Abstract
Malaria parasites proliferate by repeated invasion of and multiplication within erythrocytes in the vertebrate host. Sexually committed intraerythrocytic parasites undergo sexual stage differentiation to become gametocytes. After ingestion by the mosquito, male and female gametocytes egress from erythrocytes and fertilize within the mosquito midgut. A complex signaling pathway likely responds to environmental events to trigger gametogenesis and regulate fertilization; however, such knowledge remains limited for malaria parasites. Several pseudokinases are highly transcribed at the gametocyte stage and are possible multi-functional regulators controlling critical steps of the life cycle. Here we characterized one pseudokinase, termed PypPK1, in Plasmodium yoelii that is highly expressed in schizonts and male gametocytes. Immunofluorescence assays for parasites expressing Myc-tagged PypPK1 confirmed that PypPK1 protein is expressed in schizonts and sexual stage parasites. Transgenic ΔpPK1 parasites, in which the PypPK1 gene locus was deleted by the CRISPR/Cas9 method, showed significant growth defect and reduced virulence in mice. In the blood stage, ΔpPK1 parasites were able to egress from erythrocytes similar to wild type parasites; however, erythrocyte invasion efficacy was significantly reduced. During sexual stage development, no clear changes were seen in male and female gametocytemias as well as gametocyte egress from erythrocytes; but, the number of exflagellation centers and oocysts were significantly reduced in ΔpPK1 parasites. Taken together, PypPK1 has an important role for both erythrocyte invasion and exflagellation center formation.
Highlights
Malaria remains a heavy burden on human society worldwide, causing > 200 million cases and > 400,000 deaths annually [1]
We designated this protein as P. yoelii pseudo Protein Kinase 1 (PypPK1)
Deletion of PypPK1 resulted in a significant growth defect and lower erythrocyte invasion efficacy compared to the parental parasite line
Summary
Malaria remains a heavy burden on human society worldwide, causing > 200 million cases and > 400,000 deaths annually [1]. Malaria parasites proliferate by repeated invasion of and multiplication within erythrocytes in mammalian hosts. This asexual blood stage is responsible for malaria clinical symptoms and pathogenesis. Sexual stage parasites do not contribute to malaria pathogenesis, they are essential for transmission to other hosts by mosquito vectors. Wide-scale proteomics and exhaustive phosphor-proteome analysis has revealed key roles of phosphorylation in both asexual and sexual stage malaria parasites [4,5,6]. Malaria parasites possess 8 pseudokinases [18], all of which are poorly characterized with the exception of one pseudotyrosine kinase which was reported to be an exported protein to the infected erythrocyte and has a scaffolding role for serine repeat antigen 5 (SERA5) and protein phosphatase type 1 (PP1) [19]. To further characterize the role of this pseudokinase we took advantage that it is dispensable during the asexual stage, and evaluated PY17X_1220300 for its expression using epitope-tagged transgenic P. yoelii lines and its biological roles in the asexual and gametocyte stages using gene deletion parasite lines
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