The Role Of H-type Thioredoxin In The Pathogenesis Of Edwardsiella Piscicida

Thioredoxin(Trx),a highly conserved protein,exists in a variety of organisms and plays an important role in biological antioxidant stress and maintains disulfide bonds to maintain the correct function of proteins.Edwardsiella piscicida is a serious pathogen of fish,but its pathogenesis is still unclear.Genomic analysis showed that Edwardsiella piscicida encoded three kinds of thioredoxin,namely Trx A,Trx C and Trx H,but their biological functions were still unclear.Especially,there is no report on the role of Trx H in pathogenic bacteria.Based on this,the purpose of this study was to elucidate the role of Trx H in bacterial resistance and pathogenicity.By analyzing the sequence and structure of TrxH protein of Edwardsiella piscicida,we found that Trx H had the conserved motif WCXXC and QSTL of H-type thioredoxin,and the secondary structure analysis showed that there were 5 ?-helix and 4 ?-fold in the protein.The expression of trx H gene was studied by quantitative real-time reverse transcriptase-PCR.The results showed that the expression of trx H was significantly up-regulated under oxidative stress,iron deficiency,low p H and infected host cells,and these results suggested that Trx H may play an important role in bacterial adaptation to stress environment and host intracellular environment.At the same time,we found that the expression of trx H was negatively regulated by the iron regulatory factor Fur,a global regulator of Edwardsiella piscicida.This was the first time found that Trx H was regulated by Fur.To investigate the biological role of Trx H,we used homologous recombination technique to carry out a markerless trx H in-frame mutant strain,TX01(35)trx H,was constructed.Compared to the wild type TX01,TX01(35)trx H exhibited markedly compromised growth status,biofilm formation,stress resistance and antiserum bactericidal ability.The results showed that the absence of trx H had no effect on the growth of bacteria,but significantly weakened the resistance of bacteria to iron deficiency,acid environment and oxidation environment,and significantly decreased the ability of biofilm formation and antiserum sterilization.In vivo experiments showed that compared with wild strain TX01,the ability of trx H mutant TX01(35)trx H to infect host tissue was significantly decreased,and the mortality of host was also significantly decreased.We constructed mutant TX01(35)trx HC,a complementary strain of TX01(35)trx H,and found that the stress tolerance and pathogenicity of complementary strains were similar to those of wild strains.This is a study about the function of H-type thioredoxin in the pathogenic bacteria of fish,and the results show that Edwardsiella piscicida Trx H plays an important role in the stress-resistant environment and pathogenicity of the bacteria and is an important virulence factor of the bacterium.The results of this study can help to further understand its pathogenic mechanism,and can provide a reference for the study of thioredoxin in other pathogenic bacteria,also can provide some theoretical basis for the biological control of Edwardsiosis.It lays a foundation for the green,healthy and sustainable development of aquatic industry.