Skip to main content

In Situ Detection of Dormant Trypanosoma cruzi Amastigotes Using Bioluminescent-Fluorescent Reporters

  • Protocol
  • First Online:
T. cruzi Infection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1955))

Abstract

Chagas disease agent, Trypanosoma cruzi, is capable to persist after prolonged drug treatment using effective drugs. The reason of treatment failure is not known, but recent development of highly sensible bioluminescence imaging coupled to tissue clarification techniques has made possible the detection of individual amastigotes within chronically infected murine tissues and the study of their replicative status. In this chapter, we provide a step-by-step explanation for these protocols that allowed the visualization of nonproliferating amastigotes in tissues of chronically infected mice for the first time.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Rassi A Jr, Rassi A, Marcondes de Rezende J (2012) American trypanosomiasis (Chagas disease). Infect Dis Clin N Am 26:275–291

    Article  Google Scholar 

  2. Sanchez-Valdez FJ, Padilla A, Wang W, Orr D, Tarleton RL (2018) Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure. eLife 7:e34039

    Article  Google Scholar 

  3. Lewis MD, Fortes Francisco A, Taylor MC, Burrell-Saward H, McLatchie AP, Miles MA et al (2014) Bioluminescence imaging of chronic Trypanosoma cruzi infections reveals tissue-specific parasite dynamics and heart disease in the absence of locally persistent infection. Cell Microbiol 16:1285–1300

    Article  CAS  Google Scholar 

  4. Lewis MD, Francisco AF, Taylor MC, Kelly JM (2015) A new experimental model for assessing drug efficacy against Trypanosoma cruzi infection based on highly sensitive in vivo imaging. J Biomol Screen 20:36–43

    Article  Google Scholar 

  5. Costa FC, Francisco AF, Jayawardhana S, Calderano SG, Lewis MD, Olmo F et al (2018) Expanding the toolbox for Trypanosoma cruzi: a parasite line incorporating a bioluminescence-fluorescence dual reporter and streamlined CRISPR/Cas9 functionality for rapid in vivo localisation and phenotyping. PLoS Negl Trop Dis 12:e0006388

    Article  Google Scholar 

  6. Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, Ueda HR (2015) Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc 10:1709–1727

    Article  CAS  Google Scholar 

  7. Pan C, Cai R, Quacquarelli FP, Ghasemigharagoz A, Lourbopoulos A, Matryba P et al (2016) Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat Methods 13:859–867

    Article  CAS  Google Scholar 

  8. Canavaci AM, Bustamante JM, Padilla AM, Perez Brandan CM, Simpson LJ, Xu D et al (2010) In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds. PLoS Negl Trop Dis 4:e740

    Article  Google Scholar 

  9. Tyler KM, Engman DM (2000) Flagellar elongation induced by glucose limitation is preadaptive for Trypanosoma cruzi differentiation. Cell Motil Cytoskeleton 46:269–278

    Article  CAS  Google Scholar 

  10. Bourguignon SC, de Souza W, Souto-Padron T (1998) Localization of lectin-binding sites on the surface of Trypanosoma cruzi grown in chemically defined conditions. Histochem Cell Biol 110:527–534

    Article  CAS  Google Scholar 

  11. Vazquez MP, Levin MJ (1999) Functional analysis of the intergenic regions of TcP2beta gene loci allowed the construction of an improved Trypanosoma cruzi expression vector. Gene 239:217–225

    Article  CAS  Google Scholar 

  12. Yeo M, Lewis MD, Carrasco HJ, Acosta N, Llewellyn M, da Silva Valente SA et al (2007) Resolution of multiclonal infections of Trypanosoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium. Int J Parasitol 37:111–120

    Article  Google Scholar 

  13. Isola EL, Lammel EM, Gonzalez Cappa SM (1986) Trypanosoma cruzi: differentiation after interaction of epimastigotes and Triatoma infestans intestinal homogenate. Exp Parasitol 62:329–335

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. Rick Tarleton, Wei Wang, Dylan Orr, as well as Julie Nelson from the CTEGD Flow Cytometry Core and Muthugapatti Kandasamy from the Biomedical Microscopy Core. This work was supported by US National Institutes of Health grants AI108265 and AI124692 to Rick Tarleton.

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sánchez-Valdéz, F., Padilla, A. (2019). In Situ Detection of Dormant Trypanosoma cruzi Amastigotes Using Bioluminescent-Fluorescent Reporters. In: Gómez, K., Buscaglia, C. (eds) T. cruzi Infection. Methods in Molecular Biology, vol 1955. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9148-8_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9148-8_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9147-1

  • Online ISBN: 978-1-4939-9148-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics