Genetic and biochemical characterization of the cytochrome P450, CYP6P9, associated with pyrethroid resistance in the African malaria vector Anopheles funestus
Anopheles funestus Giles is a major vector of malaria in Africa and pyrethroid resistance observed in this species has disrupted malaria control in southern Africa. Metabolic detoxification, based on the overproduction of cytochrome P450s, specifically CYP6P9 and CYP6P13, was identified as the principal resistance mechanism in both field and laboratory populations. This project aimed to characterize this resistance mechanism further, both on a molecular as well as a biochemical level. Biochemical analysis on total P450 activity levels revealed a 25.5-fold increase in the resistant strain compared to a pyrethroid susceptible strain. Analysis of the effect of pyrethroids on mRNA expression of three P450 genes showed that two of them (CYP6P9 and CYP6P13) as well as Cytochrome oxidase I (COI) was induced. HPLC analysis using a heterologously (recombinant) expressed CYP6P9 enzyme, showed that CYP6P9 was able to metabolize the pyrethroid permethrin and that it was catalytically efficient. Immunoblotting revealed no significant variation in CYP6P9 protein abundance between the different An. funestus colonies. Although an approximate molecular weight (≈Mr) of 58kDa was predicted for CYP6P9, two fragments were detected at ≈Mr 52,000 and ≈45,000. The smaller fragment was very likely a result of proteolytic degradation. Statistical analysis revealed there was no significant difference in CYP6P9 protein expression between strains or sexes. Although CYP6P9 mRNA is over-expressed it is important to assess the abundance of protein as well when elucidating whether a gene and its protein are important candidates in resistance. Differences in pyrethroid resistant or susceptible profiles of An. funestus colonies could be related to enzyme affinity for substrate and stability of CYP6P9 protein however; it is recommended that further studies need to be done before any conclusions can be drawn. CYP6P9 in An. funestus is a major candidate in conferring pyrethroid resistance and the pyrethroid resistant strain is able to metabolize the pyrethroid permethrin.