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AutorReis, Flavia Coelho Garcia dos
AutorGimenez, Bianca
AutorJozefowicz, Luísa J.
AutorCastelli, Rafael Fernando
AutorMartins, Sharon de Toledo
AutorAlves, Lysangela Ronalte
AutorOliveira, Haroldo Cesar de
AutorRodrigues, Marcio Lourenço
Fecha de acceso2022-01-19T11:58:49Z
Fecha de disponibilización2022-01-19T11:58:49Z
Fecha de publicación2021
ReferenciaREIS, 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.pt_BR
ISSN2165-0497pt_BR
URIhttps://www.arca.fiocruz.br/handle/icict/50806
Idiomaporpt_BR
EditorAmerican Society for Microbiologypt_BR
Derechos de autoropen accesspt_BR
Palabras clave en PortuguésCryptococcuspt_BR
TítuloAnalysis 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 cellspt_BR
Tipo del documentoArticlept_BR
DOI10.1128/Spectrum.00125-21.
Resumen en InglésExtracellular 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.pt_BR
AfiliaciónFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.pt_BR
AfiliaciónFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.pt_BR
AfiliaciónFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.pt_BR
AfiliaciónFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.pt_BR
AfiliaciónFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.pt_BR
AfiliaciónFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.pt_BR
AfiliaciónFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.pt_BR
AfiliaciónFundação 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.pt_BR
Palavras clave en InglêsExtracellular Vesiclespt_BR
Palavras clave en InglêsPathogenicitypt_BR
Palavras clave en InglêsFungal Cellspt_BR
Palavras claveVesículas Extracelularespt_BR
Palavras clavePatogenicidadpt_BR
Palavras claveCélulas Fúngicaspt_BR
Palavras clave en FrancêsVésicules extracellulairespt_BR
Palavras clave en FrancêsPathogénicitépt_BR
Palavras clave en FrancêsCellules Fongiquespt_BR
DeCSVesículas Extracelularespt_BR
DeCSPatogenicidadept_BR
DeCSCélulas Fúngicaspt_BR


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