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Identifier 000451829
Title The Achilles’ heel of the malaria vector Anopheles coluzzii
Alternative Title H αχίλλειος πτέρνα του κουνουπιού-φορέα της ελονοσίας Anopheles coluzzi
Author Κεφή, Μαρία Α
Thesis advisor Χαλεπάκης, Γεώργιος
Reviewer Βόντας, Ιωάννης
Δουρής, Βασίλης
Δελιδάκης, Χρήστος
Γκουρίδης, Γiώργος
Καλαντίδης, Κρίτων
Van Leeuwen, Thomas
Abstract Malaria is one of the most life-threatening infectious diseases representing a global health concern, with Africa carrying the vast burden. The most effective tools against malaria are insecticides; yet their scale up has resulted in severe insecticide resistance. In the lack of new compounds understanding insecticide resistance molecular mechanisms is fundamental. Based on vector control implementation, legs comprise the first tissue the insecticide bypasses to enter the body. Interestingly, African resistant populations have slower insecticide penetration due to their thicker cuticles. Cuticle, the insect exoskeleton, is a non-cellular layer above the epidermis rich in cuticular proteins (CPs), chitin and lipids, mainly cuticular hydrocarbons (CHCs). During my PhD entitled “The Achilles heel of the malaria vector Anopheles coluzzii’ : I) I analyzed the legs of insecticide resistant An. Coluzzii compared to susceptible controls, II) studied role of an AΤP-Binding Cassette (ABC-transporter) leg transporter in insecticide toxicity and III) provided insights into enzymes implicated in CHC biosynthesis in An. coluzzii and D. melanogaster. Comparative transcriptome analyses of legs from resistant and susceptible mosquitoes and transcriptome analysis of induced legs with deltamethrin (widely-used insecticide) were performed. The findings highlighted that legs of resistant mosquitoes have cuticles, with enhanced expression of cuticular proteins CPs and elevated detoxification processes. Additionally, transcriptomic data of deltamethrin-induced mosquitoes, underlined the divergent response upon insecticide induction with plethora of up-regulated genes including ABC transporters, G-protein coupled receptors (GPCRs) and odorant binding proteins (OBPs) among others. Interestingly, an ATP-binding cassette transporter (ABCH2), up-regulated upon induction was shown to be involved in deltamethrin toxicity. RNAi-silencing followed by deltamethrin toxicity assays almost completely restored susceptibility of the resistant mosquitoes. We found ABCH2 mainly in legs and head appendages and specifically on apical epidermis. Additionally, we provided evidence on the dimeric nature of this half-transporter, based on in silico modelling and in vitro data both indicating that it most probably acts as a homodimer. Further, protein-ligand docking analysis, demonstrate that deltamethrin could be a potential substrate, while silencing this gene in mosquitoes followed by deltamethrin exposure, results in increased penetration of insecticides. These lines of evidence suggest that this transporter is implicated in deltamethrin toxicity, perhaps via pumping out deltamethrin. As CHCs are enhanced in resistant mosquito legs, I studied proteins implicated in their biosynthesis. This takes place in oenocytes, where cytochrome P450 enzymes of 4G family (CYP4Gs) catalyze the last decarbonylation step. We provided the distinct developmental localization patterns of the two An. coluzzii CYP4Gs (AcCYP4G16 and AcCYP4G17), revealed they act as decarbonylases and provided their CHC blends by ectopically expressing them in D. melanogaster, while simultaneously silencing the endogenous oenocyte gene (DmCyp4g1). The data revealed that An. coluzzii CYP4Gs, both exhibit slightly different substrate specificities, with flies expressing CYP4G17 transgene producing more dimethyl-branched hydrocarbon species. This feature was studied in respect to desiccation and toxicity and the aforementioned flies were shown to cope better with these stresses. Since, CYP4Gs are not only found in oenocytes, with the most characteristic example D. melanogaster CYP4G (DmCYP4G15), previously reported in larval heads/brains, we were interested in characterization of this enzyme too. We revealed its decarbonylase activity and the distinct CHCs it produces when ectopically expressed in oenocytes. CYP4Gs of interest (AcCYP4G16, AcCYP4G17, DmCYP4G15) were also functionally expressed in insect cells using Baculovirus system. This is a valuable tool for in vitro substrate screenings. Finally, as all CYP4Gs share a specific, acidic-rich insertion, with unknown contribution to CYP4G function, I generated tools using reverse genetics in D. melanogaster to study its contribution in essentiality. The two deletions we generated, resulted in different phenotypes; the short one resulted in survival, while the longer one in mortality. These results perhaps dictate an essential region on this loop, which probably mediates useful interactions with proteins or with the lipid substrates. Overall, the thesis supports the understanding of the legs of resistant mosquitoes providing an informative, comparative dataset (RNA sequencing data) and going deeper into the mechanisms underlying the resistance phenotype by studying a leg transporter and biosynthetic enzymes of the cuticular hydrocarbons which are the major components of An. coluzzii leg epicuticles. This knowledge may contribute to identifying the Achilles’ heel of the major malaria vector, to tackle insecticide resistance and improve vector control.
Language English
Subject Insecticides
Legs
Mosquito
Ελονοσία
Εντομοκτόνα
Κουνούπι
Πόδια
Issue date 2022-11-23
Collection   School/Department--School of Sciences and Engineering--Department of Biology--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/e/6/f/metadata-dlib-1666776800-63344-8425.tkl Bookmark and Share
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