title: The complex mechanism of antimycobacterial action of 5-fluorouracil.
abstract: A combination of chemical genetic and biochemical assays was applied to investigate the mechanism of action of the anticancer drug 5-fluorouracil (5-FU), against Mycobacterium tuberculosis (Mtb). 5-FU resistance was associated with mutations in upp or pyrR. Upp-catalyzed conversion of 5-FU to FUMP was shown to constitute the first step in the mechanism of action, and resistance conferred by nonsynonymous SNPs in pyrR shown to be due to derepression of the pyr operon and rescue from the toxic effects of FUMP and downstream antimetabolites through de novo production of UMP. 5-FU-derived metabolites identified in Mtb were consistent with the observed incorporation of 5-FU into RNA and DNA and the reduced amount of mycolyl arabinogalactan peptidoglycan in 5-FU-treated cells. Conditional depletion of the essential thymidylate synthase ThyX resulted in modest hypersensitivity to 5-FU, implicating inhibition of ThyX by fluorodeoxyuridylate as a further component of the mechanism of antimycobacterial action of this drug.
authors.affiliation: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research, Institute of Infectious Disease and Molecular Medicine and Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory 7925, Rondebosch, Cape Town 7700, South Africa.
authors.forename: Vinayak
authors.initials: V
authors.lastname: Singh
authors.affiliation: Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-1, 84125 Bratislava, Slovak Republic.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Brecik
authors.affiliation: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research, Institute of Infectious Disease and Molecular Medicine and Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory 7925, Rondebosch, Cape Town 7700, South Africa.
authors.forename: Raju
authors.initials: R
authors.lastname: Mukherjee
authors.affiliation: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research, Institute of Infectious Disease and Molecular Medicine and Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory 7925, Rondebosch, Cape Town 7700, South Africa.
authors.forename: Joanna C
authors.initials: JC
authors.lastname: Evans
authors.affiliation: Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-1, 84125 Bratislava, Slovak Republic.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Svetlíková
authors.affiliation: Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH01, 84125 Bratislava, Slovak Republic.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Blaško
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: Sachin
authors.initials: S
authors.lastname: Surade
authors.affiliation: Institute of Infectious Disease and Molecular Medicine and Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Rondebosch, Cape Town 7701, South Africa.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Blackburn
authors.affiliation: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research, Institute of Infectious Disease and Molecular Medicine and Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory 7925, Rondebosch, Cape Town 7700, South Africa.
authors.forename: Digby F
authors.initials: DF
authors.lastname: Warner
authors.affiliation: Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-1, 84125 Bratislava, Slovak Republic.
authors.forename: Katarína
authors.initials: K
authors.lastname: Mikušová
authors.affiliation: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of Excellence for Biomedical TB Research, Institute of Infectious Disease and Molecular Medicine and Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory 7925, Rondebosch, Cape Town 7700, South Africa. Electronic address: valerie.mizrahi@uct.ac.za.
authors.forename: Valerie
authors.initials: V
authors.lastname: Mizrahi
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000963:Antimetabolites / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002250:Carbon Radioisotopes / Q000737:chemistry; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism; D005472:Fluorouracil / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D007553:Isotope Labeling; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000378:metabolism; D009876:Operon; D010430:Pentosyltransferases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D000963:Antimetabolites; D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012097:Repressor Proteins; D013940:Thymidylate Synthase; D010430:Pentosyltransferases; C098851:PyrR protein, bacteria; C023269:uracil phosphoribosyltransferase; D005472:Fluorouracil