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.

The Human Pathogen Paracoccidioides brasiliensis Has a Unique 1-Cys Peroxiredoxin That Localizes Both Intracellularly and at the Cell Surface.

Paracoccidioides brasiliensis is a temperature-dependent dimorphic fungus that causes systemic paracoccidioidomycosis, a granulomatous disease. The massive production of reactive oxygen species (ROS) by the host's cellular immune response is an essential strategy to restrain the fungal growth. Among the ROS, the hydroperoxides are very toxic antimicrobial compounds and fungal peroxidases are part of the pathogen neutralizing antioxidant arsenal against the host's defense. Among them, the peroxiredoxins are highlighted, since some estimates suggest that they are capable of decomposing most of the hydroperoxides generated in the host's mitochondria and cytosol. We presently characterized a unique P. brasiliensis 1-Cys peroxiredoxin (PbPrx1). Our results reveal that it can decompose hydrogen peroxide and organic hydroperoxides very efficiently. We showed that dithiolic, but not monothiolic compounds or heterologous thioredoxin reductant systems, were able to retain the enzyme activity. Structural analysis revealed that PbPrx1 has an α/ß structure that is similar to the 1-Cys secondary structures described to date and that the quaternary conformation is represented by a dimer, independently of the redox state. We investigated the PbPrx1 localization using confocal microscopy, fluorescence-activated cell sorter, and immunoblot, and the results suggested that it localizes both in the cytoplasm and at the cell wall of the yeast and mycelial forms of P. brasiliensis, as well as in the yeast mitochondria. Our present results point to a possible role of this unique P. brasiliensis 1-Cys Prx1 in the fungal antioxidant defense mechanisms.