Rapidly-correcting Frameshift Mutations in the Mycobacterium tuberculosis orn Gene Produce Reversible Ethambutol Resistance and Small Colony Variant Morphology.

We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB)-resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosis orn gene. A frameshift mutation in orn produces small colony variant (SCV) phenotype, but this mutation does not change the minimal inhibitory drug concentrations (MICs) of any drug in wild-type M. tuberculosis However, the same orn mutation in a low level EMB resistant double embB-aftA mutant (MIC=8μg/ml) produces an SCV with an EMB MIC of 32μg/ml. Reversible-resistance is indistinguishable from a drug-persistent phenotype because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size and reversing the EMB MIC back to the 8μg/ml MIC of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to the parental embB-aftA mutant, whereas large colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug-resistance in human tuberculosis.