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Analysis of Yeast Extracellular Vesicles

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Unconventional Protein Secretion

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1459))

Abstract

Extracellular vesicles (EV) are important carriers of biologically active components in a number of organisms, including fungal cells. Experimental characterization of fungal EVs suggested that these membranous compartments are likely involved in the regulation of several biological events. In fungal pathogens, these events include mechanisms of disease progression and/or control, suggesting potential targets for therapeutic intervention or disease prophylaxis. In this manuscript we describe methods that have been used in the last 10 years for the characterization of EVs produced by yeast forms of several fungal species. Experimental approaches detailed in this chapter include ultracentrifugation methods for EV fractionation, chromatographic approaches for analysis of EV lipids, microscopy techniques for analysis of both intracellular and extracellular vesicular compartments, interaction of EVs with host cells, and physical chemical analysis of EVs by dynamic light scattering.

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References

  1. Colombo M, Raposo G, Thery C (2014) Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30:255–289. doi:10.1146/annurev-cellbio-101512-122326

    Article  CAS  PubMed  Google Scholar 

  2. Lo Cicero A, Stahl PD, Raposo G (2015) Extracellular vesicles shuffling intercellular messages: for good or for bad. Curr Opin Cell Biol 35:69–77. doi:10.1016/j.ceb.2015.04.013

    Article  CAS  PubMed  Google Scholar 

  3. Rodrigues ML, Nimrichter L, Oliveira DL, Frases S, Miranda K, Zaragoza O, Alvarez M, Nakouzi A, Feldmesser M, Casadevall A (2007) Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport. Eukaryot Cell 6(1):48–59. doi:10.1128/EC.00318-06

    Article  CAS  PubMed  Google Scholar 

  4. Vargas G, Rocha JD, Oliveira DL, Albuquerque PC, Frases S, Santos SS, Nosanchuk JD, Gomes AM, Medeiros LC, Miranda K, Sobreira TJ, Nakayasu ES, Arigi EA, Casadevall A, Guimaraes AJ, Rodrigues ML, Freire-de-Lima CG, Almeida IC, Nimrichter L (2015) Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans. Cell Microbiol 17(3):389–407. doi:10.1111/cmi.12374

    Article  CAS  PubMed  Google Scholar 

  5. Wolf JM, Espadas J, Luque-Garcia J, Reynolds T, Casadevall A (2015) Lipid biosynthetic genes affect Candida albicans extracellular vesicle morphology, cargo, and immunostimulatory properties. Eukaryot Cell 14(8):745–754. doi:10.1128/EC.00054-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gil-Bona A, Llama-Palacios A, Parra CM, Vivanco F, Nombela C, Monteoliva L, Gil C (2015) Proteomics unravels extracellular vesicles as carriers of classical cytoplasmic proteins in Candida albicans. J Proteome Res 14(1):142–153. doi:10.1021/pr5007944

    Article  CAS  PubMed  Google Scholar 

  7. Peres da Silva R, Puccia R, Rodrigues ML, Oliveira DL, Joffe LS, Cesar GV, Nimrichter L, Goldenberg S, Alves LR (2015) Extracellular vesicle-mediated export of fungal RNA. Sci Rep 5:7763. doi:10.1038/srep07763

    Article  CAS  PubMed  Google Scholar 

  8. Albuquerque PC, Nakayasu ES, Rodrigues ML, Frases S, Casadevall A, Zancope-Oliveira RM, Almeida IC, Nosanchuk JD (2008) Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol 10(8):1695–1710. doi:10.1111/j.1462-5822.2008.01160.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gehrmann U, Qazi KR, Johansson C, Hultenby K, Karlsson M, Lundeberg L, Gabrielsson S, Scheynius A (2011) Nanovesicles from Malassezia sympodialis and host exosomes induce cytokine responses--novel mechanisms for host-microbe interactions in atopic eczema. PLoS One 6(7), e21480. doi:10.1371/journal.pone.0021480

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Albuquerque PC, Cordero RJ, Fonseca FL, Peres da Silva R, Ramos CL, Miranda KR, Casadevall A, Puccia R, Nosanchuk JD, Nimrichter L, Guimaraes AJ, Rodrigues ML (2012) A Paracoccidioides brasiliensis glycan shares serologic and functional properties with cryptococcal glucuronoxylomannan. Fungal Genet Biol 49(11):943–954. doi:10.1016/j.fgb.2012.09.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Vallejo MC, Nakayasu ES, Longo LV, Ganiko L, Lopes FG, Matsuo AL, Almeida IC, Puccia R (2012) Lipidomic analysis of extracellular vesicles from the pathogenic phase of Paracoccidioides brasiliensis. PLoS One 7(6), e39463. doi:10.1371/journal.pone.0039463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Vallejo MC, Nakayasu ES, Matsuo AL, Sobreira TJ, Longo LV, Ganiko L, Almeida IC, Puccia R (2012) Vesicle and vesicle-free extracellular proteome of Paracoccidioides brasiliensis: comparative analysis with other pathogenic fungi. J Proteome Res 11(3):1676–1685. doi:10.1021/pr200872s

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Vallejo MC, Matsuo AL, Ganiko L, Medeiros LC, Miranda K, Silva LS, Freymuller-Haapalainen E, Sinigaglia-Coimbra R, Almeida IC, Puccia R (2011) The pathogenic fungus Paracoccidioides brasiliensis exports extracellular vesicles containing highly immunogenic alpha-galactosyl epitopes. Eukaryot Cell 10(3):343–351. doi:10.1128/EC.00227-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Oliveira DL, Nakayasu ES, Joffe LS, Guimaraes AJ, Sobreira TJ, Nosanchuk JD, Cordero RJ, Frases S, Casadevall A, Almeida IC, Nimrichter L, Rodrigues ML (2010) Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis. PLoS One 5(6), e11113. doi:10.1371/journal.pone.0011113

    Article  PubMed  PubMed Central  Google Scholar 

  15. Silva BM, Prados-Rosales R, Espadas-Moreno J, Wolf JM, Luque-Garcia JL, Goncalves T, Casadevall A (2014) Characterization of Alternaria infectoria extracellular vesicles. Med Mycol 52(2):202–210. doi:10.1093/mmy/myt003

    Article  CAS  PubMed  Google Scholar 

  16. Rizzo J, Oliveira DL, Joffe LS, Hu G, Gazos-Lopes F, Fonseca FL, Almeida IC, Frases S, Kronstad JW, Rodrigues ML (2014) Role of the Apt1 protein in polysaccharide secretion by Cryptococcus neoformans. Eukaryot Cell 13(6):715–726. doi:10.1128/EC.00273-13

    Article  PubMed  PubMed Central  Google Scholar 

  17. Oliveira DL, Freire-de-Lima CG, Nosanchuk JD, Casadevall A, Rodrigues ML, Nimrichter L (2010) Extracellular vesicles from Cryptococcus neoformans modulate macrophage functions. Infect Immun 78(4):1601–1609. doi:10.1128/IAI.01171-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Rodrigues ML, Nakayasu ES, Oliveira DL, Nimrichter L, Nosanchuk JD, Almeida IC, Casadevall A (2008) Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence. Eukaryot Cell 7(1):58–67. doi:10.1128/EC.00370-07

    Article  CAS  PubMed  Google Scholar 

  19. Rodrigues ML, Nimrichter L, Oliveira DL, Nosanchuk JD, Casadevall A (2008) Vesicular trans-cell wall transport in fungi: a mechanism for the delivery of virulence-associated macromolecules? Lipid Insights 2:27–40

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226(1):497–509

    CAS  PubMed  Google Scholar 

  21. Cantin R, Diou J, Belanger D, Tremblay AM, Gilbert C (2008) Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants. J Immunol Methods 338(1-2):21–30. doi:10.1016/j.jim.2008.07.007

    Article  CAS  PubMed  Google Scholar 

  22. Oliveira DL, Nimrichter L, Miranda K, Frases S, Faull KF, Casadevall A, Rodrigues ML (2009) Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronoxylomannan. Fungal Genet Biol 46(12):956–963. doi:10.1016/j.fgb.2009.09.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Meli F, Klein T, Buhr E, Frase CG, Gleber G, Krumrey M, Duta A, Duta S, Korpelainen V, Bellotti R, Picotto GB, Boyd RD, Cuenat A (2012) Traceable size determination of nanoparticles, a comparison among European metrology institutes. Meas Sci Technol 23(12)

    Google Scholar 

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Acknowledgement

We are grateful to Jorge José-Jó Bastos Ferreira for helpful suggestions. This work was supported by grants from the Brazilian agencies CNPq, CAPES, and FAPERJ. The authors also acknowledge support from the Instituto Nacional de Ciência e Tecnologia de Inovação em Doenças Negligenciadas (INCT-IDN). M.L.R. is the recipient of a Pathfinder Award from the Wellcome Trust (UK).

Author information

Authors and Affiliations

  1. Fundação Oswaldo Cruz (Fiocruz), Centro de Desenvolvimento Tecnológico em Saúde (CDTS) Fundação Oswaldo Cruz, Avenida Brasil, 4036, Sala 814, Rio de Janeiro, RJ, Brazil

    Marcio L. Rodrigues & Debora L. Oliveira

  2. Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

    Marcio L. Rodrigues, Gabriele Vargas & Leonardo Nimrichter

  3. Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil

    Wendell Girard-Dias

  4. Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho and Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

    Wendell Girard-Dias, Susana Frasés & Kildare Miranda

  5. Laboratório de Tecnologia em Cultura de Células, Centro Universitário Estadual da Zona Oeste, Rio de Janeiro, RJ, Brazil

    Anderson J. Franzen

  6. Laboratório de Biologia Estrutural, Diretoria de Programas, Instituto Nacional de Metrologia, Normalização e Qualidade Industrial (INMETRO), Xerém, Rio de Janeiro, RJ, Brazil

    Susana Frasés

Authors
  1. Marcio L. Rodrigues
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  2. Debora L. Oliveira
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  3. Gabriele Vargas
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  4. Wendell Girard-Dias
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  5. Anderson J. Franzen
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  6. Susana Frasés
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  7. Kildare Miranda
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  8. Leonardo Nimrichter
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Corresponding author

Correspondence to Marcio L. Rodrigues .

Editor information

Editors and Affiliations

  1. Instituto di Bioscienze e Biorisorse, Perugia, Italy

    Andrea Pompa

  2. Instituto di Bioscienze e Biorisorse, Perugia, Italy

    Francesca De Marchis

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© 2016 Springer Science+Business Media New York

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Rodrigues, M.L. et al. (2016). Analysis of Yeast Extracellular Vesicles. In: Pompa, A., De Marchis, F. (eds) Unconventional Protein Secretion. Methods in Molecular Biology, vol 1459. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3804-9_12

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  • DOI: https://doi.org/10.1007/978-1-4939-3804-9_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3802-5

  • Online ISBN: 978-1-4939-3804-9

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