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Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony

Nature Microbiology volume 2, Article number: 17017 (2017) Cite this article

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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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Figure 1: Conditional destabilization identified P. falciparum CRK4 as essential for asexual blood-stage development.
Figure 2: Nuclear-localized PfCRK4 is essential for the trophozoite-to-schizont transition and DNA replication.
Figure 3: PfCRK4 regulates S phase.
Figure 4: PfCRK4 is essential throughout schizogony and critical for transmission.

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References

  1. Francia, M. E. & Striepen, B. Cell division in apicomplexan parasites. Nat. Rev. Microbiol. 12, 125–136 (2014).

    Article  CAS  Google Scholar 

  2. Read, M., Sherwin, T., Holloway, S. P., Gull, K. & Hyde, J. E. Microtubular organization visualized by immunofluorescence microscopy during erythrocytic schizogony in Plasmodium falciparum and investigation of post-translational modifications of parasite tubulin. Parasitology 106, 223–232 (1993).

    Article  Google Scholar 

  3. Arnot, D. E., Ronander, E. & Bengtsson, D. C. The progression of the intra-erythrocytic cell cycle of Plasmodium falciparum and the role of the centriolar plaques in asynchronous mitotic division during schizogony. Int. J. Parasitol. 41, 71–80 (2011).

    Article  CAS  Google Scholar 

  4. Farrell, J. A. & O'Farrell, P. H. From egg to gastrula: how the cell cycle is remodeled during the Drosophila mid-blastula transition. Annu. Rev. Genet. 48, 269–294 (2014).

    Article  CAS  Google Scholar 

  5. Tewari, R. et al. The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission. Cell Host Microbe 8, 377–387 (2010).

    Article  CAS  Google Scholar 

  6. Solyakov, L. et al. Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum. Nat. Commun. 2, 565 (2011).

    Article  Google Scholar 

  7. Dvorin, J. D. et al. A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes. Science 328, 910–912 (2010).

    Article  CAS  Google Scholar 

  8. Farrell, A. et al. A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis. Science 335, 218–221 (2012).

    Article  CAS  Google Scholar 

  9. Paul, A. S. et al. Parasite calcineurin regulates host cell recognition and attachment by apicomplexans. Cell Host Microbe 18, 49–60 (2015).

    Article  CAS  Google Scholar 

  10. Armstrong, C. M. & Goldberg, D. E. An FKBP destabilization domain modulates protein levels in Plasmodium falciparum. Nat. Methods. 4, 1007–1009 (2007).

    Article  CAS  Google Scholar 

  11. Chu, B. W., Banaszynski, L. A., Chen, L.-C. & Wandless, T. J. Recent progress with FKBP-derived destabilizing domains. Bioorg. Med. Chem. Lett. 18, 5941–5944 (2008).

    Article  CAS  Google Scholar 

  12. Taylor, H. M. et al. The malaria parasite cyclic GMP-dependent protein kinase plays a central role in blood-stage schizogony. Eukaryotic Cell 9, 37–45 (2010).

    Article  CAS  Google Scholar 

  13. Collins, C. R. et al. Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog. 9, e1003344 (2013).

    Article  CAS  Google Scholar 

  14. Muralidharan, V., Oksman, A., Pal, P., Lindquist, S. & Goldberg, D. E. Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers. Nat. Commun. 3, 1310 (2012).

    Article  Google Scholar 

  15. Beck, J. R., Muralidharan, V., Oksman, A. & Goldberg, D. E. PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes. Nature 511, 592–595 (2014).

    Article  CAS  Google Scholar 

  16. Buchholz, K. et al. A high-throughput screen targeting malaria transmission stages opens new avenues for drug development. J. Infect. Dis. 203, 1445–1453 (2011).

    Article  Google Scholar 

  17. Doerig, C., Endicott, J. & Chakrabarti, D. Cyclin-dependent kinase homologues of Plasmodium falciparum. Int. J. Parasitol. 32, 1575–1585 (2002).

    Article  CAS  Google Scholar 

  18. Ward, P., Equinet, L., Packer, J. & Doerig, C. Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote. BMC Genomics 5, 79 (2004).

    Article  Google Scholar 

  19. Coudreuse, D. & Nurse, P. Driving the cell cycle with a minimal CDK control network. Nature 468, 1074–1079 (2010).

    Article  CAS  Google Scholar 

  20. Hydbring, P., Malumbres, M. & Sicinski, P. Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases. Nat. Rev. Mol. Cell Biol. 17, 280–292 (2016).

    Article  CAS  Google Scholar 

  21. Aikawa, M., Huff, C. G. & Sprinz, H. Fine structure of the asexual stages of Plasmodium elongatum. J. Cell Biol. 34, 229–249 (1967).

    Article  CAS  Google Scholar 

  22. Russo, I., Oksman, A., Vaupel, B. & Goldberg, D. E. A calpain unique to alveolates is essential in Plasmodium falciparum and its knockdown reveals an involvement in pre-S-phase development. Proc. Natl Acad. Sci. USA 106, 1554–1559 (2009).

    Article  CAS  Google Scholar 

  23. Theron, M., Hesketh, R. L., Subramanian, S. & Rayner, J. C. An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites. Cytometry A 77, 1067–1074 (2010).

    Article  Google Scholar 

  24. Songyang, Z. et al. Use of an oriented peptide library to determine the optimal substrates of protein kinases. Curr. Biol. 4, 973–982 (1994).

    Article  CAS  Google Scholar 

  25. Lowe, E. D. et al. Specificity determinants of recruitment peptides bound to phospho-CDK2/cyclin A. Biochemistry 41, 15625–15634 (2002).

    Article  CAS  Google Scholar 

  26. Yeeles, J. T. P., Deegan, T. D., Janska, A., Early, A. & Diffley, J. F. X. Regulated eukaryotic DNA replication origin firing with purified proteins. Nature 519, 431–435 (2015).

    Article  CAS  Google Scholar 

  27. Ubersax, J. A. et al. Targets of the cyclin-dependent kinase Cdk1. Nature 425, 859–864 (2003).

    Article  CAS  Google Scholar 

  28. Hornbeck, P. V. et al. Phosphositeplus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 43, D512–D520 (2015).

    Article  CAS  Google Scholar 

  29. Bozdech, Z. et al. The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol. 1, e5 (2003).

    Article  Google Scholar 

  30. Roques, M. et al. Plasmodium P-Type cyclin CYC3 modulates endomitotic growth during oocyst development in mosquitoes. PLoS Pathog. 11, e1005273 (2015).

    Article  Google Scholar 

  31. Trager, W. & Jensen, J. B. Human malaria parasites in continuous culture. Science 193, 673–675 (1976).

    Article  CAS  Google Scholar 

  32. Fidock, D. A. & Wellems, T. E. Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil. Proc. Natl Acad. Sci. USA 94, 10931–10936 (1997).

    Article  CAS  Google Scholar 

  33. Boyle, M. J. M. et al. Isolation of viable Plasmodium falciparum merozoites to define erythrocyte invasion events and advance vaccine and drug development. Proc. Natl Acad. Sci. USA 107, 14378–14383 (2010).

    Article  CAS  Google Scholar 

  34. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Article  CAS  Google Scholar 

  35. Johnson, J. D. et al. Assessment and continued validation of the malaria SYBR green I-based fluorescence assay for use in malaria drug screening. Antimicrob. Agents Chemother. 51, 1926–1933 (2007).

    Article  CAS  Google Scholar 

  36. Neuhauser, C. Calculus For Biology and Medicine: Pearson New International Edition (Pearson, 2013).

    Google Scholar 

  37. Tonkin, C. J. et al. Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method. Mol. Biochem. Parasitol. 137, 13–21 (2004).

    Article  CAS  Google Scholar 

  38. Dinglasan, R. R. et al. Plasmodium falciparum ookinetes require mosquito midgut chondroitin sulfate proteoglycans for cell invasion. Proc. Natl Acad. Sci. USA 104, 15882–15887 (2007).

    Article  CAS  Google Scholar 

  39. Flueck, C. et al. A major role for the Plasmodium falciparum ApiAP2 protein PfSIP2 in chromosome end biology. PLoS Pathog. 6, e1000784 (2010).

    Article  Google Scholar 

  40. Gallagher, J. R., Matthews, K. A. & Prigge, S. T. Plasmodium falciparum apicoplast transit peptides are unstructured in vitro and during apicoplast import. Traffic 12, 1124–1138 (2011).

    Article  CAS  Google Scholar 

  41. Billker, O. et al. Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito. Nature 392, 289–292 (1998).

    Article  CAS  Google Scholar 

  42. Thathy, V. et al. Levels of circumsporozoite protein in the Plasmodium oocyst determine sporozoite morphology. EMBO J. 21, 1586–1596 (2002).

    Article  CAS  Google Scholar 

  43. Pasini, E. M., van den Ierssel, D., Vial, H. J. & Kocken, C. H. M. A novel live-dead staining methodology to study malaria parasite viability. Malar. J. 12, 190 (2013).

    Article  Google Scholar 

  44. Coppens, I. & Joiner, K. A. Host but not parasite cholesterol controls toxoplasma cell entry by modulating organelle discharge. Mol. Biol. Cell 14, 3804–3820 (2003).

    Article  CAS  Google Scholar 

  45. Villén, J. & Gygi, S. P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat. Protoc. 3, 1630–1638 (2008).

    Article  Google Scholar 

  46. Paulo, J. A. et al. Effects of MEK inhibitors GSK1120212 and PD0325901 in vivo using 10-plex quantitative proteomics and phosphoproteomics. Proteomics 15, 462–473 (2014).

    Article  Google Scholar 

  47. Wessel, D. & Flügge, U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143 (1984).

    Article  CAS  Google Scholar 

  48. Paulo, J. A. & Gygi, S. P. A comprehensive proteomic and phosphoproteomic analysis of yeast deletion mutants of 14-3-3 orthologs and associated effects of rapamycin. Proteomics 15, 474–486 (2015).

    Article  CAS  Google Scholar 

  49. Kettenbach, A. N. & Gerber, S. A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments. Anal. Chem. 83, 7635–7644 (2011).

    Article  CAS  Google Scholar 

  50. Paulo, J. A., Gaun, A. & Gygi, S. P. Global analysis of protein expression and phosphorylation levels in nicotine-treated pancreatic stellate cells. J. Proteome Res. 14, 4246–4256 (2015).

    Article  CAS  Google Scholar 

  51. McAlister, G. C. et al. Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses. Anal. Chem. 84, 7469–7478 (2012).

    Article  CAS  Google Scholar 

  52. Huttlin, E. L. et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–1189 (2010).

    Article  CAS  Google Scholar 

  53. Beausoleil, S. A., Villén, J., Gerber, S. A., Rush, J. & Gygi, S. P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24, 1285–1292 (2006).

    Article  CAS  Google Scholar 

  54. Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).

    Article  CAS  Google Scholar 

  55. Elias, J. E. & Gygi, S. P. Target-decoy search strategy for mass spectrometry-based proteomics. Methods Mol. Biol. 604, 55–71 (2010).

    Article  CAS  Google Scholar 

  56. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    Article  CAS  Google Scholar 

  57. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. 30, 1312–1313 (2014).

  58. Finn, R. D. et al. Pfam: the protein families database. Nucleic Acids Res. 42, D222–D230 (2014).

    Article  CAS  Google Scholar 

  59. Finn, R. D. et al. HMMER web server: 2015 update. Nucleic Acids Res. 43, W30–W38 (2015).

    Article  CAS  Google Scholar 

  60. Webb, B. & Sali, A. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinformatics 47, 5–32 (2014).

    Article  Google Scholar 

  61. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  62. Hartigan, J. A. Clustering. Annu. Rev. Biophys. Bioeng. 2, 81–101 (1973).

    Article  CAS  Google Scholar 

  63. Fischer, S. et al. Using OrthoMCL to assign proteins to OrthoMCL-DB groups or to cluster proteomes into new ortholog groups. Curr. Protoc. Bioinformatics 35, 6.12.1–6.12.19 (2011).

    Google Scholar 

  64. Bauer, S., Grossmann, S., Vingron, M. & Robinson, P. N. Ontologizer 2.0—a multifunctional tool for GO term enrichment analysis and data exploration. 24, 1650–1651 (2008).

  65. Gene Ontology Consortium. Gene Ontology Consortium: going forward. Nucleic Acids Res. 43, D1049–D1056 (2015).

    Article  Google Scholar 

  66. Friedl, J. E. F. Mastering Regular Expressions (O'Reilly Media, 2006).

    Google Scholar 

  67. Crooks, G. E., Hon, G., Chandonia, J.-M. & Brenner, S. E. Weblogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    Article  CAS  Google Scholar 

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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.).

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Author notes

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

    Present address: †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.).,

Authors and Affiliations

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

    Jeffrey D. Dvorin

  3. Department of Cell Biology, Harvard Medical School, Boston, 02115, Massachusetts, 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, 21205, Maryland, 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.

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Correspondence to Manoj T. Duraisingh.

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Supplementary information

Supplementary Information

Supplementary Figures 1–9, Supplementary Table 1, Supplementary References (PDF 6951 kb)

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. (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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