Studies On The Antioxidant Function Of Type Ⅵ Secretion System In Yersinia Pseudotuberculosis

Bacterial Type VI Secretion System is a new secretion system which was found inGram-negative bacteria in recent years. T6SS is involved in virulence, anti-virulence,bacteria-bacteria interaction and environmental adaptability.We havestudied the T6SS4in the entericpathogen Yersinia pseudotuberculosis.Westudied its antioxidant function and regulation mechanism, and preliminarily illuminated itsantioxidant mechanism. The specific research results are as follows:1. We found that there is a binding site of the antioxidant regulatory protein OxyR in theT6SS4promoter region by the analysis of the promoter of T6SS4in Yersiniapseudotuberculosis. By in-vitro EMSA experiment,we affirmed the OxyR can directly bind toT6SS4promoter, and found the binding site sequences by footprint assay. We confirmedT6SS4was positively regulated by OxyR using LacZ reporter gene and Western blot.2. Expression of T6SS4gradually increased in response to the increased H2O2concentration, and the dose-dependent induction of T6SS4by H2O2challenge was apparentlymediated by OxyR, as its expression was not altered inΔoxyR.3. Through the construction of mutant strains of T6SS4’s core genes clpV4, hcp4, vgrG4and icmF4, we found a significant reduction of survival rate in various mutant strains exposedto H2O2compared with wild type. We also found the content of intracellular hydroxyl radicalsand damage degree of DNA in T6SS4mutant strains were apparently higher than in the wildtype strains by detecting hydroxyl radicals levels of different strains using HPF probe andbacterial DNA damage degree using TUNEL under oxidative stress.4. We discovered the survival rates of T6SS4mutant strains reduced obviously comparedto wild type under many primary and secondary oxidative stresses, including Cumenehydroperoxide,Gentamicin, Diamide, cadmium(II) and42℃.In conclusion, in this research, we discovered that under primary and secondaryoxidative stresses, OxyR in Yersinia pseudotuberculosis had the most sensitive response, andthen bound to the T6SS4promoter to increase the expression of T6SS4, ultimately causing the reduction of hydroxyl radicals level and DNA damage degree under oxidative stress.