Abstract
Transmission of Plasmodium falciparum and other malaria parasites requires their differentiation from asexual blood stages into gametocytes, the non-replicative sexual stage necessary to infect the mosquito vector. This transition involves changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. However, the genomes of malaria parasites have been noted for their limited number of transcriptional and chromatin regulators, and the molecular mediators of these changes remain largely unknown. We recently identified homeodomain protein 1 (HDP1) as a DNA-binding protein, first expressed in gametocytes, that enhances the expression of key genes critical for early sexual differentiation. The discovery of HDP1 marks a new class of transcriptional regulator in malaria parasites outside of the better-characterized ApiAP2 family. Here, using molecular biology, biochemistry and microscopy techniques, we show that HDP1 is essential for gametocyte maturation, facilitating the necessary upregulation of inner membrane complex components during early gametocytogenesis that gives P. falciparum gametocytes their characteristic shape.
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Data availability
Processed and raw high-throughput sequencing data have been deposited in the NCBI Gene Expression Omnibus under accession nos. GSE189197 (RNA-seq) and GSE189151 (ChIP–seq). The pipelines for RNA-seq and ChIP–seq data analysis are available at https://github.com/KafsackLab/HDP1. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Information. Additional data related to this paper may be requested from the authors. P. falciparum lines generated in this study are available at cost on request. Source data are provided with this paper.
Change history
08 February 2022
A Correction to this paper has been published: https://doi.org/10.1038/s41564-022-01082-3
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Acknowledgements
We thank P. Malhotra for the generous gift of anti-PhIL1 antibodies, V. Carruthers for the generous gift of anti-Ty1 antibodies and the Weill Cornell Medicine genomics core for technical support; and K. Deitsch and J. King for valuable feedback on the manuscript. This work was supported by startup funds from Weill Cornell Medicine: to B.F.C.K., nos. 1R01 AI141965 and 1R01 AI138499; to M.L., no. 1R01 AI125565; and to K.G.L.R., nos. 1R01 AI136511 and R21 AI142506-01; by the University of California, Riverside (no. NIFA-Hatch-225935, to K.G.L.R.); and by support from the Mathers Foundation (to D.J.P.).
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Conceptualization was the responsibility of B.F.C.K. Methodology was performed by B.F.C.K., R.A.C.M., W.X. and K.G.L.R. R.A.C.M., X.T., W.X., L.M.O. and W.D. carried out investigations. Software, formal analysis and data curation were provided by B.F.C.K. R.A.C.M. wrote the original draft. B.F.C.K. and R.A.C.M. wrote, reviewed and edited the article. Visualization was conducted by R.A.C.M. and B.F.C.K. B.F.C.K., K.G.L.R., M.L. and D.J.P. supervised the project. Project administration and funding acquisition were carried out by B.F.C.K.
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Extended data
Extended Data Fig. 1 The predicted DNA-binding protein HDP1 is expressed in gametocytes.
(a) The single exon locus Pf3D7_1466200 encodes a large 3078aa protein with a predicted C-terminal Helix-Turn-Helix DNA-binding domain (DBD) and two nuclear localization signals (NLS). Multiple AP2-G binding sites are found in its 2 kb promoter region. Black bracket indicates the antigen used for generating antisera used in29. (b) Alignment of the helix-turn-helix domain for homologs from other apicomplexan parasites. (c) Quantitative RT-PCR of hdp1 transcripts found minimal expression in asexual blood stages with upregulation during gametocyte development (data are presented as mean values of n = 2 biologically independent samples). Transcript levels were normalized to Seryl-tRNA synthetase.
Extended Data Fig. 2 Validation of engineered parasite lines.
(a) Generation of halo-hdp1 parasites by CAS9 genome editing. Insertion of the N-terminal HALO tag at the 5’ end of hdp1 coding sequence was confirmed by PCR and checked for mutations by Sanger sequencing of the 3.8 kb PCR product (not shown). (b) Generation of hdp1-gfp parasites by CAS9 genome editing. Insertion of the C-terminal GFP tag at the 3’ end the hdp1 coding sequence was confirmed by PCR and checked for mutations by Sanger sequencing of the 1.9 kb PCR product (not shown). (c) Generation of hdp1-Ty1 parasites by CAS9 genome editing. Insertion of the C-terminal triple Ty1 epitope tag at the 3’ end the hdp1 coding sequence was confirmed by PCR and checked for mutations by Sanger sequencing of the 1.1 kb PCR product (not shown). (d) Generation of ∆hdp1 parasites by CAS9 genome editing. Replacement of 1.4 kb flanking the hdp1 start codon by a hDHFR selectable marker cassette was confirmed by PCR. (e) Generation of hdp1-glmS parasites by CAS9 genome editing. Insertion of the C-terminal triple Ty1 epitope tag and the glmS ribozyme at the 3’ end the hdp1 coding sequence was confirmed by PCR and checked for mutations by Sanger sequencing of the 1.4 kb PCR product (not shown).
Extended Data Fig. 3 Loss of HDP1 does not alter the sexual commitment frequency or Stage I gametocyte viability.
(a) The sexual commitment frequency (day 5 gametocytes per day 1 ring stages) is not significantly affected in halo-hdp1 and ∆hdp1 parasites. Data are presented as mean values ± s.e.m. of n = 3 biologically independent samples. (b) Mitochondrial membrane potential of gametocytes (GCs), as measured by DilC(1)5 staining, indicates similar viability on day 2, but not days 5 or 10, for NF54 (orange) and ∆hdp1 (green) gametocytes. Flow cytometry plots are representative of n = 2 biologically independet samples.
Extended Data Fig. 4 Inducible knockdown of HDP1 reduces gametocyte maturation in early but not late gametocytes.
(a) Percentage of falciform gametocytes on Day 10 in response to 5 mM glucosamine on days 1-5, 3-8, 5-10, or in the absence of glucosamine for hdp1-Ty1 or hdp1-glmS parasites. Data are presented as mean values ± s.e.m of n = 2 biologically independent samples. (b) Representative morphology of hdp1-glmS gametocytes in response to 5 mM glucosamine on days 1-5, 3-8, 5-10, or in the absence of glucosamine, among two independent experiments. Scale bar, 3 μm.
Extended Data Fig. 5 Immunofluorescence microscopy localizes the HA-tagged ortholog TGME49_233160 to the nucleus of Toxoplasma gondii tachyzoites.
Staining of TgME49_233160-HA intracellular tachyzoites with anti-HA (TgME49_23316, green), anti-GAP45 (inner membrane complex, red), and DAPI (DNA, blue). Scale bar, 5 μm. Image is representative of n = 2 independent experiments.
Extended Data Fig. 6 HDP1-GFP localized to the nucleus of hdp1-gfp gametocytes.
No signal was observed in hdp1-gfp asexual blood stages or gametocytes of the untagged NF54 parent line. Exposure and brightness/contrast settings are uniform across the images shown. Images are representative of n = 2 biologically independent experiments. Scale bar, 3 µm.
Extended Data Fig. 7 The HDP1 DNA-binding domain tandem GC-rich motif.
(a) Coomassie stain of the recombinant GST-HDP1-DBD used for PBM analysis. (b) The three most highly enriched DNA motifs for the GST-HDP1 DBD domain on the protein-binding microarray. (c) Top three secondary motif hits after removal of primary hits.
Extended Data Fig. 8 Genes encoding inner membrane complex genes with significantly reduced expression in HDP1 knockout parasites have upstream HDP1 binding sites.
Remaining HDP1 binding sites upstream of genes encoding inner membrane complex proteins. Histogram track shows the significance of enrichment by position. Regions of significant enrichment are shown as boxes with black vertical lines indicating peak summits within each peak. Instances of Motif A, Motif B, or overlapping motifs within peaks are shown in red, blue and purple, respectively. Genes encoded in forward or reverse orientation are shown in blue or red, respectively. Combined estimate of two biologically independent samples.
Extended Data Fig. 9 Gating schema for viable gametocytes.
Populations were gated for single cells based on forward (FSC) and side scatter (SSC). Viable gametocytes were identified based on DNA content and mitochondrial membrane potential based on Hoechst33342 and DilC(1)5 staining.
Supplementary information
Supplementary Data
Processed RNA-seq and ChIP–seq data.
Supplementary Table 1
List of primers used.
Source data
Source Data Fig. 1
Numerical source data.
Source Data Fig. 1
Unprocessed immunoblots.
Source Data Fig. 2
Unprocessed immunoblots.
Source Data Fig. 4
Unprocessed EMSA.
Source Data Fig. 5
Numerical source data.
Source Data Fig. 5
Unprocessed immunoblots.
Source Data Extended Data Fig. 1
Numerical source data.
Source Data Extended Data Fig. 2
Unprocessed agarose gels.
Source Data Extended Data Fig. 3
Numerical source data.
Source Data Extended Data Fig. 4
Numerical source data.
Source Data Extended Data Fig. 7
Unprocessed acrylamide gel stained with Coomassie blue.
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Campelo Morillo, R.A., Tong, X., Xie, W. et al. The transcriptional regulator HDP1 controls expansion of the inner membrane complex during early sexual differentiation of malaria parasites. Nat Microbiol 7, 289–299 (2022). https://doi.org/10.1038/s41564-021-01045-0
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DOI: https://doi.org/10.1038/s41564-021-01045-0
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