Analysis of cryptococcal extracellular vesicles: experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells

Detalhes bibliográficos
Autor(a) principal: Reis, Flavia Coelho Garcia dos
Data de Publicação: 2021
Outros Autores: Gimenez, Bianca, Jozefowicz, Luísa J., Castelli, Rafael Fernando, Martins, Sharon de Toledo, Alves, Lysangela Ronalte, Oliveira, Haroldo Cesar de, Rodrigues, Marcio Lourenço
Tipo de documento: Artigo
Idioma: por
Título da fonte: Repositório Institucional da FIOCRUZ (ARCA)
Texto Completo: https://www.arca.fiocruz.br/handle/icict/50806
Resumo: Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.
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spelling Reis, Flavia Coelho Garcia dosGimenez, BiancaJozefowicz, Luísa J.Castelli, Rafael FernandoMartins, Sharon de ToledoAlves, Lysangela RonalteOliveira, Haroldo Cesar deRodrigues, Marcio Lourenço2022-01-19T11:58:49Z2022-01-19T11:58:49Z2021REIS, Flavia Coelho Garcia dos et al. Analysis of cryptococcal extracellular vesicles: Experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells. Microbiol Spectr, v. 9, n. 1, p. 1–15, 2021.2165-0497https://www.arca.fiocruz.br/handle/icict/5080610.1128/Spectrum.00125-21.porAmerican Society for MicrobiologyCryptococcusExtracellular VesiclesPathogenicityFungal CellsVesículas ExtracelularesPatogenicidadCélulas FúngicasVésicules extracellulairesPathogénicitéCellules FongiquesVesículas ExtracelularesPatogenicidadeCélulas FúngicasAnalysis of cryptococcal extracellular vesicles: experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cellsinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Rio de Janeiro, RJ, Brasil.Extracellular vesicles (EVs) produced by members of the Cryptococcus genus are associated with fundamental processes of fungal physiology and virulence. However, several questions about the properties of cryptococcal EVs remain unanswered, mostly because of technical limitations. We recently described a fast and efficient protocol of high-yield EV isolation from solid medium. In this study, we aimed at using the solid medium protocol to address some of the open questions about EVs, including the kinetics of EV production, the diversity of EVs produced by multiple isolates under different culture conditions, the separation of vesicles in a density gradient followed by the recovery of functional EVs, the direct detection of EVs in culture supernatants, and the production of vesicles in solid cultures of Titan cells. Our results indicate that the production of EVs is directly impacted by the culture medium and time of growth, resulting in variable detection of EVs per cell and a peak of EV detection at 24 h of growth. Nanoparticle tracking analysis (NTA) of EV samples revealed that multiple isolates produce vesicles with variable properties, including particles of diverging dimensions. EVs were produced in the solid medium in amounts that were separated on a centrifugation density gradient, resulting in the recovery of functional EVs containing the major cryptococcal capsular antigen. We also optimized the solid medium protocol for induction of the formation of Titan cells, and analyzed the production of EVs by NTA and transmission electron microscopy. This analysis confirmed that EVs were isolated from solid cultures of cryptococcal enlarged cells. With these approaches, we expect to implement simple methods that will facilitate the analysis of EVs produced by fungal cells. Fungal extracellular vesicles (EVs) are considered to be important players in the biology of fungal pathogens. However, the limitations in the methodological approaches to studying fungal EVs impair the expansion of knowledge in this field. In the present study, we used the Cryptococcus genus as a model for the study of EVs. We explored the simplification of protocols for EV analysis, which helped us to address some important, but still unanswered, questions about fungal EVs.info:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZLICENSElicense.txtlicense.txttext/plain; charset=utf-83084https://www.arca.fiocruz.br/bitstream/icict/50806/1/license.txt783568c2893d2e25a99990b126be1772MD51ORIGINALSpectrum.00125-21Ok.pdfSpectrum.00125-21Ok.pdfapplication/pdf2388121https://www.arca.fiocruz.br/bitstream/icict/50806/2/Spectrum.00125-21Ok.pdf388691a2e8b2d5b917e66bc1337917c7MD52icict/508062022-01-19 08:58:49.836oai:www.arca.fiocruz.br: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ório InstitucionalPUBhttps://www.arca.fiocruz.br/oai/requestrepositorio.arca@fiocruz.bropendoar:21352022-01-19T11:58:49Repositório Institucional da FIOCRUZ (ARCA) - 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dc.title.pt_BR.fl_str_mv Analysis of cryptococcal extracellular vesicles: experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells
title Analysis of cryptococcal extracellular vesicles: experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells
spellingShingle Analysis of cryptococcal extracellular vesicles: experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells
Reis, Flavia Coelho Garcia dos
Cryptococcus
Extracellular Vesicles
Pathogenicity
Fungal Cells
Vesículas Extracelulares
Patogenicidad
Células Fúngicas
Vésicules extracellulaires
Pathogénicité
Cellules Fongiques
Vesículas Extracelulares
Patogenicidade
Células Fúngicas
title_short Analysis of cryptococcal extracellular vesicles: experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells
title_full Analysis of cryptococcal extracellular vesicles: experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells
title_fullStr Analysis of cryptococcal extracellular vesicles: experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells
title_full_unstemmed Analysis of cryptococcal extracellular vesicles: experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells
title_sort Analysis of cryptococcal extracellular vesicles: experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells
author Reis, Flavia Coelho Garcia dos
author_facet Reis, Flavia Coelho Garcia dos
Gimenez, Bianca
Jozefowicz, Luísa J.
Castelli, Rafael Fernando
Martins, Sharon de Toledo
Alves, Lysangela Ronalte
Oliveira, Haroldo Cesar de
Rodrigues, Marcio Lourenço
author_role author
author2 Gimenez, Bianca
Jozefowicz, Luísa J.
Castelli, Rafael Fernando
Martins, Sharon de Toledo
Alves, Lysangela Ronalte
Oliveira, Haroldo Cesar de
Rodrigues, Marcio Lourenço
author2_role author
author
author
author
author
author
author
dc.contributor.author.fl_str_mv Reis, Flavia Coelho Garcia dos
Gimenez, Bianca
Jozefowicz, Luísa J.
Castelli, Rafael Fernando
Martins, Sharon de Toledo
Alves, Lysangela Ronalte
Oliveira, Haroldo Cesar de
Rodrigues, Marcio Lourenço
dc.subject.other.pt_BR.fl_str_mv Cryptococcus
topic Cryptococcus
Extracellular Vesicles
Pathogenicity
Fungal Cells
Vesículas Extracelulares
Patogenicidad
Células Fúngicas
Vésicules extracellulaires
Pathogénicité
Cellules Fongiques
Vesículas Extracelulares
Patogenicidade
Células Fúngicas
dc.subject.en.pt_BR.fl_str_mv Extracellular Vesicles
Pathogenicity
Fungal Cells
dc.subject.es.pt_BR.fl_str_mv Vesículas Extracelulares
Patogenicidad
Células Fúngicas
dc.subject.fr.pt_BR.fl_str_mv Vésicules extracellulaires
Pathogénicité
Cellules Fongiques
dc.subject.decs.pt_BR.fl_str_mv Vesículas Extracelulares
Patogenicidade
Células Fúngicas
description Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.
publishDate 2021
dc.date.issued.fl_str_mv 2021
dc.date.accessioned.fl_str_mv 2022-01-19T11:58:49Z
dc.date.available.fl_str_mv 2022-01-19T11:58:49Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.citation.fl_str_mv REIS, Flavia Coelho Garcia dos et al. Analysis of cryptococcal extracellular vesicles: Experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells. Microbiol Spectr, v. 9, n. 1, p. 1–15, 2021.
dc.identifier.uri.fl_str_mv https://www.arca.fiocruz.br/handle/icict/50806
dc.identifier.issn.pt_BR.fl_str_mv 2165-0497
dc.identifier.doi.none.fl_str_mv 10.1128/Spectrum.00125-21.
identifier_str_mv REIS, Flavia Coelho Garcia dos et al. Analysis of cryptococcal extracellular vesicles: Experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of Titan cells. Microbiol Spectr, v. 9, n. 1, p. 1–15, 2021.
2165-0497
10.1128/Spectrum.00125-21.
url https://www.arca.fiocruz.br/handle/icict/50806
dc.language.iso.fl_str_mv por
language por
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv American Society for Microbiology
publisher.none.fl_str_mv American Society for Microbiology
dc.source.none.fl_str_mv reponame:Repositório Institucional da FIOCRUZ (ARCA)
instname:Fundação Oswaldo Cruz (FIOCRUZ)
instacron:FIOCRUZ
instname_str Fundação Oswaldo Cruz (FIOCRUZ)
instacron_str FIOCRUZ
institution FIOCRUZ
reponame_str Repositório Institucional da FIOCRUZ (ARCA)
collection Repositório Institucional da FIOCRUZ (ARCA)
bitstream.url.fl_str_mv https://www.arca.fiocruz.br/bitstream/icict/50806/1/license.txt
https://www.arca.fiocruz.br/bitstream/icict/50806/2/Spectrum.00125-21Ok.pdf
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repository.name.fl_str_mv Repositório Institucional da FIOCRUZ (ARCA) - Fundação Oswaldo Cruz (FIOCRUZ)
repository.mail.fl_str_mv repositorio.arca@fiocruz.br
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