title: The Role of Folate Transport in Antifolate Drug Action in Trypanosoma brucei.
abstract: The aim of this study was to identify and characterize mechanisms of resistance to antifolate drugs in African trypanosomes. Genome-wide RNAi library screens were undertaken in bloodstream form Trypanosoma brucei exposed to the antifolates methotrexate and raltitrexed. In conjunction with drug susceptibility and folate transport studies, RNAi knockdown was used to validate the functions of the putative folate transporters. The transport kinetics of folate and methotrexate were further characterized in whole cells. RNA interference target sequencing experiments identified a tandem array of genes encoding a folate transporter family, TbFT1-3, as major contributors to antifolate drug uptake. RNAi knockdown of TbFT1-3 substantially reduced folate transport into trypanosomes and reduced the parasite's susceptibly to the classical antifolates methotrexate and raltitrexed. In contrast, knockdown of TbFT1-3 increased susceptibly to the non-classical antifolates pyrimethamine and nolatrexed. Both folate and methotrexate transport were inhibited by classical antifolates but not by non-classical antifolates or biopterin. Thus, TbFT1-3 mediates the uptake of folate and classical antifolates in trypanosomes, and TbFT1-3 loss-of-function is a mechanism of antifolate drug resistance.
authors.affiliation: From the Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Simon
authors.initials: S
authors.lastname: Dewar
authors.affiliation: From the Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Natasha
authors.initials: N
authors.lastname: Sienkiewicz
authors.affiliation: From the Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Han B
authors.initials: HB
authors.lastname: Ong
authors.affiliation: From the Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Wall
authors.affiliation: From the Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Horn
authors.affiliation: From the Division of Biological Chemistry and Drug Discovery, Wellcome Trust Building, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom. Electronic address: a.h.fairlamb@dundee.ac.uk.
authors.forename: Alan H
authors.initials: AH
authors.lastname: Fairlamb
mesh_terms: D005492:Folic Acid / Q000378:metabolism*; D058972:Folic Acid Transporters / Q000235:genetics / Q000378:metabolism*; D055106:Genome-Wide Association Study; D008727:Methotrexate / Q000493:pharmacokinetics* / Q000494:pharmacology; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*
chemical_list: D058972:Folic Acid Transporters; D015800:Protozoan Proteins; D005492:Folic Acid; D008727:Methotrexate
keywords: RIT-seq; RNA interference (RNAi); Trypanosoma brucei; antifolate; drug resistance; folate; human African trypanosomiasis; methotrexate; transport; uptake