The emergence of resistance to the antiparasitic selamectin in <i>Mycobacterium smegmatis</i> is improbable and contingent on cell wall integrity

Tuberculosis remains the deadliest infectious disease of the 21^st century. New antimicrobials are needed to improve treatment outcomes and enable therapy shortening. Drug repurposing is an alternative to the traditional drug discovery process. The avermectins are a family of macrocyclic lactones with anthelmintic activity active against <i>Mycobacterium tuberculosis</i>. However, their mode of action in mycobacteria remains unknown. In this study, we employed traditional mutant isolation approaches using <i>Mycobacterium smegmatis</i>, a non-pathogenic <i>M. tuberculosis</i> surrogate. We were only able to isolate mutants with decreased susceptibility to selamectin using the ∆<i>nucS</i> mutator <i>M. smegmatis</i> strain. This phenotype was caused by mutations in <i>mps1</i> and <i>mmpL11</i>. Two of these mutants were used for a second experiment in which high-level selamectin-resistant mutants were isolated; however, specific mutations driving the phenotypic change to high-level resistance could not be identified. The susceptibility to selamectin in these mutants was restored to the basal level by subinhibitory concentrations of ethambutol. The selection of ethambutol resistance in a high-level selamectin-resistant mutant also resulted in multiple colonies becoming susceptible to selamectin again. These colonies carried mutations in <i>embB</i>, suggesting that the integrity of the cell envelope is a prerequisite for selamectin resistance. The absence of increased susceptibility to selamectin in an <i>embB</i> deletion strain demonstrated that the target of selamectin is not cytosolic. Our data show that the concurrence of specific multiple mutations and complete integrity of the mycobacterial envelope are necessary for selamectin resistance. Our studies provide first-time insights into the antimycobacterial mode of action of the antiparasitic avermectins.IMPORTANCETuberculosis is the deadliest infectious disease of the 21^st century. New antibiotics are needed to improve treatment. However, developing new drugs is costly and lengthy. Drug repurposing is an alternative to the traditional drug discovery process. The avermectins are a family of drugs used to treat parasitic infections that are active against <i>Mycobacterium tuberculosis</i>, the bacterium that causes tuberculosis. However, their mode of action in mycobacteria remains unknown. Understanding how avermectins kill mycobacteria can facilitate its development as an anti-mycobacterial drug, including against <i>M. tuberculosis</i>.In this study, we used <i>Mycobacterium smegmatis</i>, a non-pathogenic <i>M. tuberculosis</i> surrogate model to understand the molecular mechanisms of how selamectin (a drug of the avermectin family selected for this study as a model) acts against mycobacteria. Our data show that the generation of resistance to selamectin is unlikely and that complete integrity of the mycobacterial envelope is necessary for selamectin resistance, providing first-time insights into the antimycobacterial mode of action of the avermectins.