Extracellular vesicle-mediated export of fungal RNA

Extracellular vesicles (EVs) play an important role in the biology of various organisms, including fungi, in which they are required for the trafficking of molecules across the cell wall. Fungal EVs contain a complex combination of macromolecules, including proteins, lipids and glycans. in this work, we aimed to describe and characterize RNA in EV preparations from the human pathogens Cryptococcus neoformans, Paracoccidiodes brasiliensis and Candida albicans, and from the model yeast Saccharomyces cerevisiae. the EV RNA content consisted mostly of molecules less than 250 nt long and the reads obtained aligned with intergenic and intronic regions or specific positions within the mRNA. We identified 114 ncRNAs, among them, six small nucleolar (snoRNA), two small nuclear (snRNA), two ribosomal (rRNA) and one transfer (tRNA) common to all the species considered, together with 20 sequences with features consistent with miRNAs. We also observed some copurified mRNAs, as suggested by reads covering entire transcripts, including those involved in vesicle-mediated transport and metabolic pathways. We characterized for the first time RNA molecules present in EVs produced by fungi. Our results suggest that RNA-containing vesicles may be determinant for various biological processes, including cell communication and pathogenesis.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Instituto Nacional de Ciencia e Tecnologia de Inovacao em Doencas Negligenciadas (INCT-IDN)Brazilian agency Fundacao Araucaria - PRONEXBrazilian agency Papes-FiocruzUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFundacao Oswaldo Cruz, CDTS, Rio de Janeiro, RJ, BrazilUniv Fed Rio de Janeiro, Inst Microbiol Prof Paulo de Goes, BR-21941 Rio de Janeiro, RJ, BrazilFiocruz PR, Fundacao Oswaldo Cruz, Inst Carlos Chagas, Curitiba, PR, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

Perceptions of consent, permission structures and approaches to the community: a rapid ethical assessment performed in North West Cameroon

BACKGROUND Understanding local contextual factors is important when conducting international collaborative studies in low-income country settings. Rapid ethical assessment (a brief qualitative intervention designed to map the ethical terrain of a research setting prior to recruitment of participants), has been used in a range of research-naïve settings. We used rapid ethical assessment to explore ethical issues and challenges associated with approaching communities and gaining informed consent in North West Cameroon. METHODS This qualitative study was carried out in two health districts in the North West Region of Cameroon between February and April 2012. Eleven focus group discussions (with a total of 107 participants) were carried out among adult community members, while 72 in-depth interviews included health workers, non-government organisation staff and local community leaders. Data were collected in English and pidgin, translated where necessary into English, transcribed and coded following themes. RESULTS Many community members had some understanding of informed consent, probably through exposure to agricultural research in the past. Participants described a centralised permission-giving structure in their communities, though there was evidence of some subversion of these structures by the educated young and by women. Several acceptable routes for approaching the communities were outlined, all including the health centre and the Fon (traditional leader). The importance of time spent in sensitizing the community and explaining information was stressed. CONCLUSIONS Respondents held relatively sophisticated understanding of consent and were able to outline the structures of permission-giving in the community. Although the structures are unique to these communities, the role of certain trusted groups is common to several other communities in Kenya and Ethiopia explored using similar techniques. The information gained through Rapid Ethical Assessment will form an important guide for future studies in North West Cameroon

Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

Background: Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown. Methodology/Principal Findings: We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicleassociated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100–300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasm

Potential Roles of Fungal Extracellular Vesicles during Infection

Submitted by Fabricia Pimenta ([email protected]) on 2018-06-29T19:43:36Z No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2016a.pdf: 1107301 bytes, checksum: ff6553fccea25073308ca3455a19b55e (MD5)Approved for entry into archive by Fabricia Pimenta ([email protected]) on 2018-07-26T17:25:44Z (GMT) No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2016a.pdf: 1107301 bytes, checksum: ff6553fccea25073308ca3455a19b55e (MD5)Made available in DSpace on 2018-07-26T17:25:45Z (GMT). No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2016a.pdf: 1107301 bytes, checksum: ff6553fccea25073308ca3455a19b55e (MD5) Previous issue date: 2016-06-29Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Departamento de Microbiologia Geral. Rio de Janeiro, RJ, Brasil / University Stony Brook. Department of Molecular Genetics and Microbiology. Stony Brook, New York, USA.University Stony Brook. Department of Molecular Genetics and Microbiology. Stony Brook, New York, USA.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Rio de Janeiro, RJ, Brasil.University, Stony Brook. Department of Molecular Genetics and Microbiology. Stony Brook, New York, USA / Veterans Administration Medical Center. Northport, NY, USA.Extracellular vesicles (EVs) are produced by virtually all cell types. Within the past few years, work in this field has revealed more information about fungal EVs. Fungal EVs have been shown to carry proteins, lipids, pigments, polysaccharides, and RNA; these components are known virulence factors, a fact which supports the hypothesis that fungal EVs concentrate pathogenic determinants. Additionally, recent studies have demonstrated that fungal EVs stimulate the host immune system. In this review, putative roles of fungal EVs are discussed, including their potential as vaccination tools and their possible contribution to pathogenesis in invasive fungal diseases

Where Do They Come from and Where Do They Go : Candidates for Regulating Extracellular Vesicle Formation in Fungi

In the past few years, extracellular vesicles (EVs) from at least eight fungal species were characterized. EV proteome in four fungal species indicated putative biogenesis pathways and suggested interesting similarities with mammalian exosomes. Moreover, as observed for mammalian exosomes, fungal EVs were demonstrated to be immunologically active. Here we review the seminal and most recent findings related to the production of EVs by fungi. Based on the current literature about secretion of fungal molecules and biogenesis of EVs in eukaryotes, we focus our discussion on a list of cellular proteins with the potential to regulate vesicle biogenesis in the fungi.Science, Faculty ofNon UBCMicrobiology and Immunology, Department ofReviewedFacult

The Anti-helminthic Compound Mebendazole Has Multiple Antifungal Effects against Cryptococcus neoformans

Submitted by Fabricia Pimenta ([email protected]) on 2018-06-29T19:03:08Z No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2017a.pdf: 3873194 bytes, checksum: 99621cb2d1cf68a20e492eae8b6b1757 (MD5)Approved for entry into archive by Fabricia Pimenta ([email protected]) on 2018-07-26T16:28:40Z (GMT) No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2017a.pdf: 3873194 bytes, checksum: 99621cb2d1cf68a20e492eae8b6b1757 (MD5)Made available in DSpace on 2018-07-26T16:28:40Z (GMT). No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2017a.pdf: 3873194 bytes, checksum: 99621cb2d1cf68a20e492eae8b6b1757 (MD5) Previous issue date: 2017-03-28Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Laboratório de Biologia Celular de Leveduras Patogênicas Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.Veterans Administration Medical Center. Northport, NY, USA / Stony Brook University. Department of Molecular Genetics and Microbiology. Stony Brook, NY, USA.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Laboratório de Biologia Celular de Leveduras Patogênicas Rio de Janeiro, RJ, Brasil.Cryptococcus neoformans is the most lethal pathogen of the central nervous system. The gold standard treatment of cryptococcosis, a combination of amphotericin B with 5-fluorocytosine, involves broad toxicity, high costs, low efficacy, and limited worldwide availability. Although the need for new antifungals is clear, drug research and development (R&D) is costly and time-consuming. Thus, drug repurposing is an alternative to R&D and to the currently available tools for treating fungal diseases. Here we screened a collection of compounds approved for use in humans seeking for those with anti-cryptococcal activity. We found that benzimidazoles consist of a broad class of chemicals inhibiting C. neoformans growth. Mebendazole and fenbendazole were the most efficient antifungals showing in vitro fungicidal activity. Since previous studies showed that mebendazole reaches the brain in biologically active concentrations, this compound was selected for further studies. Mebendazole showed antifungal activity against phagocytized C. neoformans, affected cryptococcal biofilms profoundly and caused marked morphological alterations in C. neoformans, including reduction of capsular dimensions. Amphotericin B and mebendazole had additive anti-cryptococcal effects. Mebendazole was also active against the C. neoformans sibling species, C. gattii. To further characterize the effects of the drug a random C. gattii mutant library was screened and indicated that the antifungal activity of mebendazole requires previously unknown cryptococcal targets. Our results indicate that mebendazole is as a promising prototype for the future development of anti-cryptococcal drugs

Role of the Apt1 protein in polysaccharide secretion by Cryptococcus neoformans

Submitted by Fabricia Pimenta ([email protected]) on 2019-02-01T15:47:25Z No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013f.pdf: 1895669 bytes, checksum: 6dea90228d254715230bf0e6f7e4def6 (MD5)Approved for entry into archive by Fabricia Pimenta ([email protected]) on 2019-03-07T20:40:52Z (GMT) No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013f.pdf: 1895669 bytes, checksum: 6dea90228d254715230bf0e6f7e4def6 (MD5)Made available in DSpace on 2019-03-07T20:40:52Z (GMT). No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013f.pdf: 1895669 bytes, checksum: 6dea90228d254715230bf0e6f7e4def6 (MD5) Previous issue date: 2014-06Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.The University of British Columbia. Faculty of Land and Food Systems. Department of Microbiology and Immunology. Michael Smith Laboratories. Vancouver, Canada.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.The University of British Columbia. Faculty of Land and Food Systems. Department of Microbiology and Immunology. Michael Smith Laboratories. Vancouver, Canada.The University of Texas at El Paso. Department of Biological Sciences. Border Biomedical Research Center. El Paso, Texas, USA.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.The University of Texas at El Paso. Department of Biological Sciences. Border Biomedical Research Center. El Paso, Texas, USA.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Ultraestrutura Celular Hertha Meyer. Rio de Janeiro, RJ, BrazilThe University of British Columbia. Faculty of Land and Food Systems. Department of Microbiology and Immunology. Michael Smith Laboratories. Vancouver, Canada.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.Flippases are key regulators of membrane asymmetry and secretory mechanisms. Vesicular polysaccharide secretion is essential for the pathogenic mechanisms of Cryptococcus neoformans. On the basis of the observations that flippases are required for polysaccharide secretion in plants and the putative Apt1 flippase is required for cryptococcal virulence, we analyzed the role of this enzyme in polysaccharide release by C. neoformans, using a previously characterized apt1Δ mutant. Mutant and wild-type (WT) cells shared important phenotypic characteristics, including capsule morphology and dimensions, glucuronoxylomannan (GXM) composition, molecular size, and serological properties. The apt1Δ mutant, however, produced extracellular vesicles (EVs) with a lower GXM content and different size distribution in comparison with those of WT cells. Our data also suggested a defective intracellular GXM synthesis in mutant cells, in addition to changes in the architecture of the Golgi apparatus. These findings were correlated with diminished GXM production during in vitro growth, macrophage infection, and lung colonization. This phenotype was associated with decreased survival of the mutant in the lungs of infected mice, reduced induction of interleukin-6 (IL-6) cytokine levels, and inefficacy in colonization of the brain. Taken together, our results indicate that the lack of APT1 caused defects in both GXM synthesis and vesicular export to the extracellular milieu by C. neoformans via processes that are apparently related to the pathogenic mechanisms used by this fungus during animal infection

The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans

Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of zinc finger containing transcriptional regulators known as GATA-factors. These factors activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. Cryptococcus neoformans GAT1 is an ortholog to the Aspergillus nidulans AreA and Candida albicans GAD genes. In an attempt to define the function of this transcriptional regulator in C. neoformans, we generated null mutants (gat1 Delta) of this gene. The gat 1 mutant exhibited impaired growth on all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore, the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently mimics a low-quality nitrogen source. Gal is not required for C. neoformans survival during macrophage infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identification of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitrogen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans. (C) 2010 Elsevier Inc. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, Brazil)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Amparo a Pesquisa no Estado do Rio de Janeiro (FAPERJ, Brazil)Fundacao de Amparo a Pesquisa no Estado do Rio de Janeiro (FAPERJ, Brazil)Financiadora de Estudos e Projetos (FINEP)Financiadora de Estudos e Projetos (FINEP, Brazil

Golgi Reassembly and Stacking Protein (GRASP) Participates in Vesicle-Mediated RNA Export in Cryptococcus neoformans

Golgi reassembly and stacking protein (GRASP) is required for polysaccharide secretion and virulence in Cryptococcus neoformans. In fungal species, extracellular vesicles (EVs) participate in the export of polysaccharides, proteins and RNA. In the present work, we investigated if EV-mediated RNA export is functionally connected with GRASP in C. neoformans using a graspΔ mutant. Since GRASP-mediated unconventional secretion involves autophagosome formation in yeast, we included the atg7Δ mutant with defective autophagic mechanisms in our analysis. All fungal strains exported EVs but deletion of GRASP or ATG7 profoundly affected vesicular dimensions. The mRNA content of the graspΔ EVs differed substantially from that of the other two strains. The transcripts associated to the endoplasmic reticulum were highly abundant transcripts in graspΔ EVs. Among non-coding RNAs (ncRNAs), tRNA fragments were the most abundant in both mutant EVs but graspΔ EVs alone concentrated 22 exclusive sequences. In general, our results showed that the EV RNA content from atg7Δ and WT were more related than the RNA content of graspΔ, suggesting that GRASP, but not the autophagy regulator Atg7, is involved in the EV export of RNA. This is a previously unknown function for a key regulator of unconventional secretion in eukaryotic cells

Cryptococcus neoformans glucuronoxylomannan and sterylglucoside are required for host protection in an animal vaccination model

© 2019 Colombo et al. Cryptococcus neoformans is an encapsulated fungal pathogen that causes meningoencephalitis. There are no prophylactic tools for cryptococcosis. Previously, our group showed that a C. neoformans mutant lacking the gene encoding sterylglucosidase (Δsgl1) induced protection in both immunocompetent and immunocompromised murine models of cryptococcosis. Since sterylglucosidase catalyzes degradation of sterylglucosides (SGs), accumulation of this glycolipid could be responsible for protective immunity. In this study, we analyzed whether the activity of SGs is sufficient for the protective effect induced by the Δsgl1 strain. We observed that the accumulation of SGs impacted several properties of the main polysaccharide that composes the fungal capsule, glucuronoxylomannan (GXM). We therefore used genetic manipulation to delete the SGL1 gene in the acapsular mutant Δcap59 to generate a double mutant (strain Δcap59/Δsgl1) that was shown to be nonpathogenic and cleared from the lung of mice within 7 days post-intranasal infection. The inflammatory immune response triggered by the Δcap59/Δsgl1 mutant in the lung differed from the response seen with the other strains. The double mutant did not induce protection in a vaccination model, suggesting that SG-related protection requires the main capsular polysaccharide. Finally, GXM-containing extracellular vesicles (EVs) enriched in SGs delayed the acute lethality of Galleria mellonella against C. neoformans infection. These studies highlighted a key role for GXM and SGs in inducing protection against a secondary cryptococcal infection, and, since EVs notoriously contain GXM, these results suggest the potential use of Δsgl1 EVs as a vaccination strategy for cryptococcosis. IMPORTANCE The number of deaths from cryptococcal meningitis is around 180,000 per year. The disease is the second leading cause of mortality among individuals with AIDS. Antifungal treatment is costly and associated with adverse effects and resistance, evidencing the urgency of development of both therapeutic and prophylactic tools. Here we demonstrate the key roles of polysaccharide- and glycolipidcontaining structures in a vaccination model to prevent cryptococcosis