title: An Atypical Mitochondrial Carrier That Mediates Drug Action in Trypanosoma brucei.
abstract: Elucidating the mechanism of action of trypanocidal compounds is an important step in the development of more efficient drugs against Trypanosoma brucei. In a screening approach using an RNAi library in T. brucei bloodstream forms, we identified a member of the mitochondrial carrier family, TbMCP14, as a prime candidate mediating the action of a group of anti-parasitic choline analogs. Depletion of TbMCP14 by inducible RNAi in both bloodstream and procyclic forms increased resistance of parasites towards the compounds by 7-fold and 3-fold, respectively, compared to uninduced cells. In addition, down-regulation of TbMCP14 protected bloodstream form mitochondria from a drug-induced decrease in mitochondrial membrane potential. Conversely, over-expression of the carrier in procyclic forms increased parasite susceptibility more than 13-fold. Metabolomic analyses of parasites over-expressing TbMCP14 showed increased levels of the proline metabolite, pyrroline-5-carboxylate, suggesting a possible involvement of TbMCP14 in energy production. The generation of TbMCP14 knock-out parasites showed that the carrier is not essential for survival of T. brucei bloodstream forms, but reduced parasite proliferation under standard culture conditions. In contrast, depletion of TbMCP14 in procyclic forms resulted in growth arrest, followed by parasite death. The time point at which parasite proliferation stopped was dependent on the major energy source, i.e. glucose versus proline, in the culture medium. Together with our findings that proline-dependent ATP production in crude mitochondria from TbMCP14-depleted trypanosomes was reduced compared to control mitochondria, the study demonstrates that TbMCP14 is involved in energy production in T. brucei. Since TbMCP14 belongs to a trypanosomatid-specific clade of mitochondrial carrier family proteins showing very poor similarity to mitochondrial carriers of mammals, it may represent an interesting target for drug action or targeting.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
authors.forename: Juan P
authors.initials: JP
authors.lastname: de Macêdo
authors.affiliation: Institute of Cell Biology, University of Bern, Bern, Switzerland.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Schumann Burkard
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Moritz
authors.initials: M
authors.lastname: Niemann
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, and Glasgow Polyomics, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Barrett
authors.affiliation: Dynamique Moléculaire des Interactions Membranaires, CNRS UMR 5235, Université Montpellier II, Montpellier, France.
authors.forename: Henri
authors.initials: H
authors.lastname: Vial
authors.affiliation: Swiss Tropical and Public Health Institute, Basel, Switzerland; University of Basel, Basel, Switzerland.
authors.forename: Pascal
authors.initials: P
authors.lastname: Mäser
authors.affiliation: Institute of Cell Biology, University of Bern, Bern, Switzerland.
authors.forename: Isabel
authors.initials: I
authors.lastname: Roditi
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: André
authors.initials: A
authors.lastname: Schneider
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland.
authors.forename: Peter
authors.initials: P
authors.lastname: Bütikofer
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D018432:Drug Resistance, Multiple; D004734:Energy Metabolism / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D055786:Gene Knockout Techniques; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D055432:Metabolomics; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021261:Parasitic Sensitivity Tests; D010419:Pentamidine / Q000494:pharmacology; D011392:Proline / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011758:Pyrroles / Q000378:metabolism; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D015800:Protozoan Proteins; D011758:Pyrroles; D011994:Recombinant Proteins; D014344:Trypanocidal Agents; C015485:delta-1-pyrroline-5-carboxylate; D010419:Pentamidine; D011392:Proline