Studies of a ring-cleaving dioxygenase illuminate the role of cholesterol metabolism in the pathogenesis of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis, the etiological agent of TB, possesses a cholesterol catabolic pathway implicated in pathogenesis. This pathway includes an iron-dependent extradiol dioxygenase, HsaC, that cleaves catechols. Immuno-compromised mice infected with a DeltahsaC mutant of M. tuberculosis H37Rv survived 50% longer than mice infected with the wild-type strain. In guinea pigs, the mutant disseminated more slowly to the spleen, persisted less successfully in the lung, and caused little pathology. These data establish that, while cholesterol metabolism by M. tuberculosis appears to be most important during the chronic stage of infection, it begins much earlier and may contribute to the pathogen's dissemination within the host. Purified HsaC efficiently cleaved the catecholic cholesterol metabolite, DHSA (3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione; k(cat)/K(m) = 14.4+/-0.5 microM(-1) s(-1)), and was inactivated by a halogenated substrate analogue (partition coefficient<50). Remarkably, cholesterol caused loss of viability in the DeltahsaC mutant, consistent with catechol toxicity. Structures of HsaC:DHSA binary complexes at 2.1 A revealed two catechol-binding modes: bidentate binding to the active site iron, as has been reported in similar enzymes, and, unexpectedly, monodentate binding. The position of the bicyclo-alkanone moiety of DHSA was very similar in the two binding modes, suggesting that this interaction is a determinant in the initial substrate-binding event. These data provide insights into the binding of catechols by extradiol dioxygenases and facilitate inhibitor design.

Figure 1. The role of HsaC in the cholesterol degradation pathway.

Cholesterol is transformed to DHSA (3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione) via multiple enzymatic steps. HsaC catalyzes the extradiol ring-cleavage of DHSA to DSHA (4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-diene-4-oic acid).

Figure 2. The structural fold of HsaC, molecule A (DHSA bidentate bound).

The Cα traces of the structurally similar N- and C-terminal domains are colored in silver and dark green, respectively. As in other two-domain type I extradiol dioxygenases, the active site is located in the C-terminal domain. The iron ion is colored orange. The C and O atoms of the bound DHSA are grey and red, respectively.

Figure 3. The binding modes of DHSA in HsaC.

(A) DHSA in molecule A. (B) DHSA in molecule B. The (2F _o−F _c) electron density (blue, contour level = 1σ) was calculated without ligand to remove bias. (C) Stereo view of a structural superposition of DHSA in the two molecules in the asymmetric unit. HsaC:DHSA in molecule A is colored in dark green/yellow. HsaC:DHSA in molecule B is colored in light green/orange.

Figure 4. The design of a ΔhsaC mutant of M. tuberculosis.

(A) Genetic organization of the hsaC locus in wild-type H37Rv and the ΔhsaC mutant. The size of the XhoI fragments as well as the location of the probe relevant for Southern analysis are indicated. γδres, res-sites of the γδ-resolvase; hyg, hygromycin resistance gene. (B) Southern analysis of XhoI digested genomic DNA from wild-type H37Rv and three independent ΔhsaC mutant clones. Gene deletion was confirmed employing a [α-³²P]dCTP-labeled probe hybridizing to the position indicated in A.

Figure 5. Growth of a ΔhsaC mutant of M. tuberculosis on cholesterol and in mice.

(A) Growth of H37Rv strains in minimal media containing 0.1% (v/v) glycerol, 0.8% (v/v) isopropanol (solvent control), 0.02% (w/v) cholesterol with 0.8% (v/v) isopropanol, or no added carbon source. The plotted values represent the means of triplicates, with error bars indicating standard deviation. (B) Accumulation of a colored metabolite during cholesterol utilization by the ΔhsaC mutant. (C) Survival of SCID mice after intravenous infection with 10⁵ CFU of wild-type H37Rv, the ΔhsaC mutant or the complemented ΔhsaC mutant, respectively (n = 10 mice per group).

Figure 6. Growth of ΔhsaC mutant in the guinea pig model of tuberculosis.

(A) Growth kinetics in the lung and spleen of guinea pigs aerosol-infected with H37Rv, ΔhsaC mutant, or the complemented ΔhsaC mutant (n = 15 guinea pigs per group). Asterisks indicate significant (*p<0.05) or highly significant (**p<0.01) differences found between guinea pigs infected with the H37Rv wild-type and ΔhsaC mutant strain. (B) Gross pathology of guinea pig lungs infected with wild-type, mutant, and complemented strains at week 4 and week 8. (C) Histopathological appearance of same lung specimens as those depicted in (B).

Figure 7. Preparation of 2,3-dihydroxy-6-methyl-7,8-dihydro-10-Cl-stilbene (DHDS) via directed ortho metalation.