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A transcriptional switch underlies commitment to sexual development in malaria parasites

Nature volume 507, pages 248–252 (2014)Cite this article

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Abstract

The life cycles of many parasites involve transitions between disparate host species, requiring these parasites to go through multiple developmental stages adapted to each of these specialized niches. Transmission of malaria parasites (Plasmodium spp.) from humans to the mosquito vector requires differentiation from asexual stages replicating within red blood cells into non-dividing male and female gametocytes. Although gametocytes were first described in 1880, our understanding of the molecular mechanisms involved in commitment to gametocyte formation is extremely limited, and disrupting this critical developmental transition remains a long-standing goal1. Here we show that expression levels of the DNA-binding protein PfAP2-G correlate strongly with levels of gametocyte formation. Using independent forward and reverse genetics approaches, we demonstrate that PfAP2-G function is essential for parasite sexual differentiation. By combining genome-wide PfAP2-G cognate motif occurrence with global transcriptional changes resulting from PfAP2-G ablation, we identify early gametocyte genes as probable targets of PfAP2-G and show that their regulation by PfAP2-G is critical for their wild-type level expression. In the asexual blood-stage parasites pfap2-g appears to be among a set of epigenetically silenced loci2,3 prone to spontaneous activation4. Stochastic activation presents a simple mechanism for a low baseline of gametocyte production. Overall, these findings identify PfAP2-G as a master regulator of sexual-stage development in malaria parasites and mark the first discovery of a transcriptional switch controlling a differentiation decision in protozoan parasites.

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Figure 1: pfap2-g transcript levels mirror gametocyte production.
Figure 2: Disrupting PfAP2-G function results in loss of gametocyte production.
Figure 3: Identification of PfAP2-G targets.
Figure 4: Activation of PfAP2-G.

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Accession codes

Accessions

Gene Expression Omnibus

Sequence Read Archive

Data deposits

Microarray data was submitted to the NCBI GEO repository (series accession GSE52030). Next generation sequencing data was submitted to the NCBI Sequence Read Archive (SRA) (study number ERP000190 for samples F12 (ERS011445), 3D7A (ERS011446) and GNP-A4 (ERS011447) and study number SRP035432 for samples E5 (SRS529791) and Δpfap2-g (SRS529811)).

References

  1. Wells, T. N. C., Alonso, P. L. & Gutteridge, W. E. New medicines to improve control and contribute to the eradication of malaria. Nature Rev. Drug Discov. 8, 879–891 (2009)

    Article  CAS  Google Scholar 

  2. Flueck, C. et al. Plasmodium falciparum heterochromatin protein 1 marks genomic loci linked to phenotypic variation of exported virulence factors. PLoS Pathog. 5, e1000569 (2009)

    Article  Google Scholar 

  3. Lopez-Rubio, J. J., Mancio-Silva, L. & Scherf, A. Genome-wide analysis of heterochromatin associates clonally variant gene regulation with perinuclear repressive centers in malaria parasites. Cell Host Microbe 5, 179–190 (2009)

    Article  CAS  Google Scholar 

  4. Cortés, A., Crowley, V. M., Vaquero, A. & Voss, T. S. A view on the role of epigenetics in the biology of malaria parasites. PLoS Pathog. 8, e1002943 (2012)

    Article  Google Scholar 

  5. Alano, P. Plasmodium falciparum gametocytes: still many secrets of a hidden life. Mol. Microbiol. 66, 291–302 (2007)

    Article  CAS  Google Scholar 

  6. Dixon, M. W. A., Thompson, J., Gardiner, D. L. & Trenholme, K. R. Sex in Plasmodium: a sign of commitment. Trends Parasitol. 24, 168–175 (2008)

    Article  Google Scholar 

  7. Rovira-Graells, N. et al. Transcriptional variation in the malaria parasite Plasmodium falciparum. Genome Res. 22, 925–938 (2012)

    Article  CAS  Google Scholar 

  8. Painter, H. J., Campbell, T. L. & Llinás, M. The Apicomplexan AP2 family: integral factors regulating Plasmodium development. Mol. Biochem. Parasitol. 176, 1–7 (2011)

    Article  CAS  Google Scholar 

  9. Yuda, M. et al. Identification of a transcription factor in the mosquito-invasive stage of malaria parasites. Mol. Microbiol. 71, 1402–1414 (2009)

    Article  CAS  Google Scholar 

  10. Yuda, M., Iwanaga, S., Shigenobu, S., Kato, T. & Kaneko, I. Transcription factor AP2-Sp and its target genes in malarial sporozoites. Mol. Microbiol. 75, 854–863 (2010)

    Article  CAS  Google Scholar 

  11. Iwanaga, S., Kaneko, I., Kato, T. & Yuda, M. Identification of an AP2-family protein that is critical for malaria liver stage development. PLoS ONE 7, e47557 (2012)

    Article  CAS  ADS  Google Scholar 

  12. Alano, P. et al. Plasmodium falciparum: parasites defective in early stages of gametocytogenesis. Exp. Parasitol. 81, 227–235 (1995)

    Article  CAS  Google Scholar 

  13. Day, K. P. et al. Genes necessary for expression of a virulence determinant and for transmission of Plasmodium falciparum are located on a 0.3-megabase region of chromosome 9. Proc. Natl Acad. Sci. USA 90, 8292–8296 (1993)

    Article  CAS  ADS  Google Scholar 

  14. Eksi, S. et al. Plasmodium falciparum gametocyte development 1 (Pfgdv1) and gametocytogenesis early gene identification and commitment to sexual development. PLoS Pathog. 8, e1002964 (2012)

    Article  CAS  Google Scholar 

  15. Ikadai, H. et al. Transposon mutagenesis identifies genes essential for Plasmodium falciparum gametocytogenesis. Proc. Natl Acad. Sci. USA 110, E1676–E1684 (2013)

    Article  CAS  Google Scholar 

  16. Manske, M. et al. Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing. Nature 487, 375–379 (2012)

    Article  CAS  ADS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Banaszynski, L. A., Chen, L.-C., Maynard-Smith, L. A., Ooi, A. G. L. & Wandless, T. J. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 995–1004 (2006)

    Article  CAS  Google Scholar 

  19. Pradel, G. Proteins of the malaria parasite sexual stages: expression, function and potential for transmission blocking strategies. Parasitology 134, 1911–1929 (2007)

    Article  CAS  Google Scholar 

  20. Silvestrini, F. et al. Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum. Mol. Cell. Proteomics 9, 1437–1448 (2010)

    Article  CAS  Google Scholar 

  21. Campbell, T. L., De Silva, E. K., Olszewski, K. L., Elemento, O. & Llinás, M. Identification and genome-wide prediction of DNA binding specificities for the ApiAP2 family of regulators from the malaria parasite. PLoS Pathog. 6, e1001165 (2010)

    Article  Google Scholar 

  22. Guizetti, J. & Scherf, A. Silence, activate, poise, and switch! Mechanisms of antigenic variation in Plasmodium falciparum. Cell. Microbiol. 15, 718–726 (2013)

    Article  CAS  Google Scholar 

  23. Pérez-Toledo, K. et al. Plasmodium falciparum heterochromatin protein 1 binds to tri-methylated histone 3 lysine 9 and is linked to mutually exclusive expression of var genes. Nucleic Acids Res. 37, 2596–2606 (2009)

    Article  Google Scholar 

  24. Bruce, M. C., Alano, P., Duthie, S. & Carter, R. Commitment of the malaria parasite Plasmodium falciparum to sexual and asexual development. Parasitology 100, 191–200 (1990)

    Article  Google Scholar 

  25. Avraham, I., Schreier, J. & Dzikowski, R. Insulator-like pairing elements regulate silencing and mutually exclusive expression in the malaria parasite Plasmodium falciparum. Proc. Natl Acad. Sci. USA 109, 52 (2012)

    Article  Google Scholar 

  26. Sinha, A. et al. A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium. Nature http://dx.doi.org/10.1038/nature12970 (this issue)

  27. Fivelman, Q. L. et al. Improved synchronous production of Plasmodium falciparum gametocytes in vitro. Mol. Biochem. Parasitol. 154, 119–123 (2007)

    Article  CAS  Google Scholar 

  28. Kafsack, B. F. C., Painter, H. J. & Llinás, M. New Agilent platform DNA microarrays for transcriptome analysis of Plasmodium falciparum and Plasmodium berghei for the malaria research community. Malar. J. 11, 187 (2012)

    Article  CAS  Google Scholar 

  29. Straimer, J. et al. Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases. Nature Methods 9, 993–998 (2012)

    Article  CAS  Google Scholar 

  30. Robinson, T. et al. Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients. PLoS ONE 6, e23204 (2011)

    Article  CAS  ADS  Google Scholar 

  31. Cortés, A., Benet, A., Cooke, B. M., Barnwell, J. W. & Reeder, J. C. Ability of Plasmodium falciparum to invade Southeast Asian ovalocytes varies between parasite lines. Blood 104, 2961–2966 (2004)

    Article  Google Scholar 

  32. Walliker, D. et al. Genetic analysis of the human malaria parasite Plasmodium falciparum. Science 236, 1661–1666 (1987)

    Article  CAS  ADS  Google Scholar 

  33. Taylor, C. J. The role of two cyclic nucleotide phosphodiesterases in the sexual development of Plasmodium falciparum and Plasmodium berghei. (PhD thesis, Univ. London, 2007)

  34. Taylor, C. J., McRobert, L. & Baker, D. A. Disruption of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene causes aberrant gametogenesis. Mol. Microbiol. 69, 110–118 (2008)

    Article  CAS  Google Scholar 

  35. Maier, A. G., Braks, J. A. M., Waters, A. P. & Cowman, A. F. Negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum for targeted gene deletion by double crossover recombination. Mol. Biochem. Parasitol. 150, 118–121 (2006)

    Article  CAS  Google Scholar 

  36. Ménard, R. Malaria: Methods and Protocols. Methods in Molecular Biology Vol. 923, 2nd edn, 3–15 (Humana Press, Springer, 2013)

    Book  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  38. Calderwood, M. S., Gannoun-Zaki, L., Wellems, T. E. & Deitsch, K. W. Plasmodium falciparum var genes are regulated by two regions with separate promoters, one upstream of the coding region and a second within the intron. J. Biol. Chem. 278, 34125–34132 (2003)

    Article  CAS  Google Scholar 

  39. Cortés, A. et al. Epigenetic silencing of Plasmodium falciparum genes linked to erythrocyte invasion. PLoS Pathog. 3, e107 (2007)

    Article  Google Scholar 

  40. Crowley, V. M., Rovira-Graells, N., Ribas de Pouplana, L. & Cortés, A. Heterochromatin formation in bistable chromatin domains controls the epigenetic repression of clonally variant Plasmodium falciparum genes linked to erythrocyte invasion. Mol. Microbiol. 80, 391–406 (2011)

    Article  CAS  Google Scholar 

  41. Jiang, L. et al. Epigenetic control of the variable expression of a Plasmodium falciparum receptor protein for erythrocyte invasion. Proc. Natl Acad. Sci. USA 107, 2224–2229 (2010)

    Article  CAS  ADS  Google Scholar 

  42. SMALT - Wellcome Trust Sanger Institute. (http://www.sanger.ac.uk/resources/software/smalt/)

  43. The Wellcome Trust Sanger Institute SRA Study ERP000190. (http://www.ebi.ac.uk/ena/data/view/ERP000190)

Download references

Acknowledgements

We would like to thank C. Klein, T. Campbell and A. Schieler for technical assistance and are grateful to O. Billker, C. Flueck, J. Kelly, C. Sutherland, A. Vaidya and A. Waters for discussion and reading of the manuscript. We would also like to thank P. Alano for providing P. falciparum clone F12, C. Taylor for providing the P. falciparum GNP-A4 clone, E. Thompson for isolating P. falciparum DNA for whole genome analysis, Z. Gorvett for assistance with confirming single nucleotide polymorphisms in gametocyte non-producing clones, M. Duraisingh for the ddFKBP tagging construct pJDD145, C. Ben Mamoun for the anti-PP2c antibody and D. Goldberg for Shld1. M.L. is funded by National Institutes of Health (NIH) grant R01 AI076276 with support from the Centre for Quantitative Biology (P50GM071508). B.F.C.K. was supported by a Howard Hughes Medical Institute fellowship of the Damon Runyon Cancer Research Foundation. D.A.B. is funded by Wellcome Trust grant ref. 094752 and European Commission FP7 ‘MALSIG’ (ref. 223044). L.G.D. is supported by a Biotechnology and Biological Sciences Research Council CASE PhD studentship with Pfizer as the Industrial partner. A.C. is funded by the Spanish Ministry of Science and Innovation grant SAF2010-20111. V.M.C. was supported by a fellowship from IRB Barcelona. T.G.C. is supported by the Medical Research Council UK (J005398). D.P.K. and S.G.C. are supported through the Wellcome Trust (098051; 090532/Z/09/Z) and the Medical Research Council UK (G0600230). C.B. is supported by the Catalan Government fellowship 2011-BP-B 00060 (AGAUR, Catalonia, Spain).

Author information

Author notes

  1. Björn F. C. Kafsack, Valerie M. Crowley & Manuel Llinás

    Present address: Present addresses: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA (B.F.C.K.); Department of Molecular Biology and Center for Infectious Disease Dynamics, The Pennsylvania State University, State College, Pennsylvania 16802, USA (V.M.C., M.L.).,

Authors and Affiliations

  1. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, 08544, New Jersey, USA

    Björn F. C. Kafsack, Valerie M. Crowley & Manuel Llinás

  2. Barcelona Centre for International Health Research (CRESIB, Hospital Clínic-Universitat de Barcelona), Barcelona, 08036 Catalonia, Spain,

    Núria Rovira-Graells, Cristina Bancells & Alfred Cortés

  3. Institute for Research in Biomedicine (IRB), Barcelona, 08028 Catalonia, Spain,

    Núria Rovira-Graells, Valerie M. Crowley & Alfred Cortés

  4. Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK,

    Taane G. Clark, Laura G. Drought & David A. Baker

  5. Faculty of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK,

    Taane G. Clark

  6. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK,

    Susana G. Campino & Dominic P. Kwiatkowski

  7. Department of Molecular Biology, Princeton University, Princeton, 08544, New Jersey, USA

    April E. Williams & Manuel Llinás

  8. Wellcome Trust Sanger Centre for Human Genetics, Oxford OX3 7BN, UK,

    Dominic P. Kwiatkowski

  9. Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, 08010 Catalonia, Spain,

    Alfred Cortés

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Contributions

M.L. managed the overall project with input from B.F.C.K., D.A.B. and A.C. B.F.C.K. generated the Δpfap2-g knockout, PfAP2-G–ddFKBP and luciferase lines and designed, performed and analysed the microarray, gel shift, luciferase and ligand-regulatable gametocytogenesis experiments. V.M.C. performed qRT–PCR validation. A.E.W. prepared Δpfap2-g sequencing libraries and together with B.F.C.K. analysed the sequencing data. D.A.B., T.G.C. and S.G.C. conceived the sequencing of gametocyte non-producer lines F12 and GNP-A4. T.G.C. analysed the gametocyte non-producer sequencing data and L.G.D. confirmed the SNPs by PCR. S.G.C. and D.P.K. carried out and supervised sequencing of gametocyte non-producer lines, respectively. A.C. and N.R.-G. generated E5 and other 3D7-B subclones and respectively supervised and performed the experiments presented in Figs. 1 and 2b, and provided the analysis presented in Supplementary Fig. 1. V.M.C. and N.R.-G. performed and A.C. supervised chromatin immunoprecipitation experiments. C.B. and A.C. generated the PfAP2-G–HA×3 line and carried out immunofluorescence assays and correlations with gametocyte formation. B.F.C.K. wrote the manuscript with major input from M.L., D.A.B. and A.C.

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Correspondence to Manuel Llinás.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10, Supplementary References and Supplementary Table 3. (PDF 9157 kb)

Supplementary Table 1

This table shows coding sequence mutations affecting F12, GNP-A4, and Δpfap2‐g. (XLSX 39 kb)

Supplementary Table 2

This table shows differentially expressed genes in F12 and Δpfap2-g. (XLSX 72 kb)

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Kafsack, B., Rovira-Graells, N., Clark, T. et al. A transcriptional switch underlies commitment to sexual development in malaria parasites. Nature 507, 248–252 (2014). https://doi.org/10.1038/nature12920

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