The Effect Of MMR System Key Gene MutS On ROS Resistance In Vibrio Cholerae

Vibrio cholerae is a Gram-negative pathogen,which is curved rod and adhered single flagella,infecting human body usually begins with the oral ingestion of polluted food or water,and results in cholera disease.Cholera is an acute intestinal infection deadly infectious disease.The disease which is old and prevalent sweepingly,is still a public health issue needed to be solved in developing countries.The studies have shown that the ability of V.cholerae strains to infect humans depends on the expression of cholera toxin and toxin-coregulated pilus.Maintaining genomic integrity and stability is important for several bacterial pathogens to infect their hosts.There are several types of DNA damage repair mechanisms in bacteria:the SOS response,base excision repair,base mismatch repair etc.DNA base mismatch repair(MMR)system repairs DNA damage after cell replication,to maintain the fidelity of DNA replication,controling genetic variation.MutS is the key protein to recognize and combine with mismatched base in the MMR system.So MutS defect will have a serious impact on MMR system.When pathogenic bacteria infect the host,the host cell will produce ROS(Reactive oxygen species)to kill incursive bacteria by destroying their membrane lipids,proteins,DNA and so on.Meanwhile,pathogenic bacteria will also rise resistance by producing a variety of enzymes to clear ROS in vivo,and ensure the infection efficiency.We constructed mutS(vc0535)mutant in V cholerae though in-frame deletion to study if mutS affects colonization.It showed that colonization ability of AmutS is better than wild type in adult mouse.If we cleaned up peroxide press in adult mouse,the colonization of?mutS will back to the similar level of wild type strains.Analysis speculates that gene mutS may be related to ROS resistance in V.cholerae.To define role of gene mutS on ROS resistance in V.cholerae,we tested wild type and?mutS mutant through ROS killing assays.The data shows that mutS didn't work on ROS resistance ability.In mutation rate detection,gene mutS mutation has high spontaneous mutation frequency obviously with or without ROS.H2O2 can induce V.cholerae mutation frequency raise in vitro.In competition between ?mutS and WT in vitro,?mutS adapts better in environment change by its higher mutation rate,in order to get better growth than WT along with time gone.And along with continuous ROS selective pressure can enlarge this growth preponderance.To ascertain how gene mutS improve the colonization ability of V.cholerae,we complemented gene mutS in ?mutS separated from adult mouse.Physiologic studies showed that ?mutS didn't have advantage on growth,toxin expression and colonization factors.But?mutS can produce more ROS tolerance strengthened strains.It suggests that those mutations which help V.cholerae to survive quickly in vivo usually sacrifice part ability at a cost.Above all,?mutS has higher mutation rate than WT,which help ?mutS evolve better.When ?mutS and WT compete in vitro,?mutS will have better growth advantage.Meanwhile?mutS can acquire more ROS tolerance strengthened ability in vivo,to enhance its colonization in adult mouse.