Biochemical characterization of cytochrome P450 enzyme (CYP6P9) involvement in insecticide resistance in the major malaria vector Anopheles funestus

Matambo, Tonderayi Sylvester
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Anopheles funestus is one of three major malaria vectors in Africa. During 2000 malaria cases increased in Kwazulu/Natal, South Africa. This was a result of pyrethroid resistance in this vector. Biochemical studies showed elevated levels of Cytochrome P450 (monooxygenases) in the resistant mosquitoes. Monooxygenases are one of the three major metabolic based resistance mechanisms used by mosquitoes as a defence mechanism against insecticides. Further studies identified one gene in the CYP6 class, CYP6P9, highly overexpressed. Microsomal cytochrome P450s were isolated from pyrethroid resistant (FUMOZ-R) larval midguts and headless adults. High enzyme activity of 7-ethoxycoumaorin was noted in both larval midguts and adults (FUMOZ-R) compared to susceptible (FANG) strain (3.6 and 6.4 fold increases respectively). HPLC analysis further confirmed that microsomal cytochrome P450s from FUMOZ-R were involved in permethrin (pyrethroid) detoxification. In order the understand the mode of action of CYP6P9 as a metabolizing enzyme, this study aimed to isolate and characterize full length CYP6P9 as well as develop a suitable expression system in E. coli. Sequence comparisons of full-length cDNA’s between CYP6P9 from resistant and susceptible strains revealed several mutations. Redox partners Cytochrome b5 and Cytochrome P450 reductase genes were also isolated, sequenced and cloned as these proteins plays an integral part in detoxification. These were almost identical between the two strains analysed. analysed. iii A second cytochrome P450, CYP6P13, was isolated only from FANG genomic DNA. This gene shared a 93.7% identity with that of CYP6P9. Its absence in FUMOZ-R and FANG cDNA indicated that it was not expressed at the protein level. This gene is most likely the result of gene duplication. In order to enzymatically characterize the metabolic activity of CYP6P9, recombinant CYP6P9 protein was expressed in E. coil. Expression of CYP6P9 was problematic in E. coli even when the expression vector was changed from pIX4.0 to pCWori+ in the presence of a bacterial signal sequence (ompA). Expression was successful when eight of the first amino acid sequences were replaced by the 17-α hydroxylase sequence. This work is the first to provide evidence of permethrin detoxification by microsomal P450 from a resistant mosquito strain. It is also the first to express An. funestus P450 in E. coli.