Recombinant expression and characterization of GSTd3 from a resistant population of Anopheles arabiensis and comparison of DDTase activity with GSTe2

The development of insecticide resistance in malaria vectors is a challenge for the global effort to control and eradicate malaria. Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the detoxification of many classes of insecticides. For mosquitoes, it is known that overexpression of an epsilon GST, GSTe2, confers resistance towards DDT and pyrethroids. In addition to GSTe2, consistent overexpression of a delta class GST, GSTd3, has been observed in insecticide resistant populations of different malaria vector species. However, the functional role of GSTd3 towards DDT resistance has not yet been investigated. Here, we recombinantly expressed both GSTe2 and GSTd3 from Anopheles arabiensis and compared their metabolic activities against DDT. Both AaGSTd3 and AaGSTe2 exhibited CDNB-conjugating and glutathione peroxidase activity and DDT metabolism was observed for both GSTs. However, the DDT dehydrochlorinase activity exhibited by AaGSTe2 was much higher than for AaGSTd3, and AaGSTe2 was also able to eliminate DDE although the metabolite could not be identified. Molecular modeling revealed subtle differences in the binding pocket of both enzymes and a better fit of DDT within the H-site of AaGSTe2. The overexpression but much lower DDT metabolic activity of AaGSTd3, might suggest that AaGSTd3 sequesters DDT. These findings highlight the complexity of insecticide resistance in the major malaria vectors and the difficulties associated with control of the vectors using DDT, which is still used for indoor residual spraying.