Catabolism of the Last Two Steroid Rings in <italic toggle="yes">Mycobacterium tuberculosis</italic> and Other Bacteria
ABSTRACT 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 b...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2017-05-01
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| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.00321-17 |
| _version_ | 1818739481927221248 |
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| author | Adam M. Crowe Israël Casabon Kirstin L. Brown Jie Liu Jennifer Lian Jason C. Rogalski Timothy E. Hurst Victor Snieckus Leonard J. Foster Lindsay D. Eltis |
| author_facet | Adam M. Crowe Israël Casabon Kirstin L. Brown Jie Liu Jennifer Lian Jason C. Rogalski Timothy E. Hurst Victor Snieckus Leonard J. Foster Lindsay D. Eltis |
| author_sort | Adam M. Crowe |
| collection | DOAJ |
| description | ABSTRACT 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. IMPORTANCE 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. |
| first_indexed | 2024-12-18T01:25:32Z |
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| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-18T01:25:32Z |
| publishDate | 2017-05-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-8a695f8dfbf74f748bcfad84bcde434f2022-12-21T21:25:44ZengAmerican Society for MicrobiologymBio2150-75112017-05-018210.1128/mBio.00321-17Catabolism of the Last Two Steroid Rings in <italic toggle="yes">Mycobacterium tuberculosis</italic> and Other BacteriaAdam M. Crowe0Israël Casabon1Kirstin L. Brown2Jie Liu3Jennifer Lian4Jason C. Rogalski5Timothy E. Hurst6Victor Snieckus7Leonard J. Foster8Lindsay D. Eltis9Department of Biochemistry and Molecular Biology, Life Sciences Institute, The University of British Columbia, Vancouver, CanadaDepartment of Microbiology and Immunology, The University of British Columbia, Vancouver, CanadaDepartment of Microbiology and Immunology, The University of British Columbia, Vancouver, CanadaDepartment of Microbiology and Immunology, The University of British Columbia, Vancouver, CanadaDepartment of Microbiology and Immunology, The University of British Columbia, Vancouver, CanadaCentre for High-Throughput Biology (CHiBi), Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, CanadaDepartment of Chemistry, Queen’s University, Kingston, CanadaDepartment of Chemistry, Queen’s University, Kingston, CanadaCentre for High-Throughput Biology (CHiBi), Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, CanadaDepartment of Microbiology and Immunology, The University of British Columbia, Vancouver, CanadaABSTRACT 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. IMPORTANCE 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.https://journals.asm.org/doi/10.1128/mBio.00321-17CoA thioesterMycobacterium tuberculosiscatabolismcholesterolring opening |
| spellingShingle | Adam M. Crowe Israël Casabon Kirstin L. Brown Jie Liu Jennifer Lian Jason C. Rogalski Timothy E. Hurst Victor Snieckus Leonard J. Foster Lindsay D. Eltis Catabolism of the Last Two Steroid Rings in <italic toggle="yes">Mycobacterium tuberculosis</italic> and Other Bacteria mBio CoA thioester Mycobacterium tuberculosis catabolism cholesterol ring opening |
| title | Catabolism of the Last Two Steroid Rings in <italic toggle="yes">Mycobacterium tuberculosis</italic> and Other Bacteria |
| title_full | Catabolism of the Last Two Steroid Rings in <italic toggle="yes">Mycobacterium tuberculosis</italic> and Other Bacteria |
| title_fullStr | Catabolism of the Last Two Steroid Rings in <italic toggle="yes">Mycobacterium tuberculosis</italic> and Other Bacteria |
| title_full_unstemmed | Catabolism of the Last Two Steroid Rings in <italic toggle="yes">Mycobacterium tuberculosis</italic> and Other Bacteria |
| title_short | Catabolism of the Last Two Steroid Rings in <italic toggle="yes">Mycobacterium tuberculosis</italic> and Other Bacteria |
| title_sort | catabolism of the last two steroid rings in italic toggle yes mycobacterium tuberculosis italic and other bacteria |
| topic | CoA thioester Mycobacterium tuberculosis catabolism cholesterol ring opening |
| url | https://journals.asm.org/doi/10.1128/mBio.00321-17 |
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