title: A glutathione-S-transferase (TuGSTd05) associated with acaricide resistance in Tetranychus urticae directly metabolizes the complex II inhibitor cyflumetofen.
abstract: Cyflumetofen is a recently introduced acaricide with a novel mode of action, acting as an inhibitor of complex II of mitochondrial electron transport chain. It is activated by hydrolysis and the resulting de-esterified metabolite is a much stronger inhibitor. Cyflumetofen represents a great addition for the control of mite species including Tetranychus urticae, a major agricultural pest, which has the ability to develop resistance to most classes of pesticides rapidly. A resistant strain (Tu008R) was recently described and synergism experiments pointed towards the involvement of GSTs. Here, we conducted genome-wide gene expression analysis, comparing Tu008R with its parental susceptible strain, and identified the delta GST TuGSTd05 as the prime resistance-conferring candidate. Docking analysis suggests that both cyflumetofen and its de-esterified metabolite are potential substrates for conjugation by TuGSTd05. Several amino acids were identified that might be involved in the interaction, with Y107 and N103 possibly having an important role. To further investigate interaction as well as the role of Y107 and N103 in vitro, we recombinantly expressed and kinetically characterized the wild type TuGSTd05, TuGSTd05 Y107F and TuGSTd05 N103L mutants. While cyflumetofen was not found to act as a strong inhibitor, the de-esterified metabolite showed strong affinity for TuGSTd05 (IC50= 4 μM), which could serve as a mechanism of rapid detoxification. Y107 and N103 might contribute to this interaction. HPLC-MS analysis provided solid indications that TuGSTd05 catalyzes the conjugation of ionized glutathione (GS-) to cyflumetofen and/or its de-esterified metabolite and the resulting metabolite and possible site of attack were identified.
authors.affiliation: Department of Biology, University of Crete, Vassilika Vouton, 71409, Heraklion, Crete, Greece; Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam (UvA), Science Park 904, 1098 XH, Amsterdam, The Netherlands.
authors.forename: Nena
authors.initials: N
authors.lastname: Pavlidi
authors.affiliation: Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, B-9000, Ghent, Belgium.
authors.forename: Mousaalreza
authors.initials: M
authors.lastname: Khalighi
authors.affiliation: Environmental Chemical Processes Laboratory (ECPL), Department of Chemistry, University of Crete, 71003, Heraklion, Greece.
authors.forename: Antonis
authors.initials: A
authors.lastname: Myridakis
authors.affiliation: Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, B-9000, Ghent, Belgium.
authors.forename: Wannes
authors.initials: W
authors.lastname: Dermauw
authors.affiliation: Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam (UvA), Science Park 904, 1098 XH, Amsterdam, The Netherlands.
authors.forename: Nicky
authors.initials: N
authors.lastname: Wybouw
authors.affiliation: Department of Biology, University of Crete, Vassilika Vouton, 71409, Heraklion, Crete, Greece; Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, GR-700 13, Heraklion, Crete, Greece.
authors.forename: Dimitra
authors.initials: D
authors.lastname: Tsakireli
authors.affiliation: Environmental Chemical Processes Laboratory (ECPL), Department of Chemistry, University of Crete, 71003, Heraklion, Greece.
authors.forename: Euripides G
authors.initials: EG
authors.lastname: Stephanou
authors.affiliation: Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos Street, Athens, GR-11855, Greece.
authors.forename: Nikolaos E
authors.initials: NE
authors.lastname: Labrou
authors.affiliation: Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, GR-700 13, Heraklion, Crete, Greece; Department of Crop Science, Agricultural University of Athens, 75 Iera Odos Street, Athens, GR-11855, Greece. Electronic address: vontas@imbb.forth.gr.
authors.forename: John
authors.initials: J
authors.lastname: Vontas
authors.affiliation: Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam (UvA), Science Park 904, 1098 XH, Amsterdam, The Netherlands; Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, B-9000, Ghent, Belgium. Electronic address: thomas.vanleeuwen@ugent.be.
authors.forename: Thomas
authors.initials: T
authors.lastname: Van Leeuwen
mesh_terms: D056810:Acaricides; D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics*; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008658:Inactivation, Metabolic; D011422:Propionates / Q000378:metabolism*; D016415:Sequence Alignment; D040102:Tetranychidae / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
chemical_list: D056810:Acaricides; D060829:Arthropod Proteins; D011422:Propionates; D005982:Glutathione Transferase; C572153:cyflumetofen
keywords: Cyflumetofen resistance; Gene-expression microarray; Glutathione S-transferase; Metabolic detoxification; Molecular docking; Site-directed mutagenesis