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Extracellular vesicles (EVs) are cellular components involved in cargo delivery to the extracellular environment, including the fungal cell wall. Their importance in cell–cell communication, cell wall remodeling, and fungal virulence is starting to be better explored. In the human pathogenic Paracoccidioides spp., our group has pioneered the description of the EV secretome, carbohydrate cargo, surface oligosaccharide ligands, lipid, and RNA content. Presently, we studied the role of fungal EVs in the context of the virulent/attenuated model of the P. brasiliensis Pb18 isolate, which consists of variants transiently displaying higher (vPb18) or attenuated (aPb18) virulence capacity. In this model, the virulence traits can be recovered through passages of aPb18 in mice. Here, we have been able to revert the aPb18 sensitivity to growth under oxidative and nitrosative stress upon previous co-incubation with vEVs from virulent vPb18. That was probably due to the expression of antioxidant molecules, considering that we observed increased gene expression of the alternative oxidase AOX and peroxiredoxins HYR1 and PRX1 , in addition to higher catalase activity. We showed that aEVs from aPb18 stimulated macrophages of the RAW 264.7 and bone marrow-derived types to express high levels of inflammatory mediators, specifically, TNF-α, IL-6, MCP-1, and NO. In our experimental conditions, subcutaneous treatment with EVs (three doses, 7-day intervals) before vPb18 challenge exacerbated murine PCM, as concluded by higher colony-forming units in the lungs after 30 days of infection and histopathology analysis. That effect was largely pronounced after treatment with aEVs, probably because the lung TNF-α, IFN-γ, IL-6, and MCP-1 concentrations were specially increased in aEV-treated when compared with vEV-treated mice. Our present studies were performed with EVs isolated from yeast cell washes of confluent cultures in Ham’s F-12 defined medium. Under these conditions, vEVs and aEVs have similar sizes but probably distinct cargo, considering that vEVs tended to aggregate upon storage at 4°C and −20°C. Additionally, aEVs have decreased amounts of carbohydrate and protein. Our work brings important contribution to the understanding of the role of fungal EVs in cell–cell communication and on the effect of EVs in fungal infection, which clearly depends on the experimental conditions because EVs are complex and dynamic structures.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by a group of cryptic species embedded in the Paracoccidioides brasiliensis complex and Paracoccidioides lutzii . Four species were recently inferred to belong to the P. brasiliensis complex, but the high genetic diversity found in both human and environmental samples have suggested that the number of lineages may be higher. This study aimed to assess the 43-kilodalton glycoprotein genotypes (Pb GP43 ) in paraffin-embedded samples from PCM patients to infer the phylogenetic lineages of the P. brasiliensis complex responsible for causing the infection. Formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients with histopathological diagnosis of PCM were analyzed. DNAs were extracted and amplified for a region of the second exon of the Pb GP43 gene. Products were sequenced and aligned with other Pb GP43 sequences available. A haplotype network and the phylogenetic relationships among sequences were inferred. Amino acid substitutions were investigated regarding the potential to modify physicochemical properties in the proteins. Six phylogenetic lineages were identified as belonging to the P. brasiliensis complex. Two lineages did not group with any of the four recognized species of the complex, and, interestingly, one of them comprised only FFPE samples. A coinfection involving two lineages was found. Five parsimony-informative sites were identified and three of them showed radical non-synonymous substitutions with the potential to promote changes in the protein. This study expands the knowledge regarding the genetic diversity existing in the P. brasiliensis complex and shows the potential of FFPE samples in species identification and in detecting coinfections.

Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Man p main chain linked to β-Gal f (1,3) and α-Man p (1,6) end units. The results also suggested the presence of small amounts of a (1→6)-Man p polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. The role of these newly identified components in the interaction with the host remains to be unraveled.

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. 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.

Fungal extracellular vesicles are able to cross the cell wall and transport molecules that help in nutrient acquisition, cell defense, and modulation of the host defense machinery. Here we present a detailed lipidomic analysis of extracellular vesicles released by Paracoccidioides brasiliensis at the yeast pathogenic phase. We compared data of two representative isolates, Pb3 and Pb18, which have distinct virulence profiles and phylogenetic background. Vesicle lipids were fractionated into different classes and analyzed by either electrospray ionization- or gas chromatography-mass spectrometry. We found two species of monohexosylceramide and 33 phospholipid species, including phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Among the phospholipid-bound fatty acids in extracellular vesicles, C181 predominated in Pb3, whereas C18:2 prevailed in Pb18. The prevalent sterol in Pb3 and Pb18 vesicles was brassicasterol, followed by ergosterol and lanosterol. Inter-isolate differences in sterol composition were observed, and also between extracellular vesicles and whole cells. The extensive lipidomic analysis of extracellular vesicles from two P. brasiliensis isolates will help to understand the composition of these fungal components/organelles and will hopefully be useful to study their biogenesis and role in host-pathogen interactions.

The fungal APSES protein family of transcription factors is characterized by a conserved DNA-binding motif facilitating regulation of gene expression in fungal development and other biological processes. However, their functions in the thermally dimorphic fungal pathogen Histoplasma capsulatum are unexplored. Histoplasma capsulatum switches between avirulent hyphae in the environment and virulent yeasts in mammalian hosts. We identified five APSES domain-containing proteins in H. capsulatum homologous to Swi6, Mbp1, Stu1 and Xbp1 proteins and one protein found in related Ascomycetes (APSES-family protein 1; Afp1). Through transcriptional analyses and RNA interference-based functional tests we explored their roles in fungal biology and virulence. Mbp1 serves an essential role and Swi6 contributes to full yeast cell growth. Stu1 is primarily expressed in mycelia and is necessary for aerial hyphae development and conidiation. Xbp1 is the only factor enriched specifically in yeast cells. The APSES proteins do not regulate conversion of conidia into yeast and hyphal morphologies. The APSES-family transcription factors are not individually required for H. capsulatum infection of cultured macrophages or murine infection, nor do any contribute significantly to resistance to cellular stresses including cell wall perturbation, osmotic stress, oxidative stress or antifungal treatment. Further studies of the downstream genes regulated by the individual APSES factors will be helpful in revealing their functional roles in H. capsulatum biology.

Extracellular vesicles (EVs) are bilayer-membrane cellular components that deliver protected cargo to the extracellular environment and can mediate long-distance signaling. We have previously reported that EVs isolated from the virulent fungal pathogen Paracoccidioides brasiliensis Vpb18 can revert the expression, in the attenuated variant Apb18, of stress-related virulence traits. We presently show that the Vev and Aev, respectively, produced by these variants display distinct proteomes, with prevalent functional enrichment in Vev related to oxidative stress response, signal transduction, transport, and localization, in addition to richer protein–protein interaction. Proteome sequences were obtained by nanoflow liquid chromatography coupled with tandem mass spectrometry (nano LC-ESI-MS/MS). The Vev and corresponding Vpb18 proteomes also differed, suggesting a selective bias in vesicle protein cargo. Moreover, sublethal oxidative (VevOxi) and nitrosative (VevNO) stress modulated the Vev proteome and a positive correlation between VevOxi/VevNO-enriched and Vev-enriched (relative to Aev) proteins was observed. Out of 145 fungal virulence factors detected in Vev, 64% were enriched, strongly suggesting that molecules with virulence roles in Paracoccidioides are selectively concentrated in Vev. Our study significantly advanced the field by exploring protein N-terminal acetylation to a dimension rarely investigated in fungal EV proteomics. The proportion of N-terminally acetylated proteins in Vev was higher than in Vpb18 and the presence of Nt-acetylation in Vev-enriched virulence factors varied across the samples, suggesting that it might interfere with protein sorting into EVs and/or protein functionality. Our findings highlight the relevance of our fungal model to unraveling the significance of fungal EVs in pathogenesis and phenotypic transfer.

Paracoccidioidomycosis is a systemic fungal disease caused by Paracoccidioides spp., predominantly affecting populations in Latin America, with Brazil reporting the highest number of cases. The infection is associated with severe pulmonary and systemic manifestations. Previous studies have highlighted the role of fungal proteases in adhesion, invasion, and the modulation of host immune responses, implicating them as key virulence factors. Our group previously demonstrated that Paracoccidioides restrepiensis secretes proteases that activate protease‐activated receptors (PAR‐1 and PAR‐2) in human lung epithelial cells, stimulating the secretion of proinflammatory cytokines, including IL‐6 and IL‐8. We hypothesized that P. restrepiensis secretes proteases that are capable of degrading key host cytokines, such as IL‐6, thereby contributing to modulate the host immune response during infection. This study is aimed at identifying and characterizing proteases secreted by P. restrepiensis that degrade human IL‐6. Proteases secreted by P. restrepiensis were isolated using a p ‐aminomethylbenzamidine ( p ABA)‐Sepharose affinity column. Protease‐containing fractions were incubated with recombinant human IL‐6 and further analyzed by Western blot to evaluate their ability to degrade this cytokine. Fractions were submitted to liquid chromatography and mass spectrometry to characterize the proteome content, focusing on the identification of fungal proteases. The hydrolysis of IL‐6 in the presence of different protease inhibitors was also analyzed to confirm the specific activity of the fungal proteases. Enzymatic assays revealed proteases that hydrolyze human IL‐6, suggesting a mechanism by which P. restrepiensis modulates the host immune response. In addition, mass spectrometry analysis confirmed the presence of a serine protease in the protease activity–containing fractions. These findings indicate that Paracoccidioides proteases may modulate host immune response by degrading key cytokines involved in inflammation and host defense.

Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available. This is the first study of gp43 using genetically modified P. brasiliensis.