Autophagy-related genes affect drug resistance of mycobacteria by regulating autophagy.

ABSTRACT

OBJECTIVE: To investigate the effect of autophagy-related gene (ATG) on the drug resistance of mycobacteria by regulating autophagy. METHODS: In the present study, primary macrophages were selected as objects of study. The cell lines with ATG13 and ATG6 interference and stable overexpression were constructed with Crisp/Case technique and verified by fluorescence quantitative polymerase chain reaction (PCR) and western blotting, and the qualified cells were used for subsequent experiments. Then the above different mutant and wild-type cells were cultured in Dulbecco's Modified Eagle medium (DMEM) containing fetal bovine serum for 5 h, and Mycobacterium tuberculosis H37Rv was added, followed by co-culture for 4 h. The cells were treated and co-cultured with isoniazid (INH, 0.05 mg/L), rifampicin (RFP, 0.4 mg/L) and ethambutol (EMB, 25 mg/L) for 3 d. Then the cells were sampled and stained with monodansylcadaverine (MDC), and autophagy was observed. Finally, an appropriate number of cells were taken and cultured in the modified L-G medium, and the bacteria were counted. RESULTS: The results of fluorescence quantitative PCR and western blotting revealed that the messenger ribonucleic acid (mRNA) transcription levels and protein expression levels of ATG13 and ATG6 in cells significantly declined after using Crisp/Case. The MDC staining showed that ATG13 and ATG6 interference could significantly reduce the number of autophagosomes in cells, while ATG13 and ATG6 overexpression could significantly increase the number of autophagosomes in cells. Compared with wild-type cells, the number of mycobacteria was obviously increased in mycobacterium-infected cells with ATG13 and ATG6 interference after they were treated with INH, RFP and EMB, displaying a significant difference (P<0.05), while the number of mycobacteria was obviously decreased in mycobacterium-infected cells with ATG13 and ATG6 overexpression after they were treated with INH, RFP and EMB, also a significant difference (P<0.05). CONCLUSION: ATG and other autophagy-related genes can affect the drug resistance of mycobacteria through regulating autophagy.