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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
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
Alano, P. Plasmodium falciparum gametocytes: still many secrets of a hidden life. Mol. Microbiol. 66, 291–302 (2007).
Baker, D. A. Malaria gametocytogenesis. Mol. Biochem. Parasitol. 172, 57–65 (2010).
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).
Josling, G. A. & Llinas, M. Sexual development in Plasmodium parasites: knowing when it’s time to commit. Nat. Rev. Microbiol. 13, 573–587 (2015).
Meibalan, E. & Marti, M. Biology of malaria transmission. Cold Spring Harb. Perspect. Med. 7, a025452 (2017).
Dixon, M. W., Thompson, J., Gardiner, D. L. & Trenholme, K. R. Sex in Plasmodium: a sign of commitment. Trends. Parasitol. 24, 168–175 (2008).
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).
Inselburg, J. Gametocyte formation by the progeny of single Plasmodium falciparum schizonts. J. Parasitol. 69, 584–591 (1983).
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).
Kafsack, B. F. et al. A transcriptional switch underlies commitment to sexual development in malaria parasites. Nature 507, 248–252 (2014).
Sinha, A. et al. A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium. Nature 507, 253–257 (2014).
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).
Poran, A. et al. Single-cell RNA sequencing reveals a signature of sexual commitment in malaria parasites. Nature 551, 95–99 (2017).
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).
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).
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).
Rovira-Graells, N. et al. Transcriptional variation in the malaria parasite Plasmodium falciparum. Genome Res. 22, 925–938 (2012).
Cortes, A. & Deitsch, K. W. Malaria epigenetics. Cold Spring Harb. Perspect. Med. 7, a025528 (2017).
Brancucci, N. M. et al. Heterochromatin protein 1 secures survival and transmission of malaria parasites. Cell Host Microbe 16, 165–176 (2014).
Coleman, B. I. et al. A Plasmodium falciparum histone deacetylase regulates antigenic variation and gametocyte conversion. Cell Host Microbe 16, 177–186 (2014).
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).
Filarsky, M. et al. GDV1 induces sexual commitment of malaria parasites by antagonizing HP1-dependent gene silencing. Science 359, 1259–1263 (2018).
Carter, R. et al. Plasmodium falciparum: an abundant stage-specific protein expressed during early gametocyte development. Exp. Parasitol. 69, 140–149 (1989).
Gupta, S. K., Schulman, S. & Vanderberg, J. P. Stage-dependent toxicity of N-acetyl-glucosamine to Plasmodium falciparum. J. Protozool 32, 91–95 (1985).
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).
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).
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).
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).
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).
Alano, P. et al. Plasmodium falciparum: parasites defective in early stages of gametocytogenesis. Exp. Parasitol. 81, 227–235 (1995).
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).
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).
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).
Otto, T. D. New insights into the blood-stage transcriptome of Plasmodium falciparum using RNA-seq. Mol. Microbiol. 76, 12–24 (2010).
Waters, A. P. Epigenetic roulette in blood stream Plasmodium: gambling on sex. PLoS. Pathog. 12, e1005353 (2016).
Fivelman, Q. L. et al. Improved synchronous production of Plasmodium falciparum gametocytes in vitro. Mol. Biochem. Parasitol. 154, 119–123 (2007).
Brancucci, N. M. B. et al. Lysophosphatidylcholine regulates sexual stage differentiation in the human malaria parasite Plasmodium falciparum. Cell 171, 1532–1544 (2017).
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).
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).
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).
Alonso, P. L. et al. A research agenda to underpin malaria eradication. PLoS Med. 8, e1000406 (2011).
Sinden, R. E. Developing transmission-blocking strategies for malaria control. PLoS Pathog. 13, e1006336 (2017).
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).
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).
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).
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).
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).
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).
Lim, M. Y. et al. UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes. Nat. Microbiol. 1, 16166 (2016).
Ghorbal, M. et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat. Biotechnol. 32, 819–821 (2014).
Moelans, I. I. M. D. Pfs16, A Potential Vaccine Candidate Against the Human Malaria Parasite Plasmodium falciparum. PhD thesis, Katholieke Universiteit Nijmegen (1995).
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).
Mira-Martinez, S. et al. Expression of the Plasmodium falciparum clonally variant clag3 genes in human infections. J. Infect. Dis. 215, 938–945 (2017).
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).
Aguilar, R. et al. Molecular evidence for the localization of Plasmodium falciparum immature gametocytes in bone marrow. Blood 123, 959–966 (2014).
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).
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
Authors and Affiliations
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
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.
Rights and permissions
About this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41564-018-0291-7
This article is cited by
-
Epigenetic regulation as a therapeutic target in the malaria parasite Plasmodium falciparum
Malaria Journal (2024)
-
High-throughput analysis of the transcriptional patterns of sexual genes in malaria
Parasites & Vectors (2023)
-
Protocols for Plasmodium gametocyte production in vitro: an integrative review and analysis
Parasites & Vectors (2022)
-
The transcriptional regulator HDP1 controls expansion of the inner membrane complex during early sexual differentiation of malaria parasites
Nature Microbiology (2022)
-
A transcriptional switch controls sex determination in Plasmodium falciparum
Nature (2022)