| Edwardsiella piscicida is one of the main pathogens threatening the global aquaculture industry.It can infect a variety of marine and freshwater animals such as flounder,turbot and tilapia,which severely restricts the healthy development of aquaculture.It has been considered as a model organism for studying infection of intracellular pathogens.Type Ⅱ Toxin-Antitoxin（TA）system plays an important role in participating in the process of bacterial pathogens adapting to environmental pressure and infecting the host.It can help pathogens to improve their pathogenicity.At present,the type Ⅱ TA system is widely studied in a variety of clinical pathogens,but the research reports on the type Ⅱ TA system of E.piscicida are relatively rare.In this study,through bioinformatics analysis and co-transcription analysis,we determined that yefM-yoeB of E.piscicida belongs to the type Ⅱ TA system,where yefM is the antitoxin gene and yoeB is the toxin gene.By constructing in-frame deletion mutants of yoeB and yefM-yoeB genes,TX01ΔyoeB and TX01ΔyefM-yoeB,and the complementary strain TX01ΔyefM-yoeBC of yefM-yoeB,the effects of survival ability of yoeB and yefM-yoeB on E.piscicida in H2O2,high temperature and serum were studied.The experimental results showed that the survival rate of the wild strain TX01 in H2O2 was 78.30%,while the survival rate of TX01ΔyefM-yoeB was only 30.31%,which was significantly lower than that of the wild strain,but the survival rate of TX01ΔyoeB was 87.56%,which was no significant difference with TX01.Compared with the wild strain TX01,TX01ΔyefM-yoeB had significantly enhanced high temperature tolerance and antiserum killing ability,and it was not killed in the serum but increased by 20%,while the survival rate of TX01ΔyoeB was 77.52%,no significant difference with TX01.The survival rate of the complementary strain TX01ΔyefM-yoeBC in H2O2,high temperature and serum was not significantly different from that of the wild strain TX01.The above results preliminarily confirmed that yefM-yoeB is related to the stress resistance of E.piscicida.Through testing the biofilm formation ability of TX01,TX01ΔyoeB,TX01ΔyefM-yoeB and TX01ΔyefM-yoeBC,in vivo infection experiment of tilapia,and in vitro cell infection experiment,the role of yoeB and yefM-yoeB in the pathogenicity of E.piscicida was studied.Experiments showed that the biofilm formation ability of TX01ΔyefM-yoeB was significantly enhanced than that of TX01.In vitro infection experiments showed that the ability of bacteria to adhere to host FG cells was enhanced by the deletion of yefM-yoeB.Through the experiment of infecting tilapia to carry out the bacteria transmission and virulence analysis,it was found that compared with the wild strain TX01,the number of bacteria in the spleen of the fish infected with TX01ΔyefM-yoeB was increased,and the mortality rate of the tilapia infected with TX01ΔyefM-yoeB was significantly increased,could reach 100%.However,the number of bacteria in the spleen of fish infected with TX01ΔyoeB did not change significantly,and the survival rate after 20 days of infection was 20%compared with TX01.The complementary strain TX01ΔyefM-yoeBC was not significantly different from the wild strain TX01 in either in vivo or in vitro infection experiments.The experimental results confirmed the role of yefM-yoeB in the pathogenicity of E.piscicida.Throughβ-galactosidase activity experiment and EMSA experiment,the relationship between yefM-yoeB own promoter P345 and yefM-yoeB was analyzed.The results showed that the activity value of DH5α/p SCP345/p T was 2197.07 Miller Units,the activity value of DH5α/p SCP345/p TYefM was 2080.47 Miller Units,which had no significant difference.While the activity value of DH5α/p SCP345/p TYefM-YoeB was significantly reduced,only 8.28 Miller Units.EMSA experiments showed that YefM protein could directly bind to the yefM-yoeB promoter P345.The experimental results confirmed that YefM protein can directly bind to its own promoter P345 and inhibit the activity of promoter P345,while the binding of YoeB protein to YefM protein strengthened the inhibitory effect of YefM protein on promoter P345.In summary,this study reveals that yefM-yoeB of E.piscicida is a type Ⅱ TA system and an important participant in the resistance and pathogenicity of the bacteria.This will help us to further clarify the pathogenic mechanism of the bacteria on fish.It provides new methods and new ideas for the prevention and treatment of fish-killing Edwardsirosis. |
