Plasmodium falciparum sexual stage gametocytes are critical for parasite transmission from the human host to the mosquito vector. Mature gametocytes generate fertile male (micro-) or female (macro-) gametes upon activation inside the mosquito midgut. While a number of parasite genes have been described that are critical for P. falciparum gametogenesis and fertility, no parasite gene has been shown to have a unique function in macrogametes. The genome of P. falciparum encodes numerous RNA-binding proteins. We identified a novel protein containing a putative RNA-binding domain, which we named Macrogamete-Contributed Factor Essential for Transmission (MaCFET). This protein is expressed in the asexual and sexual stages. Parasites that carry a deletion of MaCFET (Pfmacfet¯), developed normally as asexual stages, indicating that its function is not essential for the asexual proliferation of the parasite in vitro. Furthermore, Pfmacfet¯ male and female gametocytes developed normally and underwent activation to form microgametes and macrogametes. However, by utilizing genetic crosses, we demonstrate that Pfmacfet¯ parasites suffer a complete female-specific defect in successful fertilization. Therefore, PfMaCFET is a critical female-contributed factor for parasite transmission to the mosquito. Based on its putative RNA-binding properties, PfMaCFET might be in involved in the regulation of mRNAs that encode female-specific functions for fertilization or female-contributed factors needed post fertilization.

Cell fusion of female and male gametes is the climax of sexual reproduction. In many organisms, the Hapless 2 (HAP2) family of proteins play a critical role in gamete fusion. We find that Plasmodium falciparum , the causative agent of human malaria, expresses two HAP2 proteins: Pf HAP2 and Pf HAP2p. These proteins are present in stage V gametocytes and localize throughout the flagellum of male gametes. Gene deletion analysis and genetic crosses show that Pf HAP2 and Pf HAP2p individually are essential for male fertility and thereby, parasite transmission to the mosquito. Using a cell fusion assay, we demonstrate that Pf HAP2 and Pf HAP2p are both authentic plasma membrane fusogens. Our results establish nonredundant essential roles for Pf HAP2 and Pf HAP2p in mediating gamete fusion in Plasmodium and suggest avenues in the design of novel strategies to prevent malaria parasite transmission from humans to mosquitoes. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-022-04583-w.

Plasmodium sexual stages represent a critical bottleneck in the parasite life cycle. Gametocytes taken up in an infectious blood meal by female anopheline mosquito get activated to form gametes and fuse to form short-lived zygotes, which transform into ookinetes to infect mosquitoes.

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