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Toxoplasma gondii: Bradyzoite Differentiation In Vitro and In Vivo

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Toxoplasma gondii

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2071))

Abstract

Toxoplasma gondii, a member of the Apicomplexa, is known for its ability to infect an impressive range of host species. It is a common human infection that causes significant morbidity in congenitally infected children and immunocompromised patients. This parasite can be transmitted by bradyzoites, a slowly replicating life stage found within intracellular tissue cysts, and oocysts, the sexual life cycle stage that develops in domestic cats and other Felidae. T. gondii bradyzoites retain the capacity to revert back to the quickly replicating tachyzoite life stage, and when the host is immune compromised unrestricted replication can lead to significant tissue destruction. Bradyzoites are refractory to currently available Toxoplasma treatments. Improving our understanding of bradyzoite biology is critical for the development of therapeutic strategies to eliminate latent infection. This chapter describes a commonly used protocol for the differentiation of T. gondii tachyzoites into bradyzoites using human foreskin fibroblast cultures and a CO2-limited alkaline cell media, which results in a high proportion of differentiated bradyzoites for further study. Also described are methods for purifying tissue cysts from chronically infected mouse brain using isopycnic centrifugation and a recently developed approach for measuring bradyzoite viability.

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References

  1. Frenkel JK, Smith DD (2003) Determination of the genera of cyst-forming coccidia. Parasitol Res 91:384–389

    Article  CAS  Google Scholar 

  2. Dubey JP, Lindsay DS, Speer CA (1998) Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clin Microbiol Rev 11:267–299

    Article  CAS  Google Scholar 

  3. Montoya JG, Liesenfeld O (2004) Toxoplasmosis. Lancet 363:1965–1976

    Article  CAS  Google Scholar 

  4. Di Cristina M, Marocco D, Galizi R, Proietti C, Spaccapelo R, Crisanti A (2008) Temporal and spatial distribution of Toxoplasma gondii differentiation into bradyzoites and tissue cyst formation in vivo. Infect Immun 76:3491–3501

    Article  Google Scholar 

  5. Rougier S, Montoya JG, Peyron F (2017) Lifelong persistence of Toxoplasma cysts: a questionable dogma? Trends Parasitol 33:93–101

    Article  Google Scholar 

  6. Sahm M, Fischer HG, Gross U, Reiter-Owona I, Seitz HM (1997) Cyst formation by Toxoplasma gondii in vivo and in brain-cell culture: a comparative morphology and immunocytochemistry study. Parasitol Res 83:659–665

    Article  CAS  Google Scholar 

  7. Watts E, Zhao Y, Dhara A, Eller B, Patwardhan A, Sinai AP (2015) Novel approaches reveal that Toxoplasma gondii bradyzoites within tissue cysts are dynamic and replicating entities in vivo. MBio 6:e01155–e01115

    Article  CAS  Google Scholar 

  8. Dou Z, McGovern OL, Di Cristina M, Carruthers VB (2014) Toxoplasma gondii ingests and digests host cytosolic proteins. MBio 5:e01188–e01114

    Article  Google Scholar 

  9. Caffaro CE, Boothroyd JC (2011) Evidence for host cells as the major contributor of lipids in the intravacuolar network of Toxoplasma-infected cells. Eukaryot Cell 10:1095–1099

    Article  CAS  Google Scholar 

  10. Ferguson DJ, Hutchison WM (1987) An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice. Parasitol Res 73:483–491

    Article  CAS  Google Scholar 

  11. Ferguson DJ, Hutchison WM (1987) The host-parasite relationship of Toxoplasma gondii in the brains of chronically infected mice. Virchows Arch A Pathol Anat Histopathol 411:39–43

    Article  CAS  Google Scholar 

  12. Fortier B, Coignard-Chatain C, Soete M, Dubremetz JF (1996) Structure and biology of Toxoplasma gondii bradyzoites. Comptes rendus des seances de la Societe de biologie et de ses filiales 190:385–394

    CAS  PubMed  Google Scholar 

  13. Sibley LD, Niesman IR, Parmley SF, Cesbron-Delauw MF (1995) 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

    CAS  PubMed  Google Scholar 

  14. Tu V, Yakubu R, Weiss LM (2017) Observations on bradyzoite biology. Microbes Infect 20:466–476

    Article  Google Scholar 

  15. Bohne W, Gross U, Ferguson DJ, Heesemann J (1995) Cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants. Mol Microbiol 16:1221–1230

    Article  CAS  Google Scholar 

  16. Parmley SF, Weiss LM, Yang S (1995) Cloning of a bradyzoite-specific gene of Toxoplasma gondii encoding a cytoplasmic antigen. Mol Biochem Parasitol 73:253–257

    Article  CAS  Google Scholar 

  17. Buchholz KR, Bowyer PW, Boothroyd JC (2013) Bradyzoite pseudokinase 1 is crucial for efficient oral infectivity of the Toxoplasma gondii tissue cyst. Eukaryot Cell 12:399–410

    Article  CAS  Google Scholar 

  18. Tomita T, Bzik DJ, Ma YF, Fox BA, Markillie LM, Taylor RC, Kim K, Weiss LM (2013) The Toxoplasma gondii cyst wall protein CST1 is critical for cyst wall integrity and promotes bradyzoite persistence, e1003823. PLoS Pathog 9

    Google Scholar 

  19. Sethi KK, Rahman A, Pelster B, Brandis H (1977) Search for the presence of lectin-binding sites on Toxoplasma gondii. J Parasitol 63:1076–1080

    Article  CAS  Google Scholar 

  20. Caffaro CE, Koshy AA, Liu L, Zeiner GM, Hirschberg CB, Boothroyd JC (2013) A nucleotide sugar transporter involved in glycosylation of the Toxoplasma tissue cyst wall is required for efficient persistence of bradyzoites. PLoS Pathog 9:e1003331

    Article  CAS  Google Scholar 

  21. Tomita T, Sugi T, Yakubu R, Tu V, Ma Y, Weiss LM (2017) Making home sweet and sturdy: Toxoplasma gondii ppGalNAc-Ts glycosylate in hierarchical order and confer Cyst Wall rigidity. MBio 8:e02048–e02016

    Article  CAS  Google Scholar 

  22. Yang S, Parmley SF (1997) Toxoplasma gondii expresses two distinct lactate dehydrogenase homologous genes during its life cycle in intermediate hosts. Gene 184:1–12. 23

    Article  CAS  Google Scholar 

  23. Unno A, Suzuki K, Batanova T, Cha SY, Jang HK, Kitoh K, Takashima Y (2009) Visualization of Toxoplasma gondii stage conversion by expression of stage-specific dual fluorescent proteins. Parasitology 136:579–588

    Article  CAS  Google Scholar 

  24. Kim SK, Boothroyd JC (2005) Stage-specific expression of surface antigens by Toxoplasma gondii as a mechanism to facilitate parasite persistence. J Immunol 174:8038–8048

    Article  CAS  Google Scholar 

  25. Milligan-Myhre K, Wilson SK, Knoll LJ (2016) Developmental change in translation initiation alters the localization of a common microbial protein necessary for Toxoplasma chronic infection. Mol Microbiol 102:1086–1098

    Article  CAS  Google Scholar 

  26. Dubey JP, Speer CA, Shen SK, Kwok OC, Blixt JA (1997) Oocyst-induced murine toxoplasmosis: life cycle, pathogenicity, and stage conversion in mice fed Toxoplasma gondii oocysts. J Parasitol 83:870–882

    Article  CAS  Google Scholar 

  27. Weiss LM, Kim K (2000) The development and biology of bradyzoites of Toxoplasma gondii. Front Biosci 5:D391–D405

    Article  CAS  Google Scholar 

  28. Lyons RE, McLeod R, Roberts CW (2002) Toxoplasma gondii tachyzoite-bradyzoite interconversion. Trends Parasitol 18:198–201

    Article  CAS  Google Scholar 

  29. Luder CGK, Rahman T (2017) Impact of the host on Toxoplasma stage differentiation. Microb Cell 4:203–211

    Article  Google Scholar 

  30. Sullivan WJ Jr, Jeffers V (2012) Mechanisms of Toxoplasma gondii persistence and latency. FEMS Microbiol Rev 36:717–733

    Article  CAS  Google Scholar 

  31. Luder CG, Giraldo-Velasquez M, Sendtner M, Gross U (1999) Toxoplasma gondii in primary rat CNS cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation. Exp Parasitol 93:23–32

    Article  CAS  Google Scholar 

  32. Swierzy IJ, Luder CG (2015) Withdrawal of skeletal muscle cells from cell cycle progression triggers differentiation of Toxoplasma gondii towards the bradyzoite stage. Cell Microbiol 17:2–17

    Article  CAS  Google Scholar 

  33. Cabral CM, Tuladhar S, Dietrich HK, Nguyen E, MacDonald WR, Trivedi T, Devineni A, Koshy AA (2016) Neurons are the primary target cell for the brain-tropic intracellular parasite Toxoplasma gondii. PLoS Pathog 12:e1005447

    Article  Google Scholar 

  34. Melzer TC, Cranston HJ, Weiss LM, Halonen SK (2010) Host cell preference of Toxoplasma gondii cysts in murine brain: a confocal study. J Neuroparasitology 1:pii: N100505

    Article  Google Scholar 

  35. Hong DP, Radke JB, White MW (2017) Opposing transcriptional mechanisms regulate Toxoplasma development. mSphere 2:e00347-16

    Article  Google Scholar 

  36. Holmes MJ, Augusto LDS, Zhang M, Wek RC, Sullivan WJ Jr (2017) Translational control in the latency of apicomplexan parasites. Trends Parasitol 33:947–960

    Article  CAS  Google Scholar 

  37. Watts EA, Dhara A, Sinai AP (2017) Purification Toxoplasma gondii tissue cysts using percoll gradients. Curr Protoc Microbiol 45:20c.2.1–20c.2.19

    Article  Google Scholar 

  38. Tobin C, Pollard A, Knoll L (2010) Toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions. J Vis Exp 12(42):2091

    Google Scholar 

  39. Cornelissen AW, Overdulve JP, Hoenderboom JM (1981) Separation of Isospora (Toxoplasma) gondii cysts and cystozoites from mouse brain tissue by continuous density-gradient centrifugation. Parasitology 83:103–108

    Article  CAS  Google Scholar 

  40. Bohne W, Roos DS (1997) Stage-specific expression of a selectable marker in Toxoplasma gondii permits selective inhibition of either tachyzoites or bradyzoites. Mol Biochem Parasitol 88:115–126

    Article  CAS  Google Scholar 

  41. Szabo EK, Finney CA (2017) Toxoplasma gondii: one organism multiple models. Trends Parasitol 33:113–127

    Article  Google Scholar 

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Acknowledgments

This work was supported by 1F31AI136401 (J.M.), R01AI134753 (L.M.W.), and R21AI123495 (L.M.W.).

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Authors and Affiliations

  1. Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA

    Joshua Mayoral & Louis M. Weiss

  2. Department of Chemistry, Biology, and Biotechnology, University of Perugia, Perugia, Italy

    Manlio Di Cristina

  3. Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA

    Vern B. Carruthers

  4. Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

    Louis M. Weiss

Authors
  1. Joshua Mayoral
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  2. Manlio Di Cristina
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  3. Vern B. Carruthers
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  4. Louis M. Weiss
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Corresponding author

Correspondence to Louis M. Weiss .

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Editors and Affiliations

  1. Division of Infectious Disease and Immune Defense, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia

    Christopher J. Tonkin

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Mayoral, J., Di Cristina, M., Carruthers, V.B., Weiss, L.M. (2020). Toxoplasma gondii: Bradyzoite Differentiation In Vitro and In Vivo. In: Tonkin, C. (eds) Toxoplasma gondii. Methods in Molecular Biology, vol 2071. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9857-9_15

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  • DOI: https://doi.org/10.1007/978-1-4939-9857-9_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9856-2

  • Online ISBN: 978-1-4939-9857-9

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