| Campylobacter jejuni is a very important food-borne pathogen, it remains one of the major pathogens of cause infectious diarrhea worldwide. And it is also a symbiotic bacteria of many animal species. The main sources of Campylobacter jejuni infection in humans are contaminated water and animal foods, such as dairy products and uncooked meat. Infection with Campylobacter jejuni often results in acute, self-limiting enteritis, but it also can lead to systemic infection and even cause the most serious complications Guillain-Barre Syndrome. Antibiotic therapy is necessary when Campylobacter infections are prolonged or severe or when they occur in immunocompromised persons. Macrolides and fluoroquinolones are the drugs of choice for clinical treatment of Campylobacter infection. Resistance to fluoroquinolones is on the rise in recent years, it is particularly important for marolides to the treatment of infections in this case. Erythromycin is widely used on the treatment of intestinal infections; tylosin, an antibiotics specific for animal use, also widely used in veterinary clinical. Epidemiological data show a trend of rising marolides resistance in Campylobacter jejuni. But the mechanisms of resistance are not yet completely clear, while research for fitness is still at its initial stage. This is a disadvantage for surveillance of resistance generation and dissemination. Dr. Haihong Hao in our laboratory screened differentially expressed genes in different levels of erythromycin-resistant Campylobactert jejuni NCTC11168and81-176by microarray. There are five genes co-expressed present in set of two types bacteria with unknown function, in which cj1199expression increased while drug resistance level higher. Cj1199is a putative iron/ascorbate-dependent oxidoreductase, connect with the analysis of the protein net we suspected that this gene might be involved in amino acid biosynthesis and response to the stimulus of environment and then contributed to the resistance and fitness of erythromycin. Therefore we selected cj1199as the target gene, study for its function and its role in resistance for erythromycin, provide the theoretical guidance and support to prevent resistance occurrence and spread, in order to protect the safety and effectiveness of drug. To construct cj1199knockout mutant in our study we constructed vector first and deleted cj1199by homologous recombination; analyzed the impact of bacteria by absence of cj1199though phenotypes. To deeply understand the function of this gene and the effect on resistance, microarray was used for screening differentially expressed genes in deletion mutant.Inverse PCR and overlap extension PCR for vector construction. Amplified gene fragments about2000bp from upstream to downstream which containing cj1199in middle, then connected with vector pGEM-T easy. Inverse amplified vector using the primers which carrying the restriction sites BamH I and Sma I, while amplified cat using the primers which carrying the same restriction sites. After recovered, these two fragments were digested by BamH I and Sma I, then recovered by agarose electrophoresis, connected with T4DNA ligase, and transformed into Escherichia coli competent cells, amplified culture and extract plasmid after verification. For overlap extension PCR, fragments of cj1199carrying the homologous of cat gene were amplified, connected these two fragment with cat using overlap extension, obtained a long fragment, and then connected with vector pGEM-T easy, transformed into Escherichia coli competent cells, amplified culture and extracted plasmid after verification. We obtained target vector successfully by inverse PCR, while the overlap extension PCR was failed.NCTC11168was incubated with the vector for natural transformation. Mutants were selected on MH agar with chloromycetin. Vector was added to electrocompetent bacteria which prepared for electroporation at the same time, pulsed under2450V、200Ω25μF for5msec, added preheated SOC, gently mixed, then recovered on MH agar medium with chloromycetin for screening mutant. The results indicate that mutant had constructed successfully by these two methods and verified by PCR and sequencing.Screened the genes differentially expressed in mutant by microarray. Then Real-time RT-PCR was used to verify the results. Set four biological replicates, a total of23differential expression genes were screened after the SAM analysis. In which only2genes up-regulated expression, all of these involved in transport;21genes down-regulated expression:6related with transport, involved in aspartate/glutamate and iron transport respectively;5involved with amino acid biosynthesis, participated in leucine and tryptophan biosynthesis;5related with cell surface structure, two in which associated with iron stimulate;2hypothetical proteins,3were with no significant similarity with others. Based on the functions of differentially expressed fragments, we infer that cj1199had impact on the biosynthesis of leucine, and then had an impact on the synthesis of short peptide, it can contribute to the initial survival rate of the Campylobacter jejuni during antibiotic treatment and therefore increase the chances of acquiring one of the major resistance mechanisms; it also play an important role in aspartate/glutamate transport, affecting the supply of energy and carbon, contributed to the colonization and survival in the gut.Then a series of phenotypic tests were done. We measured single growth, competitive growth of the resistance comparing with the susceptible strains, competitive growth in the culture with erythromycin and single growth in medium absence of leucine. The results show that there’s no significant change in mutant for single growth, but competitive growth declined markedly, especially in erythromycin. And in medium lack of leucine mutant growth is inferior to that in complete medium, and lower than the standard strain in the same medium too. Mutant was tested in antibiotic-resistance, the results indicate that there’s no significant different between mutant and standard strain. Permeability changes of the mutant after erythromycin treatment were detected by flow cytometer (FCM), indicate that permeability of mutant increased in this condition, we could speculate that the tolerance of mutant to erythromycin is decrease. Observed the effect of cj1199during induced resistance to erythromycin. Mutant showed more difficult to induce, and more difficult to obtain high-level resistant strains.In conclusion, we successfully constructed a vector used for construction of Campylobacter jejuni deletion mutants, and a mutant was successfully constructed used this vector by homologous recombination, then differential expressed genes were selected by microarray connected with related phenotype tests. The mutant showed a weak competitive in a variety of competitive environments. The mutant has hyperpermeability, and it’s tolerance to erythromycin is reduced, so it is difficult to produce drug-resistant strains. It shows that mutant will soon be replaced by other dominant microorganisms, especially in the environment with erythromycin mutant possesses lower resistance development risk. Based on functions of differential expression genes, cj1199had impact on the biosynthesis of leucine and utilization of amino acid in gut, and then play an important role in the synthesis of short peptide and the provision of energy and carbon, it contribute to the initial survival rate of the Campylobacter jejuni during antibiotic treatment and the colonization in the gut. This study provides scientific accordance for the research of erythromycin resistance in Campylobacter jejuni. It widened the thread for molecular mechanism of resistance and it provides scientific reference for clinical drug research and application. |
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