A transcriptional switch underlies commitment to sexual development in malaria parasites

Journal name:
Nature
Volume:
507,
Pages:
248–252
Date published:
DOI:
doi:10.1038/nature12920
Received
Accepted
Published online

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.

At a glance

Figures

  1. pfap2-g transcript levels mirror gametocyte production.
    Figure 1: pfap2-g transcript levels mirror gametocyte production.

    a, pfap2-g relative transcript abundance in synchronized (early schizont stage) cultures as measured by qPCR varies significantly between 3D7-A and 3D7-B populations as well as the 3D7-B subclones E5, A7 and B11. Values are normalized against seryl transfer RNA synthetase (PF07_0073) (n = 3, standard deviation shown). b, Per cent commitment to gametocyte differentiation in these lines mirrors relative pfap2-g transcript levels (mean of n = 2).

  2. Disrupting PfAP2-G function results in loss of gametocyte production.
    Figure 2: Disrupting PfAP2-G function results in loss of gametocyte production.

    a, Positions of pfap2-g mutations in the gametocyte non-producer lines F12 and GNP-A4 and the targeted deletion of Δpfap2-g. b, Southern blot showing successful disruption of the pfap2-g locus by homologous recombination (also see Supplementary Fig. 4). Single replicate. c, pfap2-g mutants fail to produce gametocytes (n = 3, standard error shown). d, Ligand-regulatable gametocyte formation in PfAP2-G–ddFKBP (bottom row images) but not in the E5 parent (top row images). Representive of n = 4. Scale bars, 5μm. e, Quantification of ligand-regulatable gametocyte formation (n = 4, standard error shown).

  3. Identification of PfAP2-G targets.
    Figure 3: Identification of PfAP2-G targets.

    a, PfAP2-G–HA×3 localizes to the nuclei of schizonts in asexually growing parasites (see Supplementary Fig. 7 for additional stages). Scale bar, 1μm. Representative of n = 8. BF, bright field; DAPI, 4′,6-diamidino-2-phenylindole. b, Relative abundance of transcripts with greater than twofold average difference in both Δpfap2-g and F12 with respect to 3D7-B clone E5 across the intra-erythrocytic developmental cycle at 6-h intervals. Columns on the right indicate whether genes are known gametocyte markers (blue), detected in two or more gametocyte proteomes (orange), enriched in early gametocyte proteome (purple), and the number of PfAP2-G cognate motifs within 2kb upstream of the start codon. c, Binding of the recombinant PfAP2-G AP2 domain to three gametocyte promoters occurs only in the presence of the wild-type cognate motif (+). Representative of n = 3. d, Relative luciferase activity under the control of wild-type gametocyte promoters in 3D7-B E5 (blue) and Δpfap2-g (red), or in 3D7-B E5 under control of promoters lacking the PfAP2-G motif (green). (18–30h post-invasion, n = 3, standard error is shown, two-sided t-test used). NA, not tested.

  4. Activation of PfAP2-G.
    Figure 4: Activation of PfAP2-G.

    a, Only a small fraction (1–6%) of asexually growing subclone 9A schizonts (see Supplementary Fig. 7 for details) express detectable levels of PfAP2-G–HA×3 (top row). H3K4me3 staining was performed in parallel to confirm full permeabilization (bottom row). Scale bars,10μm. Representative of n = 4. b, The percentage of PfAP2-G–HA×3-positive cells is highly predictive (R2 = 0.94) of subsequent gametocyte formation levels. c, Model of PfAP2-G activation and function.

Accession codes

Referenced accessions

Gene Expression Omnibus

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Author information

Affiliations

  1. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, USA

    • Björn F. C. Kafsack,
    • Valerie M. Crowley &
    • Manuel Llinás
  2. Barcelona Centre for International Health Research (CRESIB, Hospital Clínic-Universitat de Barcelona), Barcelona, 08036 Catalonia, Spain

    • Núria Rovira-Graells,
    • Cristina Bancells &
    • Alfred Cortés
  3. Institute for Research in Biomedicine (IRB), Barcelona, 08028 Catalonia, Spain

    • Núria Rovira-Graells,
    • Valerie M. Crowley &
    • Alfred Cortés
  4. Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK

    • Taane G. Clark,
    • Laura G. Drought &
    • David A. Baker
  5. Faculty of Epidemiology and Population Health, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK

    • Taane G. Clark
  6. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK

    • Susana G. Campino &
    • Dominic P. Kwiatkowski
  7. Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA

    • April E. Williams &
    • Manuel Llinás
  8. Wellcome Trust Sanger Centre for Human Genetics, Oxford OX3 7BN, UK

    • Dominic P. Kwiatkowski
  9. Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, 08010 Catalonia, Spain

    • Alfred Cortés
  10. Present addresses: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA (B.F.C.K.); Department of Molecular Biology and Center for Infectious Disease Dynamics, The Pennsylvania State University, State College, Pennsylvania 16802, USA (V.M.C., M.L.).

    • Björn F. C. Kafsack,
    • Valerie M. Crowley &
    • Manuel Llinás

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

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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Supplementary information

PDF files

  1. Supplementary Information (8.9 MB)

    This file contains Supplementary Figures 1-10, Supplementary References and Supplementary Table 3.

Excel files

  1. Supplementary Table 1 (39 KB)

    This table shows coding sequence mutations affecting F12, GNP-A4, and Δpfap2‐g.

  2. Supplementary Table 2 (72 KB)

    This table shows differentially expressed genes in F12 and Δpfap2-g.

Additional data