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CRISPR-based functional profiling of the Toxoplasma gondii genome during acute murine infection

Nature Microbiology volume 9, pages 2323–2343 (2024)Cite this article

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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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Fig. 1: Condensed-barcoded gRNA libraries improve screening efficiency in mice.
Fig. 2: Genome-wide survey of gene fitness during mouse infection.
Fig. 3: Validation of genes required for parasite fitness during acute infection.
Fig. 4: RASP1 facilitates repeated invasion and broadens host-cell tropism.
Fig. 5: GTP cyclohydrolase I is a druggable enzyme necessary during mouse infection.

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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).

References

  1. Montoya, J. G. & Liesenfeld, O. Toxoplasmosis. Lancet 363, 1965–1976 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Lawley, T. D. et al. Genome-wide screen for Salmonella genes required for long-term systemic infection of the mouse. PLoS Pathog. 2, e11 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Keys, H. R. & Knouse, K. A. Genome-scale CRISPR screening in a single mouse liver. Cell Genom. 2, 100217 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Chen, S. et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160, 1246–1260 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Sidik, S. M. et al. A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell 166, 1423–1435.e12 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Harding, C. R. et al. Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility. Nat. Commun. 11, 4813 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chen, Y. et al. Genome-wide CRISPR/Cas9 screen identifies new genes critical for defense against oxidant stress in Toxoplasma gondii. Front. Microbiol. 12, 670705 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Wang, Y. et al. Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages. Nat. Commun. 11, 5258 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Sangaré, L. O. et al. In vivo CRISPR screen identifies TgWIP as a Toxoplasma modulator of dendritic cell migration. Cell Host Microbe 26, 478–492.e8 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Young, J. et al. A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice. Nat. Commun. 10, 3963 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Butterworth, S. et al. Toxoplasma gondii virulence factor ROP1 reduces parasite susceptibility to murine and human innate immune restriction. PLoS Pathog. 18, e1011021 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tachibana, Y., Hashizaki, E., Sasai, M. & Yamamoto, M. Host genetics highlights IFN-γ-dependent Toxoplasma genes encoding secreted and non-secreted virulence factors in in vivo CRISPR screens. Cell Rep. 42, 112592 (2023).

    Article  CAS  PubMed  Google Scholar 

  13. Bushell, E. et al. Functional profiling of a Plasmodium genome reveals an abundance of essential genes. Cell 170, 260–272.e8 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Michlits, G. et al. CRISPR-UMI: single-cell lineage tracing of pooled CRISPR-Cas9 screens. Nat. Methods 14, 1191–1197 (2017).

    Article  CAS  PubMed  Google Scholar 

  15. Wong, A. S. L. et al. Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM. Proc. Natl Acad. Sci. USA 113, 2544–2549 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhu, S. et al. Guide RNAs with embedded barcodes boost CRISPR-pooled screens. Genome Biol. 20, 20 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Schmierer, B. et al. CRISPR/Cas9 screening using unique molecular identifiers. Mol. Syst. Biol. 13, 945 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Paredes-Santos, T. C. et al. Genome-wide CRISPR screen identifies genes synthetically lethal with GRA17, a nutrient channel encoding gene in Toxoplasma. PLoS Pathog. 19, e1011543 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Barylyuk, K. et al. A comprehensive subcellular atlas of the Toxoplasma proteome via hyperLOPIT provides spatial context for protein functions. Cell Host Microbe 28, 752–766.e9 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Fichera, M. E. & Roos, D. S. A plastid organelle as a drug target in apicomplexan parasites. Nature 390, 407–409 (1997).

    Article  CAS  PubMed  Google Scholar 

  21. Kennedy, K., Crisafulli, E. M. & Ralph, S. A. Delayed death by plastid inhibition in apicomplexan parasites. Trends Parasitol. 35, 747–759 (2019).

    Article  CAS  PubMed  Google Scholar 

  22. Andenmatten, N. et al. Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat. Methods 10, 125–127 (2013).

    Article  CAS  PubMed  Google Scholar 

  23. Hullahalli, K., Pritchard, J. R. & Waldor, M. K. Refined quantification of infection bottlenecks and pathogen dissemination with STAMPR. mSystems 6, e0088721 (2021).

    Article  PubMed  Google Scholar 

  24. Wincott, C. J. et al. Cellular barcoding of protozoan pathogens reveals the within-host population dynamics of Toxoplasma gondii host colonization. Cell Rep. Methods 2, 100274 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Etheridge, R. D. et al. The Toxoplasma pseudokinase ROP5 forms complexes with ROP18 and ROP17 kinases that synergize to control acute virulence in mice. Cell Host Microbe 15, 537–550 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Behnke, M. S. et al. Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases. Proc. Natl Acad. Sci. USA 108, 9631–9636 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Butterworth, S. et al. Toxoplasma gondii ROP1 subverts murine and human innate immune restriction. PLoS Pathog. 18, e1011021 (2022).

  28. Konrad, C., Wek, R. C. & Sullivan, W. J. Jr. A GCN2-like eukaryotic initiation factor 2 kinase increases the viability of extracellular Toxoplasma gondii parasites. Eukaryot. Cell 10, 1403–1412 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Krishnan, A. et al. Functional and computational genomics reveal unprecedented flexibility in stage-specific Toxoplasma metabolism. Cell Host Microbe 27, 290–306.e11 (2020).

    Article  CAS  PubMed  Google Scholar 

  30. Chattopadhyay, R. et al. PfSPATR, a Plasmodium falciparum protein containing an altered thrombospondin type I repeat domain is expressed at several stages of the parasite life cycle and is the target of inhibitory antibodies. J. Biol. Chem. 278, 25977–25981 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. Huynh, M.-H., Boulanger, M. J. & Carruthers, V. B. A conserved apicomplexan microneme protein contributes to Toxoplasma gondii invasion and virulence. Infect. Immun. 82, 4358–4368 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Kafsack, B. F. C. et al. Rapid membrane disruption by a perforin-like protein facilitates parasite exit from host cells. Science 323, 530–533 (2009).

    Article  CAS  PubMed  Google Scholar 

  33. Behnke, M. S. et al. The polymorphic pseudokinase ROP5 controls virulence in Toxoplasma gondii by regulating the active kinase ROP18. PLoS Pathog. 8, e1002992 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Dongchao, Z., Ning, J. & Qijun, C. Loss of rhoptry protein 9 impeded Toxoplasma gondii infectivity. Acta Trop. 207, 105464 (2020).

    Article  PubMed  Google Scholar 

  35. Peixoto, L. et al. Integrative genomic approaches highlight a family of parasite-specific kinases that regulate host responses. Cell Host Microbe 8, 208–218 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Michelin, A. et al. GRA12, a Toxoplasma dense granule protein associated with the intravacuolar membranous nanotubular network. Int. J. Parasitol. 39, 299–306 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. Fox, B. A. et al. Toxoplasma gondii parasitophorous vacuole membrane-associated dense granule proteins orchestrate chronic infection and GRA12 underpins resistance to host gamma interferon. mBio 10, e00589-19 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Coffey, M. J. et al. Aspartyl protease 5 matures dense granule proteins that reside at the host–parasite interface in Toxoplasma gondii. mBio 9, e01796-18 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Shastri, A. J., Marino, N. D., Franco, M., Lodoen, M. B. & Boothroyd, J. C. GRA25 is a novel virulence factor of Toxoplasma gondii and influences the host immune response. Infect. Immun. 82, 2595–2605 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Franco Magdalena et al. A novel secreted protein, MYR1, is central to Toxoplasma’s manipulation of host cells. mBio 7, e02231-15 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Marino, N. D. et al. Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii. PLoS Pathog. 14, e1006828 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Cygan, A. M. et al. Coimmunoprecipitation with MYR1 identifies three additional proteins within the Toxoplasma gondii parasitophorous vacuole required for translocation of dense granule effectors into host cells. mSphere 5, e00858-19 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Harker, K. S., Ueno, N. & Lodoen, M. B. Toxoplasma gondii dissemination: a parasite’s journey through the infected host. Parasite Immunol. 37, 141–149 (2015).

    Article  CAS  PubMed  Google Scholar 

  44. Buguliskis, J. S., Brossier, F., Shuman, J. & Sibley, L. D. Rhomboid 4 (ROM4) affects the processing of surface adhesins and facilitates host cell invasion by Toxoplasma gondii. PLoS Pathog. 6, e1000858 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Kenthirapalan, S., Waters, A. P., Matuschewski, K. & Kooij, T. W. A. Copper-transporting ATPase is important for malaria parasite fertility. Mol. Microbiol. 91, 315–325 (2014).

  46. Xue, J. et al. Thioredoxin reductase from Toxoplasma gondii: an essential virulence effector with antioxidant function. FASEB J. 31, 4447–4457 (2017).

    Article  CAS  PubMed  Google Scholar 

  47. Rahman, K. et al. The E3 ubiquitin ligase adaptor protein Skp1 is glycosylated by an evolutionarily conserved pathway that regulates protist growth and development. J. Biol. Chem. 291, 4268–4280 (2016).

  48. Mas-Bargues, C. et al. Relevance of oxygen concentration in stem cell culture for regenerative medicine. Int. J. Mol. Sci. 20, 1195 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Vonlaufen, N., Naguleswaran, A., Coppens, I. & Sullivan, W. J. Jr. MYST family lysine acetyltransferase facilitates ataxia telangiectasia mutated (ATM) kinase-mediated DNA damage response in Toxoplasma gondii. J. Biol. Chem. 285, 11154–11161 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zhang, H. et al. Toxoplasma gondii UBL-UBA shuttle proteins contribute to the degradation of ubiquitinylated proteins and are important for synchronous cell division and virulence. FASEB J. 34, 13711–13725 (2020).

    Article  CAS  PubMed  Google Scholar 

  51. Moreira-Souza, A. C. A. et al. The P2X7 receptor mediates Toxoplasma gondii control in macrophages through canonical NLRP3 inflammasome activation and reactive oxygen species production. Front. Immunol. 8, 1257 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Tomita, T. et al. Toxoplasma gondii Matrix Antigen 1 is a secreted immunomodulatory effector. mBio 12, e00603-21 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Gold, D. A. et al. The Toxoplasma dense granule proteins GRA17 and GRA23 mediate the movement of small molecules between the host and the parasitophorous vacuole. Cell Host Microbe 17, 642–652 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Wang, J.-L. et al. Immunization with Toxoplasma gondii GRA17 deletion mutant induces partial protection and survival in challenged mice. Front. Immunol. 8, 730 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Dunn, J. D., Ravindran, S., Kim, S.-K. & Boothroyd, J. C. The Toxoplasma gondii dense granule protein GRA7 is phosphorylated upon invasion and forms an unexpected association with the rhoptry proteins ROP2 and ROP4. Infect. Immun. 76, 5853–5861 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Panas, M. W. & Boothroyd, J. C. Toxoplasma uses GRA16 to upregulate host c-Myc. mSphere https://doi.org/10.1128/msphere.00402-20 (2020).

  57. Sibley, L. D., Niesman, I. R., Parmley, S. F. & Cesbron-Delauw, M. F. Regulated secretion of multi-lamellar vesicles leads to formation of a tubulo-vesicular network in host-cell vacuoles occupied by Toxoplasma gondii. J. Cell Sci. 108, 1669–1677 (1995).

    Article  CAS  PubMed  Google Scholar 

  58. Sinai, A. P., Webster, P. & Joiner, K. A. Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction. J. Cell Sci. 110, 2117–2128 (1997).

    Article  CAS  PubMed  Google Scholar 

  59. Pernas, L. et al. Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response. PLoS Biol. 12, e1001845 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Deffieu, M. S., Alayi, T. D., Slomianny, C. & Tomavo, S. The Toxoplasma gondii dense granule protein TgGRA3 interacts with host Golgi and dysregulates anterograde transport. Biol. Open 8, bio039818 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Coppens, I. & Romano, J. D. Hostile intruder: Toxoplasma holds host organelles captive. PLoS Pathog. 14, e1006893 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Hariri, M. et al. Biogenesis of multilamellar bodies via autophagy. Mol. Biol. Cell 11, 255–268 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Coppens, I., Sinai, A. P. & Joiner, K. A. Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition. J. Cell Biol. 149, 167–180 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Romano, J. D. et al. Toxoplasma scavenges mammalian host organelles through usurpation of host ESCRT-III and Vps4. J. Cell Sci. 136, jcs260159 (2023).

  65. Pernas, L., Bean, C., Boothroyd, J. C. & Scorrano, L. Mitochondria restrict growth of the intracellular parasite Toxoplasma gondii by limiting its uptake of fatty acids. Cell Metab. 27, 886–897.e4 (2018).

    Article  CAS  PubMed  Google Scholar 

  66. Boothroyd, J. C. & Dubremetz, J.-F. Kiss and spit: the dual roles of Toxoplasma rhoptries. Nat. Rev. Microbiol. 6, 79–88 (2008).

    Article  CAS  PubMed  Google Scholar 

  67. Suarez, C. et al. A lipid-binding protein mediates rhoptry discharge and invasion in Plasmodium falciparum and Toxoplasma gondii parasites. Nat. Commun. 10, 4041 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  68. Liffner, B. et al. PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes. Nat. Commun. 11, 1411 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Liffner, B. et al. Cell biological analysis reveals an essential role for Pfcerli2 in erythrocyte invasion by malaria parasites. Commun. Biol. 5, 121 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Koshy, A. A. et al. Toxoplasma secreting Cre recombinase for analysis of host–parasite interactions. Nat. Methods 7, 307–309 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    Article  CAS  PubMed  Google Scholar 

  72. Park, J. & Hunter, C. A. The role of macrophages in protective and pathological responses to Toxoplasma gondii. Parasite Immunol. 42, e12712 (2020).

    Article  CAS  PubMed  Google Scholar 

  73. Delgado Betancourt, E. et al. From entry to early dissemination—Toxoplasma gondii’s initial encounter with its host. Front. Cell. Infect. Microbiol. 9, 46 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  74. Lodoen, M. B., Gerke, C. & Boothroyd, J. C. A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection. Cell. Microbiol. 12, 55–66 (2010).

    Article  CAS  PubMed  Google Scholar 

  75. Mageswaran, S. K. et al. In situ ultrastructures of two evolutionarily distant apicomplexan rhoptry secretion systems. Nat. Commun. 12, 4983 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Chen, X.-M. et al. Apical organelle discharge by Cryptosporidium parvum is temperature, cytoskeleton and intracellular calcium dependent and required for host cell invasion. Infect. Immun. 72, 6806–6816 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Koshy, A. A. et al. Toxoplasma co-opts host cells it does not invade. PLoS Pathog. 8, e1002825 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Mazumdar, J. & Striepen, B. Make it or take it: fatty acid metabolism of apicomplexan parasites. Eukaryot. Cell 6, 1727–1735 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Hartmann, A., Hellmund, M., Lucius, R., Voelker, D. R. & Gupta, N. Phosphatidylethanolamine synthesis in the parasite mitochondrion is required for efficient growth but dispensable for survival of Toxoplasma gondii. J. Biol. Chem. 289, 6809–6824 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Müller, S. & Kappes, B. Vitamin and cofactor biosynthesis pathways in Plasmodium and other apicomplexan parasites. Trends Parasitol. 23, 112–121 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  81. Yang, J., He, Z., Chen, C., Zhao, J. & Fang, R. Starch Branching Enzyme 1 is important for amylopectin synthesis and cyst reactivation in Toxoplasma gondii. Microbiol. Spectr. 10, e0189121 (2022).

    Article  PubMed  Google Scholar 

  82. Cantor, J. R. et al. Physiologic medium rewires cellular metabolism and reveals uric acid as an endogenous inhibitor of UMP synthase. Cell 169, 258–272.e17 (2017).

  83. Kümpornsin, K. et al. Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I. Malar. J. 13, 150 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  84. Dittrich, S. et al. An atypical orthologue of 6-pyruvoyltetrahydropterin synthase can provide the missing link in the folate biosynthesis pathway of malaria parasites. Mol. Microbiol. 67, 609–618 (2008).

    Article  CAS  PubMed  Google Scholar 

  85. Nzila, A., Ward, S. A., Marsh, K., Sims, P. F. G. & Hyde, J. E. Comparative folate metabolism in humans and malaria parasites (part I): pointers for malaria treatment from cancer chemotherapy. Trends Parasitol. 21, 292–298 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Nzila, A., Ward, S. A., Marsh, K., Sims, P. F. G. & Hyde, J. E. Comparative folate metabolism in humans and malaria parasites (part II): activities as yet untargeted or specific to Plasmodium. Trends Parasitol. 21, 334–339 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Krishnan, A., Kloehn, J., Lunghi, M. & Soldati-Favre, D. Vitamin and cofactor acquisition in apicomplexans: synthesis versus salvage. J. Biol. Chem. 295, 701–714 (2020).

    Article  PubMed  Google Scholar 

  88. Cosentino, F. & Katusić, Z. S. Tetrahydrobiopterin and dysfunction of endothelial nitric oxide synthase in coronary arteries. Circulation 91, 139–144 (1995).

    Article  CAS  PubMed  Google Scholar 

  89. Tegeder, I. et al. GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence. Nat. Med. 12, 1269–1277 (2006).

    Article  CAS  PubMed  Google Scholar 

  90. Dai, Y., Cui, J., Gan, P. & Li, W. Downregulation of tetrahydrobiopterin inhibits tumor angiogenesis in BALB/c-nu mice with hepatocellular carcinoma. Oncol. Rep. 36, 669–675 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Rembold, H. Hemmung des Crithidia- Wachstums durch 4-Amino-pyrimidine. Biol. Chem. 339, 258–259 (1964).

  92. Xie, L., Smith, J. A. & Gross, S. S. GTP cyclohydrolase I inhibition by the prototypic inhibitor 2, 4-diamino-6-hydroxypyrimidine. Mechanisms and unanticipated role of GTP cyclohydrolase I feedback regulatory protein. J. Biol. Chem. 273, 21091–21098 (1998).

    Article  CAS  PubMed  Google Scholar 

  93. Fry, M. & Pudney, M. Site of action of the antimalarial hydroxynaphthoquinone, 2-[trans-4-(4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80). Biochem. Pharmacol. 43, 1545–1553 (1992).

    Article  CAS  PubMed  Google Scholar 

  94. Abel, S. et al. Sequence tag-based analysis of microbial population dynamics. Nat. Methods 12, 223–226 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Hullahalli, K. & Waldor, M. K. Pathogen clonal expansion underlies multiorgan dissemination and organ-specific outcomes during murine systemic infection. Elife 10, e70910 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Liu, X. et al. Exploration of bacterial bottlenecks and Streptococcus pneumoniae pathogenesis by CRISPRi-seq. Cell Host Microbe 29, 107–120.e6 (2021).

    Article  CAS  PubMed  Google Scholar 

  97. Carrasquilla, M. et al. Barcoding genetically distinct Plasmodium falciparum strains for comparative assessment of fitness and antimalarial drug resistance. mBio 13, e0093722 (2022).

    Article  PubMed  Google Scholar 

  98. Ogata, M. et al. Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol. Cell. Biol. 26, 9220–9231 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Wang, T. et al. Toxoplasma gondii induce apoptosis of neural stem cells via endoplasmic reticulum stress pathway. Parasitology 141, 988–995 (2014).

    Article  CAS  PubMed  Google Scholar 

  100. Aquilini, E. et al. An Alveolata secretory machinery adapted to parasite host cell invasion. Nat. Microbiol. 6, 425–434 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Coppens, I. & Joiner, K. A. Host but not parasite cholesterol controls Toxoplasma cell entry by modulating organelle discharge. Mol. Biol. Cell 14, 3804–3820 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Sugimoto, M. et al. MMMDB: Mouse Multiple Tissue Metabolome Database. Nucleic Acids Res. 40, D809–D814 (2012).

    Article  CAS  PubMed  Google Scholar 

  103. Zhou, C.-X. et al. Metabolomic profiling of mice serum during toxoplasmosis progression using liquid chromatography-mass spectrometry. Sci. Rep. 6, 19557 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Osei, M. et al. Amplification of GTP-cyclohydrolase 1 gene in Plasmodium falciparum isolates with the quadruple mutant of dihydrofolate reductase and dihydropteroate synthase genes in Ghana. PLoS ONE 13, e0204871 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  105. Baker, N. et al. Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival. Nat. Commun. 12, 1244 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Shames, S. R. et al. Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries. Proc. Natl Acad. Sci. USA 114, E10446–E10454 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Molofsky, A. B. et al. Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection. J. Exp. Med. 203, 1093–1104 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Mukhopadhyay, D., Sangaré, L. O., Braun, L., Hakimi, M.-A. & Saeij, J. P. Toxoplasma GRA15 limits parasite growth in IFNγ-activated fibroblasts through TRAF ubiquitin ligases. EMBO J. 39, e103758 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Modrzynska, K. et al. A knockout screen of ApiAP2 genes reveals networks of interacting transcriptional regulators controlling the Plasmodium life cycle. Cell Host Microbe 21, 11–22 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Sidik, S. M., Huet, D. & Lourido, S. CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii. Nat. Protoc. 13, 307–323 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Markus, B. M., Bell, G. W., Lorenzi, H. A. & Lourido, S. Optimizing systems for Cas9 expression in Toxoplasma gondii. mSphere 4, e00386-19 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Djurković-Djaković, O. et al. Kinetics of parasite burdens in blood and tissues during murine toxoplasmosis. Exp. Parasitol. 131, 372–376 (2012).

    Article  PubMed  Google Scholar 

  114. Hegde, M., Strand, C., Hanna, R. E. & Doench, J. G. Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens. PLoS ONE 13, e0197547 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  115. Dillon, J. V. et al. TensorFlow Distributions. Preprint at https://arxiv.org/abs/1711.10604 (2017).

  116. Prokopenko, D. et al. Utilizing the Jaccard index to reveal population stratification in sequencing data: a simulation study and an application to the 1000 Genomes Project. Bioinformatics 32, 1366–1372 (2016).

    Article  CAS  PubMed  Google Scholar 

  117. Cavalli-Sforza, L. L. & Edwards, A. W. Phylogenetic analysis. Models and estimation procedures. Am. J. Hum. Genet. 19, 233–257 (1967).

    CAS  PubMed  PubMed Central  Google Scholar 

  118. Paradis, E., Claude, J. & Strimmer, K. APE: Analyses of Phylogenetics and Evolution in R language. Bioinformatics 20, 289–290 (2004).

    Article  CAS  PubMed  Google Scholar 

  119. Schliep, K. P. phangorn: phylogenetic analysis in R. Bioinformatics 27, 592–593 (2011).

    Article  CAS  PubMed  Google Scholar 

  120. Hullahalli, K. Stampr_rtisan: code for reproducing data from STAMPR experiments. GitHub https://github.com/hullahalli/stampr_rtisan (2024).

  121. Federico, A. & Monti, S. hypeR: an R package for geneset enrichment workflows. Bioinformatics 36, 1307–1308 (2020).

    Article  CAS  PubMed  Google Scholar 

  122. Trager, W. & Jensen, J. B. Human malaria parasites in continuous culture. Science 193, 673–675 (1976).

    Article  CAS  PubMed  Google Scholar 

  123. Lambros, C. & Vanderberg, J. P. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J. Parasitol. 65, 418–420 (1979).

    Article  CAS  PubMed  Google Scholar 

  124. Smith, T. A., Lopez-Perez, G. S., Herneisen, A. L., Shortt, E. & Lourido, S. Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress. Nat. Microbiol. 7, 868–881 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Markus, B. M., Waldman, B. S., Lorenzi, H. A. & Lourido, S. High-resolution mapping of transcription initiation in the asexual stages of Toxoplasma gondii. Front. Cell. Infect. Microbiol. 10, 617998 (2020).

    Article  PubMed  Google Scholar 

  126. Benevides, L. et al. Toxoplasma gondii soluble tachyzoite antigen triggers protective mechanisms against fatal intestinal pathology in oral infection of C57BL/6 mice. PLoS ONE 8, e75138 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Gazzinelli, R. T., Hakim, F. T., Hieny, S., Shearer, G. M. & Sher, A. Synergistic role of CD4+ and CD8+ T lymphocytes in IFN-gamma production and protective immunity induced by an attenuated Toxoplasma gondii vaccine. J. Immunol. 146, 286–292 (1991).

    Article  CAS  PubMed  Google Scholar 

  128. Grimwood, J., Mineo, J. R. & Kasper, L. H. Attachment of Toxoplasma gondii to host cells is host cell cycle dependent. Infect. Immun. 64, 4099–4104 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Kimura, S., Noda, T. & Yoshimori, T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452–460 (2007).

    Article  CAS  PubMed  Google Scholar 

  130. Johnson, J. D. et al. Assessment and continued validation of the malaria SYBR green I-based fluorescence assay for use in malaria drug screening. Antimicrob. Agents Chemother. 51, 1926–1933 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Smilkstein, M., Sriwilaijaroen, N., Kelly, J. X., Wilairat, P. & Riscoe, M. Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrob. Agents Chemother. 48, 1803–1806 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Giuliano, C. & Lourido, S. CRISPR-based functional profiling of the Toxoplasma gondii genome during acute murine infection. Zenodo https://doi.org/10.5281/zenodo.11218353 (2024).

  133. Giuliano, C. & Lourido, S. Crispr-based functional profiling of the Toxoplasma gondii genome during acute murine infection. Zenodo https://doi.org/10.5281/zenodo.11247744 (2024).

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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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Authors and Affiliations

  1. Whitehead Institute, Cambridge, MA, USA

    Christopher J. Giuliano, Kenneth J. Wei, Faye M. Harling, Benjamin S. Waldman, Elizabeth A. Boydston, Tammy C. T. Lan, Raina W. Thomas, Alice L. Herneisen & Sebastian Lourido

  2. Biology Department, MIT, Cambridge, MA, USA

    Christopher J. Giuliano, Kenneth J. Wei, Benjamin S. Waldman, Raina W. Thomas, Alice L. Herneisen, Allen G. Sanderlin & Sebastian Lourido

  3. Division of Infectious Diseases, Boston Children’s Hospital, Boston, MA, USA

    Madeline A. Farringer & Jeffrey D. Dvorin

  4. Biological Sciences in Public Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA

    Madeline A. Farringer

  5. Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA

    Isabelle Coppens

  6. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Jeffrey D. Dvorin

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Contributions

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.

Corresponding author

Correspondence to Sebastian Lourido.

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Competing interests

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).

Source data

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).

Source data

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.

Source data

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).

Source data

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.

Source data

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.

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Supplementary information

Reporting Summary

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 9, 2323–2343 (2024). https://doi.org/10.1038/s41564-024-01754-2

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