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A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium

Abstract

Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium) to be transmitted through mosquitoes1. The molecular mechanism(s) responsible for commitment have been hitherto unknown. Here we show that PbAP2-G, a conserved member of the apicomplexan AP2 (ApiAP2) family of DNA-binding proteins, is essential for the commitment of asexually replicating forms to sexual development in Plasmodium berghei, a malaria parasite of rodents. PbAP2-G was identified from mutations in its encoding gene, PBANKA_143750, which account for the loss of sexual development frequently observed in parasites transmitted artificially by blood passage. Systematic gene deletion of conserved ApiAP2 genes in Plasmodium confirmed the role of PbAP2-G and revealed a second ApiAP2 member (PBANKA_103430, here termed PbAP2-G2) that significantly modulates but does not abolish gametocytogenesis, indicating that a cascade of ApiAP2 proteins are involved in commitment to the production and maturation of gametocytes. The data suggest a mechanism of commitment to gametocytogenesis in Plasmodium consistent with a positive feedback loop involving PbAP2-G that could be exploited to prevent the transmission of this pernicious parasite.

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Figure 1: Identification of mutations in pbap2-g that account for the repeated spontaneous loss of commitment to gametocytogenesis.
Figure 2: Characterization of the DNA-binding specificity, expression and subcellular localization of PbAP2-G.
Figure 3: pbap2-g acts upstream of gametocyte gene transcription.

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Primary accessions

Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

Data deposits

Microarray data has been submitted to the GEO database under accession numbers GSE52859 and GSE53246.

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Acknowledgements

We thank A. McBride for technical assistance with mosquito transmissions; A. R. Gomes, J. Tripathi, D. Vaughan and the III flow cytometry facility for assistance; C. Cairney and N. Keith at the Beatson Institute for use of their Agilent microarray scanner; and A. Cortes and D. Baker for reading of the manuscript. A.P.W. is funded by the Wellcome Trust (ref. 083811/Z/07/Z). The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from the Wellcome Trust (085349). A.S. was a student of the University of Glasgow Wellcome Trust 4-year PhD Programme Molecular Functions in Disease. A.P.W., O.B. and M.B. are members of Evimalar (ref. 242095), which funds the work of T.D.O. Work at the Sanger Institute was funded by grants from the Wellcome Trust (098051) and the Medical Research Council (G0501670). M.L. is funded by the National Institutes of Health (R01 AI076276) and 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.

Author information

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Authors

Contributions

A.P.W. and O.B. directed the research. A.S. generated the GNP clones, performed some of the EMSA analyses, made pbap2-g gene knockout lines and complementation lines and analysed the latter. K.R.H. performed microarray analyses, generated reporter and minigene constructs, made transgenic parasites and analysed them, performed competition experiments. K.K.M. made the complementation construct, generated and analysed knockout and complemented lines for pbap2-g and pbap2-g2 and performed and analysed competition and microarray experiments. C.P. generated knockout lines for pbap2-g and pbap2-g2 and performed the initial parasitological analysis. E.B. generated recombinase engineered constructs for use at Wellcome Trust Sanger Institute and University of Glasgow. A.L.G. and A.A.R. performed expression analyses. N.J.D. performed statistical analyses of motif distribution and assisted with the microarray analyses. R.C. performed the complementation experiments and transmission experiments. A.E.W. performed EMSA analyses and generated constructs used in the analysis. T.D.O. and M.B. generated the GNP sequence data and SNP analyses. M.L. and B.F.C.K. performed microarray analyses, M.L. and A.E.W. performed EMSA analyses and generated recombinant PbAP2-G DBD. A.P.W., O.B., A.S., K.R.H. and K.K.M. wrote the paper.

Corresponding authors

Correspondence to Oliver Billker or Andrew P. Waters.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-13 and Supplementary Tables 8 and 10. (PDF 3749 kb)

Supplementary Table 1

Weekly parasitaemias and gametocytaemias of the 10 mechanically passaged lines of PBANKA clone 820. Parasitaemias are given as a percentage of the erythrocytes that are infected. Gametocytaemias are given as a percentage of the parasitaemia setting the parasitaemia as 100%. (XLSX 171 kb)

Supplementary Table 2

Parasitological parameters of the GNP lines developed in the study. (XLSX 9 kb)

Supplementary Table 3

Summary of whole genome sequencing data for the cloned parasite lines used in the study. (XLSX 21 kb)

Supplementary Table 4

Characterisation of GNP repair lines and complemented pbap2-g ko line. (XLSX 644 kb)

Supplementary Table 5

Energy matrices and enrichment score matrices for PBM assay of predicted DNA binding domain of PBANKA_143750 expressed as a recombinant protein. (XLSX 70 kb)

Supplementary Table 6

Microarray analysis of GNP, ap2-g and ap2-g2 knock out lines generated in the study. (XLSX 1867 kb)

Supplementary Table 7

Down regulated transcripts in ap2-g and ap2-g2 knock out schizonts relative to wild type expression. Transcripts are classified into those exclusively down in GNP lines compared to reverse genetically engineered lines; down in all lines studied, down in ap2-g or ap2-g2 KO only; down only in both or down in all GNP and either ap2-g or ap2-g2 KO lines. Red shading indicates peptides detected in female gametocytes, blue in male gametocytes and grey indicates transcripts predicted to be translationally repressed in female gametocytes. Proteome data adapted from ref. 8. Translation repression data was taken from ref. 9. (XLSX 136 kb)

Supplementary Table 9

A list of oligonucleotides used in the study. (XLSX 26 kb)

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Sinha, A., Hughes, K., Modrzynska, K. et al. A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium . Nature 507, 253–257 (2014). https://doi.org/10.1038/nature12970

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