Caractérisation génomique et fonctionnelle du rôle des glutathion s-transférases epsilon dans la résistance aux insecticides chez Anopheles funestus s.s, vecteur majeur du paludisme.

Abstract

Elucidating the resistance mechanisms of malaria vectors to insecticides is crucial for the design of new and sustainable tools to fight this pandemic. In this study, we assessed the role of glutathione s-transferases epsilon (GSTes) in conferring insecticide resistance in Anopheles funestus using a functional characterization approach. A combination of analysis associating the expression profiling of GST epsilon in An. funestus, the detection of resistance markers, the structural and functional characterization of the role of GSTs on insecticide resistance and the evaluation of the impact of L119F allelic variation of GSTe2 on the main vector control tools was used. In Cameroon, mortality rates of mosquitoes after susceptibility testing were less than 90 % for all the insecticides tested, showing that An. funestus populations from Cameroon are resistant to DDT (55.28 ± 5.95 %), permethrin (48.88 ± 2.31 %) and deltamethrin (53 ± 2.5 %). Transcriptional analysis of genes associated with DDT resistance revealed that, in addition to cytochromes P450 (example of CYP6P9a/b, CYP6P5, CYP315A1) and GSTs (GST epsilon, delta and theta), other gene families were involved in DDT resistance, including carboxylesterases, sulfotransferases, UDP-glucuronosyltransferases and ABC transporters and metalloproteins. This analysis revealed that GSTe1 ((Cameroon (Fold change, FC: 2.54), Ghana (4.20), Malawi (2.51)), GSTe2 ((Cameroon (4.47), Ghana (7.52), Malawi (2.13)), GSTe3 ((Cameroon (2.49), Uganda (2.60)), GSTe4 in Ghana (3.47), GSTe5 ((Ghana (2.94), Malawi (2, 26)), GSTe6 ((Cameroon (3.0), Ghana (3.11), Malawi (3.07), Uganda (3.78)), and GSTe7 in Ghana (2.39) were overexpressed in DDT resistant populations of An. funestus. Validation of the expression level of genes by qPCR confirmed that GSTes are differentially expressed on the continent and are more associated with DDT resistance. However, the polymorphism analysis helps to detect the G26D-GSTe3, H181Y-GSTe4 and E189A-GSTe4 alleles present in Benin and Cameroon, and T201S-GSTe6 and G201E-GSTe6 alleles present in Benin and Malawi and absent in Cameroon. Furthermore, the structural characterization of the different alleles revealed that the presence of allelic variations increases the binding cavity at the active site of the GSTes G26- GSTe3 (37.88); Y/A-GSTe4 (29.18) and E/S-GSTe6 (19.45) and allows strong affinity with DDT and permethrin. The evaluation of insecticide metabolic activities of the synthesized proteins confirms that GSTes are able to eliminate DDT and permethrin. Only GSTe4 proteins showed metabolic activity towards deltamethrin. However, the mutated proteins showed a greater ability to eliminate insecticides. Transgenic expression of GSTes in Drosophila showed that the expression of GSTe3, GSTe4 and GSTe6 and the presence of allelic variations confer resistance to DDTs and pyrethroids. Similarly, the inhibition of the expression of GSTe2, 3, 4, 5 and GSTe6 in mosquitoes by RNA interference technique increases their susceptibility to insecticides, confirming that the overexpression of GSTes contributes to insecticide resistance observed in An. funestus. This study showed that the presence of L119F-GSTe2 allelic variation increases the ability of An. funestus to survive in the presence of pyrethroids, DDT, PermaNet 3.0, and PermaNet 2.0 mosquito nets. The opposite was observed with the Olyset Plus mosquito net. This study was able to demonstrate that overexpression of GSTe1, 2, 3, 4, 5, 6, 7 and 8 as well as the presence of allelic variations in An. funestus increase insecticide resistance and consequently reduces the effectiveness of vector control tools used in Cameroon.