Successful completion of the
Plasmodium
life cycle requires formation of mature gametocytes and their uptake by the female
Anopheles
mosquito vector in an infected blood meal. Inside the mosquito midgut the parasite undergoes gametogenesis and sexual reproduction.
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.
What genes determine in vitro growth and nutrient utilization in asexual blood-stage malaria parasites? Competition experiments between NF54, clone 3D7, a lab-adapted African parasite, and a recently isolated Asian parasite (NHP4026) reveal contrasting outcomes in different media: 3D7 outcompetes NHP4026 in media containing human serum, while NHP4026 outcompetes 3D7 in media containing AlbuMAX, a commercial lipid-rich bovine serum formulation. To determine the basis for this polymorphism, we conducted parasite genetic crosses using humanized mice and compared genome-wide allele frequency changes in three independent progeny populations cultured in media containing human serum or AlbuMAX. This bulk segregant analysis detected three quantitative trait loci (QTL) regions [on chromosome (chr) 2 containing aspartate transaminase AST; chr 13 containing EBA-140; and chr 14 containing cysteine protease ATG4] linked with differential growth in serum or AlbuMAX in each of the three independent progeny pools. Selection driving differential growth was strong (s = 0.10 – 0.23 per 48-hour lifecycle). We conducted validation experiments for the strongest QTL on chr 13: competition experiments between ΔEBA-140 and 3D7 wildtype parasites showed fitness reversals in the two medium types as seen in the parental parasites, validating this locus as the causative gene. These results (i) demonstrate the effectiveness of bulk segregant analysis for dissecting fitness traits in P. falciparum genetic crosses, and (ii) reveal intimate links between red blood cell invasion and nutrient composition of growth media. Use of parasite crosses combined with bulk segregant analysis will allow systematic dissection of key nutrient acquisition/metabolism and red blood cell invasion pathways in P. falciparum.
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.
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