The Influence Of Rugose Phenotype On The Adaptability In Interaction Between Vibrio Cholerae And Host

Vibrio cholerae is a Gram-negative and comma-shaped bacteria.It’s the pathogen of cholera,an acute infectious disease.The colony phenotype of Vibrio cholerae is usually smooth.But in some situations,the phenotype will transform to rugose.The main cause of rugose phenotype is the overproduction of VPS(Vibrio polysaccharide).This makes the rugose phenotype strains get the stronger ability of biofilm formation and become more resistant to environmental stress.And the ROS(reactive oxygen species)producing by the host cells is one important environmental stress that Vibrio cholerae need to face.mutS is key gene of MMR(DNA mismatch repair system).In previous work of our laboratory,△mutS mutants had a competitive advantage over WT in high ROS level mouse intestine,And many △mutS mutants isolated from mouse intestine showed phenotype change(from smooth to rugose).We do many experiments to investigate the relationship between the phenotype change and ROS resistance.Then we find the ratio of phenotype change in high ROS level mouse intestine is higher than that in low ROS level mouse intestine.It suggests that rugose phenotype can help Vibrio cholerae survive in high ROS level intestinal environment,and increase the ROS resistance of Vibrio cholerae in vivo.But ROS doesn’t increase the phenotype change ratio in vitro obviously.Next,we do experiments about ΔmutS mutants isolated from intestine directly to further study the cause of the phenomenon above.Firstly,two kinds of different phenotype△mutS mutants(smooth and rugose)are isolated from mouse intestine Next,mutS is complemented(marked as ΔmutS*).Then test the ROS resistance in vitro and CAT(catalase)activity of them.The results show that smooth ΔmutS*mutants have higher CAT activity than WT and △mutS mutants.And they are also more resistant to ROS in vitro.It is same as the result that the △mutS mutants have a competitive advantage over WT in high ROS level mouse intestine.But most of the rugose ΔmutS*mutants don’t show increasing CAT activity and resistance to ROS.Because the rugose phenotype strains have stronger biofilm formation ability,and biofilm is one strategy of the microorganism to defense environmental stress.So we carry out relevant researches from the perspective of biofilm.Results show that ROS resistance of biofilm-associated cells is higher than that of planktonic cells.When the structure of biofilm is disrupted,the ROS resistance of cells will reduce.This means the higher ROS resistance of biofilm is mainly caused by the physiological structure.The result of the competition experiment in vivo between rugose and smooth strains indicate the rugose strains have a competitive advantage over smooth strains.Then we construct mutS in-frame deletion in △vpsA mutants(responsible for the production of VPS).And do the competition experiment in vivo(ΔmutSΔvpsA and WT,ΔmutS and WT).According to the result,the competitive advantage of △mutS△vpsA mutants(defective in biofilm formation)over WT is smaller than that of ΔmutS mutants.Results of sequencing and reversion(from rugose to smooth)experiment show that the phenotype change may be not strongly associated with the mutation of hapR and vpvC.In the reversion experiment(from rugose to smooth)of rugose ΔmutS*,the reversion rate in aerobic is higher than that in anaerobic.It means the lacking of oxygen in the intestine can induce the phenotype change.It’s likely reversible phenotype change.Above all,when Vibrio cholerae encounters ROS in the intestine,it will mutate in CAT activity on the one hand;on the other hand,its phenotype will change(from smooth to rugose)to resist ROS.The increasing biofilm formation ability of rugose strains can enhance ROS resistance by biofilm physiological structure.It won’t increase individual cells’ ROS resistance.This phenotype is reversible.Except for increasing Vibrio cholera’s ROS resistance directly,it also can enhance ROS resistance resulting from gene mutation to enhance Vibrio cholera’s adaptability in the interaction between Vibrio cholerae and host.Furthermore,it will assist Vibrio cholerae to colonize in small intestine.The experiments mainly find two kinds of ROS resistance mechanisms spontaneously generated by Vibrio cholerae when exposed to oxidative stress caused by ROS in vivo.In this experiment,we delete the mutS to increase the spontaneous mutation rate.Then make△mutS mutants live under environmental stress to enrich the mutations which are benefited for surviving.Next,we complement the mutS for further research of mutations in genes or phenotype.This experimental model can be used for the study of pathogens under specific conditions,as well as for research under multiple complex conditions.