Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Revisiting the initial steps of sexual development in the malaria parasite Plasmodium falciparum

Abstract

Human to vector transmission of malaria requires that some blood-stage parasites abandon asexual growth and convert into non-replicating sexual forms called gametocytes. The initial steps of gametocytogenesis remain largely uncharacterized. Here, we study this part of the malaria life cycle in Plasmodium falciparum using PfAP2-G, the master regulator of sexual conversion, as a marker of commitment. We demonstrate the existence of PfAP2-G-positive sexually committed parasite stages that precede the previously known committed schizont stage. We also found that sexual conversion can occur by two different routes: the previously described route in which PfAP2-G-expressing parasites complete a replicative cycle as committed forms before converting into gametocytes upon re-invasion, or a direct route with conversion within the same cycle as initial PfAP2-G expression. The latter route is linked to early PfAP2-G expression in ring stages. Reanalysis of published single-cell RNA-sequencing (RNA-seq) data confirmed the presence of both routes. Consistent with these results, using plaque assays we observed that, in contrast to the prevailing model, many schizonts produced mixed plaques containing both asexual parasites and gametocytes. Altogether, our results reveal unexpected features of the initial steps of sexual development and extend the current view of this part of the malaria life cycle.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Gametocytes can form at the same cycle of PfAP2-G activation.
Fig. 2: Plaque assays reveal that some individual schizonts generate mixed sexual and asexual plaques.
Fig. 3: Single-cell RNA-seq identification and characterization of SCC gametocytes.
Fig. 4: Temporal dynamics of pfap2-g transcript levels.
Fig. 5: PfAP2-G expression dynamics.

Data availability

The single-cell RNA-sequencing data analysed in this study have been deposited at the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under study accession code SRP116718. The authors declare that all other relevant data generated or analysed during this study are included in the Article or its Supplementary Information. Materials and protocols are available from the corresponding author on reasonable request.

References

  1. 1.

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

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Baker, D. A. Malaria gametocytogenesis. Mol. Biochem. Parasitol. 172, 57–65 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Bousema, T. & Drakeley, C. Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination. Clin. Microbiol. Rev. 24, 377–410 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Josling, G. A. & Llinas, M. Sexual development in Plasmodium parasites: knowing when it’s time to commit. Nat. Rev. Microbiol. 13, 573–587 (2015).

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Meibalan, E. & Marti, M. Biology of malaria transmission. Cold Spring Harb. Perspect. Med. 7, a025452 (2017).

    Article  PubMed  Google Scholar 

  6. 6.

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

    Article  PubMed  Google Scholar 

  7. 7.

    Carter, R. & Miller, L. H. Evidence for environmental modulation of gametocytogenesis in Plasmodium falciparum in continuous culture. Bull. World Health Organ. 57, 37–52 (1979).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Inselburg, J. Gametocyte formation by the progeny of single Plasmodium falciparum schizonts. J. Parasitol. 69, 584–591 (1983).

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    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  PubMed  Google Scholar 

  10. 10.

    Kafsack, B. F. et al. A transcriptional switch underlies commitment to sexual development in malaria parasites. Nature 507, 248–252 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Sinha, A. et al. A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium. Nature 507, 253–257 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Campbell, T. L., De Silva, E. K., Olszewski, K. L., Elemento, O. & Llinas, 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  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Poran, A. et al. Single-cell RNA sequencing reveals a signature of sexual commitment in malaria parasites. Nature 551, 95–99 (2017).

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    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  PubMed  PubMed Central  Google Scholar 

  15. 15.

    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).

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Fraschka, S. A. et al. Comparative heterochromatin profiling reveals conserved and unique epigenome signatures linked to adaptation and development of malaria parasites. Cell Host Microbe 23, 407–420 (2018).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Cortes, A. & Deitsch, K. W. Malaria epigenetics. Cold Spring Harb. Perspect. Med. 7, a025528 (2017).

    Article  PubMed  Google Scholar 

  19. 19.

    Brancucci, N. M. et al. Heterochromatin protein 1 secures survival and transmission of malaria parasites. Cell Host Microbe 16, 165–176 (2014).

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Coleman, B. I. et al. A Plasmodium falciparum histone deacetylase regulates antigenic variation and gametocyte conversion. Cell Host Microbe 16, 177–186 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Filarsky, M. et al. GDV1 induces sexual commitment of malaria parasites by antagonizing HP1-dependent gene silencing. Science 359, 1259–1263 (2018).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Carter, R. et al. Plasmodium falciparum: an abundant stage-specific protein expressed during early gametocyte development. Exp. Parasitol. 69, 140–149 (1989).

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Gupta, S. K., Schulman, S. & Vanderberg, J. P. Stage-dependent toxicity of N-acetyl-glucosamine to Plasmodium falciparum. J. Protozool 32, 91–95 (1985).

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Ponnudurai, T., Lensen, A. H., Meis, J. F. & Meuwissen, J. H. Synchronization of Plasmodium falciparum gametocytes using an automated suspension culture system. Parasitology 93, 263–274 (1986).

    Article  PubMed  Google Scholar 

  26. 26.

    Bruce, M. C., Carter, R. N., Nakamura, K., Aikawa, M. & Carter, R. Cellular location and temporal expression of the Plasmodium falciparum sexual stage antigen Pfs16. Mol. Biochem. Parasitol. 65, 11–22 (1994).

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Eksi, S. Identification of a subtelomeric gene family expressed during the asexual-sexual stage transition in Plasmodium falciparum. Mol. Biochem. Parasitol. 143, 90–99 (2005).

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Lopez-Barragan, M. J. et al. Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum. BMC Genomics 12, 587 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

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

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Baker, D. A. et al. A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission. Nat. Commun. 8, 430 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Tiburcio, M. et al. Specific expression and export of the Plasmodium falciparum Gametocyte EXported Protein-5 marks the gametocyte ring stage. Malar. J. 14, 334 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Painter, H. J., Carrasquilla, M. & Llinas, M. Capturing in vivo RNA transcriptional dynamics from the malaria parasite Plasmodium falciparum. Genome Res. 27, 1074–1086 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Otto, T. D. New insights into the blood-stage transcriptome of Plasmodium falciparum using RNA-seq. Mol. Microbiol. 76, 12–24 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Waters, A. P. Epigenetic roulette in blood stream Plasmodium: gambling on sex. PLoS. Pathog. 12, e1005353 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

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

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Brancucci, N. M. B. et al. Lysophosphatidylcholine regulates sexual stage differentiation in the human malaria parasite Plasmodium falciparum. Cell 171, 1532–1544 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Modrzynska, K. et al. A knockout screen of ApiAP2 genes reveals networks of interacting transcriptional regulators controlling the Plasmodium life cycle. Cell Host Microbe 21, 11–22 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Yuda, M., Iwanaga, S., Kaneko, I. & Kato, T. Global transcriptional repression: an initial and essential step for Plasmodium sexual development. Proc. Natl Acad. Sci. USA 112, 12824–12829 (2015).

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Kent, R. S. et al. Inducible developmental reprogramming redefines commitment to sexual development in the malaria parasite Plasmodium berghei. Nat. Microbiol 3, 1206–1213 (2018).

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Alonso, P. L. et al. A research agenda to underpin malaria eradication. PLoS Med. 8, e1000406 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Sinden, R. E. Developing transmission-blocking strategies for malaria control. PLoS Pathog. 13, e1006336 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    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  PubMed  Google Scholar 

  44. 44.

    Delves, M. J. et al. Male and female Plasmodium falciparum mature gametocytes show different responses to antimalarial drugs. Antimicrob. Agents Chemother. 57, 3268–3274 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Delves, M. J. et al. Routine in vitro culture of P. falciparum gametocytes to evaluate novel transmission-blocking interventions. Nat. Protoc. 11, 1668–1680 (2016).

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Roncales, M., Vidal-Mas, J., Leroy, D. & Herreros, E. Comparison and optimization of different methods for the in vitro production of Plasmodium falciparum gametocytes. J. Parasitol. Res. 2012, 927148 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Brancucci, N. M., Goldowitz, I., Buchholz, K., Werling, K. & Marti, M. An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development. Nat. Protoc. 10, 1131–1142 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Knuepfer, E., Napiorkowska, M., van Ooij, C. & Holder, A. A. Generating conditional gene knockouts in Plasmodium—a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9. Sci. Rep. 7, 3881 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Lim, M. Y. et al. UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes. Nat. Microbiol. 1, 16166 (2016).

    CAS  Article  Google Scholar 

  50. 50.

    Ghorbal, M. et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat. Biotechnol. 32, 819–821 (2014).

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Moelans, I. I. M. D. Pfs16, A Potential Vaccine Candidate Against the Human Malaria Parasite Plasmodium falciparum. PhD thesis, Katholieke Universiteit Nijmegen (1995).

  52. 52.

    Rovira-Graells, N., Aguilera-Simon, S., Tinto-Font, E. & Cortes, A. New assays to characterise growth-related phenotypes of Plasmodium falciparum reveal variation in density-dependent growth inhibition between parasite lines. PLoS ONE 11, e0165358 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Mira-Martinez, S. et al. Expression of the Plasmodium falciparum clonally variant clag3 genes in human infections. J. Infect. Dis. 215, 938–945 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Crowley, V. M., Rovira-Graells, N., de Pouplana, L. R. & 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).

    CAS  Article  PubMed  Google Scholar 

  55. 55.

    Aguilar, R. et al. Molecular evidence for the localization of Plasmodium falciparum immature gametocytes in bone marrow. Blood 123, 959–966 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Suárez-Cortés, P., Silvestrini, F. & Alano, P. A fast, non-invasive, quantitative staining protocol provides insights in Plasmodium falciparum gamete egress and in the role of osmiophilic bodies. Malar. J. 13, 389 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank P. Alano (Istituto Superiore di Sanità) for the F12 line, M. J. Delves (Imperial College London) for the 3D7 line from Imperial College London (3D7-Imp.), R. Carter (University of Edinburgh) for the anti-Pfg27 monoclonal antibody, R. W. Sauerwein (Radboud University) for the anti-Pfs16 monoclonal antibody, M. Lee (Wellcome Sanger Institute) for plasmid pDC2-Cas9-U6-hdhfr, J.-J. López-Rubio (University of Montpellier) for plasmid pL6-eGFP-yFCU, M. Llinás (Pennsylvannia State University) for providing the E5-HA-DD line and a plasmid containing the eyfp gene followed by a P. falciparum terminator and S. Osborne (LifeArc) and D. Baker (LSHTM) for providing the compound ML10 and advice on its use. The authors also thank J. Romero Ortolà and C. Sànchez Guirado for assistance with the generation of plasmids and S. Pagans (Universitat de Girona) for critical reading of the manuscript. The authors acknowledge the Flow Cytometry core facility of the IDIBAPS for technical help. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO)/Agencia Estatal de Investigación (AEI) (SAF2013-43601-R and SAF2016-76190-R to A.C.), co-funded by the European Regional Development Fund (ERDF, European Union), and the Secretary for Universities and Research under the Department of Economy and Knowledge of the Catalan Government (2014 SGR 485 to A.C.). C.B. was supported by postdoctoral fellowship 2011-BP-B 00060 from the Secretary for Universities and Research. O.L.-B. is supported by an FPU fellowship from the Spanish Ministry of Education, Culture and Sports (FPU014/02456). ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya. Single-cell experiments were supported by WCM internal startup funds (B.F.C.K.) and NSF CAREER award (DBI-10549646 to O.E.), LLS SCOR (7006-13 and 7012016 to O.E.), Hirschl Trust Award (O.E.), Starr Cancer Consortium (I6-A618 to O.E.) and NIH (1R01CA194547 to O.E.). A.P. and C.N. were supported by WCM graduate fellowships.

Author information

Affiliations

Authors

Contributions

C.B. and A.C. conceived the project. C.B., O.L.-B. and A.C. designed and interpreted the experiments. C.B., O.L.-B., N.R.-G. and A.C. performed the experiments. A.P. and B.F.C.K. analysed single-cell RNA-seq data. A.P., C.N., O.E. and B.F.C.K. contributed resources or data. C.B. and A.C. wrote the article, with major input from O.L.-B. and B.F.C.K.

Corresponding author

Correspondence to Alfred Cortés.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1–4, Supplementary Figures 1–13, Supplementary References.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bancells, C., Llorà-Batlle, O., Poran, A. et al. Revisiting the initial steps of sexual development in the malaria parasite Plasmodium falciparum. Nat Microbiol 4, 144–154 (2019). https://doi.org/10.1038/s41564-018-0291-7

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing