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

Rapid and accurate molecular detection of bedaquiline resistant tuberculosis is difficult as only a few of the many reported variants in candidate bedaquiline resistance genes have been statistically associated with phenotypic bedaquiline resistance. Several approaches are thus needed to better understand the mechanisms underlying bedaquiline resistance. In this study, we performed site-directed mutagenesis to investigate the phenotypic effect of the presence of two variants: atpE Ile66Vale and Rv0678 Thr33Ala. We introduced these variants in the Mycobacterium tuberculosis H37Rv reference strain using homologous recombineering or recombination. The genotype of the resulting strains was confirmed by Sanger sequencing and whole genome sequencing, and minimal inhibitory concentration (MIC) assays were performed to assess bedaquiline susceptibility. Although introduction of secondary unwanted variants could not be avoided, we successfully generated the mutants of interest in Mycobacterium tuberculosis H37Rv. Phenotypic analysis showed that the atpE Ile66Val variant did not raise the MIC above the critical concentration (MIC 0.25 – 0.5 µg/ml), confirming the observation in a single clinical strain and contradicting in silico predictions made in a prior study. The MIC of the Rv0678 Thr33Ala mutant strains (>1.0 µg/ml) classifies the strain as resistant, confirming both clinical findings and the predicted disruption of the DNA binding affinity of the MmpR protein. We also performed in silico analyses to predict the impact of the atpE Ile66Vale and Rv0678 Thr33Ala on protein stability and interactions. The structure of Mycobacterium tuberculosis ATP synthase subunit c was obtained through homology modelling with MODELLER and the impact of the mutations on their protein structure and function was assessed using multiple established mCSM tools. Analyses confirmed that the atpE Ile66Val variant minimally disrupts the bedaquiline – ATP Synthase interaction and predicted that the Rv0678 Thr33Ala variant substantially affects the DNA binding affinity of the MmpR transcriptional repressor. This study demonstrates that both in vitro site-directed mutagenesis of Mycobacterium tuberculosis and structural modelling are important and complementary methods that help improve our understanding of bedaquiline resistance.