Study On The Effect Of Different Carbon Source Metabolism Of Vibrio Cholerae On Host Microflora

Vibrio cholerae as one of pathogens of the Class A infectious diseases in China can lead to a global epidemic and outbreak.Ingesting water and food with V.cholerae can cause watery diarrhea and rapid dehydration,resulting in death.On the other hand,when V.cholerae enters small intestine of host,needs to confront a series of stresses from host,such as stomach acid,reactive oxygen species(ROS),reactive nitrogen species(RNS),antimicrobial peptides,gut microbiota and nutrients competition.V.cholerae needs to overcome these stresses to successfully colonize.For example,V.cholerae can induce the host to produce ROS to kill intestinal bacteria to obtain colonization advantages.The effects of carbon source on the interaction between V.cholerae and intestinal flora needs to be further studied.Therefore,in the first part of this paper,the effect of citrate fermentation on V.cholerae and intestinal flora was investigated.The second part explored the effect of mannitol utilization on V.cholerae and intestinal flora.In recent years,studies have shown that intestinal flora plays an important role in resisting pathogenic bacterial infections,and has been applied to clinical treatments including Clostridium difficile infections.In this study,an adult mouse model treated with antibiotics and high-throughput sequencing(RNA-seq)were used to find that the expression of citA gene(sensing citrate)of V.cholerae in adult mice treated with antibiotic was 16 times higher than that in the group without antibiotic treatment.Amino acid homology alignment indicates that the corresponding genes of citrate fermentation between V.cholerae and Klebsiella pneumoniae are highly conserved,indicating that they metabolize citrate likely using the similar fermentation pathway.Although the mechanism involved in citrate utilization have been extensively characterized in some enteric bacteria,their roles in pathogenesis is still not clear.Therefore,in this study,we investigated regulatory mechanism of citrate utilization and physiological significance in V.cholerae.We first explored and verified whether the citB,citC and citS genes of V.cholerae are related to anaerobic citrate fermentation in V.cholerae.Gene knockout assays show that these cit genes are essential for V.cholerae growth when citrate is the sole carbon source.For the study of the regulatory mechanism,we constructed luminescence fusion reporter plasmids with promoters of citA,citC and citS genes and introduced them into V.cholerae different variants.Results indicate that the expression of citC and citS operons is dependent on the presence of citrate and CitAB,however,these signals will be not necessary when overexpressed CitB in these strains.In addition,transcription of CitAB two-component system not only is autorepressed,but also negatively regulated by another TCS regulator,ArcA.Global regulator CRP participates in the utilization of multiple nutrients has been reported.Further experimental results show that citrate fermentation was under the control of catabolite repression and was directly modulated by carbon metabolite repressor CRP(cyclic AMP receptor protein).Infant and adult mouse colonization experiments indicated that V.cholerae can utilize citrate in vivo using the citrate fermentation pathway and that V.cholerae likely needs to compete with other members of the gut microbiota to access citrate in the gut.In vitro the commensal Escherichia coli MP1 and V.cholerae co-cultivation assays confirmed assumption above.After that,we studied the second part from the perspective of mannitol utilization.Changes in gut microbes to a large extent influenced by environmental factors,for instance,the preference of intestinal microbes utilizing carbon sources and the utilization of short-chain fatty acids.The sorbitol fermentation assay was used to classify EI Tor biotype V.cholerae into fast-fermenting strains(non-epidemic strains)and slow-fermenting strains epidemic strains)according to color change of fermentation liquid.Then,the popular toxin-producing strains are slow-fermenting strains,that non-toxin-producing strains as fast-fermenting strains do not cause disease or occasionally cause,which has became a gold standard for rapid diagnosis of V.cholerae infection in the past.Mannitol and sorbitol are each other isomers,fermentation test was almost the same characteristics,while mannitol used only needs 1%of the concentration of sorbitol used.In this study,we investigated the mechanism behind their differences in fermentation speed and their evolutionary significance.In order to study this mechanism,we first focused on the mannitol specific phosphotransferase system(PTS),the slow-fermenting strain V.cholerae N16961 and the fast-fermenting strain 93097 were used as representative strains.The activity of mtlA gene promoter in the two strains was examined and found that the activity of the mannitol fermentation promoter in the non-producing CT strain 93097 was obviously higher than that in the toxin-producing strain(N16961).Promoter sequence alignment and recombinant luminescence reporter plasmid assays demonstrated that the 115 site base A frameshift mutation of mtlA promoter(namely mtl115 ins A)caused a conversion form fast fermentation rate to slow fermentation rate.In order to prove whether this point mutation is widely distributed in environmental strains,we analyzed mannitol fermentation experiments and the promoter region sequence of over 100 strains of El Tor type strains of V.cholerae O1 serogroup from different places and times around the world,and found all the toxin-producing strains are of slow-fermentation promoter sequence-type and slow-fermentation phenotype.Most of the non-toxin-producing strains are of fast-fermentation promoter sequence-type and slow-fermentation phenotype,and a small part are of slow-fermentation promoter sequence type and slow-fermentation phenotype.In addition,result of the phylogenetic tree uncovered that the slow-fermenting strains in the environmental strains are evolutionarily close to the genetic distance of the toxin-producing strains,indicating that the toxin-producing strains may have potentiality evolved from the slow-fermenting environmental strains.To prove whether this mutation has physiological significance for V.cholerae,we constructed a mtl 115 ins A mutation to obtain+A strain based on replacement of the N16961 mtlA promoter region.Under the consistent genetic background of two strains,we used the FMT model to study the in vivo colonization capability of these two strains.We found that colonization results are various in each fecal samples.16Sr DNA amplicons sequencing of the small intestine genome DNA in infant mice identified that the differences between V.cholerae WT and+A strains are correlated with abundance of lactic acid bacteria at family and genus level,we speculate that lactic acid bacteria of intestinal flora may inhibit the colonization of V.cholerae+A strain.In animal model,we detected that the change of metabolites of small intestines of suckling mice infected with V.cholerae WT and+A strain by metablomics,and found that acetate,lactate and ethanol of mannitol fermentation products in the group infected with+A strain were higher than that of the group infected with wild type.Further research showed that acetic acid can stimulate the growth of Lactobacillus.The acetic acid metabolism pathway has been described in many bacterium.To understand how acetic acid stimulates the growth of lactic acid bacteria,we select to delete the ackA gene of Lactobacillus plantarum WCFS1.In vitro growth assays indicated that the ackA gene is related to acetic acid utilization in Lactic acid bacteria.In the suckling mouse model,under the condition of pre-colonization of Lactobacillus,the effect of Lactobacillus ackA gene on the colonization of V cholerae wild type and+A strains was detected.The results showed that the expression of Lactobacillus ackA gene inhibited the rapid fermentation strain of V cholerae colonization.Studies have reported that lactic acid bacteria can produce antibacterial peptides to inhibit the growth of pathogenic Escherichia coli.Therefore,we also examined the effect of the supernatant of the Lactobacillus plantarum WCFS1 culture on the growth of V.cholerae and found that the growth of V.cholerae was not affected.however,through co-cultivation experiments with lactic acid bacteria and V.cholerae,we found that the decrease in pH partially inhibited the growth of V.cholerae.In summary,in this study we confirmed that human fecal transplantation in suckling mice model can significantly reduce the colonization of a toxin-producing strain with a fast-fermenting promoter sequence.Further studies show that one base A mutation in the mannitol-specific PTS promoter of toxigenic V.cholerae strains slow down their mannitol fermentation rate,preventing bloom of Lactobacillus species of the gut microbiota from utilizing fermentation products,this allows V cholerae to successfully outcompete commensal gut microbes and establish colonization.Studying the interaction between intestinal microbes and V.cholerae has important significance for our understanding the pathogenic mechanism of V cholerae.Currently,clinical treatment for cholera is still electrolyte supplementation mainly because of antibiotic treatment will bring potentially negative effects that disorder of the intestinal flora.Therefore,starting from the research field of the interaction between the flora and V.cholerae,this paper found that Escherichia coli.and Lactobacillus can affect the colonization of V.cholerae in vivo from the perspective of the use of carbon sources.This provides an important theoretical basis for the treatment and prevention of cholera outbreak from the perspective of flora.