| Campylobacter jejuni(C.jejuni)is an important foodborne zoonotic pathogenic bacterium,which can be transmitted to by animals,food,water,milk,etc.Infections caused by this microbe often leads to acute gastroenteritis with diarrhea in human being.In recent years,the reported infection case of C.jejuni has being increased all over the world,posing a serious threat to public health and food safety.The flagella play an important role in the pathogenesis of C.jejuni.It not only mediates the motility of the bacteria,helps adherence and invasion of the cells,colonization of the host,but also participates in the secretion of virulence proteins and evasion of the innate immune system.In addition,some flagellar elements are found able to co-regulate the expression of other virulence factors of this bacterium,making flagella be an important pathogenic factor of C.jejuni.FlhF is a recently identified protein that required for the spatial and numerical control of the synthesized flagella,which is mainly found in polarly flagellated bacteria.FlhF was found play various roles in flagella biosynthesis process in different species,In C.jejuni,the mutation of FlhF led to a complete loss of motility and flagellum structure,but the underlying mechanism is not well elucidated so far.In this study,the pathways that FlhF influenced and its related effects were systematically analyzed at the whole genome level by using the method of microarray,and its function was further studied in the most related flagellar system.The influence of mutation of FlhF on C.jejuni virulence and the capacity to induce diarrhea were also analyzed,which provide reference for further study of the function of FlhF and also its relevance to C.jejuni pathogenesis.1.Comparative analysis of the whole genome expression profiles of C.jejuni after inactivation of flhFThe relative expression of flagella genes of C.jejuni in different growth stages was evaluated using qRT-PCR,and the time point when expression of most flagellar genes were relatively high was selected for microarray,to evaluate the influence of FlhF on whole genome expression profiles.Cluster and enrichment analysis were performed on differentially expressed genes to determining the primary pathway that influenced by mutation of FlhF.The function of FlhF in the flagellar system and co-regulation of C.jejuni virulence were also analyzed using the microarray data,based on the collection of related genes.The results showed that C.jejuni which was cultured in vitro for 20 h grows well.The relative expressions of most flagellar genes at this time point were high,and the RNA of the wild-type and flhF mutant strain were following extracted for microarray analysis.The expression profile of C.jejuni was truly changed after mutation of FlhF,by cluster analysis of the microarray data between wild-type and flhF mutant strain.A total of 10 differentially expressed genes were screened by setting the fold changes>2,and the p value<0.01 as the threshold.Gene Ontology and Pathway analysis found that flagella and motility were the significantly affected pathways.Further analysis in the flagellar pathway showed that FlhF plays a global role in the flagellar system,64%of flagellar related genes were down-regulated after its mutation.FlhF influences flagellar gene transcription from an early stage,this effect persisted during the synthesis process and had a remarkable impact on the later flagellar genes.The flagellar genes were classified according to their functional correlation and transcriptional cascade,and the results indicated that the transcription of flagellin-related genes and class ? flagellar genes were significantly down-regulated when compared with the others.The flagellar genes of C.jejuni are scattered through its genome,the microarray data showed that 60%of chemotactic genes,51.4%of capsular polysaccharide genes,31.8%of lipopolysaccharide genes,16.7%of iron ion uptake genes,52%of adhesion and invasion genes,and 39.2%of colonization genes were down-regulated along with the mutation of FlhF.The significance analysis indicated that the effects of FlhF on the colonization,adhesion,invasion and chemotaxis were more obvious than those of iron ion uptake,lipid oligosaccharides and capsular polysaccharides.2.Molecular mechanisms of FlhF in regulating flagella biosynthesis of C.jejuniThe expression curve and subcellular localization of FlhF were analyzed for determination of its biological characteristics.The ability of FlhF to mediate the subcellular localization of flagellar elements was also explored using the method of fluorescence labeling,to analyze the possible mechanism by which flagellar biosynthesis is blocked.The interaction targets of FlhF protein were screened by immunoprecipitation and verified GST pull-down assay.Finally,whether FlhF could directly regulate flagellar gene transcription was investigated by EMSA.The results showed that FlhF was continuously expressed in C.jejuni,in a way as class ? flagellar gene does.Through the way of cell components separation and GFP fusion,FlhF was found to be primarily located in the pole of C.jejuni inner membrane,this location is the site where flagella originally generated,suggesting that FlhF may play a role in the early stage of flagella biosynthesis.FlhB-GFP and FlgB-GFP were found not localized to the cell pole in the flhF mutant strain,indicating that the assembly of flagellar export apparatus,as well as its transportation ability was greatly impeded.Thus,C.jejuni flagellation was blocked in the initial stage after mutation of FlhF.The screened proteins that interacted with FlhF suggested that there may be a functional correlation between FlhF with flagella assembly,chemotaxis,transcription and translation,substrate recognition and transportation,and metabolism.Further verification revealed that FlhF interacts directly with FlhG,CheA,DnaK and GlyA.The FlhF protein itself can be divided into B,N and G domains.N domain is the structural basis for FlhF to interact with other candidates,FlhG was found to bind the B domain mostly,and GlyA can interact with FlhF through NG domain,as the same with FlhF dimers.Whereas the CheA and DnaK was found to only bind the N domain of FlhF.Ten flagellar promoter sequence that distributed in the three flagellar cascades were selected,and the results of EMSA showed that FlhF could bind the promoter sequence of sigma 70 and sigma 28,this indicates that FlhF can directly regulate the transcription of flagellar genes as a transcription factor and influences the expression of class I and III flagellar genes.3.Effects of FlhF mutation on the virulence and the capacity to induce diarrhea of C.jejuniThe FlhF mutant and complemented strains were constructed by molecular biological method,and the effects of FlhF mutation on the virulence and the capacity to induce diarrhea of C.jejuni were following studied.The results showed that the flhF mutant strain could only grow at the inoculation site and completely lost its motility when compared with the wild-type strain.The normal flagellation phenotype of the flhF mutant strain was decreased from 94%to 0%,and the flagella structure could not be observed.The flagella related virulence phenotypes were following analyzed,which showed that the autoagglutination and biofilm-formation abilities of the flhF mutant strain were significantly decreased.The chemotactic ability of C.jejuni strains were evaluated using 3 chemoattractants,the chemotaxis of the flhF mutant strain was significantly lower than those of the wild strains in the malic acid and a-ketoglutaric acid induction system.The adhesion and invasion abilities of the C.jejuni flhF mutant strain were reduced to approximately 20.15%and 2.90%of those of the wild-type strain,respectively.The relative adhesion of the flhF mutant strain could be restored to 74.78%of the wild-type capacity after the centrifugation treatment,but this has less effect on the restoration of the invasion defect,still only 3.09%of the invasion ability relative to the wild-type level was observed for the flhF mutant strain.This indicated that the adhesion defect of flhF mutant strain was mainly caused by the loss of motility,while the decreased invasiveness was more likely caused by the absence of flagella.The chicks and mice were selected as representatives for birds and mammals respectively to evaluate the role of FlhF in C.jejuni colonization.At 1 day post inoculation,the number of recovered flhF mutant was significantly lower than that observed for the wild-type strain for all the intestinal tissues except colon in chicks.However,no significant difference was observed in mice at this time point.On day 7,the wild-type C.jejuni strain was found could consistently colonized in both chicks and mice,while the flhF mutant strain was completely cleared by the mice,and this situation was similar in chicks,as the mutant strains could be isolated only in the cecum of one chick,and the bacterial load was only 104 CFU/g.This indicates the importance of FlhF in mediating the continuous colonization of C.jejuni in the host.The capacity of C.jejuni strains to induce diarrhea were compared in the infant rabbit model by scoring the diarrhea index,the results showed that the average diarrhea index of the infant rabbits infected by the flhF mutant strain were all lower than those of wild-type strain infected groups at 1,2 and 3 days post inoculation,which indicated a role of FlhF in mediating C.jejuni pathogenesis. |
PDF Full Text Request