| Aeromonas veronii has gradually become a human-beast-aquatic comorbidity pathogen.In recent years,the prevalence,virulence or drug resistance of A.veronii showed an increasing trend.It is difficult to explain the complex pathogenic mechanism by routine pathogenic analysis.Therefore,the study on unknown virulence factors is particularly important.It has been proved that the preA gene is a periplasmic protein control gene involved in the global regulation of the strain,and dotU is an intimal protein component control gene of the VI-type secretion system,and both of them are novel unknown virulence factors.However,there is no report available on whether A.veronii has similar functions.Therefore,the study on these two virulence factors will help clarify the complex pathogenic mechanism of A.veronii.In this experiment,the homologous arm sequence of the preA gene of A.veronii TH0426 strain was amplified,and the deletion mutant ?preA and complement strain C-preA was successfully constructed by co-culture so as to promote homologous recombination.Moreover,the results of genetic stability test showed that the deletion strain ?preA and the complementary strain C-preA had good genetic stability and could inherit steadily for more than 50 generations.The physiological and biochemical results showed that deletion strain ?preA became positive for L010-salicyside metabolism,and the complement strain C-preA became negative again.The sensitivity of mutant strain ?preA to carbenicillin and piperacillin decreased.In addition,the results of EPC cell adhesion and invasion test showed that the adhesion and invasion ability of deleted strain ?preA to EPC cells was 2.1 times higher than that of wild strains.The animal pathogenicity test and cytotoxicity test were increased by 7.6-fold and 1.4-fold respectively compared with the wild-type strain.But tolerance test results showed that oxidative stress tolerance of deletion strain ?preA was significantly lower than that of wild-type strain,and the expression of related genes was up-regulated.The results showed that the tolerance of oxidative stress of A.veronii decreased after preA gene deletion,but the virulence showed an increasing trend.Similarly,a gene knockout method was successfully conducted to construct dotU gene deletion strain and corresponding complementary strain with the ability of stable inheritance.Drug sensitivity test and physiological and biochemical results analysis found that there was no significant change in the drug sensitivity and physiological and biochemical analysis of the deletion strain ?dotU,and test results on adhesion and invasion ability to EPC cells showed that the adhesion and invasive ability of deletion strain ?dotU to EPC cells decreased significantly by 1.9-fold.Animal pathogenicity test and cytotoxicity test results showed that animal pathogenicity and cytotoxicity decreased by 50.4-fold and 2.1-fold compared with that of the wild type.Biofilm formation ability test result revealed that the biofilm formation ability of mutant strain ?dotU was enhanced by 2.1-fold.But the relevant tolerance was not significantly changed.The results showed that after the deletion of the dotU gene,the virulence of A.veronii decreased significantly.In summary,this experiment characterized the difference in biological characteristics between the wild strain and constructed the deletion strains ?preA and ?dotU and the corresponding complementary strains C-preA and C-dotU.Our analysis concluded that the high-pH induced periplasmic protein encoded by the preA gene was closely related to the pathogenicity,virulence and stress tolerance of A.veronii.The membrane protein of the type VI secretion system encoded by the dotU gene was closely related to the pathogenicity and virulence of TH0426.The research results in this section will provide some references for further clarifying the complex pathogenesis of A.veronii. |
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