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

has been observed to develop resistance to the frontline anti-tuberculosis drug rifampicin, primarily through mutations in the rifampicin resistance-determining region (RRDR) of . While these mutations have been determined to confer a fitness cost, compensatory mutations in and that may enhance the fitness of resistant strains have been demonstrated. Recent genomic studies identified several non-RRDR mutations that co-occurred with RRDR mutations in clinical isolates without mutations and may confer fitness compensation. In this study, we identified 33 evolutionarily convergent non-RRDR mutations through phylogenomic analysis of public genomic data for clinical isolates. We found that none of these mutations, except V170F and I491F, can cause rifampin resistance in . The compensatory effects of five representative mutations across were evaluated by an 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 non-RRDR mutations, which could be utilized as additional molecular markers for predicting the fitness of clinical rifampin-resistant strains.