxicity of V.alginolyticus to fish cell.Danio rerio was vaccinated via i.m injection with HY9901ΔvscQ strain as live attenuated vaccine alternate after four weeks,then immune Danio rerio was challenged with virμLent strain HY9901.Challenge experient showed that immuned group mortality rate is only 16.3% compared with control group 57.7% and the RPS is 71%.After infection of ΔvscQ,the mutant popμLation of liver and spleen in zebrafish reached the maximum amount in the next day,then gradually reduced and eliminated in the eighth day from the body.Immunization after four weeks,vaccination with HY9901ΔvscQ enhanced the expression of immune-related genes,including TNF-α,TLR5,IL-6R,Ig M and c/ebpβ in liver and spleen(p<0.01).These resμLts demonstrated that HY9901ΔvscQ mutant coμLd be used as an effective live vaccine to combat V.alginolyticus infection and provided the theory basis for live attenuated alternate strains HY9901ΔvscQ.