Abstract
Mycobacterium tuberculosis 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 desA1 and desA2, controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A madR-null mutant of M. smegmatis 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 madR 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 desA1/desA2 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.
Highlights
The etiological agent of tuberculosis, Mycobacterium tuberculosis, has a distinct cell envelope among bacteria that influences its virulence and survival within the host
We performed RNA sequencing of the madR mutant along with the parental wild type (WT) and complemented strain (Dataset S1). Both desA1 and desA2 were significantly up-regulated (P < 0.01) in M. smegmatis ΔmadR compared to WT by 3.0 and 2.2 log2 fold change, respectively, while they were not significantly differentially expressed in the complemented strain compared to WT (Fig. 2A and SI Appendix, Table S2)
The results here inform a picture of mycolic acid desaturase regulator (MadR) as a regulator of mycolic acid biosynthesis, the desaturation step
Summary
The etiological agent of tuberculosis, Mycobacterium tuberculosis, has a distinct cell envelope among bacteria that influences its virulence and survival within the host. FasR regulates FAS-I by activation of the fas-acpS cluster, and MabR is an activator of the FAS-II gene cluster consisting of fabD-acpM-kasA-kasB, responsible for the biosynthesis of the longer mero-chain of mycolic acids [6, 7] Both transcription factors (TFs) have been shown to coordinate target promoter binding in response to long-chain acyl-CoA binding in vitro and upon uptake of exogenous fatty acids [1, 7, 8]. DesA1 and desA2 were found to be up-regulated (4.7-fold and 2.5-fold) early in the transition to NRP and repressed during stationary phase [16] Taken together, these implicate the regulation of desA1/desA2 as having a dual role both responsible for driving the remodeling of mycolates early in infection (via modulating desaturation) and for limiting mycolate synthesis for persistence (by reducing overall mycolic acid biosynthesis via desA1/desA2 repression). We queried the molecular mechanism of MadR-mediated repression of desA1 and desA2
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