A pseudokinase pPK4 is required for efficient red blood cell invasion and exflagellation center formation in Plasmodium yoelii

Human malaria, caused by the parasite Plasmodium , is a global health burden, with an estimated 263 million cases and 597,000 deaths in 2023 (WHO, 2024). In vertebrate hosts, malaria parasites multiply by recurrent invasion of the RBCs. Within RBCs, some asexual parasites undergo sexual differentiation to become gametocytes. After a female mosquito ingests a blood meal, micro- and macrogametocytes egress from RBCs and fertilize within the mosquito's midgut. Pseudokinases are a class of proteins that resemble typical kinases, but cannot catalyze phosphorylation reactions. Several Plasmodium pseudokinases have high transcript levels at both the schizont and sexual stages and may play critical roles within both junctures of the lifecycle. We previously reported that one pseudokinase, pPK1, is involved in invading RBCs, as well as exflagellation center formation, using the rodent malaria parasite Plasmodium yoelii . In the present study, we characterized two additional pseudokinases in P. yoelii , pPK3 and pPK4. Both pseudokinases have high transcript levels at the schizont and gametocyte stages; however, pPK3 transcripts are more abundantly detected in macrogametocytes than in microgametocytes. The pattern of pPK4 expression was opposite, with more abundant transcripts observed in microgametocytes. Immunofluorescence assay of transgenic parasites expressing Myc-tagged pPK4 revealed that pPK4 appeared to be expressed in the cytoplasm of schizonts and sexual stages. P. yoelii pPK3 knockout (KO) lines showed no significant growth defects in mice and no significant reduction in the number of oocysts following transmission to mosquitoes. However, the P. yoelii pPK4-KO lines exhibited a significant defect in growth, decreased virulence in mice, and a significant reduction in the number of oocysts in mosquitoes. An in vivo RBC invasion assay for pPK4-KO lines revealed that invasion, but not egress, was affected. There were no significant differences in gametocytemia and egress from RBC for either micro- or macrogametes; however, there was a significant reduction in the number of exflagellation centers. Thus, we conclude that pPK4 plays an important role in RBC invasion and exflagellation center formation. In contrast, pPK3 is not essential in the blood stage and subsequent parasite lifecycle development up to the oocyst stage. • P. yoelii pPK3 and pPK4 are pseudokinases missing catalytic Asp and Mg 2+ binding site. • pPK3-deleted P. yoelii show no distinct phenotype from blood stage to oocyst stage. • pPK4 protein is expressed at schizont and gametocyte stages. • pPK4-deleted P. yoelii show reduced RBC invasion efficiency and attenuated virulence. • pPK4-deleted P. yoelii show reduced exflagellation center formation and fewer oocysts.