A thiolase of Mycobacterium tuberculosis is required for virulence and production of androstenedione and androstadienedione from cholesterol

Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that shifts to a lipid-based metabolism in the host. Moreover, metabolism of the host lipid cholesterol plays an important role in M. tuberculosis infection. We used transcriptional profiling to identify genes transcriptionally regulated by cholesterol and KstR (Rv3574), a TetR-like repressor. The fadA5 (Rv3546) gene, annotated as a lipid-metabolizing thiolase, the expression of which is upregulated by cholesterol and repressed by KstR, was deleted in M. tuberculosis H37Rv. We demonstrated that fadA5 is required for utilization of cholesterol as a sole carbon source in vitro and for full virulence of M. tuberculosis in the chronic stage of mouse lung infection. Cholesterol is not toxic to the fadA5 mutant strain, and, therefore, toxicity does not account for its attenuation. We show that the wild-type strain, H37Rv, metabolizes cholesterol to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) and exports these metabolites into the medium, whereas the fadA5 mutant strain is defective for this activity. We demonstrate that FadA5 catalyzes the thiolysis of acetoacetyl-coenzyme A (CoA). This catalytic activity is consistent with a beta-ketoacyl-CoA thiolase function in cholesterol beta-oxidation that is required for the production of androsterones. We conclude that the attenuated phenotype of the fadA5 mutant is a consequence of disrupted cholesterol metabolism that is essential only in the persistent stage of M. tuberculosis infection and may be caused by the inability to produce AD/ADD from cholesterol.

FIG. 1.

Role of Hsd, KstD, KshA/KshB and HsaA-HsaD in M. tuberculosis cholesterol degradation. KstD, KshA/KshB, and HsaA-HsaD most likely accept side chain metabolites as substrates. Only the 17-keto (AD) metabolite is shown for clarity.

FIG. 2.

The Cho-region of the M. tuberculosis genome. The region of the M. tuberculosis genome (Rv3492c to Rv3574) identified by microarray analysis that includes a large cluster of genes regulated by cholesterol and KstR. In the shaded columns, black shading indicates at least 1.5-fold induction, and light gray shading indicates no change in induction: c3, induced by cholesterol in M. tuberculosis after 3 h; c24, induced by cholesterol in M. tuberculosis after 24 h; k, induced by mutation of kstR in M. tuberculosis. White indicates missing data. CoA, coenzyme A; CHP, conserved hypothetical protein; HP, hypothetical protein. *, genes that have orthologs in the Rhodococcus RHA1 Cho-region.

FIG. 3.

fadA5 is required for growth on cholesterol as the sole carbon source. The strains were grown in 7H9 medium containing 1 mg ml⁻¹ (2.6 mM) cholesterol in tyloxapol or 7H9 medium containing only tyloxapol at 37°C. The wild-type H37Rv (Rv), fadA5 mutant, and complemented fadA5 (fadA5comp) strains were grown with and without cholesterol as indicated on the graph. Data represent results of each experiment run in duplicate.

FIG. 4.

fadA5 is required for virulence in the mouse model of infection. Mice were infected with 100 to 300 CFU/lung of the respective strain of M. tuberculosis wild-type H37Rv, fadA5 mutant, or fadA5 complement, as indicated. Data represent the results of three mice at each time point. This profile is representative of three independent time courses performed.

FIG. 5.

Mutation of fadA5 does not cause cholesterol toxicity. Strains were grown to exponential phase in 7H9 medium supplemented with glycerol and dextrose. Cultures were diluted into 7H9 medium containing glycerol and dextrose with or without cholesterol (1 mg ml⁻¹ in 20% Tween). Values are for the wild-type H37Rv strain without cholesterol, wild-type H37Rv with cholesterol, the fadA5 mutant without cholesterol, and fadA5 mutant with cholesterol, as indicated. Data represent the results of the experiment done in triplicate.

FIG. 6.

SDS-PAGE analysis of purified FadA5. Lane 1, Ni²⁺-IMAC-purified FadA5; lane 2, molecular mass markers. Numbers represent the mass in kDa.

FIG. 7.

Two-substrate steady-state kinetics of FadA5. Initial velocities were measured over a range of CoA and AcAcCoA concentrations in a mixture of 100 mM Tris-HCl, pH 8.1, 25 mM MgCl₂, and 5 mM Tris(2-carboxyethyl)phosphine at 30°C. The data were fit to equation 1. The data shown are the average of two independent experiments, and the errors are the standard deviation of the measurements. (A) Hyperbolic plot of initial velocity versus [CoA] at a fixed [AcAcCoA] of 150 μM. (B) Hyperbolic plot of initial velocity versus [AcAcCoA] at a fixed [CoA] of 50 μM.

FIG. 8.

fadA5 is required for the production of AD and ADD from cholesterol by M. tuberculosis. Data are from liquid chromatography with detection at 240 nm for analysis of H37Rv, fadA5, and complemented fadA5 culture supernatants that were prepared as described in Materials and Methods. The chromatographic profile from 1.3 to 2.0 min is shown. The full profile and mass spectral analysis are shown in Fig. S1 and S2 in the supplemental material. These profiles are representative of three independent biological replicates. AU, arbitrary units.

FIG. 9.

Proposed catalytic role or roles for FadA5 in the pathway for metabolism of the cholesterol side chain.