Gluconeogenic Key Genes PpsA And PckA Affect Vibrio Cholerae Function In Vivo

Vibrio cholerae,the causative agent of severe disease cholera,is a Gram-negative bacteria with single flagella.In the natural environment,V.cholerae grows in water of low salinity.V cholerae infects the human host through polluted water and food and colonize in intestinal epithelial cell surface.In the process,V.cholerae need to overcome host challenges such as gastric acid and immune resistance,also faces the defense from the intestinal flora which known as"colonization resistance".Human and other mammals harbor a complex gastrointestinal microbiota that the predominant bacteria are firmicutes phyla,protebacteria,bacteroidetes and actinobacteria.The sources of nutrient that support to microbiota are shed epithelial cells and store in mucus layers.Different species bacteria modify their metabolisms to compete nutrients by regulating catabolic pathways so that they can keep the gastrointestinal microbiota relative balance.After pathogenic enterobacteria colonize successfully,the ability to use different metabolism pathways is important for further grows.In this study,we select 11 genes that relative to metabolism to study the effect of these genes on vibrio cholerae colonization.We found that the key genes of gluconeogenesis ppsA and pckA have significant influence colonization a lot.So,we investigated the role of gluconeogenesis pathway in vibrio cholerae.At first,we constructed ppsA and pckA in-frame deletion mutants and double deletion mutant by homologous recombination.In vitro,we detected the utilization ability of mutants and wide-type strains for different carbon sources.We found that the double deletion mutant unable to utilize tricarboxylic acid cycle intermediates and gluconeogenic substrates to growth,but it can utilize glycolytic substrates to growth.The gluconeogenic deletion strain can not utilize tryptone componented in LB medium which results in growth defection.The double deletion mutant can utilize monosaccharide that contain in mucin like wild-type,but not mucin.Through promoter expression assays,we comfirmed that phosphoenolpyruvate carboxykinase PckA plays the main role in gluconeogenesis.To examine the role of vibrio cholerae gluconeogenesis colonization in mouse samll intestine,we used infant mouse,adult mouse and M-E-A-T model to study it.Animal experiments indicated that the colonization ability of ApckA and AppsA/?pckA are inferior to wild-type and ?ppsA is same as wild-type.As time goes on,the difference between ?pckA and WT arose from 5-fold to 500-fold,while the difference between ?ppsA/?pckA and WT from 10-fold to 1000-fold.It illustrates that vibrio cholerae use gluconeogenesis to compete nutrients for colonization in mouse intestine.Furthermore,we made some researches about native gut flora effect on V cholerae colonization.We treated mice with antibiotic cocktail to eliminate gut flora and set streptomycin as control to confirm the gap variation between mutant and wild-type.The data showed that the gap between wild type and the mutant?ppsA/?pckA decrease ten-fold after antibiotic cocktail treatment.It suggests that vibrio cholerae uses gluconeogenesis more efficient to compete other gut flora.In addition,we studied the influence of gluconeogenesis on vibrio cholerae motility,biofilm formation and virulence gene expression.We found that gluconeogenesis affects vibrio cholerae motility instead of biofilm formation and virulence genes expression.Gluconeogenesis is important for bacteria to utilize Non-sugar carbonaceous substances.In general,gluconeogenesis plays an important role for vibrio cholerae not only in natural environment but also in colonization in vivo.It indicates that vibrio cholerae using gluconeogenesis to compete with intestinal flora,and get advantage in colonization.Gluconeogenesis also influences swimming ability of vibrio cholera.