Deletion Of The AscP Gene In Aeromonas Veronii TH0426 Strain And Preliminary Study On Its Gene Function

Aeromonas veronii is a kind of pathogenic bacteria that can cause diseases to human beings,animals and aquatic organisms.The diseases caused by A.veronii have the characteristics of rapid oneset,strong infectivity and high mortality.In recent years,the outbreak of A.veronii has caused great economic losses to aquaculture and seriously restricted the development of public health.According to the epidemiological investigation of A.veronii,the virulence,harmfulness and drug resistance and of this pathogen are increasing day by day,and the reports of its cases are gradually increasing.At present,the research direction of A.veronii is relatively single,which is mostly limited to its isolation,identification and drug sensitivity detection.However,the research on its pathogenic mechanism and virulence factors is relatively rare,which limits the research progress of this bacterium to some extent,and at the same time,it also makes some researchers realize that the research on its virulence factors is more important.Based on the previous research results of our research group,the ascP gene which may be related to the virulence of A.veronii TH0426 was screened out.This gene was annotated as a protein gene which can regulate the needle length of type three secretion system(T3SS)device in A.veronii.To understand the specific function of ascP gene in A.veronii and whether it is related to pathogenicity,In this study,suicide plasmid(pRE112)-mediated homologous recombination technique was used to delete the ascP gene in A.veronii TH0426,and the deleted strain △ ascP was successfully constructed,then,its complementary strain C-ascP was constructed by using broad-spectrum host plasmid(pBBRl-MCS),and the related biological characteristics of the deleted strain,complementary strain and wild strains were analyzed.According to the detection and analysis of drug sensitivity test,Compared with wild strains TH0426,the drug sensitivity of deletion strain △ ascP to the cefalexin,carbenicillin and ampicillin increased;The physiological and biochemical results showed that the metabolism of L-arginine-AMC was negative by the deletion strain △ ascP,while the complementary strain C-ascP was positive.The results of athletic ability test showed that the athletic ability of deletion strain △ ascP was significantly lower than that of the wild strains(p<0.01).The results of cell adhesion and invasion showed that the adhesion and invasion ability of deletion strain △ascP to EPC cells was 0.82 times lower than that of wild strains.The results of cytotoxicity and animal pathogenicity test showed that the cytotoxicity of deletion strain △ascP to EPC cells decreased by 2.91 times(1h)and 1.74 times(2h)compared with wild strains,and the median lethal dose to Carassius auratus increased by about 19.94 times.The detection result of that expression amount of effector protein relate genes show that the deletion of ascP gene makes the expression of related effector factors in A.veronii significantly lower than that in wild strains.In order to further explore the regulatory relationship between ascP gene and pathogenicity of strains and its influence on host inflammatory reaction,the deletion strain △ ascP and wild strains TH0426 were used to infect Carassius auratus.The bacterial load in blood,heart,spleen,liver and kidney,the concentration of related inflammatory factors in serum and the expression level of inflammatory factors in liver and spleen were detected at 12h,24h and 48h after infection.The results of bacterial load in blood and tissue showed that the bacterial load of deletion strain △ascP in spleen was significantly lower than that of wild strains TH0426 after 12 hours infection.After 24h infection,compared with wild strains,the number of bacteria in liver and spleen decreased significantly.After 48h infection,the number of bacteria in blood,liver and spleen was significantly lower than that in wild strains;Compared with TH0426 group,the concentrations of TNF-α,IL-β and IL-8 in serum of Carassius auratus infected with A.veronii were detected by ELISA.it was found that the concentrations of TNF-α,IL-β and IL-8 in △ ascP group were significantly lower,which indicated that the gene deletion of ascP affected A.veronii induces the production of inflammatory factors;The expression level of inflammatory factors in liver and spleen of Carassius auratus showed that within 48 hours after infection,the expression levels of TNF-α,IL-β,IL-8 and NF-kB in △ ascP group were all down-regulated in different degrees compared with TH0426 group,but their significant differences were different in different tissues at different time periods.Based on the above research results,ascP gene is related to the toxicity of A.veronii TH0426 strain and its inflammation to the host,and we speculate that the ascP gene can influence the virulence of the strain and the production of related pro-inflammatory cytokines by affecting the secretion of three type secretion system virulence factors of A.veronii strain.The results of this study can provide some theoretical reference for the follow-up study on the pathogenic mechanism of A.veronii.