Influence Of Intestinal Flora On Vibrio Cholerae Colonization In Vivo

Vibrio cholerae is the pathogen of cholera.Cholera is an epidemic intestinal infection caused by accidental consumption of water or food contaminated with Vibrio cholerae.After infection,watery diarrhea or dehydration cramps,even cause death in some serious cases.On the other hand,Vibrio cholerae will confront a series of stresses from host,such as stomach acid,reactive oxygen species(ROS),reactive nitrogen species(RNS),antimicrobial peptides,gut flora and nutrients competition.Key factors are important to completing the process of infection,involving ToxT and ToxRS,the factors that help Vibrio cholerae colonize;Cholera toxin(CT)that causes diarrhea;HapR,a signaling molecule that regulates group behavior,and some key detoxification enzymes such as HmpA that resist host environmental stress.In recent years,studies have shown that gut flora plays an important role in resisting pathogenic bacterial infections,and gut flora has been applied to clinical treatments including Clostridium difficile infections.However,it is unclear whether the gut flora can affect the colonization of Vibrio cholerae in the host and its mechanism of action.In this study,an adult mice gut colonization model and Real-Time quantitative PCR technology were used to detect faecal flora.This study also clarified that streptomycin treatment is benefit for the colonization of Vibrio cholerae by significantly reducing gut microbes in adult mice.After removing streptomycin,the indigenous bacteria in the intestines of the mice could be recovered.Meanwhile,the colonized Vibrio cholerae was significantly reduced.We collected the stool of 5 volunteers to prepare the transplanted intestine flora(Fecal microbiota transplantation,FMT)(denoted as FMTS).Through the experiment of FMTS in infant mice,it was found that FMTS can significantly inhibit the colonization of Vibrio cholerae in suckling mice.These results indicate that the gut flora in adult mice can not only directly inhibit the colonization of Vibrio cholerae in adult mice,but also clear Vibrio cholerae in adult mice.In addition,the gut flora of human being can also inhibit Vibrio cholerae colonization in suckling mice.First of all,this dissertation explores from the perspective of virulence genes to explores the impact of gut flora on the colonization of Vibrio cholerae in the host,AphB,ToxT and ToxRS which participate in the expression of pathogenic factors Cholera toxin(CT)and toxin co-regulated pili(TCP).The expression of CT and TCP play an important role in the pathogenicity of Vibrio cholerae.Therefore,ΔaphB mutant,ΔtoxT mutant and ΔtoxRS mutant were selected for competitive colonization experiments with the wildtype strain.As a result,compared with WT strains,ΔaphB mutant had no colonization defects in infant mice,ΔtoxT showed attenuated colonization ability,and ΔtoxRS mutant could hardly colonize in infant mice.After transplantation of human gut flora(FMTS),it was found that the colonization ability of ΔaphB mutant,ΔtoxT mutant and ΔtoxRS mutant did not change significantly,which showed that the absence of virulence genes did not influence Vibrio cholerae colonization with the coexistence of gut flora in infant mice.Next,from the perspective of quorum sensing(QS),this paper explores the effect of QS on the colonization of Vibrio cholerae under the pressure of gut flora.We found that ΔhapR mutant,the most important regulatory protein in the QS system of Vibrio cholerae,had no colonization defects in infant mice,but in FMTS mice,it appeared to be defected compared with the wild type.It demonstrated that the quorum sensing system of Vibrio cholerae plays an important role in the successful colonization of the gut flora of infant mice.Besides,in order to investigate the effect of the anaerobic induction two-component control system(ArcAB)on the colonization of Vibrio cholerae under the pressure of gut flora,ΔarcA::Km mutant,ΔarcAC173S::Km mutant and ΔarcAC233S::Km mutant were chosen to competitive colonization experiment with the wildtype strain in infant mice.Results showed that ΔarcAC233S::Km mutant had colonization defects in infant mice,ΔarcA::Km mutant and ΔarcAC173S::Km mutant showed weak colonization disadvantage to WT.Also,in FMTS mice,ΔarcA::Km mutant,ΔarcAC173S::Km mutant and ΔarcAC233S::Km mutant did not chage the colonization ability.As a result,the mutation of arcA did not affect colonization of Vibrio cholerae after the gavage of gut flora.Finally,this paper explores from the perspective of the environmental pressure of reactive nitrogen species(RNS)in the host.hmpA is a key detoxification gene for detoxifying nitric oxide in the host.hnoX and nosP are importance genes for sensing the concentration of nitric oxide in the host.Therefore,We choose ΔhmpA mutant,ΔhnoX mutant and AnosP mutant to do the competitive colonization with the wild type strains in FMTS mice.Results showed that FMTS can recover the colonization of ΔhmpA mutant,however,it did not change the colonization of ΔhnoX mutant and ΔnosP mutant.Accordingly,gut flora may help ΔhmpA mutant to resist RNS stress in infant mice.To further explore the main gut bacteria in FMTS,this study isolated three representative gut bacteria from different human faecal samples,namely five strains of Escherichia coli,and after overnight activation,mix the same amount marked as E.coli group,five strains of Lactobacillus(Marked as Lactobacillus group)and five strains of Enterococcus faecalis(Marked as Enterococcus faecalis group).Through the representative gut flora-suckling animal model experiment,it was found that there was no colonization defect in ΔhapR mutant when the representative gut bacteria was separately inoculated in the infant mice.The defected ΔhapR mutant would appear when mixed bacteria of E.coli,Lactobacillus and Enterococcus faecalis existed in infant mice.This indicated that the cooperation of various gut bacteria resulted in colonization defect of ΔhapR mutant.we found that ΔhmpA mutant had a colonization disadvantage in infant mice and adult mice.What’s more,in adult mice,the disadvantage enlarged along with time.The Escherichia coli,Lactobacillus,and Enterococcus faecalis isolated from stool samples did not change the colonization disadvantage of ΔhmpA mutant in infant mice,but GX69 which was isolated from early stool samples in this laboratory can make up for the disadvantage of colonization of ΔhmpA mutant in infant and adult mice.In addition,we found that GX69 can reduce the inflammatory response produced by measuring the concentration of tumor necrosis factor alpha(TNFα)in the host.It is suggested that the gut flora may affect the colonization of Vibrio cholerae via an immune system.In summary,starting from the research direction of the interaction between the gut flora and Vibrio cholerae,it was found that the gut flora of different species can inhibit the colonization of Vibrio cholerae in the host.Further investigation found that the gut flora may affect the colonization of Vibrio cholerae in the host through quorum sensing system,or it may affect the colonization of Vibrio cholerae in the host by changing the RNS environment in the host.This discovery is of great significance for understanding the pathogenic mechanism and clinical treatment of Vibrio cholerae.