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.

Highlights

  • Human paracoccidioidomycosis (PCM) is currently defined as a systemic mycosis caused by isolates of the Paracoccidioides brasiliensis complex, P. brasiliensis sensu strictu (S1 main group), P. americana (PS2 group), P. restrepiensis (PS3 group), and P. venezuelensis (PS4 group), and by Paracoccidioides lutzii, formerly referred to as “Pb01-like” (Matute et al, 2006; Teixeira et al, 2014; Turissini et al, 2017)

  • We found that the lung tumor necrosis factor-a (TNF-a), Interferon g (IFN-g), and MCP-1 concentrations were specially increased in aEV/vPb18 animals when compared with non-treated controls and vEV/vPb18

  • The virulent/attenuated Pb18 model of P. brasiliensis proved to be useful to study the roles of fungal extracellular vesicles (EVs) because it consists of variants from the same isolate transiently displaying higher or attenuated virulence capacity in mice

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Summary

Introduction

Human paracoccidioidomycosis (PCM) is currently defined as a systemic mycosis caused by isolates of the Paracoccidioides brasiliensis complex, P. brasiliensis sensu strictu (S1 main group), P. americana (PS2 group), P. restrepiensis (PS3 group), and P. venezuelensis (PS4 group), and by Paracoccidioides lutzii, formerly referred to as “Pb01-like” (Matute et al, 2006; Teixeira et al, 2014; Turissini et al, 2017). Protection against PCM is provided by Th1driven pro-inflammatory immune responses initiated by dendritic cells expressing interleukin (IL)-12 (Gow et al, 2017; Burger, 2021; Ferná ndez-Garcıa and Cuellar-Rodrıguez, 2021). Interferon g (IFN-g) activates macrophages to express tumor necrosis factor-a (TNF-a) that supports granuloma persistence. The production of effector molecules such as reactive oxygen (ROS) and nitrogen (NOS) species of the respiratory burst by the immune cells is a key effector mechanism in fungal clearance (Gow et al, 2017; Burger, 2021; Fernández-Garcıá and Cuellar-Rodrıǵ uez, 2021)

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