UbiA (Rv3806c) encoding DPPR synthase involved in cell wall synthesis is associated with ethambutol resistance in Mycobacterium tuberculosis

Abstract

Ethambutol (EMB) is a frontline antituberculosis drug for the treatment of tuberculosis (TB). The embB gene is responsible for EMB resistance in only about 50-60% clinical isolates of Mycobacterium tuberculosis, and the mechanism of resistance in the remaining EMB-resistant strains is not clear. We assessed the role of the ubiA gene encoding 5-phospho-α-d-ribose-1-diphosphate: decaprenyl-phosphate 5-phosphoribosyltransferase (DPPR synthase, UbiA) involved in decaprenylphosphoryl-d-arabinose (DPA) pathway for cell wall synthesis in EMB resistance. Antimicrobial susceptibility testing was performed using broth colorimetric method or an agar proportion method to compare the MICs of recombinant M.tuberculosis strains that overexpressed the wild type or mutant ubiA gene. PCR and DNA sequencing were used to detect ubiA mutations in EMB-resistant M.tuberculosis clinical isolates with or without embB mutations. ubiA mRNA expression levels were measured by qRT-PCR. Enzymatic activity of UbiA in different M.tuberculosis strains was analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Overexpression of the wild type M.tuberculosis ubiA led to increased resistance to EMB in M.tuberculosis. In addition, novel ubiA mutations were found in some EMB-resistant XDR-TB isolates without common embB mutations. Topological modeling analysis showed that the mutation sites in ubiA were mainly localized in the sixth transmembrane domain of the UbiA protein. Moreover, mutated ubiA when overexpressed led to higher level of EMB resistance and increased DPA levels, compared to wild type ubiA in M.tuberculosis. Our results indicate that ubiA is involved in EMB resistance in M.tuberculosis and that ubiA mutations that caused elevated DPA levels may be responsible for EMB resistance. The essentiality of UbiA, its involvement in EMB resistance, and lack of human homologs make UbiA an attractive target for new drug development.