Studies On The Regulation Mechanisms Of T6SS4 And Related Acid Resistance Systems In Yersinia Pseudotuberculosis

As an enteric pathogen,its transmission between humans and animals is normally through the ingestion of contaminated water or food.Gastric acidity is a barrier through which all microbial food-borne pathogens must pass.Acid resistance mechanism of enteric pathogen would be possible to provide a theoretical basis for the control of infection and spread,and it’s also one of the problems of today’s research areas focus on.Yersinia pseudotuberculosis,one of the three pathogenic Yersinia species that cause infections in humans,is an important zoonotic enteric pathogens and an important model microbe.Type VI secretion system(T6SS)is a complex macromolecular apparatus found in more than 25% of sequenced Gram-negative bacteria genomes,ranging from pathogens to environmental species.It has been found that T6 SSs have a variety of functions.In addition to the pathogenicity of pathogens,it’s also found that T6 SS play an important role on biofilm formation,endophytic bacteria symbiosis with the host,bacteria-bacteria interaction,bacterial adaptability to the environment and so on.There are four T6 SS gene clusters in Yersinia pseudotuberculosis genome,which likely confer distinct functions for specific niches in the lifecycle of the bacterium,thus representing excellent models for the study of the potentially versatile function of T6 SSs.Currently T6SS4 has been reported that play an important role in the process of acid-resistant,but research is still relatively small,its regulatory mechanism needs further study.In this study,Yersinia pseudotuberculosis T6SS4 as the research object,through a variety of molecular biology methods,analysis and study of its regulatory mechanism.1.RNA-Seq results from the wild-type strains and △rovM mutant revealed a surprisingly widespread effect of Rov M on levels of gene expression.RovM involved in the regulation of many different metabolic pathway,with a variety of biological significance,it is a global transcription factors.The most important finding is that the genes encoding T6SS4 core components were significantly down-regulated in the △rovM mutant.It’s shown that RovM plays an important role in regulating the expression of T6SS4 for the first time.In addition,genes encoding urease system,which is important in acid resistance progress,were significantly up-regulated in the △rovM mutant.Thus,RovM has great significance in the process of bacterial acid-resistant.2.Through qRT-PCR detection of the core components of T6SS4 gene cluster,the promoter activity analysis and the detection of Hcp expression and secretion,we confirmed that RovM positively regulate the expression of T6SS4.EMSA and Footprint experiments were used to determine the interaction of the RovM protein with the T6SS4 promoter region.Besides,RovM is likely to be one of the key factors that affect the growth phase-dependent expression of T6SS4.In addition,CsrA perhaps positively regulate the expression of T6SS4 through RovM,but also directly binding to the mRNA to inhibit the expression T6SS4.3.Through qRT-PCR detection of the core components of T6SS4 gene cluster,the promoter activity analysis and the detection of Hcp expression and secretion,we confirmed that RelA positively regulate the expression of T6SS4.EMSA and Footprint experiments were used to determine the interaction of the RovA protein with the T6SS4 promoter region.The complementary experiments of double mutant △rovA△relA,△rovA△relA and △rovA△rov M respectively,indicated that RelA regulates the expression of T6SS4 through RovM and RovA in response to the environmental nutrient conditions,which activates the expression of T6SS4 in nutrient-limited conditions.4.Through the study on the regulation mechanism of RovM in acid resistance process,we found that RovM coordinated regulate various acid resistance systems-T6SS4,AR3 and urease system,in response to environmental stimuli.This regulatory mechanism makes arginine-dependent AR3 and urea-dependent urease system can highly expressed under nutrient-rich conditions,while enhances the bacterial acid survival.Conversely,when nutrients are limited in the environment,the amount of RovM increased,which results in intensive repression of AR3 and urease,but high expression of T6SS4.Thus,although expression of the more efficient AR3 protection system was abolished under this condition,the less efficient T6SS4 system provides protection to the bacterial cells,albeit with lower efficiency.Hence,our data revealed a novel strategy utilized by Y.pseudotuberculosis for coordinated regulation of the expression of different acid resistance systems in response to the nutritional status in the environment,which is beneficial for adaptation of pathogenic bacteria to different acid conditions encountered in its life cycle.