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.
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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.
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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.
The authors declare no competing interests.
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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
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