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A transcriptional switch underlies commitment to sexual development in malaria parasites

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.

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)).

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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).

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Authors and Affiliations

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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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