Abstract:

Objective: To express the Mycobacterium tuberculosis membrane protein MmpL5 and MmpS5-MmpL5 efflux pump system and explore its role in the drug resistance of bedaquiline. Methods: Induced expression of proteins MmpL5 and MmpS5-MmpL5 in Mycobacterium smegmatis were achieved based on the construction of acetamide-induced expression vector. The growth characteristics and efflux capacity of the expressed strains were measured, and the change of minimum inhibitory concentration (MIC) of bedaquiline on the expressed strain and its effect on the and content of adenosine triphosphate (ATP) were investigated. Results: MmpL5 and MmpS5-MmpL5 proteins of Mycobacterium tuberculosis were successfully expressed in Mycobacterium smegmatis. The growth of expressed strains was not affected by MmpL5 and MmpS5-MmpL5, but the efflux capacity of co-expressing MmpS5-MmpL5 strains increased. Compared with the MmpL5-expressing group (△Fluoresce intensity=655) and pMV261s carrying empty plasmid group (△Fluoresce intensity=642), the accumulation of ethidium bromide in co-expressing MmpS5-MmpL5 (△Fluoresce intensity=395) was reduced by 40%. The MIC of bedaquiline to MmpS5-MmpL5 recombinant strain was increased 8-fold than the control (2 μg/ml vs. 0.25 μg/ml). The content of ATP in MmpS5-MmpL5 recombinant Mycobacterium smegmatis ((0.197±0.018) μmol/L) decreased by 36.49% ((0.125±0.010) μmol/L) after bedaquiline treatment. Single expression of MmpL5 could not form efflux pump and had no effect on bedaquiline. Conclusion: Membrane protein MmpL5 and MmpS5 of Mycobacterium tuberculosis needed to be co-expressed to reduce the drug sensitivity of bedaquiline in Mycobacterium smegmatis. The establishment of the MmpS5-MmpL5 protein expression system laid the foundation for the study of the transport mechanism of bedaquiline.

Key words: Mycobacterium tuberculosis, Drug resistance, MmpS5-MmpL5, Bedaquiline