A glutathione-S-transferase (TuGSTd05) associated with acaricide resistance in Tetranychus urticae directly metabolizes the complex II inhibitor cyflumetofen

- TuGSTd05 is over-expressed in a cyflumetofen-resistant Tetranychus urticae strain.
- Cyflumetofen de-esterified metabolite is a strong inhibitor of GST activity in vitro.
- Recombinant enzyme catalyzes the conjugation of GSH to cyflumetofen/de-esterified metabolite.
- Site of attack, metabolite structure and key amino acids involved in the interaction were identified.

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.

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