The metabolism of host cholesterol by Mycobacterium tuberculosis (Mtb) is an important factor for both its virulence and pathogenesis, although how and why cholesterol metabolism is required is not fully understood. Mtb uses a unique set of catabolic enzymes that are homologous to those required for classical β-oxidation of fatty acids but are specific for steroid-derived substrates. Here, we identify and assign the substrate specificities of two of these enzymes, ChsE4-ChsE5 (Rv3504-Rv3505) and ChsE3 (Rv3573c), that carry out cholesterol side chain oxidation in Mtb. Steady-state assays demonstrate that ChsE4-ChsE5 preferentially catalyzes the oxidation of 3-oxo-cholest-4-en-26-oyl CoA in the first cycle of cholesterol side chain β-oxidation that ultimately yields propionyl-CoA, whereas ChsE3 specifically catalyzes the oxidation of 3-oxo-chol-4-en-24-oyl CoA in the second cycle of β-oxidation that generates acetyl-CoA. However, ChsE4-ChsE5 can catalyze the oxidation of 3-oxo-chol-4-en-24-oyl CoA as well as 3-oxo-4-pregnene-20-carboxyl-CoA. The functional redundancy of ChsE4-ChsE5 explains the in vivo phenotype of the igr knockout strain of Mycobacterium tuberculosis; the loss of ChsE1-ChsE2 can be compensated for by ChsE4-ChsE5 during the chronic phase of infection. The X-ray crystallographic structure of ChsE4-ChsE5 was determined to a resolution of 2.0 Å and represents the first high-resolution structure of a heterotetrameric acyl-CoA dehydrogenase (ACAD). Unlike typical homotetrameric ACADs that bind four flavin adenine dinucleotide (FAD) cofactors, ChsE4-ChsE5 binds one FAD at each dimer interface, resulting in only two substrate-binding sites rather than the classical four active sites. A comparison of the ChsE4-ChsE5 substrate-binding site to those of known mammalian ACADs reveals an enlarged binding cavity that accommodates steroid substrates and highlights novel prospects for designing inhibitors against the committed β-oxidation step in the first cycle of cholesterol side chain degradation by Mtb.

Cholesterol is a major carbon source for Mycobacterium tuberculosis (Mtb) during infection, and cholesterol utilization plays a significant role in persistence and virulence within host macrophages. Elucidating the mechanism by which cholesterol is degraded may permit the identification of new therapeutic targets. Here, we characterized EchA19 (Rv3516), an enoyl-CoA hydratase involved in cholesterol side-chain catabolism. Steady-state kinetics assays demonstrated that EchA19 preferentially hydrates cholesterol enoyl-CoA metabolite 3-oxo-chol-4,22-diene-24-oyl-CoA, an intermediate of side-chain β-oxidation. In addition, succinyl-CoA, a downstream catabolite of propionyl-CoA that forms during cholesterol degradation, covalently modifies targeted mycobacterial proteins, including EchA19. Inspection of a 1.9 Å resolution X-ray crystallography structure of Mtb EchA19 suggests that succinylation of Lys132 and Lys139 may perturb enzymatic activity by modifying the entrance to the substrate binding site. Treatment of EchA19 with succinyl-CoA revealed that these two residues are hotspots for succinylation. Replacement of these specific lysine residues with negatively charged glutamate reduced the rate of catalytic hydration of 3-oxo-chol-4,22-diene-24-oyl-CoA by EchA19, as does succinylation of EchA19. Our findings suggest that succinylation is a negative feedback regulator of cholesterol metabolism, thereby adding another layer of complexity to Mtb physiology in the host. These regulatory pathways are potential noncatabolic targets for antimicrobial drugs.

Mycobacterium tuberculosis (Mtb) is the epitome of persistent. Mtb is the pathogen that causes tuberculosis, the leading cause of death by infection worldwide. The success of this pathogen is due in part to its clever ability to adapt to its host environment and its effective manipulation of the host immune system. A major contributing factor to the survival and virulence of Mtb is its acquisition and metabolism of host derived lipids including cholesterol. Accumulating evidence suggests that the catabolism of cholesterol during infection is highly regulated by cholesterol catabolites. We review what is known about how regulation interconnects with cholesterol catabolism. This framework provides support for an indirect approach to drug development that targets Mtb cholesterol metabolism through dysregulation of nutrient utilization pathways.

Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria species produce sterols, but their function in the bacterial domain remains unknown. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. Subsequent studies demonstrated that C-4 methylated sterols synthesized in the cytosol are localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. While proteins involved in eukaryotic sterol transport have been identified and characterized, their counterparts in bacteria remain enigmatic. In this study, we used bioinformatics, ligand binding analysis, and structural approaches to identify three novel bacterial sterol transporters from Methylococcus capsulatus. These proteins reside in the inner membrane, periplasm, and outer member of the bacterium, thereby working as a conduit to move modified sterols to the outer membrane. We report their remarkable specificity for recognizing 4-methylsterols, reveal the structural bases for substrate binding, and describe their structural divergence from eukaryotic sterol transporters. These findings provide unequivocal evidence for a distinct sterol transport system within the bacterial domain, which provide insights into our understanding of bacterial sterols and their divergence from similar lipids in higher-order organisms.SignificanceSterol lipids, such as cholesterol, were once thought to be unique to eukaryotes. However, a few bacterial species also produce sterols, albeit with structural modifications not observed in eukaryotes. While little is known about the function of bacterial sterols, it is known that sterols in certain bacteria are localized to the outer membrane. However, how bacteria transport these lipids from the cytoplasm to the outer membrane remains unknown. Here, we identify three novel bacterial sterol transporter proteins in the bacterium Methylococcus capsulatus that bind sterols methylated at the C-4 position. Through structural characterization of these proteins, we reveal insights into the mechanisms of sterol transport in bacteria with implications for understanding how bacterial cells interact with sterol lipids more broadly.

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