A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages

Abstract

Rhodococcus sp. strain RHA1, a soil bacterium related to Mycobacterium tuberculosis, degrades an exceptionally broad range of organic compounds. Transcriptomic analysis of cholesterol-grown RHA1 revealed a catabolic pathway predicted to proceed via 4-androstene-3,17-dione and 3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3,4-DHSA). Inactivation of each of the hsaC, supAB, and mce4 genes in RHA1 substantiated their roles in cholesterol catabolism. Moreover, the hsaC(-) mutant accumulated 3,4-DHSA, indicating that HsaC(RHA1), formerly annotated as a biphenyl-degrading dioxygenase, catalyzes the oxygenolytic cleavage of steroid ring A. Bioinformatic analyses revealed that 51 rhodococcal genes specifically expressed during growth on cholesterol, including all predicted to specify the catabolism of rings A and B, are conserved within an 82-gene cluster in M. tuberculosis H37Rv and Mycobacterium bovis bacillus Calmette-Guérin. M. bovis bacillus Calmette-Guérin grew on cholesterol, and hsaC and kshA were up-regulated under these conditions. Heterologously produced HsaC(H37Rv) and HsaD(H37Rv) transformed 3,4-DHSA and its ring-cleaved product, respectively, with apparent specificities approximately 40-fold higher than for the corresponding biphenyl metabolites. Overall, we annotated 28 RHA1 genes and proposed physiological roles for a similar number of mycobacterial genes. During survival of M. tuberculosis in the macrophage, these genes are specifically expressed, and many appear to be essential. We have delineated a complete suite of genes necessary for microbial steroid degradation, and pathogenic mycobacteria have been shown to catabolize cholesterol. The results suggest that cholesterol metabolism is central to M. tuberculosis's unusual ability to survive in macrophages and provide insights into potential targets for novel therapeutics.

Fig. 1.

The deduced cholesterol catabolic pathway of Rhodococcus sp. RHA1, M. tuberculosis H37Rv, and M. bovis bacillus Calmette–Guérin. The enzymatic steps of side-chain degradation and ring opening are depicted. The latter are important for H37Rv survival in the macrophage (Fig. 2). Dashed arrows indicate multiple enzymatic steps. The compound in brackets undergoes nonenzymatic hydrolysis. Genes responsible for the degradation of rings C and D in RHA1 are not conserved in H37Rv or bacillus Calmette–Guérin. ADD, 1,4-androstadiene-3,17-dione; 9OHADD, 9α-hydroxy-1,4-androstadiene-3,17-dione; KshAB, 3-ketosteroid 9α-hydroxylase.

Fig. 2.

The cholesterol catabolic genes of Rhodococcus sp. RHA1 and M. tuberculosis H37Rv: comparison of their organization and their activities in different studies. (A) Genes in the physical map are color-coded according to assigned function: purple, uptake; red, side-chain degradation; blue, cleavage of rings A and B; orange, degradation of the DOHNAA propionate moiety; green, degradation of rings C and D. White arrows represent genes for which no reciprocal homologue is present. The nucleotide sequences of the M. tuberculosis H37Rv and M. bovis bacillus Calmette–Guérin clusters share 96% identity. (B) Heat map indicating correlation between gene expression (fold difference) during growth of RHA1 on cholesterol versus pyruvate (a), effect of gene disruption on H37Rv survival in IFN-γ-activated macrophages according to TraSH analysis (reciprocal of ratio) (16) (b), and gene expression in H37Rv after 48 h of growth in IFN-γ-activated macrophages (18) (c). M. tuberculosis genes predicted as essential for survival in the macrophage (16, 32) and in vivo in mice (17) are indicated by * and #, respectively.