Frey, A. M.; Babunovic, G. H.; Culviner, P. H.; Wang, X.; Meirav, E.; Gan, M.; Zhu, J.; Moody, D. B.; Liu, Q.; Fortune, S. M.

Antibiotic pressure causes pathogens to evolve many forms of altered drug susceptibility. In addition to target or activator mutations conferring canonical drug resistance, mutations can serve as steppingstones to or enhancers of resistance. In clinical strains of Mycobacterium tuberculosis (Mtb), we find that idsA2, which encodes an isoprenyl pyrophosphate synthase involved in the synthesis of precursors for essential components of the cell wall and electron transport chain, is undergoing diversifying selection and that these mutations are associated with the acquisition of first-line antibiotic resistance. By engineering isogenic Mtb strains that express clinically prevalent variants of idsA2, we show that clinical variants increase the minimum inhibitory concentration of ethambutol and, to a lesser extent, of isoniazid. Targeted lipid analyses reveal that disrupting IdsA2 function redirects limited resources in the isoprenoid synthesis pathway, leading to increased production of decaprenylphosporyl pentose which can compete with ethambutol for binding to arabinosyltransferases. IdsA2 mutations most often occur after embB mutation and lead to a multiplicative increase in ethambutol resistance. Thus, identification of idsA2 mutations can be utilized to improve the specificity of genotypic ethambutol susceptibility testing. Together, this work defines idsA2 as an ethambutol resistance gene and demonstrates how metabolic remodeling can augment drug resistance.