Cloning and characterization of two glutathione S‐transferases from pyrethroid‐resistant Culex pipiens

Abstract

The Marin strain of Culex pipiens Say is a pyrethroid-resistant population that was collected in Marin County, California, in 2001 and subsequently maintained in the laboratory under regular permethrin exposure. In this study, two cDNAs, CpGSTd1 and CpGSTd2, encoding glutathione S-transferase (GST) were cloned from Cx. pipiens Marin. Phylogenetic analysis of the deduced amino acid sequences, CpGSTD1 and CpGSTD2, of these genes indicated that they belong to the Delta class of insect GSTs. The nucleotide and deduced amino acid sequences of CpGSTd1 and CpGSTd2 were 59 and 48% identical respectively. CpGSTD1 and CpGSTD2 were expressed in Escherichia coli and purified by affinity chromatography. The recombinant GSTs exhibited unique selectivity towards the general GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB), and also differed in their sensitivity to known inhibitors of GSTs. CpGSTD1 exhibited peroxidase activity with cumene hydroperoxide, while CpGSTD2 appeared to lack this activity. CpGSTD1 was able to metabolize 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), while DDT metabolism by CpGSTD2 was not detectable. CpGSTD1 and CpGSTD2 showed no detectable metabolism of permethrin. Gene expression of CpGSTd1 and CpGSTd2 in Marin mosquitoes was elevated about twofold in comparison with that found in a pyrethroid-sensitive mosquito strain. The results indicate that CpGSTD1 and CpGSTD2 have unique biochemical characteristics, but they do not appear to play major roles in permethrin resistance in Marin mosquitoes.