Plasmodium parasites, the causative agents of malaria, have evolved a unique cell division cycle in the clinically relevant asexual blood stage of infection1. DNA replication commences approximately halfway through the intracellular development following invasion and parasite growth. The schizont stage is associated with multiple rounds of DNA replication and nuclear division without cytokinesis, resulting in a multinucleated cell. Nuclei divide asynchronously through schizogony, with only the final round of DNA replication and segregation being synchronous and coordinated with daughter cell assembly2,3. However, the control mechanisms for this divergent mode of replication are unknown. Here, we show that the Plasmodium-specific kinase PfCRK4 is a key cell-cycle regulator that orchestrates multiple rounds of DNA replication throughout schizogony in Plasmodium falciparum. PfCRK4 depletion led to a complete block in nuclear division and profoundly inhibited DNA replication. Quantitative phosphoproteomic profiling identified a set of PfCRK4-regulated phosphoproteins with greatest functional similarity to CDK2 substrates, particularly proteins involved in the origin of replication firing. PfCRK4 was required for initial and subsequent rounds of DNA replication during schizogony and, in addition, was essential for development in the mosquito vector. Our results identified an essential S-phase promoting factor of the unconventional P. falciparum cell cycle. PfCRK4 is required for both a prolonged period of the intraerythrocytic stage of Plasmodium infection, as well as for transmission, revealing a broad window for PfCRK4-targeted chemotherapeutics.
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The authors thank the members of the Duraisingh Laboratory for discussions and critical reading of the manuscript, D.F. Wirth for continuous guidance and support and S.T. Prigge, P. Sinnis, M.T. Makler and J. C. Rayner for sharing reagents. The authors acknowledge the Microscopy Facility at the Johns Hopkins School of Medicine. The authors thank H. Hurd and P. Eggleston for the An. gambiae KEELE strain. This work was supported by the National Institutes of Health (NIH R21 1R21AIO88314-01A1 to M.T.D.), a Wellcome Trust Project grant (094752/Z/10/Z to D.A.B. and M.T.D.), a Deutsche Forschungsgemeinschaft research fellowship (GA 1668/2-1 to M.G.), a Pediatric Scientist Development Program Fellowship award (K12-HD000850 to J.D.D.), an NIH/NIDDK grant (K01 DK098285 to J.A.P.), an HFSP award (RGY0073/2012 to J.G.K. and R.R.D.), the Bloomberg Family Foundation through the Johns Hopkins Malaria Research Institute, the NIH National Center for Research Resources (UL1 RR 025005), the Malaria Research Institute of The Johns Hopkins Bloomberg School of Public Health (R.R.D.).
The authors declare no competing financial interests.
Supplementary Figures 1–9, Supplementary Table 1, Supplementary References (PDF 6951 kb)
Supplementary Data 1: Eukaryotic protein kinases used for phylogenetic analysis. Supplementary Data 2: Overview of proteomics and phosphoproteomics data. Supplementary Data 3: Proteome results and analysis. Supplementary Data 4: Phosphoproteome results and analysis. Supplementary Data 5: k-means cluster descriptions and membership counts. Supplementary Data 6: Phylogenetic profile of proteome proteins. Supplementary Data 7: Categories and descriptions of proteome GO terms in this study. (XLSX 6616 kb)
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Ganter, M., Goldberg, J., Dvorin, J. et al. Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony. Nat Microbiol 2, 17017 (2017). https://doi.org/10.1038/nmicrobiol.2017.17
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