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bioRxiv · 10.1101/2019.12.16.878082

Malaria transmission assisted by interaction between Plasmodium α-tubulin-1 and Anopheles FREP1 protein

Zhang, G.·Niu, G.·Perez, L.·Wang, X.·Li, J.
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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, -tubulin-1 and heat shock protein 70 (Hsp70) were highly conserved in Plasmodium species with >95% identity. Thus, P. falciparum -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 -tubulin-1 significantly inhibited P. falciparum transmission to An. gambiae, while Ab against P. falciparum Hsp70 serum did not. The polyclonal Ab against human -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--tubulin Ab bound to impermeable Plasmodium ookinete apical invasive apparatus. Therefore, we propose that the interaction between Anopheles FREP1 protein and Plasmodium -tubulin-1 directs the ookinete invasive apparatus towards midgut peritrophic matrix for the efficient passage of the parasite. Anopheles FREP1 and Plasmodium -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 -tubulin-1 is an FREP1 binding partner. We initially identified the -tubulin-1 through the FREP1-pulldown assay; Then we cloned P. falciparum -tubulin-1, and demonstrated that the insect cell expressed recombinant Plasmodium -tubulin-1 bound to FREP1 in vitro; Next, mouse anti-serum against P. falciparum -tubulin-1 was found to inhibit P. falciparum transmission to An. gambiae. P. falciparum -tubulin-1 shares >84% identical amino acid sequence with human -tubulin, purified Ab against human -tubulin significantly inhibited malaria transmission. Anti-human -tubulin Ab did not interfere the gametocyte-to-ookinetes conversion. Final, we found that anti--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 -tubulin-1 directs the ookinete invasive apparatus towards midgut PM for the efficient parasite invasion.

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Zhang, G., Niu, G., Perez, L., Wang, X., Li, J.. 2019-12-16. Malaria transmission assisted by interaction between Plasmodium α-tubulin-1 and Anopheles FREP1 protein. https://doi.org/10.1101/2019.12.16.878082

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