| Campylobacter jejuni is one of the most important pathogens, which can cause infectious diarrhea. Campylobacter jejuni infection will result in acute, self-limiting enteritis, and may lead to systemic infection even Guillain-Barre Syndrome in severe cases. Macrolides and fluoroquinolones are the major drugs of choice for clinical treatment of Campylobacter infection. Unfortunately, in the past decade resistance to these drugs had become a growing problem. However, there are few reports about the mechanism of macrolide-resistant Campylobacter, and it’s still unclear about the molecular mechanism involved in the fitness of macrolide-resistant Campylobacter. Dr. Haihong Hao in our laboratory screened significant genes in erythromycin-resistant Campylobacter jejuni NCTC11168by microarray. The results showed that Cj0440c is a gene of significant expression in high level of erythromycin-resistant Campylobacter jejuni. According to NCBI we are informed that Cj0440c is a hypothetical generalized regulatory protein. Cj0440c was selected as the object of this study, and the Q0440c of standard strain and erythromycin-induced resistant strain was deleted respectively with molecular biological techniques. Differentially expressed genes in mutant strains were identified by gene chip, then those differential genes directly connecting with Q0440c were analysed with bioinformatics. Correlated phenotype tests were designed to verify the role of Cj0440c. All were done to explore the role of Cj0440c in the resistance and fitness of Campylobacter jejnui. Therefore it provides favorable data for Bioinformatics Database and a theoretical guidance support to prevent resistant-Campylobacter occurrence and spreading.1. Acquistion of resistant strains and construction of respective mutants in vitro To explore the characteristics of the unknown genes Cj0440c and its role in the resistance and fitness of Campylobacter jejuni, we obtained erythromycin-resistant Campylobacter jejuni11168via erythromycin inducing stepwise in vitro; Inverse PCR was used to construct mutant vector; Cj0440c knockout mutants of Campylobacter jejuni11168and Ery-induced strain were obtained by homologous recombination; pGEM-T easy vector, as the carrier, was connected to the target fragment. The carrier fragment and the insertional kanamycin resistance fragment were amplified by respective primers designed with same restriction sites. After digestion of same enzymes, these two fragments were ligated to obtain the Cj0440c deletion mutant. Confirmed by PCR, restriction enzymes digestion and sequencing, the vector was transferred into corresponding Campylobacter cells. The results indicate that the mutant had been constructed successfully, named SM and RM respectively. However, in this study we had intended to research the characters of Cj0440c on different level. We also constructed an overexpression vector of Cj0440c with vector pRY112-pABC to overexpression and compensation of Cj0440c. But we failed to get the constructed strains via both natural transformation and electroporation. We concluded the function of Cj0440c according to characters of significant fragments from microarray and phenotypic of the mutant Campylobacters finally.2. The differentially expressed genes for the mutant with deletion of Cj0440cTo deeply understand the function of the gene Cj0440c and the mechanism of action in drug-resistant, microarray was used to screen differentially expressed genes in deleted mutants with the control of original strains, then verified by Real-time RT-PCR. The test was done with four biological replicates using SAM analysis. There was a total of19significant genes in SM group, in which only2genes are up-regulated expression, flaA and Cj1632c encoding a puptative periplasmic protein;10down-regulated genes are related with flagellar biosynthesis, involved in flagellar assembly, motility, biofilm formation, AAG, colonization and invasion;4down-regulated genes are involved in O-linked glycosylation, affecting flagellin modification;2down-regulated different genes are hypothetical protein, reported functions involved in motility and virulence. At the same time, a total of9significant genes were differentially expressed in RM group, in which no gene was up-regulated.4of9significant genes were also involved in flagellar biosynthesis; other4unknown genes were involved in flagellin or virulence factors; another significant gene is katA, a sole catalase in Campylobacter jejuni, involved in anti-oxidative stress, which contributes to intestinal colonization and survival in epithelial cells. There were2significant genes both differentially expressed in SM and RM.According to the characters of different genes in SM, it implies that Cj0440c regulate genes involve in flagellum positively, but flaA is negative regulated; Cj0440c may impact flagellar assembly and a variety biological functions of flagellar including motility, AAG, biofilm formation, colonization and invasion ability; Cj0440c may also affect glycolsyl-ation of flagellin, which is disadvantage to filament assembly. Discrepancies of genes in RM indicates that Cj0440c is likely related to flagellum synthesis, colonization and invasion, Cj0440c also helps resistant Campylobacter jejuni to fight against oxidative stress, colonizing and intracellular survival successfully.3. The phenotypic characteristics of the Cj0440c knockout mutantsTo find the consequences after Cj0440c knockout in Campylobacter jejuni on phenotypic level, a series of phenotypic tests were done, including growth of mutants, antibiotic sensitivity, flagella morphology, cell motility, colonization and pairwise-competition colonization in animals. It shows that knockout of Cj0440c didn’t affect antibiotic sensitivity, implying that Cj0440c is not involved in drug sensitivity; The mutant were slightly weaker than original strains in vitro; In electron microscopy, it showed that the filament of SM was broken, and disappeared in RM; Besides the cell motility of SM and RM were not as good as their parent strains, and intestinal colonization of them was weaker than their parent strains significantly in vivo. These indicate that Cj0440c is closely related to flagellar. The phenotypic results agree with the microarray analysis that Cj0440c affects flagellum synthesis, cell motility and colonization ability.In summary, we successfully constructed a vector used for construction of Campylobacter jejuni deletion mutants, and the mutant were successfully constructed with this vector by homologous recombination, then the differentially expressed genes were selected by microarray connected with related phenotype tests. The flagellar of mutant strains were broken or disappear, with weaker cell motility, diminished colonization and poor competitive colonization in animals. All these indicate that the mutant would be soon be replaced by other dominant strains in clinical condition. Cj0440c is closely related to flagellar biosynthesis. Cj0440c may positively regulate flaB, jlaG,flgD,flgE,flgE2,flgG,flgG2,flgH,flagI,flgK,flgL,fliD and fliK, and negatively regulate flaA and Cj1362c, thus affecting related functions of flagella. The product of Cj0440c may be connected with FlaA and FlaB closely, which are components of the flagellar filament, and is closer with FlaB in resistant Campylobacter. Cj0440c may take part in glycosylation modification of flagellin, involved in flagellin assembly and adherence and invasion of bacteria to the host cell. Cj0440c gene is up-regulated expressed in macrolide-resistant Campylobacter jejuni, which may contribute to escape drug pressure via rapid movement, colonize on epithelial cells in gastrointestinal tract rapidly and help bacteria resist of oxidative stress to invade epithelial cells smoothly and succeed in intracellular survival and reproduction. In conclution, Cj0440c may help Campylobacter jejuni survival in macrolide-environments and increase the fitness of resistant Campylobacter in the presence of macrolide.This study provides scientific basis for research of erythromycin resistance in Campylobacter jejuni. It broadens the scope of knowledge about molecular mechanism of resistance and provides scientific references for the development and application of clinical drugs. In addition, the characters of certain unknown genes are further described according with previous reports, which provid favorable evidence for completing the information of Campylobacter jejuni genome. |
