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 ΔhsaC 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 µM⁻¹ s⁻¹), and was inactivated by a halogenated substrate analogue (partition coefficienthsaC mutant, consistent with catechol toxicity. Structures of HsaC:DHSA binary complexes at 2.1 Å 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. Mycobacterium tuberculosis, the etiological agent of TB, is the most devastating infectious agent of mortality worldwide: it is carried by one-third of all humans and kills nearly two million people annually. Recent work has established that the pathogen metabolizes cholesterol, although the role of this metabolism in pathogenesis remains unclear. In the current study, we demonstrate that HsaC is a key enzyme in the cholesterol catabolic pathway and that it can be inactivated by compounds that resemble its substrate. Using molecular genetic approaches, we demonstrated that the enzyme is essential for the growth of M. tuberculosis on cholesterol and that a lack of this enzyme impairs the survival of the pathogen in each of two animal models. These studies provide definitive evidence that M. tuberculosis metabolizes cholesterol during infection and that this metabolism occurs during the early stages of infection. The oxygen-utilizing enzymes of the cholesterol catabolic pathway, of which HsaC is but one example, are intriguing potential chemotherapeutic targets, as their inhibition can lead to toxic metabolites, including reactive oxygen species. Overall, our study combines a variety of approaches to provide novel insights into a disease of global importance and into the mechanism of an interesting class of enzymes.