title: Catabolism of the Last Two Steroid Rings inMycobacterium tuberculosisand 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. InMycobacterium 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, ΔipdCand ΔipdABmutants of eitherM. tuberculosisorRhodococcus jostiistrain 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 ΔfadE32mutant ofMycobacterium smegmatisaccumulated 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 ΔipdFand ΔechA20mutants 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. tuberculosismutant 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 inM. tuberculosis.IMPORTANCEBacteria are the only known steroid degraders, but the pathway responsible for degrading the last two steroid rings has yet to be elucidated. InMycobacterium tuberculosis, this pathway includes virulence determinants. Using a series of mutants inM. tuberculosisand 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Life Sciences Institute, The University of British Columbia, Vancouver, Canada.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Crowe
authors.affiliation: Department of Microbiology and Immunology, The University of British Columbia, Vancouver, Canada.
authors.forename: Israël
authors.initials: I
authors.lastname: Casabon
authors.affiliation: Department of Microbiology and Immunology, The University of British Columbia, Vancouver, Canada.
authors.forename: Kirstin L
authors.initials: KL
authors.lastname: Brown
authors.affiliation: Department of Microbiology and Immunology, The University of British Columbia, Vancouver, Canada.
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Microbiology and Immunology, The University of British Columbia, Vancouver, Canada.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lian
authors.affiliation: Centre for High-Throughput Biology (CHiBi), Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, Canada.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Rogalski
authors.affiliation: Department of Chemistry, Queen's University, Kingston, Canada.
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Hurst
authors.affiliation: Department of Chemistry, Queen's University, Kingston, Canada.
authors.forename: Victor
authors.initials: V
authors.lastname: Snieckus
authors.affiliation: Centre for High-Throughput Biology (CHiBi), Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, Canada.
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Foster
authors.affiliation: Department of Microbiology and Immunology, The University of British Columbia, Vancouver, Canada leltis@mail.ubc.ca.|Department of Biochemistry and Molecular Biology, Life Sciences Institute, The University of British Columbia, Vancouver, Canada.
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Eltis
mesh_terms: D002784:Cholesterol / Q000378:metabolism*; D017353:Gene Deletion; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008660:Metabolism; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D012240:Rhodococcus / Q000235:genetics / Q000378:metabolism
chemical_list: D002784:Cholesterol
keywords: CoA thioester; Mycobacterium tuberculosis; catabolism; cholesterol; ring opening