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 coefficient<50). Remarkably, cholesterol caused loss of viability in the ΔhsaC 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.
Highlights
Mycobacterium tuberculosis, the leading cause of mortality among bacterial pathogens, infects one-third of the human population and is responsible for approximately 2 million deaths annually
The phenotype of the DhsaC H37Rv mutant in cholesterolcontaining medium, SCID mice and guinea pigs provides clear evidence that cholesterol metabolism contributes to the survival of M. tuberculosis in the host
The first evidence for the role of cholesterol metabolism during pathogenesis was derived from genome-wide insertional mutagenesis studies [3] and the upregulation of cholesterol catabolic genes during infection of macrophages [18]
Summary
Mycobacterium tuberculosis, the leading cause of mortality among bacterial pathogens, infects one-third of the human population and is responsible for approximately 2 million deaths annually. An important factor that contributes to the disease’s prevalence is the pathogen’s unusual ability to survive for long periods of time, and even to replicate, in the macrophage [1]. A suite of genes critical for survival of M. tuberculosis in the macrophage [3] was recently discovered to be involved in cholesterol degradation [4]. The resulting phenolic metabolite is hydroxylated, yielding a catechol, 3,4-dihydroxy-9,10seco-nandrost-1,3,5(10)-triene-9,17-dione (DHSA). Recent work by Pandey and Sassetti [5] indicates that in vitro, the pathogen uses different parts of the cholesterol molecule for energy and the biosynthesis of phthiocerol dimycocerosate (PDIM), a virulence-associated lipid, respectively. Using a mutant defective in the mce4-encoded cholesterol transporter [6], Pandey and Sassetti further demonstrated that cholesterol uptake is essential for Author Summary
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