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AutorReis, Flavia Coelho Garcia dos
AutorBorges, Beatriz Santana
AutorJozefowicz, Luísa J.
AutorSena, Bianca A. G.
AutorGarcia, Ane W. A.
AutorMedeiros, Lia Carolina Soares
AutorMartins, Sharon de Toledo
AutorHonorato, Leandro
AutorSchrank, Augusto
AutorVainstein, Marilene Henning
AutorKmetzsch, Lívia
AutorNimrichter, Leonardo
AutorAlves, Lysangela Ronalte
AutorStaats, Charley C.
AutorRodrigues, Marcio Lourenço
Data de acesso2019-07-17T15:31:35Z
Data de disponibilização2019-07-17T15:31:35Z
Data do publicação2019
CitaçãoREIS, Flávia C. G. et al. A novel protocol for the isolation of fungal extracellular vesicles reveals the participation of a putative scramblase in polysaccharide export and capsule construction in Cryptococcus gattii. mSphere, v. 4, n. 2, p. 1–15, 2019.pt_BR
ISSN2379-5042pt_BR
URIhttps://www.arca.fiocruz.br/handle/icict/34192
Idiomaporpt_BR
EditorAmerican Society for Microbiologypt_BR
Direito Autoralopen accesspt_BR
Palavras-chaveScramblasept_BR
TítuloA novel protocol for the isolation of fungal extracellular vesicles reveals the participation of a putative scramblase in polysaccharide export and capsule construction in Cryptococcus gattiipt_BR
Tipo do documentoArticlept_BR
DOI10.1128/mSphere.00080-19
Resumo em InglêsRegular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii and Cryptococcus neoformans on solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in solid media. The properties of cryptococcal vesicles varied according to the culture medium used and the EV-producing species. EV detection was reproduced with an acapsular mutant of C. neoformans, as well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces cerevisiae Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questions related to cryptococcal secretion. On the basis that polysaccharide export in Cryptococcus requires highly organized membrane traffic culminating with EV release, we analyzed the participation of a putative scramblase (Aim25; CNBG_3981) in EV-mediated GXM export and capsule formation in C. gattii EVs from a C. gattiiaim25Δ strain differed from those obtained from wild-type (WT) cells in physical-chemical properties and cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, EVs obtained from the aim25Δ mutant were more efficiently used as a source of capsular polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and increased capsular dimensions. These results associate the description of a novel protocol for the isolation of fungal EVs with the identification of a previously unknown regulator of polysaccharide release. Important information: Extracellular vesicles (EVs) are fundamental components of the physiology of cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of transfer of antifungal resistance and virulence, as well as in immune stimulation and prion transmission. However, studies on the functions of fungal EVs are still limited by the lack of efficient methods for isolation of these compartments. In this study, we developed an alternative protocol for isolation of fungal EVs and demonstrated an application of this new methodology in the study of the physiology of the fungal pathogen Cryptococcus gattii Our results describe a fast and reliable method for the study of fungal EVs and reveal the participation of scramblase, a phospholipid-translocating enzyme, in secretory processes of C. gattii.pt_BR
AfiliaçãoFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.pt_BR
AfiliaçãoFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.pt_BR
AfiliaçãoFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.pt_BR
AfiliaçãoFundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil.pt_BR
AfiliaçãoUniversidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.pt_BR
AfiliaçãoFundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil.pt_BR
AfiliaçãoFundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil.pt_BR
AfiliaçãoUniversidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.pt_BR
AfiliaçãoUniversidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.pt_BR
AfiliaçãoUniversidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.pt_BR
AfiliaçãoUniversidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.pt_BR
AfiliaçãoUniversidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.pt_BR
AfiliaçãoFundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil.pt_BR
AfiliaçãoUniversidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.pt_BR
AfiliaçãoUniversidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.pt_BR
Palavras-chave em inglêsExtracellular Vesiclespt_BR
Palavras-chave em inglêsFungipt_BR
Palavras-chave em inglêsSecretory Processpt_BR
Palavras-chave em inglêsPhospholipid Transfer Proteinspt_BR
Palavras-chave em espanholHongospt_BR
Palavras-chave em espanholVías Secretoraspt_BR
Palavras-chave em espanholProteínas de Transferencia de Fosfolípidospt_BR
DeCSCryptococcuspt_BR
DeCSVesículas Extracelularespt_BR
DeCSFungospt_BR
DeCSVia Secretóriapt_BR
DeCSProteínas de Transferência de Fosfolipídeospt_BR
e-ISSN2379-5042


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