Characterization of Genomic Variants Associated with Resistance to Bedaquiline and Delamanid in Naive Mycobacterium tuberculosis Clinical Strains

Abstract

The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants' role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.

Keywords: Mycobacterium tuberculosis; bedaquiline; delamanid; resistance to new drugs; whole-genome sequencing.

FIG 1

Amino acid sequence alignment of BDQ-susceptible frameshift mutations in rv0678. The amino acid sequences of the translated proteins with I16fs and A153fs frameshift mutations (associated with a BDQ-susceptible MIC) were aligned with the Rv0678 wild-type amino acid sequence using ClustalX. In both cases, the two insertion mutations cause the addition of new amino acid residues without altering the frame of the Rv0678 protein and functional residues of the protein.

FIG 2

Cartoon representation of Rv0678 protein structures with mutations associated with BDQ-resistant or BDQ-susceptible phenotypes. Shown is a cartoon representation of the monomer present in the X-ray unit cell (PDB accession number 4NB5). The mutations associated with a resistant (red) or susceptible (green) phenotype from BDQ MIC assays are highlighted in sticks. In silico-predicted mutations that could alter dimerization or DNA binding function and are predicted to be associated with the BDQ-susceptible phenotype are shown in orange and blue, respectively. Zoomed-in cartoon representations of the Rv0678 dimerization domain and DNA binding domain are shown.

FIG 3

Lineage and country distributions of MTBc strains with variants in rv0678, atpE (rv1305), and pepQ (rv2535c) genomic regions. The graph reports all mutations found in rv0678, atpE, and pepQ genomic regions and shows their distribution among lineages and countries of isolation. The histograms refer to the number of strains harboring mutations (y axis). The colors of the histograms represent the different countries of isolation, while the patterns inside each bar represent the different lineages. On the x axis, the results of the MIC tests for available MTBc strains are also reported: red triangles are BDQ-resistant strains (MIC > 0.25 μg/ml), yellow boxes are BDQ-low-level-resistant strains (MIC = 0.25 μg/ml), and green triangles are BDQ-susceptible strains (MIC ≤ 0.12 μg/ml).

FIG 4

Cartoon representation of Ddn and Fgd1 protein structures with mutations associated with a DLM-resistant or a DLM-susceptible phenotype. (A) Ddn protein bound to the F420 cofactor (PDB accession number 3R5R), with mutations associated with a resistant (red) or susceptible (green) phenotype highlighted in sticks. The in silico-predicted DLM-resistant mutations (orange) and DLM-susceptible mutations (blue) are also reported. F420 is shown as magenta licorice. A zoomed-in cartoon representation of Ddn bound to F420 is shown. The T140-A82-K79-F420-H₂ hydrogen bond network is shown as a dashed yellow line, and the A82 and K79 residues are shown as sticks. (B) Fgd1 cartoon representation of homodimer-Fgd1 bound to F420 (PDB accession number 3B4Y). Helices involved in protein dimerization are shown in cyan. Highlighted in sticks are the DLM-resistant (red), DLM-susceptible (green), in silico-predicted DLM-resistant (orange), and in silico-predicted DLM-susceptible (blue) mutations.

FIG 5

Lineage and country distributions of MTBc strains with variants in the ddn (rv3547), fgd1 (rv0407), and fbiA-fbiB (rv3261-rv3262) genomic regions. The graph reports all mutations found in the ddn and fgd1 (A) and the fbiA and fbiB (B) genomic regions, showing their distributions among lineages and countries of isolation. The histograms refer to the number of strains harboring mutations (y axis). The colors of the histograms represent the different countries, while the patterns inside each bar represent the different lineages. On the x axis, the results of the MIC tests for available MTBc strains are also reported: red triangles are DLM-resistant strains (MIC ≥ 0.12 μg/ml), yellow boxes are low-level-resistant strains (MIC = 0.12 μg/ml), and green triangles are DLM-susceptible strains (MIC < 0.12 μg/ml). If a mutation was previously described in the literature, it is also reported (“S” for susceptibility and “R” for resistance to DLM).

FIG 6

Lineage and country distributions of MTBc strains with variants in the fbiC (rv1173) genomic region. In this graph are all mutations found in the fbiC genomic region, showing their distribution among lineages and countries of isolation. See the Fig. 5 legend for details.