| Tuberculosis was one of the most severe infectious diseases killing more than two million people annually. One third of the world population were infected with latent TB bacilli, the progress of population resulted in tuberculosis and AIDS coinfection, and abuse of TB antibiotic led to MDR-TB and XDR-TB, it made tuberculosis precaution difficult in a few years. It was reported there were15multi-drug resistant countries in Europe, resulting in tuberculosis relapse.2%increase of the annual cases was multiple drug-resistant TB in London of England.The clinical first-line anti-TB drugs was INH (Isoniazid), rifampicin (rifampicin), ethambutol (Ethambutol) and streptomycin (straptomycin). With abuse of antibiotic, long treatment time, and the double torment of the material and spirit, they resulted in the emergence of multiple drug resistance and extensively drug resistant TB and brought about the concern of World Health Organization. Because some patients existed the compromised immune systems, leading to insecurity of BCG vaccination, the world was appealing new anti-TB drugs and therapeutic vaccines[1]. the WHO made a decision to reduce the global burden of tuberculosis by2015. In order to achieve the goal, the strategy required government to strengthen precaution and scientists to deepen understanding of pathogenesis.M. tuberculosis confronted complicated and varied environment requiring resist external pressure to maintain its balance through various pathway of which transcription regulators were very significant. The IclR family played an important role in maintaining the growth in bacteria in the particular environment, such as starvation and oxidation pressure. The IclR family was widely distributed in the diverse bacteria to participate in different biological functions, such as primary and secondary metabolism, toxicity, quorum sensing and sporulation. The production of the leucine and tryptophane were enhanced in C. glutamicum IB1486[2]. The TtgGHI efflux pump was enhanced in Pseudomonas putida ttgV mutant[3]. AttJ controlled quorum-sensing signals in Agrobacterium tumefaciens[4]. E. coli IclR controlled carbon metabolism through Icl (isocitrate lyase). Icl was key enzyme in the glyoxylate shunt. The experiment verified that Icl elevated bacterial survival within macrophages to maintain persistence infection. Further Icl was absent in mammalian, thus it acted as a potential target of new drugs[5]. Abrogation of icll and icl2genetically or chemically could impair the intracellular replication of M. tuberculosis CDC1551, even eliminate the pathogens from the lung. Chemical inhibition inhibited Icll (aceA-1) and Icl2(aceA-2) in M. tuberculosis CDC1551to block the growth on fatty acids as carbon sources and in macrophages[6]. The IclR family was important, and I hoped that finding of the IclR family in pathogenic bacteria was used to investigate IclR function in M. tuberculosis to preferably elucidate pathogenic mechanism.The latent M. tuberculosis was main reason leading to longer time, poor efficacy and relapse of chemotherapy for TB and was fairly severe factor in controlling TB. M. tuberculosis was phagocytized by macrophage into persistence where M. tuberculosis lacked the necessary nutrients such as glucose and amino acid, a variety of antimicrobial substances and low pH to cause survival crisis. M. tuberculosis utilized fatty acids as a major source of carbon and energy through the glyoxylate shunt. There were three predicted iclR genes in M. tuberculosis H37Rv(Rv2989、Rv1773c and Rv1719). the similarity of their amino acid was14.7%and nucleotide was26.3%. A library of mutant M. tuberculosis strains was generated with the transposon Tn5370, and the experiment results demonstrated Rv1773c might be involved in toxicity in M. tuberculosis. The Rv1773c mutant was more virulent than the wild for severe combined immune deficiency (SCID) mouse model. the increased virulence of this strains was due to mutations in Rv1773c. Rv1773c mutated and following Icl activity strengthened. Icl was benefit for the growth in M. tuberculosis through the glyoxylate cycle. These preliminary data implicated that further investigation into IclR and related genes were fully deserved. We expected to search analogue regulation pathway to elucidate pathogenic mechanism of M. tuberculosis by research of the IclR family in other bacteria. The diversity of IclR function and the sophisticated regulatory mechanism underlying represented unprecedented opportunity for antibiotic drug targets, not only for M. tuberculosis, but also for other drug resistant pathogens.In order to study the basic functions of Rv2989in M. tuberculosis, Rv2989was amplified from M. tuberculosis H37Rv genome and electroporated into Mycobacterium smegmatis to study the growth, anti-oxidative stress and biofilm formation. the nucleotide sequence of Rv2989gene in M. tuberculosis H37Rv was obtained from the GenBank database. According to these sequence, a pair of primers was designed by Primer Premier. M. tuberculosis H37Rv genome was used as template, gene was amplified by PCR. the PCR products was cloned into pNIT-myc shuttle plasmid. Colony PCR, restriction enzymes digestion and sequencing identified the recombinant plasmids pNIT-myc-Rv2989successfully. The recombinant plasmid was transformed into competent M. smegmatis MC2155. Colony PCR, restriction enzymes digestion,16SRNA and sequencing identified the recombinant plasmids pNIT-myc-Rv2989was transformed into M. smegmatis MC2155successfully. s-caprolactam induced the recombinant overexpression through the western-blot. We studied impact of overexpression Rv2989in host strain, including the growth, anti-oxidative stress and biofilm formation. Experimental results showed that overexpression of Rv2989decreased host bacteria into the rate of the stability. When hydrogen peroxide was low concentration, overexpression of Rv2989increased resistance to oxidation. The results suggested that Rv2989overexpression had no significant influence in biofilm formation and sliding ability. Rv2989overexpression induced the production of the leucine.When M. tuberculosis genome sequences was completed, scientists searched novel method to explore gene function. In order to simulate the growth environment in vivo, and based on the yeast two-hybrid (Y2H) method system, Amit Singh et al utilized mycobacterial protein fragment complementation (M-PFC) to explore Mtb protein-protein association in mycobacteria[8]. The main principle of the M-PFC system was trimethoprim which was a specific inhibitor of dihydrofolate reductase, but bacterial DHFR had approximately12,000-fold higher affinity for TRIM than mammalian DHFR.(a certain concentration of trimethoprim inhibited bacterial dihydrofolate reductase activity, but did not affect mammalian dihydrofolate reductase activity). Based on the M-PFC programs, we looked for protein which interacted with Rv2989, and constructed network to explore the function of Rv2989. Rv2989was amplified by PCR, and PCR products were cloned into the pUAB400integrative plasmid. We constructed pUAB400-Rv2989successfully, and transformed into competent M. smegmatis MC2155. We constructed H37RV genomic library and cloned into the pUAB300shuttle plasmid. the pUAB300recombinant plasmids were transformed into pUAB400-Rv2989recombinant M. smegmatis. Interacting proteins A and B were fused to pUAB300and pUAB400which possessed complementary mDHFR fragments F and F.Cotransformation of AF and BF fusions in Msm formed completely mammalian DHFR activity to counteract Trimethoprim and subsequently screened for interacting proteins. The experimental results showed that the system was successfully constructed, but did not find protein interacted with Rv2989temporarily. the analysis might be due to several reasons,1) the amount of library was not enough,2) stronger interactions did not emerge and action was short,3) Rv2989was a transcription factor and interacted with other protein in certain circumstances. |
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