The Study Of Physiological Regulation Fators In Vibrio Cholerae That Affect Colonization In Host

Vibrio cholerae can cause cholera,a severe disease which symptom is accurate diarrhea and dehydration.Even people could die for this.Since eighteenth century,there were seven pandemic of cholera outbreak and now it still threaten human health.People get infection by drinking containmined water and food.Bacteria flow through gastric in stomach and mucus in intestine to find a niche to colonize.Once since bacterium arrive at intestine and the virulence cassette start to be induced.Toxin-coregulated pilus?TCP?and cholera toxin?CT?are the main factors can cause virulence expression and colonization.Colonization is a complex and multiplied progress and need lots of genes involvement,Among this bacteria and its interaction with host play important roles in this process.Host environment is a diverse condition for pathogens.On the one hand,virulence could be induced by host signals.As a result,toxin-coregultated pilus is being produced.Pathogens could colonize and reproduce.At the meanwhile,the host environment could also be a stress condition for pathogens at the presence of bactericide factors such as reactive oxygen species,poor nutrient and competition with other host bacteria.Since that,bacteria have evolved a series of mechanisms to protect themselves and colonize in host such as growing biofilm,antioxidation response and toxin-antitoxin systems.In Vibrio cholerae,there are thirteen toxin-antitoxin systems?TA systems?belong to four families on small chromosome.We demonstrated six of the seven RelBE systems could encode functional toxins that inhibit cell growth.Then we investigated 13 single TA system deletion mutants on colonization,biofilm formation,antibiotic resistance and sensitivity to host-like factors.We successfully got thirteen mutants and found four TA systems may contribute to pathogen colonization and biofilm formation.RelBE-4 and RelBE-7 deletion strains showed attenuated colonization ability.In contrast,ParDE-2 and ParDE-3 deletion strains showed better colonization ability than wild type.TcpA expression level was measured in these four mutants but found nothing significant difference compared with wild type.It may suggest these four systems influence pathogenesis by other unknown mechanisms.To test these mutants on biofilm formation ability and found RelBE-1,RelBE-4,RelBE-7 and Phd/Doc mutants showed less biofilm during cultivation.All these four TA expressions were higher in biofilm cells than in planktonic cells that may indicate their involvement in biofilm formation progress.It also could be a reason for defective colonization of RelBE-4 and RelBE-7.We tested mutants on MIC to ampicillin,tetracycline,chloramphenicol,gentamicin,and nalidixic acid but found nothing significant changes for these mutants.For host-like factors,only RelBE-4 mutant showed more sensitivity to H₂O₂ than wild type.All thirteen mutants did not show any sensitivity change to curde bile and bile salts.According to the complex regulation of TA systems on gene level and function complentary of TA systmes,phenotypes could not be detected under these condtition.In future,multiple TA system deletion would be studied.In Vibrio cholerae,there are several T4P including TcpA,MshA and PilA belong to T4P.We tested survival rate of T4P mutants under H₂O₂ treatment in AKI medium in vitro and found that tcpA mutant was sensitive to H₂O₂ compared to wild type,as well as pilA mutant.Complementary of TcpA showed recovery of resistance to H₂O₂.We assumed TcpA protein might function as a catalase or antioxidation protein.Bur purified TcpA protein could not help tcpA mutant survive better under H₂O₂.We measured the catalase gene expression in tcpA mutant and found lower expression in mutants.It may indicate probably tcpA can act as a transcription inducer for antioxidation progress.We test TcpA protein in cytosol protein and membrane protein under H₂O₂ exposure.H₂O₂ did not affect TcpA protein production,secretion and localization on membrane.It showed the same amount in cytosol protein and membrane protein by western blot compared normal condition.We found the cysteine 120 and cysteine 186 are very critical for TcpA structure since once one of these was mutated,TcpA protein could not be detect anymore.It may indicate these two cysteines play very important role for its structure stability.It also may indicate that thiol disulfide bond formation would be critical for TcpA stability.This founding suggested that as virulence factor TcpA also act as an antioxidation sensor.The defective for antioxidation could also be a reason to defective colonization for TcpA mutant.It combines pathogenesis with stress response.These results may provide experimental foundation for further exploration.We investigated the interaction of mucin and vibrio polysaccharide?VPS?may influence colonization.In this study,we found most Vibrio cholerae strains with different serotype display promoted motility in mucin.We have also found surrounding medium would influence motility in mucin.Previous study showed vibrio polysaccharide might influence motility in mucin.We tested VPS overexpression strain in colonization assay,and found it showed decreased ability in small intestine.We assume the decreased ability due to the slow motility passing through mucin into epithelium and bacteria could not colonize effectively.It is a reflection of bacteria and the interaction with its host.VPS production is also regulated by mucin and quorum sensing system,so bacteria may regulate its motility passing through mucus by itself and quorum sensing systems when it reaches into host intestine.Taken all together,we studied different aspect of interactions with its host for Vibrio cholerae.It is concluded to be a complex regulation system including varieties of physiological actions.One system may control several physiological actions contributed to colonization.Toxin-antitoxin systems influence colonization and biofilm formation;at the meanwhile it may need host environment to active its expression.TcpA can act as a virulence factor and also may response to oxygen status.It connects virulence expression with oxygen status.Vibrio polysaccharide does not only affect cell adhesion in intestine by influencing biofilm formation but also affect bacteria motility in mucin.This may cause lower colonize defective for Vibrio cholerae.The aim of these studies is to illustrate the factors may affect on pathogenesis and get more details about how Vibrio cholerae colonize and live in host environment.It may help us to understand more about the interaction between bacteria and host,which could help us to invent new strategies and methods to prevent and control cholera disease.