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 lipidmetabolizing 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 ␤-ketoacyl-CoA thiolase function in cholesterol ␤-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.Circumstantial evidence implicates a role for cholesterol in M. tuberculosis infection. How this host lipid affects infection and the role of its metabolism by the bacteria is not clear. Foamy macrophages, laden with lipid droplets, are known to accumulate in lung granulomas in human infection (23) as well as in infected mouse lungs (4). Electron microscopy revealed that M. tuberculosis is in close apposition to lipid bodies in foamy macrophages and that the bacteria eventually merge with these lipid bodies and even accumulate intracellular lipids (22). In fact, since foamy macrophages contain high levels of cholesterol esters (19,32), it is possible that the lipid bodies observed to merge with M. tuberculosis bacteria are composed of cholesterol esters. Macrophages infected with Mycobacterium leprae also accumulate cholesterol esters (17) that are thought to be responsible for the conversion of macrophages into foam cells. Significantly, bacteria from the sputum of human tuberculosis (TB) patients were observed to contain lipid bodies, and transcriptional profiling of bacteria from these samples demonstrated the induction of genes proposed to encode enzymes required for cholesterol utilization (13). These studies implicate host lipids and possibly cholesterol in the development of mycobacterial disease.The first direct evidence suggesting that bacterial cholesterol metabolism is required for M. tuberculosis vir...

Characterization of the KstR-dependent promoter of the gene for the first step of the cholesterol degradative pathway in Mycobacterium smegmatis

“…Moreover, the expression level of the Rv1106c gene is similar to that of the preceding xseAB genes (Nesbitt et al, 2010), reinforcing the hypothesis that all these genes form part of the same operon. The short distance (9 bp) between the stop codon of xseB and the start codon of the Rv1106c gene in M. tuberculosis also precludes the existence of a transcription terminator in this region.…”

“…Moreover, transcriptomic analyses have demonstrated that the Rv1106c gene is expressed at the same level in the wildtype strain as in a completely derepressed KstR mutant of M. tuberculosis (Nesbitt et al, 2010). Moreover, the expression level of the Rv1106c gene is similar to that of the preceding xseAB genes (Nesbitt et al, 2010), reinforcing the hypothesis that all these genes form part of the same operon.…”

“…One of these repressors, named KstR, is encoded by the Rv3574 gene in M. tuberculosis and by the MSMEG_6042 gene in M. smegmatis. KstR controls the expression of 83 cholesterol catabolic genes (kstR regulon) (Kendall et al, 2007); many of these genes are essential for virulence, highlighting the importance of cholesterol catabolism in the pathogenicity of M. tuberculosis (Chang et al, 2007;Hu et al, 2010;Nesbitt et al, 2010;Rengarajan et al, 2005;Sassetti & Rubin, 2003;Schnappinger et al, 2003;Yam et al, 2009). In spite of the relevant role that the kstR regulon seems to play, the mechanisms involved in this regulation are still poorly understood.…”

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

“…Metabolites with a C 5 side chain have not been reported to accumulate during growth on cholesterol. Furthermore, degradation of the cholesterol side chain is predicted to proceed via thiolytic cleavage, yielding a C 5 CoA thioester intermediate not requiring activation (47). Consistent with this prediction, FadD17 does not appear to be essential for growth of M. tuberculosis on cholesterol (31).…”

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

“…The cholesterol catabolic pathway of Mtb is very similar to that of other pathogenic and environmental actinomycetes (10), as initially predicted in Rhodococcus jostii RHA1 (5). Sidechain degradation is initiated by a cytochrome P450 and proceeds via a ␤-oxidative process (11).…”