The Effects Of ExsA,smpB,tmRNA And Hfq On The Virulence Of Aeromonas Veronii And Its Application As A Live Attenuated Vaccine

Aeromonas veronii is an important pathogen of mermaid comorbidity,which causes a large number of deaths of various aquatic products and can also infect human beings.It has great harm to aquaculture industry and public safety and health.Until now,there is no good method for the control of Aeromonas veronii,so the development of its vaccine is imminent.T3SS is a ubiquitous protein secretion mechanism in Gram-negative bacteria.It is also an important way for virulence factors to produce pathogenic effects.ExsA,an AraC-type DNA binding protein,is one of the main activators of T3SS,which is essential for the expression of T3SS-related proteins.Studies have reported that deletion of the core elements of trans-translation,Smpb and tmRNA,and the RNA chaperone Hfq may lead to the formation of attenuated strains.ExsA has been reported to some extent,but it has not been studied in Aeromonas veronii.Therefore,this paper is based on the background of laboratory-preserved Aeromonas veronii C4.Firstly,according to the principle of homologous gene recombination,the exsA knockout strain△exsA was constructed by homologous double exchange method in Aeromonas veronii and the growth curve of mutant strain was determined.The exsA gene knockout had no significant effect on the growth compared with the wild type.Then the expression levels of exsB,exsC,exsD and exsE were detected by real-time fluorescence quantitative PCR(RT-qPCR)after exsA deletion.Although the expressions of exsB,exsC,exsD and exsE were changed,there were no significant differences.It might be that these genes were regulated by more complex cascade modulation instead of a single factor.Subsequently,the intraperitoneal injection of AexsA was conducted to test the toxicity of Oreochromis niloticus and LD50.The toxicity of AexsA was decreased by 28.23 times compared with that of wild type.Likewise,the activities of alkaline phosphatase(AKP)and superoxide dismutase(SOD)were measured in the viscera after the tilapia was challenged at non-lethal concentration of △exsA for one week.The two enzyme activities were increased first and then decreased,suggest that △exsA induced immune response.Therefore,it is considered to further develop into live attenuated vaccine.Except the above experiments,three deletion mutants of smpB,tmRNA and hfq were analyzed for their virulence and virulence-related genes using real-time fluorescence quantitative PCR(RT-qPCR),and the semi-lethal concentration(LD50)of the three mutants were calculated after the intraperitoneal challenge test of tilapia.The virulence of △smpB and △tmRNA and Ahfq were decreased by 2.24,6.32,20 times respectively.So Ahfq also has potential to develop as a live attenuated vaccine.These results will provide a theoretical basis for further development of live attenuated vaccine against Aeromonas veronii.