Spontaneous mutational patterns and novel mutations for bedaquiline and clofazimine resistance in Mycobacterium tuberculosis

Abstract

The 2022 World Health Organization guidelines recommend use of two core anti-tuberculosis (TB) drugs, bedaquiline (BDQ) and clofazimine (CFZ), for treatment of drug-resistant (DR)-TB. However, several mutated Mycobacterium tuberculosis (MTB) genes, conferring BDQ and CFZ resistance, have been reported that predominantly arose from sporadic mutations that have not been comprehensively characterized. Herein, MTB clinical isolates collected from drug-susceptible (DS)-, multidrug-resistant (MDR)-, and extensively drug-resistant (XDR)-TB patients were cultured in vitro with BDQ or CFZ to generate progeny strains with resistance to these drugs. Progeny strains exposed to CFZ exhibited increased CFZ minimum inhibitory concentrations (MICs) that exceeded MIC increases of BDQ-exposed progeny strains. Notably, mmpR and pepQ mutations accounted for 83% and 17% of BDQ-induced spontaneous gene mutations, respectively, and 86% and 14% of CFZ-induced spontaneous gene mutations, respectively. Analyses of predicted mutation-induced changes in amino acid sequences and structures of MmpR and PepQ mutants revealed several point mutations affected sequence conversation and functionality as an underlying mechanism for observed acquired BDQ/CFZ resistance. Moreover, our results revealed differences in patterns of BDQ- and CFZ-induced acquired spontaneous mutations that may enhance our understanding of MTB BDQ/CFZ-resistance mechanisms. IMPORTANCE This study of MTB drug resistance mechanisms revealed patterns of spontaneous MTB mutations associated with acquired BDQ and CFZ resistance that arose after clinical MTB isolates were cultured in vitro with BDQ or CFZ. Results of protein sequence and structural analyses provided insights into potential mechanisms underlying associations between MTB gene mutations and DR phenotypes. Taken together, these results revealed differences in acquired BDQ and CFZ resistance mechanisms as a new perspective that may enhance our understanding of BDQ/CFZ resistance mechanisms and facilitate the development of new methods for detecting MTB drug resistance genes.

Keywords: Mycobacterium tuberculosis; bedaquiline; clofazimine; resistance.

Fig 1

Sequence and structural analyses of MTB MmpR. (A) Multiple sequence alignments of MTB MmpR and its orthologs. According to BLAST results, the five orthologs of MmpR (UniProt entry I6Y8F7) with highest BLAST scores included a transcriptional regulator from Mycobacterium decipiens (UniProt entry A0A1 × 2M150), the putative regulatory protein MarR from Mycobacterium ulcerans (UniProt entry X8FGG6), a transcriptional regulator from Mycobacterium marinum (UniProt entry B2HSK6), a transcriptional regulator from Rhodococcus sp. WMMA185 (UniProt entry A0A1D8T546), and a MarR family protein from Sediminihabitans luteus (UniProt entry A0A2M9CD98). (B) Structures of wild-type MmpR (left) and MmpR G65E mutant (right) within MmpR-DNA complexes. MmpR protein (yellow) and DNA (gray) molecular structures are displayed in cartoon mode with residue 65 indicated in green, as based on the Corey–Pauling–Koltun color convention.