Function Of ATPase And Its Effector Hop In Vibrio Splendidus Type Ⅲ Secretion System

Vibrio splendidus is one of the most important opportunistic pathogens that could infect Apostichopus japonicus.Skin ulceration syndrome which occurrs in A.japonicus is the characterized by large area of infection and high mortality.Type Ⅲ secretion system（T3SS）from pathogenic bacteria secretes virulence factors,which can be directly transported into the host cell through the needle complex of T3SS,thereby exerting the functions of lysing cells and interfering with cell functions,eventually,leading to host disease.However,there is no functional studies on the secretion system of V.splendidus and its effectors.In this study,the identification and function of T3SS ATPase and its effectors in V.splendidus were studied,and the results were as follows:1.vscn with ATPase activity was cloned and expressd in Escherichia coli.In order to explore the correlation between Vscn and pathogenicity,the△vscn of V.splendidus was constructed.The deletion of the vscn gene had no effect on the growth of V.splendidus.The results of two weeks immersion infection experiments showed that the LD₅₀ of△vscn was 1.8×10¹⁰ CFU/ml compared to a relative lower concentration of 2.5×10⁷ CFU/ml in wild type.It indicated that Vscn played an important role in the pathogenesis of V.splendidus.2.Identification and characterization of the T3SS effector Hop of V.splendidus.After a exposure to wild type and△vscn mutant with coelomic fluids of A.japonicus,the hop gene in the△vscn mutant was hardly expressed.It indicated that Hop may be a T3SS effector.After incubation for 3 hours with the coelomocytes of A.japonicus to two strains which have a DTAF fluorescent dye tag,the wild type was more significantly enriched on the cell surface than the△vscn mutant.The recombiant protein was obtained by a construction of the p GEX-4T-2-hop in E.coli and the antibody was prepared by the injection to mouse.FISH（Fluorescence in situ hybridization）experiments showed that the wild type V.splendidus could secrete Hop into the coelomocytes of A.japonicus,but the Hop was not detected in coelomocytes challenged by the△vscn mutant,which indicated that Hop was an effector of the T3SS of V.splendidus.3.Functional study of the T3SS effector Hop of V.splendidus.The study upon Hop was performed in three aspects including its own expression,regulation of host immune gene expression and cytotoxicity.First,Hop has cytotoxicity on coelomocytes.The cell viability of coelomocytes decreased to approximately 42%,26%,32%,30%and 20%when 30,50,60,80 and100μl of purified r Hop was added,respectively.The expression levels of C1q and caspase were up-regulated 1.83-fold and 2.95-fold after injection into A.japonicus by the active Hop compared to no significant change after stimulation with the heat-denatured Hop protein.The expression of hop was also regulated by host immune factors and the its own cell density.Stimulation with the coelomic fluids of A.japonicus up-regulated the expression of hop to 4.5-fold.ELISA（Enzyme-linked immunosorbent assay）experiment showed that Hop was abundantly secreted in the stage of late logarithmic growth(OD₆₀₀=1.0),which indicated that the secretion of Hop protein was dependent on the density of cells.Our results revealed that vscn gene encoded the ATPase of V.splendidus T3SS and mediated the pathogenicity of V.splendidus.In addition,Hop,which has a plenty of functions such as cytotoxicity and activation of immune gene expression,was the effector of V.splendidus T3SS and can directly enter the coelomocytes of A.japonicus.However,the deletion of the vscn gene prevented Hop from being directly injected into the coelomocytes.The construction of△vscn mutant laid a solid foundation for the subsequent screening and identification of other effectors of V.splendidus T3SS.It provided a new target site for the pathogenicity of V.splendidus and a reference for the development of novel V.splendidus inhibitors.