Cells across biological kingdoms release extracellular vesicles (EVs) as a means of communication with other cells, be their friends or foes. This is indeed true for the intracellular malaria parasite Plasmodium falciparum (Pf), which utilizes EVs to transport bioactive molecules to various human host systems. Yet, the study of this mode of communication in malaria research is currently constrained due to limitations in high-resolution tools and the absence of commercial antibodies. Here, we demonstrate the power of an advanced spectral flow cytometry approach to robustly detect secreted EVs, isolated from Pf-infected red blood cells. By labeling both EV membrane lipids and the DNA cargo within (non-antibody staining approach), we were able to detect a subpopulation of parasitic-derived EVs enriched in DNA. Furthermore, we could quantitatively measure the DNA-carrying EVs isolated from two distinct blood stages of the parasite: rings and trophozoites. Our findings showcase the potential of spectral flow cytometry to monitor dynamic changes in nucleic acid cargo within pathogenic EVs.

Bioinformatics Unit Life Science Core Facilities Weizmann Institute of Science Rehovot Israel

Department of Biomolecular Sciences Faculty of Biochemistry Weizmann Institute of Science Rehovot Israel

Department of Chemical Research Support Weizmann Institute of Science Rehovot Israel

Flow Cytometry Unit Life Sciences Core Facilities Weizmann Institute of Science Rehovot Israel

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Prague Czech Republic; The International Institute of Molecular Mechanisms and Machines Polish Academy of Sciences Warsaw Poland

See more in PubMed

Buzas E.I. The roles of extracellular vesicles in the immune system. Nat. Rev. Immunol. 2023;23:236–250. PubMed PMC

Sánchez-López C.M., Trelis M., Bernal D., Marcilla A. Overview of the interaction of helminth extracellular vesicles with the host and their potential functions and biological applications. Mol. Immunol. 2021;134:228–235. PubMed

Gill S., Catchpole R., Forterre P. Extracellular membrane vesicles in the three domains of life and beyond. FEMS Microbiol. Rev. 2019;042:273–303. PubMed PMC

Barillas-Mury C., Ribeiro J.M.C., Valenzuela J.G. Understanding pathogen survival and transmission by arthropod vectors to prevent human disease. Science (1979) 2022;377 PubMed

Cai Q., Halilovic L., Shi T., Chen A., He B., Wu H., et al. Extracellular vesicles: cross-organismal RNA trafficking in plants, microbes, and mammalian cells. Extracell Vesicles Circ. Nucl. Acids. 2023;4:262–282. PubMed PMC

Ofir-Birin Y., Heidenreich M., Regev-Rudzki N. Pathogen-derived extracellular vesicles coordinate social behaviour and host manipulation. Semin. Cell Dev. Biol. 2017;67:83–90. PubMed

Buck A.H., Coakley G., Simbari F., McSorley H.J., Quintana J.F., Le Bihan T., et al. Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity. Nat. Commun. 2014;5:5488. PubMed PMC

Chow F.W.-N., Koutsovoulos G., Ovando-Vázquez C., Neophytou K., Bermúdez-Barrientos J.R., Laetsch D.R., et al. Secretion of an Argonaute protein by a parasitic nematode and the evolution of its siRNA guides. Nucleic Acids Res. 2019;47:3594–3606. PubMed PMC

Cruz Camacho A., Alfandari D., Kozela E., Regev-Rudzki N. Biogenesis of extracellular vesicles in protozoan parasites: the ESCRT complex in the trafficking fast lane? PLoS Pathog. 2023;19 PubMed PMC

Olajide J.S., Cai J. Perils and promises of pathogenic Protozoan extracellular vesicles. Front. Cell Infect. Microbiol. 2020;10:371. PubMed PMC

Sánchez-López C.M., González-Arce A., Ramírez-Toledo V., Bernal D., Marcilla A. Unraveling new players in helminth pathology: extracellular vesicles from Fasciola hepatica and Dicrocoelium dendriticum exert different effects on hepatic stellate cells and hepatocytes. Int. J. Parasitol. 2024;54:617–634. PubMed

da Silva Lira Filho A., Lafleur A., Marcet-Palacios M., Olivier M. Identification of potential novel proteomic markers of Leishmania spp.-derived exosomes. Front. Cell Infect. Microbiol. 2024;14 PubMed PMC

Szempruch A.J., Dennison L., Kieft R., Harrington J.M., Hajduk S.L. Sending a message: extracellular vesicles of pathogenic protozoan parasites. Nat. Rev. Microbiol. 2016;14:669–675. PubMed

Tandoh K.Z., Ibarra-Meneses A.V., Langlais D., Olivier M., Torrecilhas A.C., Fernandez-Prada C., et al. Extracellular vesicles: translational agenda questions for three Protozoan parasites. Traffic. 2024;25 PubMed

Toda H., Diaz-Varela M., Segui-Barber J., Roobsoong W., Baro B., Garcia-Silva S., et al. Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence. Nat. Commun. 2020;11:2761. PubMed PMC

Ofir-Birin Y., Regev-Rudzki N. Extracellular vesicles in parasite survival. Science (1979) 2019;363:817–818. PubMed

Alfandari D., Cadury S., Morandi M.I., Regev-Rudzki N. Transforming parasites into their own foes: parasitic extracellular vesicles as a vaccine platform. Trends Parasitol. 2023;39:913–928. PubMed

Heaton P.M. Challenges of developing novel vaccines with particular global health importance. Front. Immunol. 2020;11 PubMed PMC

Fuhrmann G., Neuer A.L., Herrmann I.K. Extracellular vesicles – a promising avenue for the detection and treatment of infectious diseases? Eur. J. Pharmaceutics Biopharmaceutics. 2017;118:56–61. PubMed

Barnadas-Carceller B., del Portillo H.A., Fernandez-Becerra C. Extracellular vesicles as biomarkers in parasitic disease diagnosis. Curr. Top Membr. 2024;94:187–223. PubMed

Razim A., Zabłocka A., Schmid A., Thaler M., Černý V., Weinmayer T., et al. Bacterial extracellular vesicles as intranasal postbiotics: detailed characterization and interaction with airway cells. J. Extracell Vesicles. 2024;13 PubMed PMC

Huang G., Lin G., Zhu Y., Duan W., Jin D. Emerging technologies for profiling extracellular vesicle heterogeneity. Lab. Chip. 2020;20:2423–2437. PubMed

Erdbrügger U., Lannigan J. Analytical challenges of extracellular vesicle detection: a comparison of different techniques. Cytometry A. 2016;89:123–134. PubMed

Bordanaba-Florit G., Royo F., Kruglik S.G., Falcón-Pérez J.M. Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles. Nat. Protoc. 2021;16:3163–3185. PubMed

Cowman A.F., Healer J., Marapana D., Marsh K. Malaria: biology and disease. Cell. 2016;167:610–624. PubMed

Cox F.E. History of the discovery of the malaria parasites and their vectors. Parasit Vectors. 2010;3:5. PubMed PMC

Brejt J.A., Golightly L.M. Severe malaria. Curr. Opin. Infect. Dis. 2019;32:413–418. PubMed

Acharya P., Garg M., Kumar P., Munjal A., Raja K.D. Host–parasite interactions in human malaria: clinical implications of basic research. Front. Microbiol. 2017;8:889. PubMed PMC

Abou Karam P., Rosenhek-Goldian I., Ziv T., Ben Ami Pilo H., Azuri I., Rivkin A., et al. Malaria parasites release vesicle subpopulations with signatures of different destinations. EMBO Rep. 2022;23 PubMed PMC

Ben Ami Pilo H., Khan Khilji S., Lühle J., Biskup K., Levy Gal B., Rosenhek Goldian I., et al. Sialylated N -glycans mediate monocyte uptake of extracellular vesicles secreted from Plasmodium falciparum -infected red blood cells. J. Extracellular Biol. 2022;1:e33. PubMed PMC

Dekel E., Yaffe D., Rosenhek-Goldian I., Ben-Nissan G., Ofir-Birin Y., Morandi M.I., et al. 20S proteasomes secreted by the malaria parasite promote its growth. Nat. Commun. 2021;12:1172. PubMed PMC

Demarta-Gatsi C., Rivkin A., Di Bartolo V., Peronet R., Ding S., Commere P., et al. Histamine releasing factor and elongation factor 1 alpha secreted via malaria parasites extracellular vesicles promote immune evasion by inhibiting specific T cell responses. Cell Microbiol. 2019;21 PubMed

Kioko M., Pance A., Mwangi S., Goulding D., Kemp A., Rono M., et al. Extracellular vesicles could be a putative posttranscriptional regulatory mechanism that shapes intracellular RNA levels in Plasmodium falciparum. Nat. Commun. 2023;14:6447. PubMed PMC

Mantel P.-Y., Hjelmqvist D., Walch M., Kharoubi-Hess S., Nilsson S., Ravel D., et al. Infected erythrocyte-derived extracellular vesicles alter vascular function via regulatory Ago2-miRNA complexes in malaria. Nat. Commun. 2016;7 PubMed PMC

Regev-Rudzki N., Wilson D.W., Carvalho T.G., Sisquella X., Coleman B.M., Rug M., et al. Cell-cell communication between malaria-infected red blood cells via exosome-like vesicles. Cell. 2013;153:1120–1133. PubMed

Ofir-Birin Y., Abou Karam P., Rudik A., Giladi T., Porat Z., Regev-Rudzki N. Monitoring extracellular vesicle cargo active uptake by imaging flow cytometry. Front. Immunol. 2018;9:1011. PubMed PMC

Ofir-Birin Y., Ben Ami Pilo H., Cruz Camacho A., Rudik A., Rivkin A., Revach O.-Y., et al. Malaria parasites both repress host CXCL10 and use it as a cue for growth acceleration. Nat. Commun. 2021;12:4851. PubMed PMC

Alfandari D., Ben Ami Pilo H., Abou Karam P., Dagan O., Joubran C., Rotkopf R., et al. Monitoring distribution dynamics of EV RNA cargo within recipient monocytes and macrophages. Front. Cell Infect. Microbiol. 2022;11 PubMed PMC

Ye W., Chew M., Hou J., Lai F., Leopold S.J., Loo H.L., et al. Microvesicles from malaria-infected red blood cells activate natural killer cells via MDA5 pathway. PLoS Pathog. 2018;14 PubMed PMC

Sisquella X., Ofir-Birin Y., Pimentel M.A., Cheng L., Abou Karam P., Sampaio N.G., et al. Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors. Nat. Commun. 2017;8:1985. PubMed PMC

Combes V., Taylor T.E., Juhan-Vague I., Mège J.-L., Mwenechanya J., Tembo M., et al. Circulating endothelial microparticles in Malawian children with severe falciparum malaria complicated with coma. JAMA: J. Am. Med. Assoc. 2004;291:2542–2544. PubMed

Couper K.N., Barnes T., Hafalla J.C.R., Combes V., Ryffel B., Secher T., et al. Parasite-derived plasma microparticles contribute significantly to malaria infection-induced inflammation through potent macrophage stimulation. PLoS Pathog. 2010;6 PubMed PMC

Pankoui Mfonkeu J.B., Gouado I., Fotso Kuaté H., Zambou O., Combes V., Raymond Grau G.E., et al. Biochemical markers of nutritional status and childhood malaria severity in Cameroon. Br. J. Nutr. 2010;104:886–892. PubMed

Nantakomol D., Dondorp A.M., Krudsood S., Udomsangpetch R., Pattanapanyasat K., Combes V., et al. Circulating red cell–derived microparticles in human malaria. J. Infect. Dis. 2011;203:700–706. PubMed PMC

Kowal J., Arras G., Colombo M., Jouve M., Morath J.P., Primdal-Bengtson B., et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc. Natl. Acad. Sci. U. S. A. 2016;113:E968–E977. PubMed PMC

Tkach M., Kowal J., Théry C. Why the need and how to approach the functional diversity of extracellular vesicles. Philosophical Trans. R. Soc. B: Biol. Sci. 2018;373 PubMed PMC

Zhang H., Freitas D., Kim H.S., Fabijanic K., Li Z., Chen H., et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat. Cell Biol. 2018;20:332–343. PubMed PMC

Filipe V., Hawe A., Jiskoot W. Critical evaluation of nanoparticle tracking analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm. Res. 2010;27:796–810. PubMed PMC

Gardiner C., Vizio D. Di, Sahoo S., Théry C., Witwer K.W., Wauben M., et al. Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey. J. Extracell Vesicles. 2016;5 PubMed PMC

Rosenhek-Goldian I., Abou Karam P., Regev-Rudzki N., Rojas A. Imaging of extracellular vesicles derived from Plasmodium falciparum-infected red blood cells using atomic force microscopy. Methods Mol. Biol. 2022;2470:133–145. PubMed

Morandi M.I., Busko P., Ozer-Partuk E., Khan S., Zarfati G., Elbaz-Alon Y., et al. Extracellular vesicle fusion visualized by cryo-electron microscopy. PNAS Nexus. 2022;1 PubMed PMC

Kozela E., Meneghetti P., Regev-Rudzki N., Torrecilhas A.C., Porat Z. Subcellular particles for characterization of host-parasite interactions. Microbes Infect. 2024;26 PubMed

Dekel E., Abou Karam P., Ohana-Daniel Y., Biton M., Regev-Rudzki N., Porat Z. Antibody-free labeling of malaria-derived extracellular vesicles using flow cytometry. Biomedicines. 2020;8:98. PubMed PMC

Nolan J.P. The evolution of spectral flow cytometry. Cytometry Part A. 2022;101:812–817. PubMed

Nolan J.P., Condello D. Spectral flow cytometry. Curr. Protoc. Cytom. 2013;1:1.27.1–1.27.13. PubMed PMC

Welsh J.A., A Arkesteijn G.J., Bremer M., Cimorelli M., Dignat-George F., Giebel B., et al. A compendium of single extracellular vesicle flow cytometry. J. Extracell Vesicles. 2023;12 PubMed PMC

Théry C., Witwer K.W., Aikawa E., Alcaraz M.J., Anderson J.D., Andriantsitohaina R., et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell Vesicles. 2018;7 PubMed PMC

Chuo S.T.-Y., Chien J.C.-Y., Lai C.P.-K. Imaging extracellular vesicles: current and emerging methods. J. Biomed. Sci. 2018;25:91. PubMed PMC

Hassdenteufel S., Schuldiner M. Show your true color: mammalian cell surface staining for tracking cellular identity in multiplexing and beyond. Curr. Opin. Chem. Biol. 2022;66 PubMed

Simonsen J.B. What are we looking at? Extracellular vesicles, lipoproteins, or both? Circ. Res. 2017;121:920–922. PubMed

Botha J., Handberg A., Simonsen J.B. Lipid-based strategies used to identify extracellular vesicles in flow cytometry can be confounded by lipoproteins: evaluations of annexin V, lactadherin, and detergent lysis. J. Extracell Vesicles. 2022;11 PubMed PMC

Buntsma N.C., Shahsavari M., Gąsecka A., Nieuwland R., van Leeuwen T.G., van der Pol E. Preventing swarm detection in extracellular vesicle flow cytometry: a clinically applicable procedure. Res. Pract. Thromb. Haemost. 2023;7 PubMed PMC

Lai R.C., Lim S.K. Membrane lipids define small extracellular vesicle subtypes secreted by mesenchymal stromal cells. J. Lipid Res. 2019;60:318–322. PubMed PMC

Fantini J. Lipid rafts and human diseases: why we need to target gangliosides. FEBS Open Bio. 2023;13:1636–1650. PubMed PMC

Ben-Hur S., Biton M., Regev-Rudzki N. Extracellular vesicles: a prevalent tool for microbial gene delivery? Proteomics. 2019;19 PubMed

Smith P.J., Wiltshire M., Errington R.J. DRAQ5 labeling of nuclear DNA in live and fixed cells. Curr Protoc Cytom. 2004;7:7.25. PubMed

Clancy J.W., Sheehan C.S., Boomgarden A.C., D’Souza-Schorey C. Recruitment of DNA to tumor-derived microvesicles. Cell Rep. 2022;38 PubMed PMC

Osteikoetxea X., Sódar B., Németh A., Szabó-Taylor K., Pálóczi K., Vukman K.V., et al. Differential detergent sensitivity of extracellular vesicle subpopulations. Org. Biomol. Chem. 2015;13:9775–9782. PubMed

Boudna M., Campos A.D., Vychytilova-Faltejskova P., Machackova T., Slaby O., Souckova K. Strategies for labelling of exogenous and endogenous extracellular vesicles and their application for in vitro and in vivo functional studies. Cell Commun. Signaling. 2024;22:171. PubMed PMC

Lai C.P., Kim E.Y., Badr C.E., Weissleder R., Mempel T.R., Tannous B.A., et al. Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters. Nat. Commun. 2015;6:7029. PubMed PMC

Adan A., Alizada G., Kiraz Y., Baran Y., Nalbant A. Flow cytometry: basic principles and applications. Crit. Rev. Biotechnol. 2017;37:163–176. PubMed

Gul B., Syed F., Khan S., Iqbal A., Ahmad I. Characterization of extracellular vesicles by flow cytometry: challenges and promises. Micron. 2022;161 PubMed

Hendrix A., Lippens L., Pinheiro C., Théry C., Martin-Jaular L., Lötvall J., et al. Extracellular vesicle analysis. Nat. Rev. Methods Primers. 2023;3:56.

McKinnon K.M. Flow cytometry: an overview. Curr Protoc Immunol. 2018;120:5.1.1–5.1.11. PubMed PMC

Morales-Kastresana A., Telford B., Musich T.A., McKinnon K., Clayborne C., Braig Z., et al. Labeling extracellular vesicles for nanoscale flow cytometry. Sci. Rep. 2017;7:1878. PubMed PMC

Simeone P., Celia C., Bologna G., Ercolino E., Pierdomenico L., Cilurzo F., et al. Diameters and fluorescence calibration for extracellular vesicle analyses by flow cytometry. Int. J. Mol. Sci. 2020;21:7885. PubMed PMC

van der Vlist E.J., Nolte-’t Hoen E.N.M., Stoorvogel W., Arkesteijn G.J.A., Wauben M.H.M. Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat. Protoc. 2012;7:1311–1326. PubMed

Kuiper M., van de Nes A., Nieuwland R., Varga Z., van der Pol E. Reliable measurements of extracellular vesicles by clinical flow cytometry. Am. J. Reprod. Immunol. 2021;85 PubMed PMC

van der Pol E., Sturk A., van Leeuwen T., Nieuwland R., Coumans F., Mobarrez F., et al. Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation. J. Thromb. Haemost. 2018;16:1236–1245. PubMed

Woud W.W., Pugsley H.R., Bettin B.A., Varga Z., van der Pol E. Size and fluorescence calibrated imaging flow cytometry: from arbitrary to standard units. Cytometry Part A. 2024;105:752–762. PubMed

de Rond L., van der Pol E., Bloemen P.R., Van Den Broeck T., Monheim L., Nieuwland R., et al. A systematic approach to improve scatter sensitivity of a flow cytometer for detection of extracellular vesicles. Cytometry A. 2020;97:582–591. PubMed PMC

van der Pol E., Coumans F.A.W., Grootemaat A.E., Gardiner C., Sargent I.L., Harrison P., et al. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J. Thromb Haemost. 2014;12:1182–1192. PubMed

van der Pol E., van Gemert M.J.C., Sturk A., Nieuwland R., van Leeuwen T.G. Single vs. swarm detection of microparticles and exosomes by flow cytometry. J. Thromb. Haemost. 2012;10:919–930. PubMed

Aibaidula A.Z., Fain C.E., Garcia L.C., Wier A., Bouchal S.M., Bauman M.M., et al. Spectral flow cytometry identifies distinct nonneoplastic plasma extracellular vesicle phenotype in glioblastoma patients. Neurooncol. Adv. 2023;5 PubMed PMC

Brandenburg B., Koudstaal W., Goudsmit J., Klaren V., Tang C., Bujny M.V., et al. Mechanisms of hemagglutinin targeted influenza virus neutralization. PLoS One. 2013;8 PubMed PMC

Schwartz A., Gaigalas A.K., Wang L., Marti G.E., Vogt R.F., Fernandez-Repollet E. Formalization of the MESF unit of fluorescence intensity. Cytometry B Clin. Cytom. 2004;57B:1–6. PubMed

Trager W., Jensen J.B. Human malaria parasites in continuous culture. Science (1979) 1976;193:673–675. PubMed

Nečas D., Klapetek P. Gwyddion: an open-source software for SPM data analysis. Open Phys. 2012;10:181–188.

Mastronarde D.N. SerialEM: a program for automated tilt series acquisition on tecnai microscopes using prediction of specimen position. Microsc. Microanalysis. 2003;9:1182–1183.

Rueden C.T., Schindelin J., Hiner M.C., DeZonia B.E., Walter A.E., Arena E.T., et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics. 2017;18:529. PubMed PMC

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–682. PubMed PMC