Related Research Between Toxin-antitoxin System And The Persister Of Mycobacterium Tuberculosis

Objective By constructing deletion strains of toxin-antitoxin system in Mycobacterium tuberculosis, we studied its ability to survive under different culture conditions, and the related research between toxin-antitoxin system and the persister of Mycobacterium tuberculosis.Methods 1. Construction and identity of deletion mutant of toxin-antitoxin system maz EF and rel BE of Mycobacterium tuberculosis: First of all, By PCR, we amplified flanking(homology arms) of maz EF and rel BE gene from H37 Rv and kan gene of kanamycin resistance gene from plasmid PUC-19 K. Second, using fusion PCR, we did the hybrid splicing of maz EF and rel BE homology arms and kan gene, obtained the desired fusion fragment, and the fragment was cloned into p MD-19T(simple) vector to form a suicide plasmid(p MD-19T-Δmaz EF-kan and p MD-19T-Δrel BE-kan), then the suicide plasmid was transformed into E. coli DH5a; At last, applying electroporation technology, the constructed plasmid was transformed into H37Rv; single colony of Mycobacterium tuberculosis was screened on L-J medium with kanamycin, and the genomic DNA of positive strain was extracted, aim fragments were amplified by PCR and sequenced. We did the genetic stability testing for the H37RvΔmaz EF and the H37RvΔrel BE deletion mutant. 2. The preliminary test of the phenotypes and the growth viability of Mycobacterium tuberculosis H37RvΔMaz EF deletion mutant under different culture conditions, the acid-fast staining was conducted for the wild-type and deletion strains respectively, then observed the changes in strains by morphological; the deletion strains and wild strains were cultured in the medium of hypoxia and nutritional deficiencies, and at different points, compareed the number colonies of wild-type strain with that of deletion strains under stress conditions to find the differences on the viability. 3.The macrophage Raw264.7 of mice were infected with H37 Rv, H37RvΔmaz EF3, H37RvΔmaz EF6 and H37RvΔmaz EF9 strains respectively, through the CFU counting to detect the viability of Mycobacterium tuberculosis after infecting macrophage; besides, we detect apoptosis of macrophages after infection by using the flow cytometry CELLQest and Win MDl2. 9 Version software.Results The study showed that H37RvΔmaz EF and H37RvΔrel BE deletion strains have certain genetic stability. Acid-fast staining showed that, compaired with the wild strain, the H37RvΔmaz EF strain was shorter; Meanwhile, CFU counting showed that the deletion strains strain slowlier growth. Compaired with the wild strain, the deletion strains has a lower viability within macrophages, and the apoptotic rate of macrophages infected with H37RvΔmaz EF group was higher than that by H37 Rv strain.Conclusion This finding suggests that the presence of the MazEF system in M. tuberculosis contributes to their strong ability to survive in tissue cells, and M. tuberculosis latency under stressful conditions is a result of many factors working together, and further exploration is necessary to best understand the precise infection mechanisms.