Malaria is a worldwide health problem that affects two thirds of the world population and kills approximately one million people annually. Infecting Anopheline mosquitoes is the essential step for malaria transmission. However, the molecular mechanisms of Plasmodium invasion of the mosquito midgut have not been fully elucidated. We identified that the genetic polymorphisms of fibrinogen-related protein 1 (FREP1) gene are significantly associated with Plasmodium falciparum infection in Anopheles gambiae and essential for P. berghei infection in An. gambiae. Moreover, we identified that FREP1 was a tetrameric oligomer and secreted outside of cells. Notably FREP1 bound to the mosquito midgut peritrophic matrix (PM) through direct interaction to Plasmodium ookinetes that invade mosquitoes. Disrupting FREP1 expression by RNAi or blocking endogenous FREP1 by antibodies significantly (p ≤ 0.01) inhibited Plasmodium infection in mosquito midguts. Based on these, we propose that FREP1 mediates Plasmodium invasion of Anopheles midguts.
Furthermore, nine P. berghei proteins were identified as candidate FREP1 binding partners (FBP) through pull-down experiments followed by mass spectrometry assays. We cloned these genes and expressed them in insect cells and E. coli. All insect cell-expressed recombinant FBPs interact with FREP1. To test the role of FBPs in malaria transmission, E. coli expressed recombinant proteins were injected into mice to generate polyclonal antibodies. Six FBPs turn out to be strongly immunogenic as evidenced from high specific titers in mouse serum. We will examine activities of these antibodies in inhibiting P. falciparum transmission to An. gambiae in vivo.
Besides FREP1-mediated pathway, multiple pathways are hypothesized to involving malaria transmission. Through computational approaches based on protein sequences and gene expression profiles, 95 An. gambiae genes were selected, and 15 of them were cloned and expressed in insect cells. Ten of the recombinant proteins bound to Plasmodium parasites. RNA interference assays confirmed four related to P. falciparum transmission to mosquitoes.
Collectively, mosquito midgut FREP1, secreted from the epithelium and functioning as tetramers, mediates Plasmodium invasion via anchoring ookinetes to the mosquito PM and facilitates parasite penetration into the epithelium. Our newly identified mosquito midgut proteins including FREP1 and parasitic binding partners will enable us to limit malaria transmission with novel intervention strategies
