A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in <italic toggle="yes">Cryptococcus gattii</italic>

ABSTRACT Regular 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 Cryptococ...

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Autores principales:	Flavia C. G. Reis, Beatriz S. Borges, Luísa J. Jozefowicz, Bianca A. G. Sena, Ane W. A. Garcia, Lia C. Medeiros, Sharon T. Martins, Leandro Honorato, Augusto Schrank, Marilene H. Vainstein, Livia Kmetzsch, Leonardo Nimrichter, Lysangela R. Alves, Charley C. Staats, Marcio L. Rodrigues
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Cryptococcus extracellular vesicles fungi secretion scramblase Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/f285e1929e83407b83579be30af37ba1
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spelling	oai:doaj.org-article:f285e1929e83407b83579be30af37ba12021-11-15T15:22:22ZA Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in <italic toggle="yes">Cryptococcus gattii</italic>10.1128/mSphere.00080-192379-5042https://doaj.org/article/f285e1929e83407b83579be30af37ba12019-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00080-19https://doaj.org/toc/2379-5042ABSTRACT Regular 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. gattii aim25Δ 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. IMPORTANCE 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.Flavia C. G. ReisBeatriz S. BorgesLuísa J. JozefowiczBianca A. G. SenaAne W. A. GarciaLia C. MedeirosSharon T. MartinsLeandro HonoratoAugusto SchrankMarilene H. VainsteinLivia KmetzschLeonardo NimrichterLysangela R. AlvesCharley C. StaatsMarcio L. RodriguesAmerican Society for MicrobiologyarticleCryptococcusextracellular vesiclesfungisecretionscramblaseMicrobiologyQR1-502ENmSphere, Vol 4, Iss 2 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Cryptococcus extracellular vesicles fungi secretion scramblase Microbiology QR1-502
spellingShingle	Cryptococcus extracellular vesicles fungi secretion scramblase Microbiology QR1-502 Flavia C. G. Reis Beatriz S. Borges Luísa J. Jozefowicz Bianca A. G. Sena Ane W. A. Garcia Lia C. Medeiros Sharon T. Martins Leandro Honorato Augusto Schrank Marilene H. Vainstein Livia Kmetzsch Leonardo Nimrichter Lysangela R. Alves Charley C. Staats Marcio L. Rodrigues A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in <italic toggle="yes">Cryptococcus gattii</italic>
description	ABSTRACT Regular 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. gattii aim25Δ 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. IMPORTANCE 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.
format	article
author	Flavia C. G. Reis Beatriz S. Borges Luísa J. Jozefowicz Bianca A. G. Sena Ane W. A. Garcia Lia C. Medeiros Sharon T. Martins Leandro Honorato Augusto Schrank Marilene H. Vainstein Livia Kmetzsch Leonardo Nimrichter Lysangela R. Alves Charley C. Staats Marcio L. Rodrigues
author_facet	Flavia C. G. Reis Beatriz S. Borges Luísa J. Jozefowicz Bianca A. G. Sena Ane W. A. Garcia Lia C. Medeiros Sharon T. Martins Leandro Honorato Augusto Schrank Marilene H. Vainstein Livia Kmetzsch Leonardo Nimrichter Lysangela R. Alves Charley C. Staats Marcio L. Rodrigues
author_sort	Flavia C. G. Reis
title	A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in <italic toggle="yes">Cryptococcus gattii</italic>
title_short	A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in <italic toggle="yes">Cryptococcus gattii</italic>
title_full	A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in <italic toggle="yes">Cryptococcus gattii</italic>
title_fullStr	A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in <italic toggle="yes">Cryptococcus gattii</italic>
title_full_unstemmed	A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in <italic toggle="yes">Cryptococcus gattii</italic>
title_sort	novel protocol for the isolation of fungal extracellular vesicles reveals the participation of a putative scramblase in polysaccharide export and capsule construction in <italic toggle="yes">cryptococcus gattii</italic>
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/f285e1929e83407b83579be30af37ba1
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A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in <italic toggle="yes">Cryptococcus gattii</italic>

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