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Compensatory effects of M. tuberculosis rpoB mutations outside the rifampicin resistance-determining region.

ABSTRACT: Mycobacterium tuberculosis has been observed to develop resistance to the frontline anti-tuberculosis drug rifampicin, primarily through mutations in the rifampicin resistance-determining region (RRDR) of rpoB. While these mutations have been determined to confer a fitness cost, compensatory mutations in rpoA and rpoC that may enhance the fitness of resistant strains have been demonstrated. Recent genomic studies identified several rpoB non-RRDR mutations that co-occurred with RRDR mutations in clinical isolates without rpoA/rpoC mutations and may confer fitness compensation. In this study, we identified 33 evolutionarily convergent rpoB non-RRDR mutations through phylogenomic analysis of public genomic data for clinical M. tuberculosis isolates. We found that none of these mutations, except V170F and I491F, can cause rifampin resistance in Mycolicibacterium smegmatis. The compensatory effects of five representative mutations across rpoB were evaluated by an in vitro competition assay, through which we observed that each of these mutations can significantly improve the relative fitness of the initial S450L mutant (0.97-1.08 vs 0.87). Furthermore, we observed that the decreased RNAP transcription efficiency introduced by S450L was significantly alleviated by each of the five mutations. Structural analysis indicated that the fitness compensation observed for the non-RRDR mutations might be achieved by modification of the RpoB active centre or by changes in interactions between RNAP subunits. Our results provide experimental evidence supporting that compensatory effects are exerted by several rpoB non-RRDR mutations, which could be utilized as additional molecular markers for predicting the fitness of clinical rifampin-resistant M. tuberculosis strains.

SUBMITTER: Ma P

PROVIDER: S-EPMC8057087 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Tuberculosis (TB), caused by the Mycobacterium tuberculosis infection, continues to be a leading cause of morbidity and mortality in developing countries. Resistance to the first-line anti-TB drugs, isoniazid (INH) and rifampicin (RIF), is a major drawback to effective TB treatment. Genetic mutations in the β-subunit of the DNA-directed RNA polymerase (rpoB) are reported to be a major reason of RIF resistance. However, the structural basis and mechanisms of these resistant mutations are insufficiently understood. In the present study, thirty drug-resistant mutants of rpoB were initially modeled and screened against RIF via a comparative molecular docking analysis with the wild-type (WT) model. These analyses prioritized six mutants (Asp441Val, Ser456Trp, Ser456Gln, Arg454Gln, His451Gly, and His451Pro) that showed adverse binding affinities, molecular interactions, and RIF binding hinderance properties, with respect to the WT. These mutant models were subsequently analyzed by molecular dynamics (MD) simulations. One-hundred nanosecond all-atom MD simulations, binding free energy calculations, and a dynamic residue network analysis (DRN) were employed to exhaustively assess the impact of mutations on RIF binding dynamics. Considering the global structural motions and protein-ligand binding affinities, the Asp441Val, Ser456Gln, and His454Pro mutations generally yielded detrimental effects on RIF binding. Locally, we found that the electrostatic contributions to binding, particularly by Arg454 and Glu487, might be adjusted to counteract resistance. The DRN analysis revealed that all mutations mostly distorted the communication values of the critical hubs and may, therefore, confer conformational changes in rpoB to perturb RIF binding. In principle, the approach combined fundamental molecular modeling tools for robust "global" and "local" level analyses of structural dynamics, making it well suited for investigating other similar drug resistance cases.

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Compensatory effects of M. tuberculosis rpoB mutations outside the rifampicin resistance-determining region.

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