A Preliminary Study On The Function Of AscO,the Core Component Of The ATPase Complex Of A.veronii Type Ⅲ Secretion System

Aeromonas veronii is an important pathogenic bacteria in aquatic products.Recent research reports show that aquatic diseases caused by Aeromonas seriously restrict the development of aquaculture.A.veronii belongs to Aeromonas,Aeromonas family,and is a typical opportunistic pathogen coexisting with humans,animals and aquatic animals.It was found that type three secretion system（T3SS）originated from flagella system,and ATPase complex played an important role in promoting the secretion of utility protein in T3SS,which was closely related to bacterial pathogenicity.And chaperone protein exists in ATPase complex,which can specifically promote secretion of some effector proteins.Our research group sequenced the whole genome of A.veronii TH0426 in the early stage,in which asc O gene has homology with the structural protein of ATPase complex in other bacteria T3SS,but there are few reports on related studies of A.veronii T3SS.it is of great significance to explore the related functions of asc O gene for revealing the pathogenic mechanism of A.veronii.Therefore,based on homologous recombination method,p RE112 suicide plasmid was used to delete the asc O gene of A.veronii TH0426,which was verified at DNA and RNA levels respectively.the results showed that A.veronii TH0426Δasc O was successfully constructed,and the biological characteristics of A.veronii th0426 and A.veronii TH0426Δasc O were detected.According to the growth curve,there is no significant difference between A.veronii TH0426 and A.veronii TH0426Δasc O,and the results of colony morphology,hemolytic activity,exercise ability and drug resistance are consistent with the growth results,but there is no significant difference between A.veronii TH0426 and A.veronii TH0426Δasc O.According to physiological and biochemical results,the reaction results of A.veronii TH0426Δasc O to malonic acid（MLO）,ribitol（ADO）,CITric acid（cit）andα-ketoglutarate（KGA）were all positive,which was different from that of A.veronii TH0426,which indirectly proved that asc O gene might participate in some metabolic pathways of A.veronii TH0426.In order to further explore the effect of asc O gene on effector protein expression in A.veronii TH0426 T3SS,the expression levels of 11 effector proteins were detected.The results showed that the expression levels of Yop H,Yop O,Yop P,Yop Q and Yop S of A.veronii TH0426Δasc O decreased significantly,while the expression levels of Yop D,Yop E and Yop N increased significantly,which may be related to bacteria adhesion and invasion ability of bacteria play an important role in the pathogenic process.in this study,carp epithelioma cell line（EPC）was used as the test cell to detect the adhesion and invasion ability of bacteria.the results showed that the adhesion and invasion ability of A.veronii TH0426Δasc O decreased significantly.EPC was incubated with A.veronii TH0426 and A.veronii TH0426Δasc O continuously for 4h,and the cell morphology was observed.it was found that when A.veronii TH0426 infected EPC for 2h,the cell appeared pathological changes,while some cells infected with A.veronii TH0426Δasc O showed no cell shedding phenomenon,which indicated that asc O gene and A.veronii TH0426infected EPC for 4h.The lactate dehydrogenase（LDH）is widely used in the detection of cytotoxicity,and the LDH content has a positive correlation with cytotoxicity.when A.veronii TH0426 and A.veronii TH0426（asc O）incubated EPC for 2h respectively,the LDH of A.veronii TH0426 infection group was significantly higher than that of A.veronii TH0426Δasc O infection group.However,after incubation of EPC for 4 hours,there was no significant difference in LDH content between A.veronii TH0426 infected group and A.veronii TH0426Δasc O infected group,which may be related to the regulation mechanism of T3SS effector protein.EPC apoptosis induced by A.veronii TH0426 and A.veronii TH0426Δasc O was observed by Heochst staining.the results showed that deletion of asc O gene could significantly reduce EPC apoptosis induced by A.veronii TH0426.Bacterial load of viscera was detected by in vivo test.Carassius auratus was used as experimental animal,and A.veronii TH0426 and A.veronii TH0426Δasc O with the same concentration were injected intraperitoneally.After infection for 48 hours,bacteria appeared in the blood of A.veronii TH0426 group,and the number of bacteria was significantly higher than that of A.veronii TH0426Δasc O group,bacterial load in liver of A.veronii TH0426 group was the highest,but the bacterial load in liver of A.veronii TH0426Δasc O group was significantly lower than that of A.veronii TH0426 group,which indicated that asc O gene was closely related to invasive ability of A.veronii TH0426,which was consistent with the results of cell adhesion test.To test the effect of A.veronii TH0426 on crucian carp cell apoptosis,A.veronii TH0426 and A.veronii TH0426Δasc O were injected intraperitoneally at the same concentration,and the liver,spleen,kidney and intestinal tissues were taken at 12h,24h and 48h,respectively.the expression levels of inflammatory factors and apoptosis-related genes were detected by q PCR.The result shows that A.veronii TH0426 can significantly increase cyt C gene expression level,induce downstream Cas3 and Cas9 expression to induce apoptosis,and at the same time increase the expression levels of inflammatory factors TNF-αand IL-1β,which leads to inflammatory reaction in the body,which is consistent with the results of apoptosis test,indicating that A.veronii TH0426may play a pathogenic role by inducing apoptosis.The result of pathogenicity showed that LD₅₀of A.veronii TH0426Δasc O was about 53 times higher than that of A.veronii TH0426.The LD₅₀of A.veronii TH0426Δasc O was about 15 times higher than that of A.veronii TH0426,which indicates that asc O gene is closely related to the pathogenicity of A.veronii TH0426.To sum up,A.veronii TH0426Δasc O was successfully constructed in this experiment.through in vitro experiments,it was found that deletion of asc O gene reduced the adhesion and toxicity of A.veronii TH0426 to EPC,and at the same time reduced the apoptosis level of EPC induced by A.veronii TH0426.In vivo experiments showed that the deletion of asc O gene resulted in a significant decrease in the pathogenicity and invasiveness of A.veronii TH0426,and at the same time reduced the apoptosis level and inflammatory reaction induced by A.veronii TH0426.Combined with the expression level of T3SS effector,it was found that the expression levels of Yop O,Yop H and Yop Q were significantly down-regulated due to the deletion of asc O gene.it is speculated that asc O gene is closely related to the secretion of T3SS effector protein.the results of this experiment have guiding significance for revealing the role of A.veronii T3SS in the pathogenic process,and lay a foundation for revealing the pathogenic mechanism of A.veronii subsequently.