Influence of Plasmodium falciparum Calcium-Dependent Protein Kinase 5 (PfCDPK5) on the Late Schizont Stage Phosphoproteome

Abstract

Protein kinases are important mediators of signal transduction in cellular pathways, and calcium-dependent protein kinases (CDPKs) compose a unique class of calcium-dependent kinases present in plants and apicomplexans, including <i>Plasmodium</i> parasites, the causative agents of malaria. During the asexual stage of infection, the human malaria parasite <i>Plasmodium falciparum</i> grows inside red blood cells, and <i>P. falciparum</i> calcium-dependent protein kinase 5 (PfCDPK5) is required for egress from the host cell. In this paper, we characterize the late-schizont-stage <i>P. falciparum</i> phosphoproteome by performing large-scale phosphoproteomic profiling on tightly synchronized parasites just prior to egress, identifying 2,704 phosphorylation sites on 919 proteins. Using a conditional knockdown of PfCDPK5, we identify 58 phosphorylation sites on 50 proteins with significant reduction in levels of PfCDPK5-deficient parasites. Furthermore, gene ontology analysis of the identified proteins reveals enrichment in transmembrane- and membrane-associated proteins and in proteins associated with transport activity. Among the identified proteins is PfNPT1, a member of the apicomplexan-specific <u>n</u>ovel <u>p</u>utative <u>t</u>ransporter (NPT) family of proteins. We show that PfNPT1 is a potential substrate of PfCDPK5 and that PfNPT1 localizes to the parasite plasma membrane. Importantly, <i>P. falciparum</i> egress relies on many proteins unique to Apicomplexa that are therefore attractive targets for antimalarial therapeutics.<b>IMPORTANCE</b> The malaria parasite <i>Plasmodium falciparum</i> is a major cause of morbidity and mortality globally. The <i>P. falciparum</i> parasite proliferates inside red blood cells during the blood stage of infection, and egress from the red blood cell is critical for parasite survival. <i>P. falciparum</i> calcium-dependent protein kinase 5 (PfCDPK5) is essential for egress; parasites deficient in PfCDPK5 remain trapped inside their host cells. We have used a label-free quantitative mass spectrometry approach to identify the phosphoproteome of schizont-stage parasites just prior to egress and identify 50 proteins that display a significant reduction in phosphorylation in PfCDPK5-deficient parasites. We show that a member of the Apicomplexan-specific transport protein family, PfNPT1 is a potential substrate of PfCDPK5 and is localized to the parasite plasma membrane. <i>P. falciparum</i> egress requires several proteins not present in human cells, thus making this pathway an ideal target for new therapeutics.