Protein kinases regulate fundamental aspects of eukaryotic cell biology, making them attractive chemotherapeutic targets in parasites like Plasmodium spp. and Toxoplasma gondii. To systematically examine the parasite kinome, we developed a high-throughput tagging (HiT) strategy to endogenously label protein kinases with an auxin-inducible degron and fluorophore. Hundreds of tagging vectors were assembled from synthetic sequences in a single reaction and used to generate pools of mutants to determine localization and function. Examining 1,160 arrayed clones, we assigned 40 protein localizations and associated 15 kinases with distinct defects. The fitness of tagged alleles was also measured by pooled screening, distinguishing delayed from acute phenotypes. A previously unstudied kinase, associated with a delayed phenotype, was shown to be a regulator of invasion and egress. We named the kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores. Despite homology to mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), SPARK lacks a lipid-binding domain, suggesting a rewiring of the pathway in parasites. HiT screening extends genome-wide approaches into complex cellular phenotypes, providing a scalable and versatile platform to dissect parasite biology.
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All oligos used in this study are available in Supplementary Table 1. All plasmids used or generated in this study are listed with their appropriate GenBank or PMID accession numbers in Supplementary Table 1. Minimally processed pooled and arrayed CRISPR screen sequencing results are available in Supplementary Table 2. Localization assignments, microscopy phenotypes, lytic assay results, and UMAP coordinates and clusters are likewise available in Supplementary Table 2. Source data are provided with this paper. Data from experimental results is available in the source data files. Additional unprocessed data is available from the corresponding author upon request.
All code is described in the methods section and available from the corresponding author upon request.
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We thank the Whitehead Institute Bioinformatics and Research Computing Core, especially B. Yuan, for assistance implementing gRNA design pipelines; L.D. Sibley for the TIR1 strain; M. Treeck for the DiCre strain; W. Salmon and the W.M. Keck Biological Imaging Facility for confocal microscopy support; P. W. Reddien for use of the Illumina MiSeq; B.S. Waldman, E.A. Boydston, C.J. Giuliano, A.W. Chan, S. Sidik and B.M. Markus for technical support in generation of the array; VEuPathDB and all contributors to this resource. This work was supported by funds from a National Institutes of Health grant (R01AI144369) to S.L. and National Science Foundation Graduate Research Fellowships to T.A.S. (2018259980) and A.L.H. (174530).
The authors declare no competing interests.
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a, Fluorescence microscopy of the tagged populations displaying the correct localization of each kinase and expression levels consistent with flow cytometry (Fig. 1b). b, Live microscopy of V5-T2A-mKate2 HiT-tagged population (merged image in Fig. 1g). c, Immunofluorescence microscopy of population tagged with the HA-U1 HiT vector following treatment with rapamycin or vehicle control (merged image in Fig. 1i). d, Flow cytometry of parasite populations tagged with the V5-mNG-mAID HiT vector targeting CDPK1 or CDPK3 and treated with either IAA or vehicle control for 24 h (excerpt shown in Fig. 1l).
a, Results from dual-indexed sequencing of the arrayed clones. A minimum of 100 reads were required to assign a given gRNA to a particular clone. Cases where a second gRNA reached >10% the abundance of the first gRNA were classified as containing multiple integrations. b, Histogram showing the distribution of gRNAs and genes contained among single-integrated wells within the array. Genes and gRNAs with no representation are omitted from the plot.
a–f, Widefield microscopy of representative clones. Maximum intensity projections for IMC1-tdTomato and mNeonGreen-tagged targets are displayed for cultures treated with either IAA or vehicle for 24 hours. All images are displayed at the same scale. Localizations to the nucleus (a), daughter cell IMC (b), parasitophorous vacuole (c), perinuclear space (d), cytosol (e) or apical end (f) were assigned to a gene if half or more of single-integrated wells for that gene displayed consistent localizations.
Extended Data Fig. 4 Additional representative images from the arrayed screen and comparisons to the pooled results.
a–c, Widefield microscopy of representative clones. Maximum intensity projections for IMC1-tdTomato and mNeonGreen-tagged targets are displayed for cultures treated with either IAA or vehicle for 24 hours. All images are displayed at the same scale. Localizations to puncta (a), the basal end (b), or peripheral structures (c) were assigned to a gene if half or more of single-integrated wells for that gene displayed consistent localizations. d, Representative confocal images of a sample of clones. mNeonGreen (green); IMC1-tdTomato (magenta). Images are maximum intensity projections. Genes are numbered based on the unique identifier from ToxoDB (for example, TGGT1_210830, labeled 210830). e, Comparison of relative gRNA abundances in the array compared to the pooled population that was subcloned. Spearman correlation coefficient = 0.77. f, Impact of the initial lytic cycles on gRNA abundance for genes with delayed or acute loss phenotypes in the HiT screen. The effect of the first lytic cycle from the HiT screen is plotted against the effect of the first or second lytic cycles for the genome-wide knockout screen (Sidik & Huet, et al. 2016). Genes are paired across their first and second lytic cycles within the genome-wide knockout screen.
a, Replication assay of SPARK-AID parasites. Parasites were treated with either IAA or vehicle at 3 hours post-invasion and imaged 24 hours later. The number of parasites per vacuole were counted for 100 vacuoles per sample. Mean ± S.E. graphed for n = 3 biological replicates. b, Extracellular parasites in basal Ca2+ buffer stimulated with vehicle or the Ca2+ ionophore ionomycin, following 24 h of treatment with vehicle or IAA. Cytosolic Ca2+ flux was measured in bulk as GCaMP6f fluorescence normalized to the initial and maximum fluorescence following aerolysin permeabilization in 2 mM Ca2+. Mean ± S.E. graphed for n = 3–6 biological replicates.
b, Competition assays of delayed-loss candidates. Provided are relative abundances of each strain relative to the WT competitor strain. Values are normalized to the starting ratio. d, Area sizes of individual plaques. Provided values are in mm2. e, Invasion efficiencies of delayed-loss candidates. Invaded parasites per nuclei for each replicate are provided, in addition to the values post-normalization to the WT vehicle-treated sample.
a, Kinase domain sequences used to generate alignments and the subsequent phylogenetic tree. e,f, Egress efficiencies following either (e) zaprinast or (f) A23187 stimulation. Provided is per cent of egress relative to the final percentage of egress of the vehicle-treated sample. h, Quantification of GCaMP6f fluorescence signal following either zaprinast or A23187 treatment. Average fluorescence of each vacuole was quantified relative to initial fluorescence until egress of the vacuole or until the end of the time-course. i, Quantification of GCaMP6f fluorescence from extracellular parasites treated with either zaprinast or A23187. Provided are background-subtracted values normalized to initial fluorescence and the final maximum fluorescence following aerolysin and Ca2+ treatment.
a, Replication assays of SPARK grown in the presence or absence of IAA. 100 vacuoles were quantified for each sample and condition. Provided are the number of occurrences of each vacuole size for each replicate. b, Quantification of GCaMP6f fluorescence from extracellular parasites treated with either vehicle (DMSO) or ionomycin. Provided are background-subtracted values normalized to initial fluorescence and the final maximum fluorescence following aerolysin and Ca2+ treatment.
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Smith, T.A., Lopez-Perez, G.S., Herneisen, A.L. et al. Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress. Nat Microbiol 7, 868–881 (2022). https://doi.org/10.1038/s41564-022-01104-0