F Brod4; A Nobrega Pitaluga2; K Miura3; S Tapanelli2; W de Jongh1; C A Long3; G K Christophides2; S Biswas4;
1 ExpreS2ion Biotechnologies, Denmark; 2 Imperial College London, UK; 3 NIH: NIAID, United States; 4 University of Oxford, UK
DiscussionThe passage through the mosquito vector is a natural bottleneck in the malaria parasite’s lifecycle, and therefore an attractive intervention target. Upon ingestion of Plasmodium gametocytes by a mosquito as part of a blood meal, male and female gametocytes fuse to form a zygote which then matures into the motile ookinete. The ookinete penetrates the mosquito peritrophic matrix, followed by specific attachment to and traversal of the mosquito midgut epithelial cell layer. A number of proteins on the surface of the ookinete and the mosquito midgut epithelium have been suggested to be involved in this process, but whether specific protein-protein interactions facilitate attachment and invasion, and which proteins could form putative ligand receptor pairs, has not yet been comprehensively investigated. Identification of ligand-receptor interactions is often complicated due to low affinities which prevent them from being detected by conventional interaction screens. To overcome this problem, a library of ookinete and mosquito midgut surface proteins was generated to screen for interactions between Plasmodium falciparum and Anopheles gambiae using an avidity based extracellular interaction screen (AVEXIS), an assay specifically designed to detect low affinity interactions. This identified a highly conserved mosquito protein as a putative receptor for the well characterised transmission blocking antigen Pfs28. The protein comprises two transferrin like domains, and is expressed in high levels in the midgut and malpighian tubules in both male and female mosquitoes. The interaction was extensively characterised in vitro, which revealed that both transferrin like domains are required together to facilitate binding to Pfs28. A dissociation constant of 13 µM was determined by surface plasmon resonance, indicating the interaction could be functionally relevant at physiological concentrations. Preliminary in vivo studies on the effect of RNAi knock-down of the putative Pfs28 midgut receptor in A. gambiae on susceptibility to P. falciparum confirmed this hypothesis and suggests an important role for these proteins in midgut invasion by the ookinete. The interaction between Pfs28 and its midgut receptor is the first interaction between proteins of the ookinete and the mosquito midgut identified in P. falciparum invasion and establishment in A. gambiae. It could therefore provide a foundation for further efforts to unravel this still largely uncharacterized process and to guide the development of new interventions that could interfere with it.