title: Binding pocket alterations in dihydrofolate synthase confer resistance to para-aminosalicylic acid in clinical isolates of Mycobacterium tuberculosis.
abstract: The mechanistic basis for the resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor para-aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance in M. tuberculosis strain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and Mycobacterium bovis. From a panel of 85 multidrug-resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in the folC gene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Zhao
authors.forename: Xu-De
authors.initials: XD
authors.lastname: Wang
authors.forename: Luke N
authors.initials: LN
authors.lastname: Erber
authors.forename: Ming
authors.initials: M
authors.lastname: Luo
authors.forename: Ai-zhen
authors.initials: AZ
authors.lastname: Guo
authors.forename: Shan-shan
authors.initials: SS
authors.lastname: Yang
authors.forename: Jing
authors.initials: J
authors.lastname: Gu
authors.forename: Breanna J
authors.initials: BJ
authors.lastname: Turman
authors.forename: Yun-rong
authors.initials: YR
authors.lastname: Gao
authors.forename: Dong-fang
authors.initials: DF
authors.lastname: Li
authors.forename: Zong-qiang
authors.initials: ZQ
authors.lastname: Cui
authors.forename: Zhi-ping
authors.initials: ZP
authors.lastname: Zhang
authors.forename: Li-jun
authors.initials: LJ
authors.lastname: Bi
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Baughn
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
authors.forename: Jiao-Yu
authors.initials: JY
authors.lastname: Deng
mesh_terms: D000483:Alleles; D010131:Aminosalicylic Acid / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D008826:Microbial Sensitivity Tests; D020125:Mutation, Missense / Q000235:genetics / Q000502:physiology; D009163:Mycobacterium bovis / Q000187:drug effects / Q000201:enzymology; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D000900:Anti-Bacterial Agents; D010131:Aminosalicylic Acid; D010453:Peptide Synthases; C020041:dihydrofolate synthetase