title: A common mechanism of inhibition of the Mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone.
abstract: Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C(18), C(20), and C(22) fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523-1682, USA.
authors.forename: Anna E
authors.initials: AE
authors.lastname: Grzegorzewicz
authors.forename: Jana
authors.initials: J
authors.lastname: Korduláková
authors.forename: Victoria
authors.initials: V
authors.lastname: Jones
authors.forename: Sarah E M
authors.initials: SE
authors.lastname: Born
authors.forename: Juan M
authors.initials: JM
authors.lastname: Belardinelli
authors.forename: Adrien
authors.initials: A
authors.lastname: Vaquié
authors.forename: Vijay A K B
authors.initials: VA
authors.lastname: Gundi
authors.forename: Jan
authors.initials: J
authors.lastname: Madacki
authors.forename: Nawel
authors.initials: N
authors.lastname: Slama
authors.forename: Françoise
authors.initials: F
authors.lastname: Laval
authors.forename: Julien
authors.initials: J
authors.lastname: Vaubourgeix
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Crew
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gicquel
authors.forename: Mamadou
authors.initials: M
authors.lastname: Daffé
authors.forename: Hector R
authors.initials: HR
authors.lastname: Morbidoni
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Brennan
authors.forename: Annaik
authors.initials: A
authors.lastname: Quémard
authors.forename: Michael R
authors.initials: MR
authors.lastname: McNeil
authors.forename: Mary
authors.initials: M
authors.lastname: Jackson
mesh_terms: D000483:Alleles; D000995:Antitubercular Agents / Q000494:pharmacology; D002473:Cell Wall / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D064429:Fatty Acid Synthases / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D016680:Genome, Bacterial; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D009163:Mycobacterium bovis / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D009171:Mycolic Acids / Q000037:antagonists & inhibitors*; D010670:Phenylthiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013828:Thioacetazone / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D000995:Antitubercular Agents; D008055:Lipids; D009171:Mycolic Acids; D011994:Recombinant Proteins; C100189:thiocarlide; D010670:Phenylthiourea; D064429:Fatty Acid Synthases; D013828:Thioacetazone