scholarly journals Extracellular Vesicle-Mediated RNA Release in Histoplasma capsulatum

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Lysangela R. Alves ◽  
Roberta Peres da Silva ◽  
David A. Sanchez ◽  
Daniel Zamith-Miranda ◽  
Marcio L. Rodrigues ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Stress Responses ◽  
Histoplasma Capsulatum ◽  
Pathogenic Fungi ◽  
Extracellular Vesicle ◽  
Fungal Species ◽  
Cellular Communication ◽  
Content Type ◽  
Rna Molecules ◽  
Rna Content

ABSTRACT Eukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNAs with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains of Histoplasma capsulatum, an important human-pathogenic fungal pathogen. A total of 124 mRNAs were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, whereas 31 were enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25 to 40 nucleotides in length) that were strain specific, with a greater number identified in EVs produced by the G217B strain. Remarkably, the most highly enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of microRNA (miRNA)-like molecules in fungal EVs. We also compared the transcriptome profiles of H. capsulatum with the RNA composition of EVs, and no correlation was observed. Taking the results together, our study provided information about the RNA molecules present in H. capsulatum EVs and about the differences in composition between the strains. In addition, we found no correlation between the most highly expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism. IMPORTANCE Extracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. EV-associated RNA classes have recently been described in pathogenic fungi; however, only a few reports of studies describing the RNAs in fungal EVs are available. Improved knowledge of EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates of Histoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA.

scholarly journals Extracellular vesicle-mediated RNA release inHistoplasma capsulatum

2019 ◽  
Author(s):  
Lysangela R. Alves ◽  
Roberta Peres da Silva ◽  
David A. Sanchez ◽  
Daniel Zamith-Miranda ◽  
Marcio L. Rodrigues ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Stress Responses ◽  
Histoplasma Capsulatum ◽  
Pathogenic Fungi ◽  
Extracellular Vesicle ◽  
Fungal Species ◽  
Cellular Communication ◽  
Rna Molecules ◽  
Rna Content ◽  
Reverse Strand

AbstractEukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNA classes with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains ofHistoplasma capsulatum, an important human fungal pathogen. A total of 124 mRNA were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, while 31 enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25-40 long) that were strain-specific, with a greater number of them identified in EVs produced by the G217B strain. Remarkably, the most enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of miRNA-like molecules in fungal EVs. We also compared the transcriptome profiles ofH. capsulatumwith the RNA composition of EVs and no correlation was observed. Altogether, our study provided information about the RNA molecules present inH. capsulatumEVs, and the differences in composition between the G186AR and G217B strains. In addition, we showed that the correlation between the most expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism.ImportanceExtracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. In pathogenic fungi, EV-associated RNA classes have recently been described; however, only a few studies describing the RNA in fungal EVs are available. An improved knowledge on EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates ofHistoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA.

scholarly journals Antibody Binding Alters the Characteristics and Contents of Extracellular Vesicles Released by Histoplasma capsulatum

mSphere ◽  
2016 ◽  
Vol 1 (2) ◽  
Author(s):  
Ludmila Matos Baltazar ◽  
Ernesto S. Nakayasu ◽  
Tiago J. P. Sobreira ◽  
Hyungwon Choi ◽  
Arturo Casadevall ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Histoplasma Capsulatum ◽  
Extracellular Vesicle ◽  
Fungal Species ◽  
Antibody Binding ◽  
Common Pathway ◽  
Fungal Cell ◽  
Content Type ◽  
Protein Loading ◽  
The Impact

ABSTRACT Diverse fungal species release extracellular vesicles, indicating that this is a common pathway for the delivery of molecules to the extracellular space. However, there has been no study reporting the impact of antibody binding to the fungal cell on extracellular vesicle release. In the present work, we observed that treatment of H. capsulatum cells with Hsp60-binding MAbs significantly changed the size and cargo of extracellular vesicles, as well as the enzymatic activity of certain virulence factors, such as laccase and phosphatase. Furthermore, this finding demonstrates that antibody binding can directly impact protein loading in vesicles and fungal metabolism. Hence, this work presents a new role for antibodies in the modification of fungal physiology. Histoplasma capsulatum produces extracellular vesicles containing virulence-associated molecules capable of modulating host machinery, benefiting the pathogen. Treatment of H. capsulatum cells with monoclonal antibodies (MAbs) can change the outcome of infection in mice. We evaluated the sizes, enzymatic contents, and proteomic profiles of the vesicles released by fungal cells treated with either protective MAb 6B7 (IgG1) or nonprotective MAb 7B6 (IgG2b), both of which bind H. capsulatum heat shock protein 60 (Hsp60). Our results showed that treatment with either MAb was associated with changes in size and vesicle loading. MAb treatments reduced vesicle phosphatase and catalase activities compared to those of vesicles from untreated controls. We identified 1,125 proteins in vesicles, and 250 of these manifested differences in abundance relative to that of proteins in vesicles isolated from yeast cells exposed to Hsp60-binding MAbs, indicating that surface binding of fungal cells by MAbs modified protein loading in the vesicles. The abundance of upregulated proteins in vesicles upon MAb 7B6 treatment was 44.8% of the protein quantities in vesicles from fungal cells treated with MAb 6B7. Analysis of orthologous proteins previously identified in vesicles from other fungi showed that different ascomycete fungi have similar proteins in their extracellular milieu, many of which are associated with virulence. Our results demonstrate that antibody binding can modulate fungal cell responses, resulting in differential loading of vesicles, which could alter fungal cell susceptibility to host defenses. This finding provides additional evidence that antibody binding modulates microbial physiology and suggests a new function for specific immunoglobulins through alterations of fungal secretion. IMPORTANCE Diverse fungal species release extracellular vesicles, indicating that this is a common pathway for the delivery of molecules to the extracellular space. However, there has been no study reporting the impact of antibody binding to the fungal cell on extracellular vesicle release. In the present work, we observed that treatment of H. capsulatum cells with Hsp60-binding MAbs significantly changed the size and cargo of extracellular vesicles, as well as the enzymatic activity of certain virulence factors, such as laccase and phosphatase. Furthermore, this finding demonstrates that antibody binding can directly impact protein loading in vesicles and fungal metabolism. Hence, this work presents a new role for antibodies in the modification of fungal physiology.

scholarly journals 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 ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Flavia C. G. Reis ◽  
Beatriz S. Borges ◽  
Luísa J. Jozefowicz ◽  
Bianca A. G. Sena ◽  
Ane W. A. Garcia ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Histoplasma Capsulatum ◽  
Chemical Properties ◽  
Pathogenic Fungi ◽  
Cryptococcus Gattii ◽  
Biologically Active ◽  
Content Type ◽  
Solid Media ◽  
Transmission Electron ◽  
Prion Transmission

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.

scholarly journals Robust Transcriptional Response to Heat Shock Impacting Diverse Cellular Processes despite Lack of Heat Shock Factor in Microsporidia

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Nora K. McNamara-Bordewick ◽  
Mia McKinstry ◽  
Jonathan W. Snow
Keyword(s):  
Heat Shock ◽  
Stress Responses ◽  
Target Genes ◽  
Treatment Strategies ◽  
Pathogenic Fungi ◽  
Fungal Species ◽  
Content Type ◽  
Novel Treatment ◽  
Novel Treatment Strategies ◽  
Insight Into

ABSTRACT The majority of fungal species prefer the 12° to 30°C range, and relatively few species tolerate temperatures higher than 35°C. Our understanding of the mechanisms underpinning the ability of some species to grow at higher temperatures is incomplete. Nosema ceranae is an obligate intracellular fungal parasite that infects honey bees and can cause individual mortality and contribute to colony collapse. Despite a reduced genome, this species is strikingly thermotolerant, growing optimally at the colony temperature of 35°C. In characterizing the heat shock response (HSR) in N. ceranae, we found that this and other microsporidian species have lost the transcriptional regulator HSF and possess a reduced set of putative core HSF1-dependent HSR target genes. Despite these losses, N. ceranae demonstrates robust upregulation of the remaining HSR target genes after heat shock. In addition, thermal stress leads to alterations in genes involved in various metabolic pathways, ribosome biogenesis and translation, and DNA repair. These results provide important insight into the stress responses of microsporidia. Such a new understanding will allow new comparisons with other pathogenic fungi and potentially enable the discovery of novel treatment strategies for microsporidian infections affecting food production and human health. IMPORTANCE We do not fully understand why some fungal species are able to grow at temperatures approaching mammalian body temperature. Nosema ceranae, a microsporidium, is a type of fungal parasite that infects honey bees and grows optimally at the colony temperature of 35°C despite possessing cellular machinery for responding to heat stress that is notably simpler than that of other fungi. We find that N. ceranae demonstrates a robust and broad response to heat shock. These results provide important insight into the stress responses of this type of fungus, allow new comparisons with other pathogenic fungi, and potentially enable the discovery of novel treatment strategies for this type of fungus.

scholarly journals Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease

2019 ◽  
Vol 85 (9) ◽  
Author(s):  
Yulong Wang ◽  
Zhangxun Wang ◽  
Wenjing Yang ◽  
Xiangyun Xie ◽  
Haiyan Cheng ◽  
...  
Keyword(s):  
Small Rna ◽  
Target Genes ◽  
Rna Binding ◽  
Fungal Species ◽  
Metarhizium Robertsii ◽  
Content Type ◽  
Rna Molecules ◽  
Significant Difference ◽  
Pol Iii ◽  
Short Tandem

ABSTRACT MicroRNAs (miRNAs) have been recognized as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. However, the functions and working mechanisms of hundreds of fungal miRNA-like (miR-like) RNAs are obscure. Here, we report that a short tandem target mimic (STTM) triggered the degradation of several fungal miR-like RNAs in two different fungal species, Metarhizium robertsii and Aspergillus flavus, and that small-RNA-degrading nucleases (SDNs) were indispensable for such degradation. STTMs were most effective when the fungal polymerase II (Pol II) promoter was used for their expression, while the Pol III promoter was less effective. The length of the STTM spacer, approximately 48 to 96 nucleotides, and the number of miR-like RNA binding sites, from 2 to 4 copies, showed no significant difference in the degradation of miR-like RNAs. STTMs modulated the miR-like RNA expression levels in at least two different fungal species, which further impacted fungal asexual growth and sporulation. Further analysis showed that the degraded miR-like RNAs in STTM mutants led to the upregulation of potential target genes involved in fungal development and conidial production, which result in different phenotypes in these mutants. The STTM technology developed in this study is an effective and powerful tool for the functional dissection of fungal miR-like RNAs. IMPORTANCE The development and application of STTM technology to block miR-like RNAs in M. robertsii and A. flavus may allow for efficient generation of miR-like RNA mutants in various fungi, providing a powerful tool for functional genomics of small RNA molecules in fungi.

scholarly journals The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes

2011 ◽  
Vol 10 (3) ◽  
pp. 343-351 ◽  
Author(s):  
Milene C. Vallejo ◽  
Alisson L. Matsuo ◽  
Luciane Ganiko ◽  
Lia C. Soares Medeiros ◽  
Kildare Miranda ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fungal Pathogens ◽  
Histoplasma Capsulatum ◽  
Paracoccidioides Brasiliensis ◽  
Pathogenic Fungus ◽  
Yeast Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dimorphic Fungus ◽  
Content Type

ABSTRACTExosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such asCryptococcus neoformansandHistoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes inParacoccidioides brasiliensis. P. brasiliensisis a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 ×g. P. brasiliensisantigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, withMarasmius oreadesagglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. InP. brasiliensiscells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role playedin vivoby vesicle components and α-galactosyl epitopes.

scholarly journals The RAM Network in Pathogenic Fungi

2012 ◽  
Vol 11 (6) ◽  
pp. 708-717 ◽  
Author(s):  
Sarah Saputo ◽  
Yeissa Chabrier-Rosello ◽  
Francis C. Luca ◽  
Anuj Kumar ◽  
Damian J. Krysan
Keyword(s):  
Fungal Pathogens ◽  
Pathogenic Fungi ◽  
Fungal Species ◽  
Specific Gene ◽  
Content Type ◽  
Network Function ◽  
Cellular Processes ◽  
Ram Network ◽  
Recent Developments ◽  
Protein Kinase Signaling

ABSTRACT The r egulation of A ce2 and m orphogenesis (RAM) network is a protein kinase signaling pathway conserved among eukaryotes from yeasts to humans. Among fungi, the RAM network has been most extensively studied in the model yeast Saccharomyces cerevisiae and has been shown to regulate a range of cellular processes, including daughter cell-specific gene expression, cell cycle regulation, cell separation, mating, polarized growth, maintenance of cell wall integrity, and stress signaling. Increasing numbers of recent studies on the role of the RAM network in pathogenic fungal species have revealed that this network also plays an important role in the biology and pathogenesis of these organisms. In addition to providing a brief overview of the RAM network in S. cerevisiae , we summarize recent developments in the understanding of RAM network function in the human fungal pathogens Candida albicans , Candida glabrata , Cryptococcus neoformans , Aspergillus fumigatus , and Pneumocystis spp.

scholarly journals tiRNA signaling via stress-regulated vesicle transfer in the hematopoietic niche

2021 ◽  
Author(s):  
Youmna S. Kfoury ◽  
Fei Ji ◽  
Michael Mazzola ◽  
David B. Sykes ◽  
Allison K. Scherer ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Stress Responses ◽  
Protein Translation ◽  
Extracellular Vesicle ◽  
Osteoblastic Cells ◽  
Myeloid Differentiation ◽  
Signaling Axis ◽  
Post Radiation ◽  
Hematopoietic Niche

AbstractExtracellular vesicles transfer complex biologic material between cells, whose role in in-vivo organismal physiology is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. Stimulating osteoblastic cells with parathyroid hormone or activating its receptor enhanced extracellular vesicle transfer, myeloid recovery post radiation and improved animal survival from Candida sepsis. The extracellular vesicles contained tiRNAs known to modulate protein translation. 5’-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and when transferred to granulocyte macrophage progenitors, increased protein translation, cell proliferation and myeloid differentiation. Therefore, EV-mediated tiRNA transfer provides a stress modulated signaling axis distinct from conventional cytokine-driven stress responses.One sentence summaryStress regulated tiRNA transfer alters hematopoiesis

scholarly journals In VitroPhotodynamic Inactivation of Plant-Pathogenic Fungi Colletotrichum acutatum and Colletotrichum gloeosporioides with Novel Phenothiazinium Photosensitizers

2013 ◽  
Vol 80 (5) ◽  
pp. 1623-1632 ◽  
Author(s):  
Henrique D. de Menezes ◽  
Gabriela B. Rodrigues ◽  
Simone de Pádua Teixeira ◽  
Nelson S. Massola ◽  
Luciano Bachmann ◽  
...  
Keyword(s):  
Methylene Blue ◽  
Colletotrichum Gloeosporioides ◽  
Light Exposure ◽  
Pathogenic Fungi ◽  
Fungal Species ◽  
Colletotrichum Acutatum ◽  
Photodynamic Treatment ◽  
Plant Host ◽  
Promising Alternative ◽  
Content Type

ABSTRACTThe increasing tolerance to currently used fungicides in both clinical and agricultural areas is of great concern. The nonconventional light-based approach of antimicrobial photodynamic treatment (APDT) is a promising alternative to conventional fungicides. We evaluated the effects of APDT with four phenothiazinium derivatives (methylene blue [MB], new methylene blue N [NMBN], toluidine blue O [TBO], and the novel pentacyclic phenothiazinium photosensitizer [PS] S137) on conidia of three fungal species (Colletotrichum acutatum,Colletotrichum gloeosporioides, andAspergillus nidulans). The efficacy of APDT with each PS was determined, initially, based on photosensitizer MICs. Additionally, the effects of APDT with two selected PSs (NMBN and S137) on survival of conidia were evaluated. The subcellular localization of the PS inC. acutatumconidia was determined. The effects of photodynamic treatments on leaves of the plant hostCitrus sinensiswere also investigated. APDT with S137 showed the lowest MIC. MICs for S137 were 5 μM for the three fungal species when a fluence of 25 J cm−2was used. APDT with NMBN (50 μM) and S137 (10 μM) resulted in a reduction in the survival of the conidia of all species of approximately 5 logs with fluences of ≥15 J cm−2. Washing of the conidia before light exposure did not prevent photodynamic inactivation. Both NMBN and S137 accumulated in cytoplasmic structures, such as lipid bodies, ofC. acutatumconidia. No damage to orange tree leaves was observed after APDT.

scholarly journals Comparison of the RNA Content of Extracellular Vesicles Derived from Paracoccidioides brasiliensis and Paracoccidioides lutzii

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 765 ◽  
Author(s):  
Roberta Peres da Silva ◽  
Larissa G. V. Longo ◽  
Julia P. C. da Cunha ◽  
Tiago J. P. Sobreira ◽  
Marcio L. Rodrigues ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Protein Modification ◽  
Paracoccidioides Brasiliensis ◽  
Pathogenic Fungi ◽  
In Vitro Translation ◽  
Rna Seq ◽  
Rna Content ◽  
Oxidation Reduction ◽  
Culture Supernatants

Paracoccidioides brasiliensis and P. lutzii cause human paracoccidioidomycosis. We have previously characterized the <200-nt RNA sub-populations contained in fungal extracellular vesicles (EVs) from P. brasiliensis Pb18 and other pathogenic fungi. We have presently used the RNA-seq strategy to compare the <200- and >200-nt RNA fractions contained in EVs isolated from culture supernatants of P. brasiliensis Pb18, Pb3, and P. lutzii Pb01. Shared mRNA sequences were related to protein modification, translation, and DNA metabolism/biogenesis, while those related to transport and oxidation-reduction were exclusive to Pb01. The presence of functional full-length mRNAs was validated by in vitro translation. Among small non-coding (nc)RNA, 15 were common to all samples; small nucleolar (sno)RNAs were enriched in P. brasiliensis EVs, whereas for P. lutzii there were similar proportions of snoRNA, rRNA, and tRNA. Putative exonic sRNAs were highly abundant in Pb18 EVs. We also found sRNA sequences bearing incomplete microRNA structures mapping to exons. RNA-seq data suggest that extracellular fractions containing Pb18 EVs can modulate the transcriptome of murine monocyte-derived dendritic cells in a transwell system. Considering that sRNA classes are involved in transcription/translation modulation, our general results may indicate that differences in virulence among fungal isolates can be related to their distinct EV-RNA content.

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