Methods for the Measurement of Early Events in Toxoplasma gondii Immunity in Mouse Cells

  • Catalina Alvarez
  • Ana Claudia Campos
  • Jonathan C. HowardEmail author
  • Joana Loureiro
  • Urs Benedikt Müller
  • Ana Lina Rodrigues
Part of the Methods in Molecular Biology book series (MIMB, volume 2071)


Critical steps in resistance of mice against Toxoplasma gondii occur in the first 2 or 3 h after the pathogen has entered a cell that has been exposed to interferon γ (IFNγ). The newly formed parasitophorous vacuole is attacked by the IFNγ-inducible IRG proteins and disrupted, resulting in death of the parasite and necrotic death of the cell. Here we describe some techniques that we have used to describe and quantify these events in different combinations of the host and the parasite.

Key words

Toxoplasma gondii Parasitophorous vacuole Mouse Innate immunity IRG proteins Interferon-γ Diaphragm-derived cells Reactive necrosis Flow cytometry Immunofluorescence 



Carbon dioxide


Dulbecco’s modified Eagle’s medium


Ethylenediaminetetraacetic acid


Fluorescence-activated cell sorting


Fetal bovine serum


Hydrogen peroxide


4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid)


Human foreskin fibroblasts = Hs27 cells


Heat-inactivated fetal bovine serum


Interferon (IFN)-gamma


Multichannel microscope slide


Multiplicity of infection


Nonessential amino acids

P/W buffer

Permeabilization/wash buffer (for intracellular staining for FACS)


Phosphate-buffered saline




Relative centrifugal force


Room temperature


Toxoplasma major surface antigen 1 or P30



T. gondii, Tg

Toxoplasma gondii


Working dilution



The authors record their thanks to previous members of the laboratory who contributed to the development of the study of early postinfection events in T. gondii immunity and pioneered the application of several of these techniques. The present work would also not have been possible without the contributions of the service facilities of the IGC, in particular the Animal Facility, supported by the research infrastructure Congento, project LISBOA-01-0145-FEDER-022170, the Transgenics Facility, and the Antibody facility, both supported by Fundação Calouste Gulenkian, the Advanced Imaging Unit, supported by the project PPBI-POCI-01-0145-FEDER-022122 and the Flow Cytometry Unit, supported by the project LISBOA-01-0145-FEDER-007654.

This work was supported by central funds of the Instituto Gulbenkian de Ciência, by the Sonderforschungsbereiche 670 and 680 and Schwerpunkt 1399 of the Deutsche Forschungsgemeinde. Joana Loureiro received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement number 708694 entitled “Toxoplasma Sensing.”

Author contributions: Subheading 1, Jonathan Howard; Subheading 2, Catalina Alvarez and Ben Mueller; Subheadings 3 and 4, Ana Lina Rodrigues and Joana Loureiro; Subheading 5, Joana Loureiro; Subheading 6, Claudia Campos. All authors contributed to the preparation and editing of the entire manuscript.


  1. 1.
    Müller UB, Howard JC (2016) The impact of Toxoplasma gondii on the mammalian genome. Curr Opin Microbiol 32:19–25PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Carruthers V, Boothroyd JC (2007) Pulling together: an integrated model of Toxoplasma cell invasion. Curr Opin Microbiol 10:83–89PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Besteiro S, Dubremetz JF, Lebrun M (2011) The moving junction of apicomplexan parasites: a key structure for invasion. Cell Microbiol 13:797–805CrossRefGoogle Scholar
  4. 4.
    Dubremetz JF (2007) Rhoptries are major players in Toxoplasma gondii invasion and host cell interaction. Cell Microbiol 9:841–848CrossRefGoogle Scholar
  5. 5.
    Boothroyd JC, Dubremetz J-F (2008) Kiss and spit: the dual roles of Toxoplasma rhoptries. Nat Rev Microbiol 6:79CrossRefGoogle Scholar
  6. 6.
    Martens S et al (2005) Disruption of Toxoplasma gondii parasitophorous vacuoles by the mouse p47-resistance GTPases. PLoS Pathog 1:0187–0201CrossRefGoogle Scholar
  7. 7.
    Zhao YO, Khaminets A, Hunn JP, Howard JC (2009) Disruption of the Toxoplasma gondii parasitophorous vacuole by IFNg-inducible immunity-related GTPases (IRG proteins) triggers necrotic cell death. PLoS Pathog 5:e1000288PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Fentress SJ et al (2011) Phosphorylation of immunity-related GTPases by a Toxoplasma gondii secreted kinase promotes macrophage survival and virulence. Cell Host Microbe 8:484–495CrossRefGoogle Scholar
  9. 9.
    Khaminets A et al (2010) Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole. Cell Microbiol 12:939–961PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Weiss LM, Kim K (2013) Toxoplasma gondii: the model apicomplexan – perspectives and methods: second edition. Elsevier, AmsterdamGoogle Scholar
  11. 11.
    Allen I (2013) Mouse models of innate immunity: methods and protocols, vol 1031. Humana Press, New York, NYCrossRefGoogle Scholar
  12. 12.
    Antony VB, Owen CL, Hadley KJ (1989) Pleural mesothelial cells stimulated by asbestos release chemotactic activity for neutrophils in vitro. Am Rev Respir Dis 139:199–206PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Lilue J, Müller UB, Steinfeldt T, Howard JC (2013) Reciprocal virulence and resistance polymorphism in the relationship between Toxoplasma gondii and the house mouse. elife 2013:1–21Google Scholar
  14. 14.
    Incella (2016) ScreenFect®A transfection reagent protocol. Incella, Baden-Württemberg, pp 1–5Google Scholar
  15. 15.
    Southern PJ, Berg P (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet 1(4):327–341PubMedPubMedCentralGoogle Scholar
  16. 16.
    Chang PL et al (1986) Transformation of human cultured fibroblasts with plasmids carrying dominant selection markers and immortalizing potential. Exp Cell Res 167:407–416PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Li LM et al (2015) ScreenFect A: an efficient and low toxic liposome for gene delivery to mesenchymal stem cells. Int J Pharm 488:1–11PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Li L et al (2012) A biomimetic lipid library for gene delivery through thiol-yne click chemistry. Biomaterials 33:8160–8166PubMedCrossRefGoogle Scholar
  19. 19.
    Fisch DH et al (2018) An artificial intelligence workflow for defining host-pathogen interactions. bioRxiv 408450.
  20. 20.
    Schindelin J et al (2012) Fiji – an open source platform for biological image analysis. Nat Methods 9:676–682CrossRefGoogle Scholar
  21. 21.
    Papic N, Hunn JP, Pawlowski N, Zerrahn J, Howard JC (2008) Inactive and active states of the interferon-inducible resistance GTPase, Irga6, in vivo. J Biol Chem 283:32143–32151PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Lotze MT, Tracey KJ (2005) High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 5:331PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Zhao YO et al (2009) Toxoplasma gondii and the immunity-related GTPase (IRG) resistance system in mice – a review. Mem Inst Oswaldo Cruz 104:234–240PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Scaffidi P, Misteli T, Bianchi ME (2002) Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418:191–195PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Bonaldi T et al (2003) Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. EMBO 22:5551–5560CrossRefGoogle Scholar
  26. 26.
    Andersson U et al (2000) High mobility group 1 protein (Hmg-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med 192:565–570PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Tang D et al (2010) Endogenous HMGB1 regulates autophagy. J Cell Biol 190:881–892PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Vanden Berghe T et al (2010) Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features. Cell Death Differ 17:922–930CrossRefGoogle Scholar
  29. 29.
    Bertho ÁL, Santiago MA, Coutinho SG (2000) Flow cytometry in the study of cell death. Mem Inst Oswaldo Cruz 95:429–433PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Holmes KL et al (2014) International society for the advancement of cytometry cell sorter biosafety standards. Citometry A 85:434–453CrossRefGoogle Scholar
  31. 31.
    Könen-Waisman S, Howard JC (2007) Cell-autonomous immunity to Toxoplasma gondii in mouse and man. Microbes Infect 9:1652–1661PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Martens S et al (2004) Mechanisms regulating the positioning of mouse p47 resistance GTPases LRG-47 and IIGP1 on cellular membranes: retargeting to plasma membrane induced by phagocytosis. J Immunol 173:2594–2606PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    Zerrahn J, Schaible UE, Brinkmann V, Guhlich U, Kaufmann SHE (2002) The IFN-inducible Golgi- and endoplasmic reticulum-associated 47-kDa GTPase IIGP is transiently expressed during listeriosis. J Immunol 168:3428–3436PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Carlow DA et al (1998) Specific antiviral activity demonstrated by TGTP, a member of a new family of interferon-induced GTPases. J Immunol 161:2348–2355PubMedPubMedCentralGoogle Scholar
  35. 35.
    Maric-Biresev J et al (2016) Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection. BMC Biol 14:1–20CrossRefGoogle Scholar
  36. 36.
    Pawlowski N et al (2011) The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii. BMC Biol 9:7PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Steinfeldt T et al (2010) Phosphorylation of mouse immunity-related gtpase (IRG) resistance proteins is an evasion strategy for virulent Toxoplasma gondii. PLoS Biol 8:e1000576PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Butcher BA et al (2005) p47 GTPases regulate Toxoplasma gondii survival in sctivated macrophages. Infect Immun 73:3278–3286PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Springer HM, Schramm M, Taylor G a, Howard JC (2013) Irgm1 (LRG-47), a regulator of cell-autonomous immunity, does not localize to mycobacterial or listerial phagosomes in IFN-γ-induced mouse cells. J Immunol 191:1765–1774PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Fleckenstein MC et al (2012) A Toxoplasma gondii pseudokinase inhibits host irg resistance proteins. PLoS Biol 10:14CrossRefGoogle Scholar
  41. 41.
    Naviaux RK, Costanzi E, Haas M, Verma IM (1996) The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J Virol 70:5701–5705PubMedPubMedCentralGoogle Scholar
  42. 42.
    Christova Y, Adrain C, Bambrough P, Ibrahim A, Freeman M (2013) Mammalian iRhoms have distinct physiological functions including an essential role in TACE regulation. EMBO Rep 14:884–890PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Catalina Alvarez
    • 1
  • Ana Claudia Campos
    • 1
  • Jonathan C. Howard
    • 1
    • 2
    Email author
  • Joana Loureiro
    • 1
  • Urs Benedikt Müller
    • 2
  • Ana Lina Rodrigues
    • 1
  1. 1.Instituto Gulbenkian de CiênciaOeirasPortugal
  2. 2.Institute for GeneticsCologneGermany

Personalised recommendations