Abstract
Paracoccidioides brasiliensis, a causative agent of paracoccidioidomycosis (PCM), should be able to adapt to dramatic environmental changes inside the infected host after inhalation of air-borne conidia and transition to pathogenic yeasts. Proteins with antioxidant functions may protect fungal cells against reactive oxygen (ROS) and nitrogen (RNS) species generated by phagocytic cells, thus acting as potential virulence factors. Ras GTPases are involved in stress responses, cell morphology, and differentiation in a range of organisms. Ras, in its activated form, interacts with effector proteins and can initiate a kinase cascade. In lower eukaryotes, Byr2 kinase represents a Ras target. The present study investigated the role of Ras in P. brasiliensis after in vitro stimulus with ROS or RNS. We have demonstrated that low concentrations of H2O2 (0.1 mM) or NO2 (0.1–0.25 µM) stimulated P. brasiliensis yeast cell proliferation and that was not observed when yeast cells were pre-incubated with farnesyltransferase inhibitor. We constructed an expression plasmid containing the Byr2 Ras-binding domain (RBD) fused with GST (RBD-Byr2-GST) to detect the Ras active form. After stimulation with low concentrations of H2O2 or NO2, the Ras active form was observed in fungal extracts. Besides, NO2 induced a rapid increase in S-nitrosylated Ras levels. This alternative posttranslational modification of Ras, probably in residue Cys123, would lead to an exchange of GDP for GTP and consequent GTPase activation in P. brasiliensis. In conclusion, low concentrations of H2O2 or NO2 stimulated P. brasiliensis proliferation through Ras activation.
Highlights
Paracoccidioides brasiliensis is a thermo-dependent dimorphic fungus responsible for paracoccidioidomycosis (PCM), a systemic mycosis that is prevalent in Latin America
The ability of pathogenic fungi to resist the deleterious effects of reactive oxygen species (ROS) and/or reactive oxygen (ROS) and nitrogen (RNS) is foreseen as an important virulence mechanism, in relation to its contact with phagocytic host cells
We have here demonstrated that sub-toxic concentrations of ROS and RNS are capable of stimulating P. brasiliensis cell proliferation in a Ras GTPase activation-dependent manner
Summary
Paracoccidioides brasiliensis is a thermo-dependent dimorphic fungus responsible for paracoccidioidomycosis (PCM), a systemic mycosis that is prevalent in Latin America. The major protective host immune response against P. brasiliensis is mediated by cells, as evidenced by granuloma formation [1]. Macrophages can engulf this microorganism and confine it within phagosomes where, by action of microbicidal molecules and/or by restriction of essential nutrients, the pathogen can be destroyed. Among the molecules that exert fungicidal action in phagosomes are hydrogen peroxide (H2O2), nitric oxide (NO) and their derivatives. These are generated by NADPH oxidase and inducible nitric oxide synthase (iNOS), respectively [3,4]. Immune system cells activate the NADPH oxidase complex to generate radical superoxide (O22), which is subsequently converted into H2O2. Macrophages express iNOS with activation of the L-arginine-nitric oxide pathway, and subsequent NO production
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