Deletion Of FeoA And FeoB Gene Of Aeromonas Veronii TH0426 Strain And Its Exploration Function

Aeromonas veronii is an important zoonotic pathogen that widely exists in the aquatic environment.Research has shown that the virulence of this bacterium has shown an upward trend in recent years,which has attracted widespread attention from various countries.However,there is still little research on the virulence of this bacterium.Therefore,studying the virulence gene of this bacterium can provide a theoretical basis for further elucidating the pathogenic mechanism of this bacterium.In this study,we successfully constructed A.veronii TH0426 feoA and feoB gene deletion strains△feoA and△feoB and corresponding complement strains C-feoA and C-feoB by homologous recombination method.The results of genetic stability experiment showed that the deletion strains△feoA and△feoB and the complement strains C-feoA and C-feoB could be stably expressed for 50 generations.The results of drug sensitivity test showed that the deletion strain△feoA was less sensitive to clindamycin and butanicana.The deletion strain△feoB showed decreased sensitivity to gentamicin.The growth curve showed that the growth rates of the deficient strains△feoA and△feoB were significantly lower than those of the wild strains in the logarithmic growth period.The exercise ability results showed that the exercise ability of△feoA and△feoB of the deletion plant was significantly decreased compared with that of the wild plant（p<0.01）.Flagellum staining and electron microscopy showed that the flagellum broke after the deletion of feoA and feoB genes.The expression of flagella-related genes was detected,and the results showed that the expression of related genes was up-regulated.The biofilm formation ability test and scanning electron microscope test showed that the flagella formation ability was significantly decreased after the deletion of feoA and feoB genes（p<0.05）.The results of cell adhesion and invasion experiment showed that the adhesion and invasion ability of△feoA and△feoB of deletion strains were significantly decreased compared with that of wild strains（p<0.01）.The cytotoxicity test results showed that the toxicity of wild strain to EPC cells was 2.20 and 1.97times that of the deletion strain△feoA,and 2.62 and 2.45 times that of the deletion strain△feoB,respectively,after 1 hour and 2 hours of infection（p<0.01）.The results of median lethal dose test of zebrafish showed that LD₅₀ of△feoA of the deletion strain was 100 times higher than that of the wild strain,and the virulence of the deletion strain decreased significantly（p<0.01）.Compared with wild strains,the△feoB decreased by 178 times,and the virulence decreased more significantly（p<0.01）.The bacterial load test results showed that the bacterial content in liver,spleen and kidney of deletion strain△feoA group and deletion strain△feoB group were significantly decreased compared with the wild strain group（p<0.05）.By testing the resistance to H₂O₂,the experimental results showed that the deletion strain△feoA and deletion strain△feoB significantly decreased the antioxidant stress ability.In conclusion,the deletion strain△feoA and deletion strain△feoB as well as corresponding complement C-feoA and C-feoB were successfully constructed in this experiment,and the biological characteristics differences between them and wild strains were analyzed.We speculate that the feoA and feoB genes in the Feo transport system are closely related to the growth and virulence of A.veronii.