| Tuberculosis(TB) is one of the chronic and infectious diseases, caused by a causative bacterial agent, Mycobaterium tuberculosis(MTB). Although the directly observed treatment short-course (DOTS) has good effct on treatment of TB, the long term of the disease and its relapse,however, are the main barrier of the control of TB. It is estimated that one third of the world population harbor persistent M.tuberculosis infection. With the human immunodeficiency virus (HIV) epidemic unabated, population aging and the emergency of multidrug resistant strains of tubercle bacillus wordwide, those latently infected individuals make the control of TB more difficult. As the persitent states have been realized a major obstacle to eradicating M.tuberculosis infection, undoubtedly, to understand the molecular mechanisms operative in persistence will lead to new and more effective strategies to control or eradicate this phathogen.It is still unclear about the molecular mechanisms of how M.tuberculosis enter the persistent stage. Recent findings showed that prokaryotic chromosomes code for toxin-antitoxin (TA) loci are stress-response elements that help cells survive unfavorable growth conditions, which offer the quality control of gene expression. Gerdes, K et al surprisingly identified that M.tuberculosis H37Rv and CDC1551 have 38 and 36 TA loci, respectively, including 3 relBE family. Researchers also found that free-living slowly growing prokaryotes had particularly many TA loci and put forward the hypothesis that the number of TA loci may be beneficial for organisms characterized by slow growth i.e. the chromosome of M.smegmatis, a fast-growing close relative of M.tuberculosis , encodes two TA loci only. Despite the large amount of TA loci in M. tuberculosis genome, little is known about its functions in the pathogensis of the organism. Futher study on the functions of these hypothetical proteins encoded by M.tuberculosis chromosomes TA loci will not only be accelerated the biological knowledge of M.tuberculosis in the post-genome era but also provide a new ideal to clarify the persistent mechanisms of the causative pathogen.According to the bioinformation analysis, there exist homologies, to some extent, between hypothetical proteins of Rv1246c and Rv1247c gene and that of relBE family, one of E. coli chromosomal TA loci. We amplified the gene Rv1246c and Rv1247c from the genome of H37Rv for the first time to make an attempt to investigate the functions of them. The recombinant fused expression plasmid pET-relB and pET-relE were constructed respectively. The recombinant plasmids could express the fusion protein stably, thus provided the basis for the further study of the gene Rv1246c and Rv1247c. After being carefully analyzed the overlap gene Rv1246c-Rv1247c was proved to be a unique sequence in the M.tuberculosis Complex, a recombinant plasmid bearing gene Rv1246c-Rv1247c was constructed using the E.coil-Mycobacterium shuttle vector pMV261. Then it was electroporated into avirulent Mycobacterium smegmatis mc~2155 which is lack of the sequence. The transformants were induced to express the predicted proteins of Rv1246c and Rv1247c. Under the heat stress and acid stress condition respectively in vitro, the viability of the Recombinant Mycobacterium smegrnatis (MCBE) was evaluated. According to the stress assays, we suggested that the functions of gene Rv1246c-Rv1247c could inhibit cell growth and reduce the number of colony-forming units, which offer advantages to bacteria when the organisms lived under limiting growth conditions in vitro. Furthermore, we employed the MCBE recombinant strian to infect mouse bone marrow macrophage line ANA-1. The viability of the infected ANA-1 cells, the intracellular survival assay of the mycobacteria, ANA-1 cells apoptosis and the expression levels of iNOS mRNA and NO in ANA-1 cells were detected at different times postinfection. The transformant containing Rv1246c-Rv1247c showed more advantages to survive and a lower speed to be eliminated by macrophages than the control group. Although the infected ANA-1 cells underwent obvious apoptosis compared with normal cells as control, but the recombinant M.smegmatis only containing the pMV261 plasmid could significantly induce more ANA-1 apoptosis than the recombinant ones bearing the gene Rv1246c-Rv1247c. Worthy of note, the results of the activity response of macrophages indicated that the MCBE strain also could inhibit the excessively activity response of the mice macrophage ANA-1 cells.To investigate the protein-protein interaction between Rv1246c and Rv1247c in vivo, the genes were amplified from M. tuberculosis H37Rv genome DNA. The Rv1247c gene and Rv1246c gene were subcloned into pGBKT7 and pGADT7-Rec respectively. The recombinant vectors were transformed into yeast cell AH109. Rv1246c and Rv1247c protein could interact with each other in yeast cells as shown from the results. It also showed that the protein pairs encoded by Rv1246c and Rv1247c have the basic proterties that are required to be the TA module.It is the first time to study the biological functions of Rv12461-Rv1247c gene of M. tuberculosis by constructing recombinant M. smegmatis. In summary, Rv1246c-Rv1247c may be the stress-response element and belong to the relBE family of TA system. It also is a new way and important information to provide insights into the mechanism that M.tuberculosis has evolved into adopt its successful highly model of persistence.
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