The Structure of the Transcriptional Repressor KstR in Complex with CoA Thioester Cholesterol Metabolites Sheds Light on the Regulation of Cholesterol Catabolism in Mycobacterium tuberculosis

Abstract

Cholesterol can be a major carbon source for Mycobacterium tuberculosis during infection, both at an early stage in the macrophage phagosome and later within the necrotic granuloma. KstR is a highly conserved TetR family transcriptional repressor that regulates a large set of genes responsible for cholesterol catabolism. Many genes in this regulon, including kstR, are either induced during infection or are essential for survival of M. tuberculosis in vivo. In this study, we identified two ligands for KstR, both of which are CoA thioester cholesterol metabolites with four intact steroid rings. A metabolite in which one of the rings was cleaved was not a ligand. We confirmed the ligand-protein interactions using intrinsic tryptophan fluorescence and showed that ligand binding strongly inhibited KstR-DNA binding using surface plasmon resonance (IC 50 for Tuberculosis remains a global threat to human health due to the emergence of extremely drug-resistant forms of Mycobacterium tuberculosis and co-infection with HIV (1). Elucidation of metabolic networks essential to the pathogenesis of M. tuberculosis is an important part of the quest to identify new drug target candidates that can be exploited to tackle extremely drug-resistant tuberculosis. In particular, M. tuberculosis possesses an unusual ability to metabolize lipids, and its genome is heavily orientated toward this task with the involvement of a remarkable number of genes, ϳ250, constituting around 6% of its genome (2).Cholesterol, the dominant lipid accumulated in M. tuberculosis-induced foamy macrophages, is a key growth substrate for M. tuberculosis in the intraphagosomal environment and is also important to the bacterium in necrotizing granulomas in late stage infection (3-5). M. tuberculosis is capable of utilizing cholesterol as a sole carbon source for both energy synthesis and assimilation into membrane lipids (3, 6). Many genes in the cholesterol degradation pathway are up-regulated or are essential for M. tuberculosis infection in various models of disease (3,4,(7)(8)(9)(10)(11). Disruption of genes encoding cholesterol catabolic enzymes, either through genetic manipulation or chemically, inhibits growth of the bacterium in both macrophage and animal models, and in vitro experiments suggest that this growth inhibition may be due to the toxicity of accumulated intermediates (4, 9, 12).The cholesterol degradation pathway is regulated by two TFRs, 7 KstR and KstR2 (13,14). These regulate two distinct regulons and negatively autoregulate their own expression. KstR regulates the expression of the genes involved in the trans-

Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis

“…The KstR regulon is controlled by the transcriptional repressor, KstR which regulates expression of the genes encoding enzymes responsible for metabolizing the side chain and A/B rings of cholesterol (Kendall et al, 2007). The KstR regulon is activated in a ‘feed forward’ manner when KstR is de-repressed by binding to the second cholesterol degradation intermediate, 3-oxocholest-4-en-26-oyl-CoA (3OCh-CoA) (Ho et al, 2016). Thus, down-regulation of the KstR regulon in the Δ lucA ::hyg mutant is consistent with a decrease in cholesterol uptake by the Δ lucA ::hyg mutant and suggests that 3OCh-CoA may not be produced to levels that are sufficient to de-repress the KstR regulon in the mutant during infection in macrophages.

10.7554/eLife.26969.005Figure 2.Transcriptional profile of the Δ lucA ::hyg mutant during infection in macrophages.Bacterial gene expression profiles were determined at day 3-post infection in resting murine macrophages.

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Molecular characterization of a new gene cluster for steroid degradation in Mycobacterium smegmatis

“…1). This behaviour cannot be attributed to regulatory constraints (Garc ıa-Fern andez et al, 2014;Ho et al, 2016) since the M. smegmatis DkstR (DMSMEG_6042) mutant can metabolize AD or ADD very inefficiently, but cannot metabolize 9OH-AD at all (Fig. 2).…”

“…AD) (Fern andez-Cabez on et al, 2017) and hence, the inability to use C-19 steroids as carbon sources cannot be due to transport problems. Therefore, we ascribed this behaviour to the fact that the cholesterol pathway might be more tightly regulated in M. smegmatis mc 2 155 than in other C-19 steroid degrading bacteria, and thus, these compounds might be metabolized only after the complete activation of the kstR regulon that only responds to some CoAderivatives of the side chain as inducers (Garc ıa-Fern andez et al, 2014;Ho et al, 2016). To check this hypothesis, we investigated whether the DkstR mutant of M. smegmatis (DMSMEG_6042), expressing constitutively the kstR regulon (Kendall et al, 2007;, was able to grow on C-19 steroids as the only carbon and energy sources.…”

A Förster Resonance Energy Transfer‐Based Ratiometric Sensor with the Allosteric Transcription Factor TetR

“…The number and type of identified aTF–analyte pairs are large and diverse. [ 32–37 ] Some compounds that can be sensed include toluene, [ 38 ] cholesterol, [ 39,40 ] parathion [ 41 ] (commonly used in insecticides), metabolites like pyruvate, [ 42 ] lactate, [ 43 ] and mevalonate, [ 44 ] and a number of ligands used as gene expression inducers in molecular biology and the synthetic biology community. [ 45,46 ] A recent report describes the optimized orthogonal responsivity of 12 aTFs for 12 different small molecules, [ 46 ] and other papers describe numerous examples of evolved and mutation‐optimized aTFs.…”