Functional analyses of male-specific early-phosphorylated proteins reveal an intrinsically disordered protein required for microgamete release in Plasmodium berghei.

In the life cycle of the malaria parasite, Plasmodium, gametogenesis occurs upon the ingestion by mosquitoes of intraerythrocytic gametocytes circulating in the host bloodstream and subsequent fertilization in the mosquito midgut. Environmental changes, such as a drop in temperature and pH, and the influence of the mosquito midgut factor xanthurenic acid, induce male gametocytes to undergo multiple processes within 15 min; including emergence from within erythrocytes, three rounds of DNA replication, and the formation of flagellar gametes. A signaling pathway initiated by calcium-dependent protein kinase 4 in part facilitates these complex processes, but the detailed downstream mechanisms remain to be fully elucidated. Here, we investigated the functions of early-phosphorylated proteins upon the activation of male gametogenesis, to characterize the unique signaling cascade which facilitates Plasmodium sexual stage progression. Specifically, we focused on three proteins that are enriched in intrinsically disordered regions (IDRs) and are highly conserved across Plasmodium spp.; herein designated as male-specific early-phosphorylated IDR-rich proteins (MPDs). Gene disruption analyses via genome editing in the rodent malaria parasite Plasmodium berghei revealed that MPD2 plays an important role in microgamete release and subsequently affects oocyst formation in mosquito midguts. The observed reduction in microgamete release in ΔMPD2 was rescued by the introduction of a centromere plasmid containing an expression cassette for MPD2 fused with an AGIA-tag. Using these complemented parasites, we detected MPD2 expression in both gametocytes and gametes. These functional characterizations provide comprehensive insights into the unique mechanisms of rapid male gametogenesis triggered by environmental changes. This unique phosphorylation cascade may represent a promising target for developing novel transmission-blocking interventions.