Skip to main content

Characterizing Cell Heterogeneity Using PCR Fingerprinting of Surface Multigene Families in Protozoan Parasites

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1745))

Abstract

Parasites counteract the action of the immune system and other environmental pressures by modulating and changing the composition of their cell surfaces. Surface multigene protein families are defined not only by highly variable regions in length and/or sequence exposed to the outer space but also by conserved sequences codifying for the signal peptide, hydrophobic C-terminal regions necessary for GPI modifications, as well as conserved UTR regions for mRNA regulation. The method here presented exploits these conserved signatures for characterizing variations in the mRNA expression of clonal cell populations of protozoan parasites using a combination of nested PCR amplification and capillary electrophoresis. With this workflow, in silico gels from isolated cell clones can be generated, thus providing an excellent tool for analyzing cellular heterogeneity in protozoan parasites.

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

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Seco-Hidalgo V, Osuna A, Pablos LM (2015) To bet or not to bet: deciphering cell to cell variation in protozoan infections. Trends Parasitol 31(8):350–356. https://doi.org/10.1016/j.pt.2015.05.004

    Article  PubMed  Google Scholar 

  2. Blythe JE, Surentheran T, Preiser PR (2004) STEVOR—a multifunctional protein? Mol Biochem Parasitol 134:11–15

    Article  CAS  PubMed  Google Scholar 

  3. Niang M, Yan Yam X, Preiser PR (2009) The Plasmodium falciparum STEVOR multigene family mediates antigenic variation of the infected erythrocyte. PLoS Pathog 5:e1000307

    Article  PubMed  PubMed Central  Google Scholar 

  4. Rovira-Graells N, Gupta AP, Planet E et al (2012) Transcriptional variation in the malaria parasite Plasmodium falciparum. Genome Res 22:925–938

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Seco-Hidalgo V, De Pablos LM, Osuna A (2015) Transcriptional and phenotypical heterogeneity of Trypanosoma cruzi cell populations. Open Biol 5:150190. https://doi.org/10.1098/rsob.150190

    Article  PubMed  PubMed Central  Google Scholar 

  6. Kyes SA, Kraemer SM, Smith JD (2007) Antigenic variation in Plasmodium falciparum: gene organization and regulation of the var multigene family. Eukaryot Cell 6:1511–1520

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ferreri LM, Brayton KA, Sondgeroth KS et al (2012) Expression and strain variation of the novel “small open reading frame” (smorf) multigene family in Babesia bovis. Int J Parasitol 42:131–138

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Schmuckli-Maurer J, Casanova C, Schmied S et al (2009) Expression analysis of the Theileria parva subtelomere-encoded variable secreted protein gene family. PLoS One 4:e4839

    Article  PubMed  PubMed Central  Google Scholar 

  9. Freitas LM, dos Santos SL, Rodrigues-Luiz GF et al (2011) Genomic analyses, gene expression and antigenic profile of the trans-sialidase superfamily of Trypanosoma cruzi reveal an undetected level of complexity. PLoS One 6:e25914

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Aguero F, Campo V, Cremona L et al (2002) Gene discovery in the freshwater fish parasite Trypanosoma carassii: identification of trans-Sialidase-like and mucin-like genes. Infect Immun 70:7140–7144

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ubeda JM, Raymond F, Mukherjee A et al (2014) Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania. PLoS Biol 12:e1001868

    Article  PubMed  PubMed Central  Google Scholar 

  12. Sterkers Y, Crobu L, Lachaud L et al (2014) Parasexuality and mosaic aneuploidy in Leishmania: alternative genetics. Trends Parasitol 30:429–435

    Article  PubMed  Google Scholar 

  13. Reis-Cunha JL, Rodrigues-Luiz GF, Valdivia HO et al (2015) Chromosomal copy number variation reveals differential levels of genomic plasticity in distinct Trypanosoma cruzi strains. BMC Genomics 16:499. https://doi.org/10.1186/s12864-015-1680-4

    Article  PubMed  PubMed Central  Google Scholar 

  14. Bartholomeu DC, Cerqueira GC, Leao ACA et al (2009) Genomic organization and expression profile of the mucin-associated surface protein (masp) family of the human pathogen Trypanosoma cruzi. Nucleic Acids Res 37:3407–3417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. dos Santos SL, Freitas LM, Lobo FP et al (2012) The MASP family of Trypanosoma cruzi: changes in gene expression and antigenic profile during the acute phase of experimental infection. PLoS Negl Trop Dis 6:e1779. https://doi.org/10.1371/journal.pntd.0001779

    Article  PubMed  PubMed Central  Google Scholar 

  16. Bailey TL, Boden M, Buske FA et al (2009) MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 37:W202–W208

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Schuler GD (1997) Sequence mapping by electronic PCR. Genome Res 7:541–550

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Schulz D, Mugnier MR, Boothroyd CE et al (2016) Detection of Trypanosoma brucei variant surface glycoprotein switching by magnetic activated cell sorting and flow cytometry. J Vis Exp 116. https://doi.org/10.3791/54715

  19. Boissière A, Arnathau C, Duperray C et al (2012) Isolation of Plasmodium falciparum by flow-cytometry: implications for single-trophozoite genotyping and parasite DNA purification for whole-genome high-throughput sequencing of archival samples. Malar J 11:163. https://doi.org/10.1186/1475-2875-11-163

    Article  PubMed  PubMed Central  Google Scholar 

  20. Nair S, Nkhoma SC, Serre D et al (2014) Single-cell genomics for dissection of complex malaria infections. Genome Res 24:1028–1038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Akagi T, Sasai K, Hanafusa H (2003) Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation. Proc Natl Acad Sci U S A 100:13567–13572

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Geislinger TM, Chan S, Moll K et al (2014) Label-free microfluidic enrichment of ring-stage Plasmodium falciparum-infected red blood cells using non-inertial hydrodynamic lift. Malar J 13:375. https://doi.org/10.1186/1475-2875-13-375

    Article  PubMed  PubMed Central  Google Scholar 

  23. Hochstetter A, Stellamanns E, Deshpande S et al (2015) Microfluidics-based single cell analysis reveals drug-dependent motility changes in trypanosomes. Lab Chip 15(8):1961–1968. https://doi.org/10.1039/c5lc00124b

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Luis Miguel de Pablos .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Seco-Hidalgo, V., Osuna, A., de Pablos, L.M. (2018). Characterizing Cell Heterogeneity Using PCR Fingerprinting of Surface Multigene Families in Protozoan Parasites. In: Barteneva, N., Vorobjev, I. (eds) Cellular Heterogeneity. Methods in Molecular Biology, vol 1745. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7680-5_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7680-5_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7679-9

  • Online ISBN: 978-1-4939-7680-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics