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

Lei He; Xiaobo Wang; Peng Cui; Jialin Jin; Jiazhen Chen; Wenhong Zhang; Ying Zhang

doi:10.1016/j.tube.2014.12.002

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

Tuberculosis (Edinb). 2015 Mar;95(2):149-54. doi: 10.1016/j.tube.2014.12.002. Epub 2014 Dec 13.

Authors

Lei He¹, Xiaobo Wang¹, Peng Cui¹, Jialin Jin¹, Jiazhen Chen¹, Wenhong Zhang², Ying Zhang³

Affiliations

¹ Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai 200040, China.
² Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai 200040, China. Electronic address: zhangwenhong@fudan.edu.cn.
³ Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai 200040, China; Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA. Electronic address: yzhang@jhsph.edu.

PMID: 25547657
DOI: 10.1016/j.tube.2014.12.002

Abstract

Objectives: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

Keywords: DPA level; Ethambutol resistance; Mechanism of resistance; Mycobacterium tuberculosis; ubiA gene.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antitubercular Agents / pharmacology*
Bacterial Proteins / biosynthesis
Bacterial Proteins / genetics*
Cell Wall / enzymology
Cell Wall / genetics
Drug Resistance, Bacterial / genetics
Ethambutol / pharmacology*
Gene Expression Regulation, Bacterial
Humans
Microbial Sensitivity Tests / methods
Mutation
Mycobacterium tuberculosis / drug effects*
Mycobacterium tuberculosis / genetics*
Phosphotransferases (Phosphate Group Acceptor) / biosynthesis
Phosphotransferases (Phosphate Group Acceptor) / genetics*
Phosphotransferases (Phosphate Group Acceptor) / physiology
Point Mutation
RNA, Messenger / genetics

Substances

Antitubercular Agents
Bacterial Proteins
RNA, Messenger
Ethambutol
Phosphotransferases (Phosphate Group Acceptor)
decaprenylphosphoryl-5-phosphoribose synthase