• An Erratum to this article was published on 06 March 2017

Abstract

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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Acknowledgements

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.).

Author information

Author notes

    • Markus Ganter
    • , Jeffrey D. Dvorin
    • , Jonas G. King
    • , Rays H. Y. Jiang
    •  & Rhoel R. Dinglasan

    Present addresses: Center for Infectious Diseases, Parasitology, Heidelberg University Hospital, INF 345, 69120 Heidelberg, Germany (M.G.); Division of Infectious Diseases, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA (J.D.D.); Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Starkville, Mississippi 39762, USA (J.G.K); Department of Global Health and Center for Drug Discovery and Innovation, University of South Florida, Tampa, Florida 33612 USA (R.H.Y.J.); Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32611, USA (R.R.D.).

Affiliations

  1. Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, 651 Huntington Avenue, FXB, Room 202, Boston, Massachusetts 02115, USA

    • Markus Ganter
    • , Jonathan M. Goldberg
    • , Jeffrey D. Dvorin
    • , Aditya S. Paul
    • , Jing Yang
    • , Rays H. Y. Jiang
    • , Brendan Elsworth
    •  & Manoj T. Duraisingh
  2. Division of Infectious Diseases, Boston Children's Hospital, Boston, Massachusetts 02115, USA

    • Jeffrey D. Dvorin
  3. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Joao A. Paulo
    •  & Steven P. Gygi
  4. W. Harry Feinstone Department of Molecular Microbiology & Immunology, The Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, USA

    • Jonas G. King
    • , Abhai K. Tripathi
    • , Isabelle Coppens
    •  & Rhoel R. Dinglasan
  5. Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK

    • David A. Baker

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Contributions

M.G. designed, performed and interpreted much of the experimental work. J.M.G. analysed the phosphoproteomic data and provided bioinformatics support. J.D.D., J.A.P., J.G.K., A.K.T., A.S.P. and I.C. designed and performed specific experimental work. J.Y. constructed plasmids. R.H.Y.J. provided bioinformatics support. B.E. performed western blots. D.A.B., R.R.D. and S.P.G. provided reagents and intellectual input into study design. M.G., J.D.D. and M.T.D. conceived the study. M.G., J.M.G. and M.T.D. wrote the manuscript. All authors commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Manoj T. Duraisingh.

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    Supplementary Information

    Supplementary Figures 1–9, Supplementary Table 1, Supplementary References

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    Supplementary Data

    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.

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DOI

https://doi.org/10.1038/nmicrobiol.2017.17

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