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 Plasmodium parasites, the causative agents of malaria. During the asexual stage of infection, the human malaria parasite Plasmodium falciparum grows inside red blood cells, and P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) is required for egress from the host cell. In this paper, we characterize the late-schizont-stage P. falciparum 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 novel putative transporter (NPT) family of proteins. We show that PfNPT1 is a potential substrate of PfCDPK5 and that PfNPT1 localizes to the parasite plasma membrane. Importantly, P. falciparum egress relies on many proteins unique to Apicomplexa that are therefore attractive targets for antimalarial therapeutics. IMPORTANCE The malaria parasite Plasmodium falciparum is a major cause of morbidity and mortality globally. The P. falciparum parasite proliferates inside red blood cells during the blood stage of infection, and egress from the red blood cell is critical for parasite survival. P. falciparum 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. P. falciparum egress requires several proteins not present in human cells, thus making this pathway an ideal target for new therapeutics.

Characterization of apicomplexan amino acid transporters (ApiATs) in the malaria parasite Plasmodium falciparum

“…Due to the low expression of PfApiAT9-GFP -in agreement with the transcript levels of apiat9 in these stages 37,42,43 -no conclusive localization could be delivered. The observed PPM localization of the investigated ApiATs is in concordance with published data, since i) the P. berghei ApiAT8 homologue was shown to be a general cationic amino acid transporter of the PPM 29,30 , ii) PfApiAT8 has recently been localized to the PPM using an overexpression approach 31 , and iii) in T. gondii several ApiATs (TgApiAT1, TgApiAT2, TgApiAT3-1, TgApiAT3-2, TgApiAT3-3, TgApiAT5-3, TgApiAT6-1 and TgApiAT6-3) have been located at the PPM as well 25,29,30,32,33,44 .…”

“…P. falciparum possesses five of the eleven ApiAT subfamilies: PfApiAT2 (PfApiAT2/MFR4: PF3D7_0914700), PfApiAT4 (MFR5: PF3D7_1129900), PfApiAT8 (NPT1: PF3D7_0104800), PfApiAT9 (MFR2: PF3D7_0104700) and PfApiAT10 (MFR3: PF3D7_0312500). Previous work in the rodent malaria species Plasmodium berghei (Pb) and the related Apicomplexan parasite Toxoplasma gondii (Tg) showed that PbApiAT8 (or PbNPT1) and several other TgApiATs are localized at the PPM [29][30][31][32] and possess amino acid transport activity 25,26,[29][30][31][32][33][34] . To date, only PbApiAT4 has been shown to play an important role in parasite proliferation within erythrocytes 35 .…”

Plasmodium falciparum Calcium-Dependent Protein Kinase 4 is Critical for Male Gametogenesis and Transmission to the Mosquito Vector

“…6B ), i.e., the “Simple 1” motif, which is common to calcium-dependent kinases ( 14 , 15 ). This motif was also found to be enriched among phosphosites that were hypophosphorylated in P. falciparum schizonts upon conditional knockdown of PfCDPK1 ( 16 ) and PfCDPK5 ( 17 ). Importantly, though, the enriched motifs were further modulated by flanking residues to produce recognition motifs specific to each kinase (see Data Set S3 in the supplemental material).…”

Integration of population and functional genomics to understand mechanisms of artemisinin resistance in Plasmodium falciparum

“…The CDPKs are unique to plants and some protists, including apicomplexans. Blomqvist et al recently identified 50 proteins with significantly reduced phosphorylation using a conditional CDPK5 knockdown ( Blomqvist et al, 2020 ). An enrichment analysis of these 50 proteins based on gene ontology showed significant enrichment for proteins with transport and transmembrane transport activity, as well as proteins located in vesicles and the cell periphery, consistent with artemisinin resistance mechanisms involving endocytosis.…”