Malaria pp 523-534 | Cite as

Screening and Evaluation of Inhibitors of Plasmodium falciparum Merozoite Egress and Invasion Using Cytometry

  • Anthony Bouillon
  • Olivier Gorgette
  • Odile Mercereau-Puijalon
  • Jean-Christophe BaraleEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 923)


Drug discovery programs heavily rely on assays adequately monitoring the activity of the drug on the ­parasite stage targeted. So far, assays used to screen molecules active against Plasmodium falciparum parasites have mostly been based on measuring growth inhibition of asexual blood stages. We have developed a robust protocol allowing for monitoring parasite egress at the late schizont stage and subsequent erythrocyte invasion. This cytometry-based methodology uses nucleic acid labelling by the dye YOYO-1 and synchronized in vitro culture of P.falciparum exposed to inhibitors during the late phase of the intraerythrocytic cycle and the reinvasion process. This cytometry-based method is quick, accurate and allows for distinguishing egress from reinvasion on thousands of events. The throughput is also increased, as the assay can be scaled up for medium throughput screening for compounds that inhibit either the egress of merozoites or their entry into host erythrocytes.

Key words

Plasmodium Cytometry Egress Invasion In vitro culture Synchronization YOYO-1 


  1. 1.
    Giemsa G (1904) Eine Vereinfachung und Vervollkommnung meiner Methylenblau–Eosin–Färbemethode zur Erzielung der Romanowsky–Nocht’schen Chromatinfärbung. Centralblatt für Bakteriol 32:307Google Scholar
  2. 2.
    Desjardins RE et al (1979) Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16:710–718PubMedCrossRefGoogle Scholar
  3. 3.
    Bennett TN et al (2004) Novel, rapid, and inexpensive cell-based quantification of antimalarial drug efficacy. Antimicrob Agents Chemother 48:1807–1810PubMedCrossRefGoogle Scholar
  4. 4.
    Smilkstein M et al (2004) Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrob Agents Chemother 48:1803–1806PubMedCrossRefGoogle Scholar
  5. 5.
    Franke-Fayard B et al (2004) A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle. Mol Biochem Parasitol 137:23–33PubMedCrossRefGoogle Scholar
  6. 6.
    Wilson DW et al (2010) Development of fluorescent Plasmodium falciparum for in vitro growth inhibition assays. Malar J 9:152PubMedCrossRefGoogle Scholar
  7. 7.
    Grimberg BT (2011) Methodology and application of flow cytometry for investigation of human malaria parasites. J Immunol Methods 367:1–16PubMedCrossRefGoogle Scholar
  8. 8.
    Deligeorgieva TG et al (2009) Intercalating cyanine dyes for nucleic acid detection. Recent Patents Mat Sci 2:1–26CrossRefGoogle Scholar
  9. 9.
    Barkan D et al (2000) Optimisation of flow cytometric measurement of parasitaemia in Plasmodium-infected mice. Int J Parasitol 30:649–653PubMedCrossRefGoogle Scholar
  10. 10.
    Xie L et al (2007) Development and validation of flow cytometric measurement for parasitaemia using autofluorescence and YOYO-1 in rodent malaria. Parasitology 134:1151–1162PubMedCrossRefGoogle Scholar
  11. 11.
    Li Q et al (2007) Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO-1. Cytometry A 71:297–307PubMedGoogle Scholar
  12. 12.
    Jiménez-Díaz MB et al (2005) Improvement of detection specificity of Plasmodium-infected murine erythrocytes by flow cytometry using autofluorescence and YOYO-1. Cytometry A 67:27–36PubMedGoogle Scholar
  13. 13.
    Campo JJ et al (2011) Feasibility of flow cytometry for measurements of Plasmodium falciparum parasite burden in studies in areas of malaria endemicity by use of bidimensional assessment of YOYO-1 and autofluorescence. J Clin Microbiol 49:968–974PubMedCrossRefGoogle Scholar
  14. 14.
    Saito-Ito A (2001) A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum. Parasitol Int 50:249–257PubMedCrossRefGoogle Scholar
  15. 15.
    Bastianelli G et al (2011) Computational reverse-engineering of a spider-venom derived peptide active against Plasmodium falciparum SUB1. PLoS One 6:e21812PubMedCrossRefGoogle Scholar
  16. 16.
    Dvorak J et al (1975) Invasion of erythrocytes by malaria merozoites. Science 187:748–750PubMedCrossRefGoogle Scholar
  17. 17.
    Abkarian M et al (2011) A novel mechanism for egress of malarial parasites from red blood cells. Blood 117:4118–4124PubMedCrossRefGoogle Scholar
  18. 18.
    Cowman AF, Crabb BS (2006) Invasion of red blood cells by malaria parasites. Cell 124:755–766PubMedCrossRefGoogle Scholar
  19. 19.
    Anders RF et al (2010) Recombinant protein vaccines against the asexual blood stages of Plasmodium falciparum. Hum Vaccin 6:39–53PubMedCrossRefGoogle Scholar
  20. 20.
    Crompton PD et al (2010) Advances and challenges in malaria vaccine development. J Clin Invest 120:4168–4178PubMedCrossRefGoogle Scholar
  21. 21.
    Salmon BL et al (2001) Malaria parasite exit from the host erythrocyte: a two-step process requiring extraerythrocytic proteolysis. Proc Natl Acad Sci USA 98:271–276PubMedCrossRefGoogle Scholar
  22. 22.
    Yeoh S et al (2007) Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes. Cell 131:1072–1083PubMedCrossRefGoogle Scholar
  23. 23.
    Blackman M (2008) Malarial proteases and host cell egress: an emerging cascade. Cell Microbiol 10:1925–1934PubMedCrossRefGoogle Scholar
  24. 24.
    Arastu-Kapur S et al (2008) Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum. Nat Chem Biol 4:203–213PubMedCrossRefGoogle Scholar
  25. 25.
    Barale J-C et al (1999) Plasmodium falciparum subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1-42 maturase. Proc Natl Acad Sci USA 96:6445–6450PubMedCrossRefGoogle Scholar
  26. 26.
    Uzureau P et al (2004) Gene targeting demonstrates that the Plasmodium berghei subtilisin PbSUB2 is essential for red cell invasion and reveals spontaneous genetic recombination events. Cell Microbiol 6:65–78PubMedCrossRefGoogle Scholar
  27. 27.
    Harris PK (2005) Molecular identification of a malaria merozoite surface sheddase. PLoS Pathog 1:241–251PubMedCrossRefGoogle Scholar
  28. 28.
    Koussis K et al (2009) A multifunctional serine protease primes the malaria parasite for red blood cell invasion. EMBO J 28:725–735PubMedCrossRefGoogle Scholar
  29. 29.
    Hadley T et al (1983) Plasmodium knowlesi: studies on invasion of rhesus erythrocytes by merozoites in the presence of protease inhibitors. Exp Parasitol 55:306–311PubMedCrossRefGoogle Scholar
  30. 30.
    Boyle MJ et al (2010) Isolation of viable Plasmodium falciparum merozoites to define erythrocyte invasion events and advance vaccine and drug development. Proc Natl Acad Sci USA 107:14378–14383PubMedCrossRefGoogle Scholar
  31. 31.
    Moll K et al. (2008) Methods in Malaria Research Fifth Edition. MR4/ATCC and BioMalPar.
  32. 32.
    Trang DT et al (2004) One-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells. Malar J 3:7PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Anthony Bouillon
    • 1
  • Olivier Gorgette
    • 1
  • Odile Mercereau-Puijalon
    • 1
  • Jean-Christophe Barale
    • 1
    Email author
  1. 1.Unité d’Immunologie Moléculaire des ParasitesInstitut PasteurParisFrance

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