Ruixue Xu

Transmission of malaria parasites to mosquito vectors relies on the successful conversion from asexual blood-stage forms into sexual gametocytes. Protein ubiquitination regulates many cellular processes, including cell differentiation in other eukaryotes, but whether specific ubiquitination machinery mediates Plasmodium sexual conversion is unclear. Here we conduct CRISPR–Cas9 mutagenesis screens to systematically profile Plasmodium yoelii E2 ubiquitin-conjugating enzymes for their impact on parasite development. We demonstrate that an E2 enzyme (Rad6B), and its partner RING-type E3 ligase (Tex1), play determinant roles in gametocytogenesis and transmission to mosquitoes. Deletion of either Rad6B or Tex1 results in severe defects in gametocyte production and oocyst formation. The Rad6B–Tex1 complex catalyses histone H2B monoubiquitination in Plasmodium yoelii and Plasmodium falciparum. H2B monoubiquitination promotes H3K4 tri-methylation occupancy at the promoter region of the gene encoding transcription factor AP2-G, a master regulator of sexual conversion, thereby inducing ap2-g expression and initiating sexual commitment. These findings reveal a key epigenetic mechanism leading to sexual differentiation in both human and rodent malaria parasites, providing potential targets for transmission-blocking interventions.

Mutations in a Plasmodium de-ubiquitinase UBP1 have been linked to antimalarial drug resistance. However, the UBP1-mediated drug-resistant mechanism remains unknown. Through drug selection, genetic mapping, allelic exchange, and functional characterization, here we show that simultaneous mutations of two amino acids (I1560N and P2874T) in the Plasmodium yoelii UBP1 can mediate high-level resistance to mefloquine, lumefantrine, and piperaquine. Mechanistically, the double mutations are shown to impair UBP1 cytoplasmic aggregation and de-ubiquitinating activity, leading to increased ubiquitination levels and altered protein localization, from the parasite digestive vacuole to the plasma membrane, of the P. yoelii multidrug resistance transporter 1 (MDR1). The MDR1 on the plasma membrane enhances the efflux of substrates/drugs out of the parasite cytoplasm to confer multidrug resistance, which can be reversed by inhibition of MDR1 transport. This study reveals a previously unknown drug-resistant mechanism mediated by UBP1 through altered MDR1 localization and substrate transport direction in a mouse model, providing a new malaria treatment strategy.