MadR mediates acyl CoA-dependent regulation of mycolic acid desaturation in mycobacteria

<i>Mycobacterium tuberculosis</i> has a lipid-rich cell envelope that is remodeled throughout infection to enable adaptation within the host. Few transcriptional regulators have been characterized that coordinate synthesis of mycolic acids, the major cell wall lipids of mycobacteria. Here, we show that the mycolic acid desaturase regulator (MadR), a transcriptional repressor of the mycolate desaturase genes <i>desA1</i> and <i>desA2</i>, controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A <i>madR</i>-null mutant of <i>M. smegmatis</i> exhibited traits of an impaired cell wall with an altered outer mycomembrane, accumulation of a desaturated α-mycolate, susceptibility to antimycobacterials, and cell surface disruption. Transcriptomic profiling showed that enriched lipid metabolism genes that were significantly down-regulated upon <i>madR</i> deletion included acyl-coenzyme A (aceyl-CoA) dehydrogenases, implicating it in the indirect control of β-oxidation pathways. Electromobility shift assays and binding affinities suggest a unique acyl-CoA pool-sensing mechanism, whereby MadR is able to bind a range of acyl-CoAs, including those with unsaturated as well as saturated acyl chains. MadR repression of <i>desA1</i>/<i>desA2</i> is relieved upon binding of saturated acyl-CoAs of chain length C₁₆ to C₂₄, while no impact is observed upon binding of shorter chain and unsaturated acyl-CoAs. We propose this mechanism of regulation as distinct to other mycolic acid and fatty acid synthesis regulators and place MadR as the key regulatory checkpoint that coordinates mycolic acid remodeling during infection in response to host-derived cell surface perturbation.