Site-directed mutagenesis of Mycobacterium tuberculosis and functional validation to investigate potential bedaquiline resistance-causing mutations

Abstract

Molecular detection of bedaquiline resistant tuberculosis is challenging as only a small proportion of mutations in candidate bedaquiline resistance genes have been statistically associated with phenotypic resistance. We introduced two mutations, atpE Ile66Val and Rv0678 Thr33Ala, in the Mycobacterium tuberculosis H37Rv reference strain using homologous recombineering or recombination to investigate the phenotypic effect of these mutations. The genotype of the resulting strains was confirmed by Sanger- and whole genome sequencing, and bedaquiline susceptibility was assessed by minimal inhibitory concentration (MIC) assays. The impact of the mutations on protein stability and interactions was predicted using mutation Cutoff Scanning Matrix (mCSM) tools. The atpE Ile66Val mutation did not elevate the MIC above the critical concentration (MIC 0.25-0.5 µg/ml), while the MIC of the Rv0678 Thr33Ala mutant strains (> 1.0 µg/ml) classifies the strain as resistant, confirming clinical findings. In silico analyses confirmed that the atpE Ile66Val mutation minimally disrupts the bedaquiline-ATP synthase interaction, while the Rv0678 Thr33Ala mutation substantially affects the DNA binding affinity of the MmpR transcriptional repressor. Based on a combination of wet-lab and computational methods, our results suggest that the Rv0678 Thr33Ala mutation confers resistance to BDQ, while the atpE Ile66Val mutation does not, but definite proof can only be provided by complementation studies given the presence of secondary mutations.

Figure 1

Structural modelling of the BDQ interaction with wild type and mutated Mtb ATP Synthase subunit C. Bedaquiline is shown in orange, the mutated residue is highlighted in blue, and the amino acids defining the binding cleft are highlighted in pink. (A) Cartoon representation of BDQ interacting with wild type ATP Synthase subunit C. (B) cartoon representation of BDQ interacting with mutated (Ile66Val) ATP Synthase subunit C. (C) Molecular surface representation of BDQ interacting with wild type ATP Synthase subunit C. (D) Molecular surface representation of BDQ interacting with mutated (Ile66Val) ATP Synthase subunit C.

Figure 2

Structural modelling of MmpR dimer in complex with its fatty acid ligand, 1,3-dihydroxypropan-2-yl octadecenoate. The dimerization domains are highlighted in blue, the DNA binding domains are highlighted in yellow, and the mutated residue is highlighted in pink. (A) Wild type MmpR dimer in complex with its fatty acid ligand. (B) Mutated (Thr33Ala) MmpR dimer in complex with its fatty acid ligand.