Abstract
Examining host–pathogen interactions in animals can capture aspects of infection that are obscured in cell culture. Using CRISPR-based screens, we functionally profile the entire genome of the apicomplexan parasite Toxoplasma gondii during murine infection. Barcoded gRNAs enabled bottleneck detection and mapping of population structures within parasite lineages. Over 300 genes with previously unknown roles in infection were found to modulate parasite fitness in mice. Candidates span multiple axes of host–parasite interaction. Rhoptry Apical Surface Protein 1 was characterized as a mediator of host-cell tropism that facilitates repeated invasion attempts. GTP cyclohydrolase I was also required for fitness in mice and druggable through a repurposed compound, 2,4-diamino-6-hydroxypyrimidine. This compound synergized with pyrimethamine against T. gondii and malaria-causing Plasmodium falciparum parasites. This work represents a complete survey of an apicomplexan genome during infection of an animal host and points to novel interfaces of host–parasite interaction.
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Data availability
All screen data are available in Supplementary Data. Screen data and plots can also be accessed through an R Shiny web app at https://toxo-mouse-screen.wi.mit.edu/. UMI barcode counts have been deposited on Zenodo at https://doi.org/10.5281/zenodo.11218353 (ref. 132). Materials including strains and plasmids are available upon reasonable request. This study made use of publicly available data from toxodb.org. Source data are provided with this paper.
Code availability
Analysis scripts have been deposited on Zenodo at https://doi.org/10.5281/zenodo.11247744 (ref. 133).
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Acknowledgements
We thank C. A. Hunter, A. A. Koshy, J. P. Saeij and all members of the Lourido Lab for helpful discussions and support; E. Shortt for additionally providing key technical support; J. P. Saeij for the ∆gra17 and GRA17-complemented strains; L. M. Weiss for the anti-MAG1 antibody; D. W. Threadgill for the CAST/EiJ and C57Bl/6J MEF cell lines; B. D. Bryson for the RAW264.7 cell line; P. J. Bradley for the anti-GRA7 and anti-ROP13 antibodies; and L. D. Sibley for hybridomas for anti-TY and anti-SAG1 antibodies. W. L. Beatty at the Molecular Microbiology Imaging facility in Washington University performed the sample processing and transmission electron microscopy for initial analysis of the ∆gra72. We also thank the technical staff of the Electron Microscopy Core Facility at the Johns Hopkins University School of Medicine for assistance with secondary analysis of GRA72 by transmission electron microscopy; K. Zichichi of the Electron Microscopy Core Facility at Yale University School of Medicine Microscopy Facility for assistance with immunogold imaging. This work relied extensively on VEuPathDB.org and we thank all contributors to this resource. This work was supported by the Burroughs Wellcome Fund PATH award and grants from the National Institutes of Health (R01 AI158501 and R01 AI144369) to S.L., a grant from the National Institutes of Health (R01 AI145941) to J.D.D., and a National Science Foundation GRFP fellowship to C.J.G.
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C.J.G., S.L. and J.D.D. conceptualized this project. C.J.G., K.J.W., F.M.H., B.S.W., M.A.F., E.A.B., T.C.T.L., A.L.H., A.G.S. and I.C. performed all experiments. C.J.G., K.J.W. and R.W.T. performed all data analysis. C.J.G. and S.L. wrote the paper with input from all authors.
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A patent application has been filed by the Whitehead Institute on the basis of the results in this paper, with C.J.G. and S.L. as inventors (application number 63/339,281). C.J.G. is a scientific advisor at Meliora Therapeutics, a company not related to this study. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Condensed gRNA library screens in cell culture and mice.
(A) Scatter plot of gene fitness scores generated from CRISPR screens with 9-gRNA and 3-gRNA libraries. Data points are colored based on published phenotype scores from Sidik, et al.5. (B) Rank-ordered plots of gene fitness scores from a genome-wide CRISPR screen published in Sidik et al.5, or screens using new 9-gRNA and 3-gRNA libraries. Control dispensable and fitness-conferring genes are highlighted in orange and blue, respectively (Supplementary Data 2). (C) Scatter plots comparing gene fitness scores from 9-gRNA or 3-gRNA library screens against fitness scores from Sidik, et al.5, Paredes-Santos and Bitew, et al.18, and Wang, et al.8. Linear regressions and Pearson correlation scores are colored in blue. (D) Scatter plots comparing gene fitness scores from screen samples harvested directly from peritoneum or liver against those derived from parasite outgrowth from infected tissues. TGGT1_242380, a gene identified as fitness-conferring in parasite infection of bone-marrow derived macrophages is highlighted in blue8.
Extended Data Fig. 2 Population diversity metrics of parasite dissemination during mouse infection.
(A) Alluvial plot depicting relative barcode abundance of a nontargeting gRNA across tissues in a representative mouse. Line graph indicates evenness of barcodes in each sample. (B) Alluvial plot depicting relative barcode abundance of a ACT1 targeting gRNA across tissues in a representative mouse. Line graph indicates evenness of barcodes in each sample. (C) Superplot of species richness for nontargeting gRNAs across tissues from six mice. Colours indicate different mice with small points for each gRNA and large points for the mean of all gRNAs in each mouse. Mean ± SEM for all mice is overplotted. (D) STAMPR estimates of founding population size (Nr) of nontargeting gRNAs with a high and low estimate of colony forming units (CFU). (E) STAMPR estimates of bottleneck population size (Nb) and sample depth (Ns) for nontargeting gRNAs across tissues from six mice. (F) Total number of all gRNA-barcode pairs from all gRNAs found in each tissue from six mice. Each mouse is represented as a point with means shown by the gray line. (G) Superplot of species richness for gRNAs targeting non-expressed genes across tissues from six mice. Colours indicate different mice with small points for each gRNA and large points for the mean of all gRNAs in each mouse. Mean ± SEM for all mice is overplotted in black. (H) Superplot of Shannon evenness for gRNAs targeting non-expressed genes across tissues from six mice. Colours indicate different mice with small points for each gRNA and large points for the mean of all gRNAs in each mouse. Mean ± SEM for all mice is overplotted in black. (I) Genetic distance of nontargeting gRNA barcode populations across tissues across six mice. Bars represent mean genetic distance. (J) Aggregation of data in Extended Data Fig. 2I contrasting genetic distance between heart or brain and all other tissues. (K) STAMPR estimates of resilient genetic distance of nontargeting gRNA barcode populations across tissues across six mice. (L) Genetic distance of barcode populations from gRNAs targeting non-expressed genes across tissues across six mice. Bars represent mean genetic distance. (M) Jaccard similarity (fraction of shared UMIs) from gRNAs targeting non-expressed genes between indicated tissue pairs across six mice. Black bars indicate mean fraction. (N) Rank-ordered spleen gRNA fold changes from six mice highlighting gRNAs targeting ROP5b. (O) Spleen gRNA fold changes plotted against evenness. Blue lines represent thresholds for gRNA outliers at two standard deviations from the probabilistic regression model. (P) Correlation of gRNA scores between two replicate mice (Pearson’s correlation r = 0.98).
Extended Data Fig. 3 Analysis of genome-wide mouse screens.
(A–D) Scatter plots comparing gene fitness scores from nine subscreens from peritoneum and cell culture (passage 8), highlighting cell culture essential controls (A), nontargeting controls (B), or two control genes required for mouse infection, ROP9 (C), and GRA45 (D). (E) Scatter plot comparing gene fitness scores from sublibrary three screened in subscreen two and subscreen nine (Pearson correlation r = 0.943). (F) Pearson correlations of gene fitness scores between mice within a subscreen for each tissue. Mean ± SD indicated. (G-I) Volcano plots of differential fitness of liver, spleen, or lung relative to peritoneum against -log10(p-value). Dotted line indicates a threshold of [(-log10(pval))(differential fitness score)] = 30 (unpaired two-tailed t-test). Genes with cell culture fitness scores <−5 are colored in red.
Extended Data Fig. 4 Phenotyping ∆tgwip parasite dissemination in mice.
(A–B) Scatter plot comparing gene fitness scores from lung or brain to cell culture (passage 8). TgWIP and nontargeting control fitness scores are highlighted in red and blue, respectively. (C–D) Rank-ordered plot of gene fitness scores generated by normalization to peritoneum abundance for lung and brain. TgWIP and nontargeting control fitness scores are highlighted in red and blue, respectively. (E) DNA gel depicting TgWip knockout generated by replacing the locus with a mNeonGreen fluorophore (representative of two replicates). (F) Fraction of Δtgwip and WT parasites from the indicated sample during a co-infection competition experiment. Mean ± SD indicated (n = 3 mice for each tissue, unpaired two-tailed t-test).
Extended Data Fig. 5 CRISPR screens of T. gondii in human and mouse cell lines.
(A–B) Scatter plots comparing gene fitness scores from parasites cultured in B6 or CAST MEF cell lines compared to parasites cultured in HFFs. Candidates with decreased and increased fitness in the mouse peritoneum are highlighted in blue and red, respectively. (C–D) Volcano plot of differential fitness between gene scores from B6 or CAST MEFs relative to HFF against -log10(p-value). Dotted line indicates a threshold of [(-log10(pval)) (differential fitness score)] = 30 (unpaired two-tailed t-test). Candidates with decreased and increased fitness in the mouse peritoneum are highlighted in blue and red, respectively. (E) Bar graph showing number of candidates identified from each sublibrary.
Extended Data Fig. 6 Strain construction and phenotyping of novel genes required for mouse infection.
(A) DNA gels depicting endogenous tagging of the indicated genes with an HA epitope and (B) DNA gels depicting knockout generation by replacing the indicated loci with a mNeonGreen fluorophore, (representative of two replicates). (C) Plaque assays from 7 days of growth of 500 parasites of the indicated genotype in HFFs. (D) Serum response to T. gondii antigens from surviving mice.
Extended Data Fig. 7 Constructing and phenotyping of ∆gra72 parasites.
(A) DNA gel depicting integration of a TY tag at the endogenous MAG1 (TGGT1_270240) locus in MAG1-TY/GRA72-HA and MAG1-TY strains (representative of two replicates). (B) Immuno-gold electron micrographs depicting the localization GRA72-HA from an endogenously tagged strain (n = 1). (C) DNA gel depicting GRA72 knockout generated by replacing the locus with a mNeonGreen fluorophore in RH parasites (representative of two replicates). (D) Plaque assay of Δgra72 and WT parasites after 7 days of growth. (E) Serum response to T. gondii antigens from surviving mice. (F) DNA gel depicting integration of GRA72-TY construct into an intergenic locus in the Δgra72 parental strain (representative of two replicates). (G) DNA gel depicting GRA72 knockout generated by replacing the locus with a mNeonGreen fluorophore in ME49 parasites (representative of two replicates). (H) Immunofluorescence images showing the localization of GRA7 in WT and Δgra72 vacuoles (representative of two replicates). (I) Immunofluorescence images of MAG1 from WT, Δgra72, or complemented parasites at 24 h post infection (representative of two replicates). (J) Immunofluorescence images showing cMyc staining of fibroblasts infected with WT, Δgra72, or GRA72-complement parasites (representative of two replicates).
Extended Data Fig. 8 Phenotyping ∆gra72 parasites by electron microscopy and live-cell imaging.
(A) Electron micrograph depicting intravacuolar network (closed arrow) in WT and Δgra72 parasite vacuoles (parasites indicated with an asterisk). (B) Electron micrograph depicting vacuole membrane irregularities (closed arrow) in Δgra72 parasite vacuoles (parasites indicated with an asterisk). (C) Electron micrograph depicting host organelle recruitment of mitochondria (closed arrow), ER (open arrow), and Golgi (asterisk) by Δgra72 and Δgra72/GRA72-TY vacuoles. (D) Live-imaging of B6 MEF cells transfected with a mRFP1-EGFP-LC3 construct and infected with the indicated parasite strain 36 h post-infection (n = 2). (E) Electron micrograph depicting host material within a developing multi-lamellar body. All electron micrographs (A–D,E) are representative of three replicates.
Extended Data Fig. 9 Constructing and phenotyping ∆rasp1 parasites.
(A) DNA gel depicting integration of an HA tag at the endogenous RASP1 (TGGT1_235130) locus. (B) DNA gel depicting RASP1 knockout generated by replacing the locus with a mNeonGreen fluorophore. (C) Immunofluorescence of RASP1-TY complement strain with CDPK1 counterstain (representative of two replicates). (D) Plaque assay of Δrasp1 and WT parasites after 7 days of growth. (E) Serum response to T. gondii antigens from surviving mice. (F) DNA gel depicting RASP1 knockout generated by replacing the locus with a TdTomato fluorophore in the Toxofilin-Cre background. (G) DNA gel depicting PDX1 (TGGT1_237140) knockout generated by replacing the locus with a TdTomato fluorophore in the Toxofilin-Cre background. (H) DNA gel depicting PDX1 (TGGT1_237140) knockout generated by replacing the locus with a TdTomato fluorophore in the Toxofilin-β-lactamase background. (I) Immunofluorescence depicting complementation with a RASP1-TY construct in Δrasp1/Toxofilin-β-lactamase strain (representative of two replicates). (J) Bar graph showing relative attachment rates of WT, Δrasp1, and complemented parasites to glutaraldehyde-fixed HFFs (n = 2, unpaired two-tailed t-test). All DNA gels are representative of two replicates.
Extended Data Fig. 10 Constructing and phenotyping ∆gch parasites.
(A) DNA gel depicting GCH knockout generated by replacing the locus with a mNeonGreen fluorophore in type I RH parasites. (B) DNA gel depicting integration of a 5’GCH-GCH-3xHA complementation construct in trans in a Δgch strain. (C) Serum response to T. gondii antigens from surviving mice. (D) DNA gel depicting GCH knockout generated by replacing the locus with a mNeonGreen fluorophore in type II ME49 parasites. (E) Plaque assay of Δgch and WT parasites after 7 days of growth in HPLM. (F) Plaque assay of WT parasites after 7 days of growth in HPLM with the indicated treatments. (G) Plaque assay of WT, Δgch/5’GCH-GCH-3xHA and Δgch/5’TUB-GCH-3xHA parasites after 7 days of growth in HPLM with the indicated treatments. (H) Population doubling of C57BL/6J immortalized MEFs in the presence of PBS vehicle or 5 mM DAHP (n = 6, unpaired two-tailed t-test). (I–J) Dose response curve of P. falciparum NF54 parasites grown in varying concentrations of pyrimethamine (I) or atovaquone (J) with and without 1 mM DAHP co-treatment in RPMI. (K) IC50 value of atovaquone for P. falciparum NF54 parasites with and without DAHP co-treatment (n = 4, unpaired two-tailed t-test). (L) Dose response curve of P. falciparum Dd2 parasites grown in varying concentrations of DAHP in RPMI media (mean ± SD indicated, n = 3). (M) Dose response curve of P. falciparum Dd2 parasites grown in varying concentrations of pyrimethamine with and without 1 mM DAHP co-treatment (n = 3). All DNA gels are representative of two replicates.
Supplementary information
Supplementary Table
List of relevant reagents, primer sequences and antibodies used in this study.
Supplementary Data
All supplementary data as referenced throughout the paper.
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Giuliano, C.J., Wei, K.J., Harling, F.M. et al. CRISPR-based functional profiling of the Toxoplasma gondii genome during acute murine infection. Nat Microbiol (2024). https://doi.org/10.1038/s41564-024-01754-2
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DOI: https://doi.org/10.1038/s41564-024-01754-2
