Abstract
Malaria is caused by unicellular parasites of the genus Plasmodium, which reside in erythrocytes during the clinically relevant stage of infection. To separate parasite from host cell material, haemolytic agents such as saponin are widely used. Previous electron microscopy studies on saponin-treated parasites reported both, parasites enclosed by the erythrocyte membrane and liberated from the host cell. These ambiguous reports prompted us to investigate haemolysis by live-cell time-lapse microscopy. Using either saponin or streptolysin O to lyse Plasmodium falciparum–infected erythrocytes, we found that ring-stage parasites efficiently exit the erythrocyte upon haemolysis. For late-stage parasites, we found that only approximately half were freed, supporting the previous electron microscopy studies. Immunofluorescence imaging indicated that freed parasites were surrounded by the parasitophorous vacuolar membrane. These results may be of interest for future work using haemolytic agents to enrich for parasite material.
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References
Aikawa M, Cook RT (1972) Plasmodium: electron microscopy of antigen preparations. Exp Parasitol 31:67–74. https://doi.org/10.1016/0014-4894(72)90048-3
Ansorge I, Benting J, Bhakdi S, Lingelbach K (1996) Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O. Biochem J 315:307–314. https://doi.org/10.1042/bj3150307
Baldi DL, Good R, Duraisingh MT, Crabb BS, Cowman AF (2002) Identification and disruption of the gene encoding the third member of the low-molecular-mass rhoptry complex in Plasmodium falciparum. Infect Immun 70:5236–5245. https://doi.org/10.1128/IAI.70.9.5236-5245.2002
Behari R, Haldar K (1994) Plasmodium falciparum: protein localization along a novel, lipid-rich tubovesicular membrane network in infected erythrocytes. Exp Parasitol 79:250–259. https://doi.org/10.1006/expr.1994.1088
Bhakdi S, Weller U, Walev I, Martin E, Jonas D, Palmer M (1993) A guide to the use of pore-forming toxins for controlled permeabilization of cell-membranes. Med Microbiol Immunol 182:167–175. https://doi.org/10.1007/bf00219946
Böttger S, Melzig MF (2013) The influence of saponin on cell membrane cholesterol. Bioorg Med Chem 21:7118–7124. https://doi.org/10.1016/j.bmc.2013.09.008
Charnaud SC, Kumarasingha R, Bullen HE, Crabb BS, Gilson PR (2018) Knockdown of the translocon protein EXP2 in Plasmodium falciparum reduces growth and protein export. PLoS One 13:e0204785. https://doi.org/10.1371/journal.pone.0204785.s003
Christophers Rickard S, Fulton JD (1939) Experiments with isolated malaria parasites (Plasmodium Knowlesi) free from red cells. Ann Trop Med Parasitol 33:161–170
Cook RT, Aikawa M, Rock RC, Little W, Sprinz H (1969) The isolation and fractionation of Plasmodium knowlesi. Mil Med 134:866–883
de Koning-Ward TF, Gilson PR, Boddey JA, Rug M, Smith BJ, Papenfuss AT, Sanders PR, Lundie RJ, Maier AG, Cowman AF, Crabb BS (2009) A newly discovered protein export machine in malaria parasites. Nature 459:945–949. https://doi.org/10.1038/nature08104
Garten M, Nasamu AS, Niles JC, Zimmerberg J, Goldberg DE, Beck JR (2018) EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX. Nat Microbiol 3:1–13. https://doi.org/10.1038/s41564-018-0222-7
Ghosh S, Kennedy K, Sanders P et al (2017) The Plasmodium rhoptry associated protein complex is important for parasitophorous vacuole membrane structure and intraerythrocytic parasite growth. Cell Microbiol 19:e12733. https://doi.org/10.1111/cmi.12287
Grüring C, Heiber A, Kruse F, Ungefehr J, Gilberger TW, Spielmann T (2011) Development and host cell modifications of Plasmodium falciparum blood stages in four dimensions. Nat Commun 2:165. https://doi.org/10.1038/ncomms1169
Hall R, McBride J, Morgan G, Tait A, Werner Zolg J, Walliker D, Scaife J (1983) Antigens of the erythrocytes stages of the human malaria parasite Plasmodium falciparum detected by monoclonal antibodies. Mol Biochem Parasitol 7:247–265
Ho C-M, Beck JR, Lai M, Cui Y, Goldberg DE, Egea PF, Zhou ZH (2018) Malaria parasite translocon structure and mechanism of effector export. Nature 561:1–24. https://doi.org/10.1038/s41586-018-0469-4
Killby VA, Silverman PH (1969) Isolated erythrocytic forms of Plasmodium berghei. An electron-microscopical study. Am J Trop Med Hyg 18:836–859. https://doi.org/10.4269/ajtmh.1969.18.836
Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65:418–420. https://doi.org/10.2307/3280287
Maguire PA, Sherman IW (1990) Phospholipid composition, cholesterol content and cholesterol exchange in Plasmodium falciparum-infected red cells. Mol Biochem Parasitol 38:105–112. https://doi.org/10.1016/0166-6851(90)90210-d
Maier AG, Rug M, O’Neill MT et al (2008) Exported proteins required for virulence and rigidity of plasmodium falciparum-infected human erythrocytes. Cell 134:48–61. https://doi.org/10.1016/j.cell.2008.04.051
Martin WF, Finerty J, Rosenthal A (1971) Isolation of Plasmodium berghei (Malaria) parasites by ammonium chloride lysis of infected erythrocytes. Nat New Biol 233:260–261. https://doi.org/10.1038/newbio233260a0
Matz JM, Beck JR, Blackman MJ (2020) The parasitophorous vacuole of the blood-stage malaria parasite. Nat Rev Microbiol 18:1–13. https://doi.org/10.1038/s41579-019-0321-3
Oehring SC, Woodcroft BJ, Moes S, Wetzel J, Dietz O, Pulfer A, Dekiwadia C, Maeser P, Flueck C, Witmer K, Brancucci NMB, Niederwieser I, Jenoe P, Ralph SA, Voss TS (2012) Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum. Genome Biol 13:R108. https://doi.org/10.1186/gb-2012-13-11-r108
Orjih AU (1994) Saponin haemolysis for increasing concentration of Plasmodium falciparum infected erythrocytes. Lancet 343:295. https://doi.org/10.1016/s0140-6736(94)91142-8
Pease BN, Huttlin EL, Jedrychowski MP, Talevich E, Harmon J, Dillman T, Kannan N, Doerig C, Chakrabarti R, Gygi SP, Chakrabarti D (2013) Global analysis of protein expression and phosphorylation of three stages of Plasmodium falciparum intraerythrocytic development. J Proteome Res 12:4028–4045. https://doi.org/10.1021/pr400394g
Riglar DT, Rogers KL, Hanssen E, Turnbull L, Bullen HE, Charnaud SC, Przyborski J, Gilson PR, Whitchurch CB, Crabb BS, Baum J, Cowman AF (2013) Spatial association with PTEX complexes defines regions for effector export into Plasmodium falciparum-infected erythrocytes. Nat Commun 4:1415. https://doi.org/10.1038/ncomms2449
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. https://doi.org/10.1038/nmeth.2019
Seeman P, Cheng D, Iles GH (1973) Structure of membrane holes in osmotic and saponin hemolysis. J Cell Biol 56:519–527. https://doi.org/10.1083/jcb.56.2.519
Semple JW, Allen D, Chang W, Castaldi P, Freedman J (1993) Rapid separation of CD4+ and CD19+ lymphocyte populations from human peripheral blood by a magnetic activated cell sorter (MACS). Cytometry 14:955–960. https://doi.org/10.1002/cyto.990140816
Sherling ES, van Ooij C (2016) Host cell remodeling by pathogens: the exomembrane system in Plasmodium-infected erythrocytes. FEMS Microbiol Rev 40:701–721. https://doi.org/10.1111/j.1365-2141.2011.08766.x
Siau A, Huang X, Weng M, Sze SK, Preiser PR (2016) Proteome mapping of Plasmodium: identification of the P. yoelii remodellome. Sci Rep 6:31055. https://doi.org/10.1038/srep31055
Siddiqui WA, Kan SC, Kramer K, Richmond-Crum SM (1979) In vitro production and partial purification of Plasmodium falciparum antigen. Bull World Health Organ 57(Suppl 1):75–82
Trager W, Jensen JB (1976) Human malaria parasites in continuous culture. Science 193:673–675. https://doi.org/10.1126/science.781840
Ward GE, Miller LH, Dvorak JA (1993) The origin of parasitophorous vacuole membrane-lipids in malaria-infected erythrocytes. J Cell Sci 106:237–248
Acknowledgements
We thank Elizabeth Egan and Catherine Moreau and past and current members of the Ganter and Guizetti laboratories for discussions. We are grateful to Jude Przyborski for the generous gift of streptolysin O and for the microscopy support from the Infectious Diseases Imaging Platform (IDIP) at the Centre for Integrative Infectious Disease Research, Heidelberg, Germany. We are grateful to the Plasmodium genomics resource PlasmoDB, which facilitated this research. X.S. was as student of the Master Program Molecular Biotechnology at Heidelberg University.
Funding
This work was supported by Zendia GmbH, the German Federal Ministry of Education and Research (KMU-Innovativ Medical technique, 13GW0065A to Zendia GmbH and KMU-Innovative Biochance BIO 326-036 to Zendia GmbH and Heidelberg University). F.F. and M.G. are members of SFB1129. F.F. is a member of the CellNetworks Cluster of Excellence at Heidelberg University and the German Centre for Infection Research, TTU Malaria.
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Conceptualisation: K.Q. and M.G.; Methodology: K.Q., M.G and GB; Formal analysis and investigation: K.Q. and X. S.; Writing—original draft preparation: K.Q. and M.G.; Writing—review and editing: K.Q., X.S., F.F., G.B. and M.G; Funding acquisition: F.F. and G.B.; Resources: F.F. and G.B.; Supervision: M.G. and G.B.
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G.B. is founder and CEO of Zendia GmbH with the goal to develop a commercially available rapid diagnostic test, holding the patents WO2018002327A1 and WO2014096462A3 and country registrations. F.F. served as an advisor for Zendia GmbH and co-directs the BMBF KMU-Innovativ grant BIO-370-036 together with G.B. since 2019. K.Q. is currently employed by Zendia GmbH and X.S. and M.G. were temporarily employed by Zendia GmbH. M.G. is listed as co-inventor on the patent WO2018002327A1.
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Online Resource 1
Live-cell imaging of a late-stage, i.e. hemozoin-containing, parasite during haemolysis with saponin in presence of anti-EXP1 antibody coupled to Alexa Fluor 488. Right, Fluorescent signal of Alexa Fluor 488; left, differential interference contrast (DIC) image. Note, only after lysis, and thus assessable antigen, a specific signal is detectable. (AVI 655 kb)
Online Resource 2a
Ring–stage parasite is freed from erythrocyte membrane after saponin lysis, corresponding movie to Fig. 1a (AVI 156 kb)
Online Resource 2b
Late–stage parasite is still enclosed in erythrocyte membrane after saponin addition, corresponding movie to Fig. 1b (AVI 64 kb)
Online Resource 2c
Late–stage parasite is freed from erythrocyte membrane after saponin addition, corresponding movie to Fig. 1c (AVI 86 kb)
Online Resource 3a
Ring–stage parasite is freed from erythrocyte membrane after SLO lysis, corresponding movie to Fig. 2a (AVI 575 kb)
Online Resource 3b
Late–stage parasite is still enclosed in erythrocyte membrane after SLO addition, corresponding movie to Fig. 2b (AVI 306 kb)
Online Resource 3c
Late–stage parasite is freed from erythrocyte membrane after SLO addition, corresponding movie to Fig. 2c (AVI 263 kb)
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Quadt, K.A., Smyrnakou, X., Frischknecht, F. et al. Plasmodium falciparum parasites exit the infected erythrocyte after haemolysis with saponin and streptolysin O. Parasitol Res 119, 4297–4302 (2020). https://doi.org/10.1007/s00436-020-06932-9
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DOI: https://doi.org/10.1007/s00436-020-06932-9