The Role Of DevR In Cytotoxicity Of Mycobacterium Tuberculosis

The mortality of tuberculosis is second only to the infection of HIV, and has been a serious threatens to human’s survival and health. China, as a country suffering from tuberculosis with heavy burden, is facing to the problem of drug-resistant of Mycobacterium tuberculosis. It is significant to do more researchs on the relationship between the drug-resistant and gene mutations of Mycobacterium tuberculosis on the prevention and control of tuberculosis. Our previous study found that the expression of dev R, which is the response regulator of Mycobacterium tuberculosis two component systems, is up regulated in clinical isolate strains. And then we constructed dev R gene knockout BCG strain by homologous recombination technology. In this study, dev R compensated strain was established. Then the cytotoxicity of dev R regulated strains to macrophages and epithelial cells were determined. It is to explore the relationship between dev R and cytotoxicity of Mycobacterium tuberculosis.1. Construction of dev R gene compensated strain.1.1 Construction of recombinant dev R express strain induced by acetamindase.By using the shuttle vector PJV53 and Pcherry 3, recombinant dev R express vector induced by acetamindase was constructed and electrotransfected into dev R knocked out BCG strain. The strain was named of Pcherry3-Acetamindase-dev R.1.2 Observation of growth characteristics of recombinant BCG strain.BCG wild strain, dev R knocked out strain and dev R compensated strain were cultured to logarithmic growth phase at OD600 = 0.6, 105 bacteria were adjusted and applied to culture again in 7H9. The growth curve by detect the OD600 at different time(1-14 d)was carried out. Comparing to BCG wild strain and dev R compensated strain, the growth of dev R knocked out strain was significantly accelerated at early(phase2-10 d)(P<0.01). There was no significant difference(P>0.05) between BCG wild strain and dev R compensated strain.1.3 Resistant rate of three strains to Isoniazid and Rifampin assay.BCG wild strain, dev R knocked out strain and dev R compensated strain were inoculated into 7H10 medium with 1 Mc Farland concentration. Each medium contains 10 times MIC of Isoniazid or Rifampin. After being cultured for 28 days. The number of colonies on the medium was counted and the resistant rate was calculated. It was showed their was no significant difference between them(P>0.05).2. Cytotoxicity of three strains to A549 cells and Raw264.7 cells.A549 cells and Raw264.7 cells were infected with the three strains at MOI=10, then apoptosis ratio was detected by Flow Cytometry, and LDH released by host cells was measured by ELISA at 0, 24, 48 72 h. The expression of apoptosis related genes bcl-2 and bad were checked by RT-PCR.Apoptosis and necrosis ratio of A549 cells and Raw264.7 cells infected by dev R knocked out strain were significantly higher than that infected by BCG wild strains and dev R compensated strain(P<0.05). All three strains could regulate the expressions of bcl-2 gene of A549 cells, and dev R knocked out strain stimulated higher level of bcl-2 gene expression than BCG wild strains and dev R compensated strain(P<0.05). Also all three strains could increase the expression of bcl-2 gene of Raw264.7 cells. dev R knocked out strain induced more bcl-2 gene expression than BCG wild strains and dev R compensated strain(P<0.05).