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

Abstract

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.

Aspergillus fumigatus-Derived Extracellular Vesicles Mitigate Fungal Keratitis by Modulating the Immune Cell Function

“…M1-polarized macrophages exhibit a propensity for phagocytosis, microbial eradication, tumor cell elimination, and the induction of adaptive immunity. In vitro studies have shown that Aspergillus flavus, Trichoderma interrogans, and Paracoccidioides brasiliensis-derived EVs induce macrophage polarization toward the M1 phenotype, enhancing the clearance of microorganisms originating from the EVs’ source. ,, Our results highlighted the bioactivity of AF-derived EVs in effectively stimulating the upregulation of the inflammation level of RAW264.7 cells. Similarly, AF-derived EVs were found to modulate the inflammatory response of PMNs, as evidenced by increased mRNA expression of proinflammatory mediators, enhanced conidia phagocytosis, and improved conidia-killing ability.…”

“…The performance of cell-derived EVs in Ophthalmology has been studied on multiple diseases, and the focus has mostly been on their regeneration function for neuro or specific component cells . Studies on EVs derived from Candida albicans, Dermatophyte Trichophyton, Cryptococcus, and others have revealed that the immunogenic components within these cargo materials impact host immune function. − Due to their cell-free nature and the presence of multiple pathogen-associated molecular patterns (PAMPs), EVs possess the ability to induce robust and sustained immune responses. − Pre-emptive application of fungal EVs to the host has shown to mitigate the lethality of fungal infections and reduce tissue destruction caused by the immune response of the host. ,− …”

Participation of Extracellular Vesicles of Extracellular Pathogens in Contact-Independent Mechanisms of Pathogenicity

“…EVs play a fundamental role in various processes such as: pathogen adhesion and invasion, modulation of the immune response, in cell damage and death, as well as in the transmission of resistance and survival factors between pathogens (Table 1) (Figure 1). The inducing effect of EVs depends on factors such as the type of target cell [20], the species and even the strain in study [7,42], as well as the amount and time in which the vesicles interact [4].…”

“…Similar results have been reported through in vivo assays, where it was demonstrated that the interaction of EVs emitted by pathogenic organisms interacting with mice is related to greater cellular invasion. For example, exposing mice to EVs days before infection with the fungi S. brasilensis [23] or P. brasilensis [42] increased the fungal load in skin and lung lesions, respectively. On the other hand, the heart and blood of mice treated with EVs prior to infection with the protozoan T. cruzi, shown a higher parasite load compared to mice without exposure to EVs [36].…”

“…Paracoccidioides brasilensis [42], Candida auris [67], Sporothrix brasilensis [23], Cryptococcus neofarmans [22].…”