Malaria transmission assisted by interaction betweenPlasmodium α-tubulin-1 andAnophelesFREP1 protein

Abstract

Passage of Plasmodium through a mosquito midgut is essential for malaria transmission. FREP1, a peritrophic matrix protein in a mosquito midgut, binds to the parasite and mediates Plasmodium infection in Anopheles. The FREP1-mediated Plasmodium invasion pathway is highly conserved across multiple species of Plasmodium and Anopheles. Through pulldown, nine P. berghei proteins were co-precipitated with FREP1-conjugated beads. After cloning these nine genes from P. berghei and expressing them in insect cells, six of them were confirmed to interact with recombinant FREP1 protein. Among them, α-tubulin-1 and heat shock protein 70 (Hsp70) were highly conserved in Plasmodium species with >95% identity. Thus, P. falciparum α-tubulin-1 and Hsp70 were cloned and expressed in E. coli to stimulate antibody (Ab) in mice. Our results showed that anti-serum against P. falciparum α-tubulin-1 significantly inhibited P. falciparum transmission to An. gambiae, while Ab against P. falciparum Hsp70 serum did not. The polyclonal Ab against human α-tubulin did not interfere formation of ookinetes, however, significantly reduced the number of P. falciparum oocysts in An. gambiae midguts. Moreover, fluorescence microscope assays showed that anti-α-tubulin Ab bound to impermeable Plasmodium ookinete apical invasive apparatus. Therefore, we propose that the interaction between Anopheles FREP1 protein and Plasmodium α-tubulin-1 directs the ookinete invasive apparatus towards midgut peritrophic matrix for the efficient passage of the parasite. Anopheles FREP1 and Plasmodium α-tubulin-1 are potential targets for blocking malaria transmission to the mosquito host.AUTHOR SUMMARYThe molecular mechanisms of malaria transmission to mosquito are not well-understood. FREP1 proteins in mosquito midget PM has been proved to mediate malaria transmission by binding to parasite ookinetes. Here we reported that Plasmodium parasite α-tubulin-1 is an FREP1 binding partner. We initially identified the α-tubulin-1 through the FREP1-pulldown assay; Then we cloned P. falciparum α-tubulin-1, and demonstrated that the insect cell expressed recombinant Plasmodium α-tubulin-1 bound to FREP1 in vitro; Next, mouse anti-serum against P. falciparum α-tubulin-1 was found to inhibit P. falciparum transmission to An. gambiae. P. falciparum α-tubulin-1 shares >84% identical amino acid sequence with human α-tubulin, purified Ab against human α-tubulin significantly inhibited malaria transmission. Anti-human α-tubulin Ab did not interfere the gametocyte-to-ookinetes conversion. Final, we found that anti-α-tubulin Ab bound to the apical end of impermeable ookinetes. Structurally, ookinete invasive apparatus locates at the apical opening. Therefore, we propose that the interaction between Anopheles midgut FREP1 protein and Plasmodium apical α-tubulin-1 directs the ookinete invasive apparatus towards midgut PM for the efficient parasite invasion.

Identification, molecular characterization and expression of aminopeptidase N-1 (APN-1) from Anopheles stephensi in SF9 cell line as a candidate molecule for developing a vaccine that interrupt malaria transmission

“…Development of the vaccines that interrupt malaria transmission (VIMTs) was emphasized by the vaccine consultative group for countries that have passed the pre-elimination step and have proceeded to the global goal [10]. VIMTs are divided into various types; one type is the classical transmissionblocking vaccine that blocks the sexual parasite development by targeting the required effector molecules in the vector, including mosquito-based transmission-blocking vaccines with targets such as Anopheles gambiae aminopeptidase N-1 (AgAPN-1) [11], CPBAg1 (Carboxy Peptidase B1) [12,13], Trypsin [14], saglin [15], FREP 1 [16,17], and SGS1 [18]. APN-1 is a candidate molecule for which the blocking efficacy against Plasmodium falciparum in An.…”

“…gambiae. Some glycoproteins, such as APN-1, are receptors for the lectin-similar structures of ookinete that trigger the attachment of ookinetes to the internal side of the epithelium and are essential for preceding the sexual development of parasite in the mosquito midgut [13,16,20].…”