Extracellular Vesicles From Paracoccidioides brasiliensis Can Induce the Expression of Fungal Virulence Traits In Vitro and Enhance Infection in Mice.

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.

iTRAQ-based proteomic analysis of Paracoccidioides brasiliensis in response to hypoxia.

Aerobic organisms require oxygen for energy. In the course of the infection, adaptation to hypoxia is crucial for survival of human pathogenic fungi. Members of the Paracoccidioides complex face decreased oxygen tensions during the life cycle stages. In Paracoccidioides brasiliensis proteomic responses to hypoxia have not been investigated and the regulation of the adaptive process is still unknown, and this approach allowed the identification of 216 differentially expressed proteins in hypoxia using iTRAQ-labelling. Data suggest that P. brasiliensis reprograms its metabolism when submitted to hypoxia. The fungus reduces its basal metabolism and general transport proteins. Energy and general metabolism were more representative and up regulated. Glucose is apparently directed towards glycolysis or the production of cell wall polymers. Plasma membrane/cell wall are modulated by increasing ergosterol and glucan, respectively. In addition, molecules such as ethanol and acetate are produced by this fungus indicating that alternative carbon sources probably are activated to obtain energy. Also, detoxification mechanisms are activated. The results were compared with label free proteomics data from Paracoccidioides lutzii. Biochemical pathways involved with acetyl-CoA, pyruvate and ergosterol synthesis were up-regulated in both fungi. On the other hand, proteins from TCA, transcription, protein fate/degradation, cellular transport, signal transduction and cell defense/virulence processes presented different profiles between species. Particularly, proteins related to methylcitrate cycle and those involved with acetate and ethanol synthesis were increased in P. brasiliensis proteome, whereas GABA shunt were accumulated only in P. lutzii. The results emphasize metabolic adaptation processes for distinct Paracoccidioides species.