Abstract
Background Paracoccidioides brasiliensis is a human thermal dimorphic pathogenic fungus. Survival of P. brasiliensis inside the host depends on the adaptation of this fungal pathogen to different conditions, namely oxidative stress imposed by immune cells.Aims and MethodologyIn this study, we evaluated the role of alternative oxidase (AOX), an enzyme involved in the intracellular redox balancing, during host-P. brasiliensis interaction. We generated a mitotically stable P. brasiliensis AOX (PbAOX) antisense RNA (aRNA) strain with a 70% reduction in gene expression. We evaluated the relevance of PbAOX during interaction of conidia and yeast cells with IFN-γ activated alveolar macrophages and in a mouse model of infection. Additionally, we determined the fungal cell's viability and PbAOX in the presence of H2O2.ResultsInteraction with IFN-γ activated alveolar macrophages induced higher levels of PbAOX gene expression in PbWt conidia than PbWt yeast cells. PbAOX-aRNA conidia and yeast cells had decreased viability after interaction with macrophages. Moreover, in a mouse model of infection, we showed that absence of wild-type levels of PbAOX in P. brasiliensis results in a reduced fungal burden in lungs at weeks 8 and 24 post-challenge and an increased survival rate. In the presence of H2O2, we observed that PbWt yeast cells increased PbAOX expression and presented a higher viability in comparison with PbAOX-aRNA yeast cells.ConclusionsThese data further support the hypothesis that PbAOX is important in the fungal defense against oxidative stress imposed by immune cells and is relevant in the virulence of P. brasiliensis.
Highlights
An essential event during cell growth and host invasion of pathogenic fungi is to avoid the toxic effects of reactive oxygen species (ROS), such as superoxide (O2), hydrogen peroxide (H2O2) and hydroxyl (OH2) radicals
These data further support the hypothesis that P. brasiliensis AOX (PbAOX) is important in the fungal defense against oxidative stress imposed by immune cells and is relevant in the virulence of P. brasiliensis
Generation of a P. brasiliensis PbAOX-antisense RNA (aRNA) strain A P. brasiliensis PbAOX-aRNA strain was generated via Agrobacterium tumefaciens-mediated transformation (ATMT) by expressing an aRNA oligonucleotide targeting the coding sequence of the PbAOX gene in PbWt yeast cells (Fig. 1A)
Summary
An essential event during cell growth and host invasion of pathogenic fungi is to avoid the toxic effects of reactive oxygen species (ROS), such as superoxide (O2), hydrogen peroxide (H2O2) and hydroxyl (OH2) radicals. Of its origin, these oxidizing agents may alter the bioenergetic status of the cell and affect essential metabolic pathways and/or its growth within host tissues, representing a toxic stimulus that decreases fungal survival [4,5]. Human pathogenic fungi such as Paracoccidioides brasiliensis, Histoplasma capsulatum, Candida albicans, among others, possess a defense mechanism against ROS that attempts to modulate the oxidative attack. Survival of P. brasiliensis inside the host depends on the adaptation of this fungal pathogen to different conditions, namely oxidative stress imposed by immune cells
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