Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria.
Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria.
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DOI：
10.1128/mbio.00321-17
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发表时间：
2017-04-04
期刊：
影响因子：
6.4
通讯作者：
Eltis LD
中科院分区：
文献类型：
--
作者：
Crowe AM;Casabon I;Brown KL;Liu J;Lian J;Rogalski JC;Hurst TE;Snieckus V;Foster LJ;Eltis LD
大多数含有霉菌酸的放线菌和一些变形菌使用类固醇作为生长底物，但最后两个类固醇环的分解代谢尚未阐明。在结核分枝杆菌中，该途径包括毒力决定因素，并被提出由kstr2调控基因编码，其中包括预测辅酶a （CoA）转移酶基因（ipdAB）和酰基辅酶a还原酶基因（ipdC）。在胆固醇存在的情况下，结核分枝杆菌或乔氏红球菌菌株RHA1的ΔipdC和ΔipdAB突变体积累了先前未描述的代谢物：3aα-H-4α（羧基- coa）-5-羟基-7aβ-甲基六氢-1-吲哚酮（5-OH HIC-CoA）和(R)-2-（2-羧乙基）-3-甲基-6-氧环-1-烯-1-羧基- coa （COCHEA-CoA）。耻垢分枝杆菌ΔfadE32突变体积累了4-甲基-5-氧辛烷二酸（MOODA）。用纯化的IpdF、IpdC和烯酰辅酶a水合酶20 （crotonase超家族成员）孵育合成5-OH HIC-CoA，得到COCHEA-CoA，再用IpdAB和辅酶a硫酶进一步孵育，得到MOODA-CoA。基于这些研究，我们提出了类固醇分解代谢最后步骤的途径，其中5元环被EchA20水解，然后6元环被IpdAB水解。ΔipdF和ΔechA20突变体积累的代谢物支持该模型。这些基因在已知的类固醇降解细菌中的保存表明，这一途径是共享的。该途径进一步预测胆固醇分解代谢产生四种丙酰辅酶a、四种乙酰辅酶a、一种丙酮酸和一种琥珀酰辅酶a。最后，ΔipdAB结核分枝杆菌突变体不能在巨噬细胞中存活，并表现出与胆固醇依赖性毒性相关的CoASH水平严重降低。我们的结果和开发的工具为进一步阐明结核分枝杆菌的细菌类固醇分解代谢和毒力决定因素提供了基础。细菌是唯一已知的类固醇降解者，但负责降解最后两个类固醇环的途径尚未阐明。在结核分枝杆菌中，这一途径包括毒力决定因素。利用结核分枝杆菌和相关细菌的一系列突变体，我们确定了一些新的辅酶a硫酯作为途径中间体。代谢产物分析结合酶学研究确定了KstR2调控子编码的酶是如何水解打开最后两个类固醇环的。我们的研究结果为新的环降解酶提供了实验证据，显著提高了我们对细菌类固醇分解代谢的理解，并确定了以前未表征的胆固醇依赖性毒性，这可能有助于开发新的结核病治疗方法。
Most mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene (ipdAB) and an acyl-CoA reductase gene (ipdC). In the presence of cholesterol, ΔipdC and ΔipdAB mutants of either M. tuberculosis or Rhodococcus jostii strain RHA1 accumulated previously undescribed metabolites: 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A ΔfadE32 mutant of Mycobacterium smegmatis accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by ΔipdF and ΔechA20 mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a ΔipdAB M. tuberculosis mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in M. tuberculosis. Bacteria are the only known steroid degraders, but the pathway responsible for degrading the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants. Using a series of mutants in M. tuberculosis and related bacteria, we identified a number of novel CoA thioesters as pathway intermediates. Analysis of the metabolites combined with enzymological studies establishes how the last two steroid rings are hydrolytically opened by enzymes encoded by the KstR2 regulon. Our results provide experimental evidence for novel ring-degrading enzymes, significantly advance our understanding of bacterial steroid catabolism, and identify a previously uncharacterized cholesterol-dependent toxicity that may facilitate the development of novel tuberculosis therapeutics.