Introduction: Paracoccidioides brasiliensis is a temperature-dependent dimorphic fungus that cause paracoccidioidomycosis (PCM). The capacity to evade the innate immune response of the host is due to its ability to respond and to survive the nitrosative stress caused by cells of the immune system. However, the regulation of signal transduction pathways associated to nitrosative stress response are poorly understood. Ras GTPases (Ras1 and Ras2) are well known to regulate antagonistically or cooperatively various cellular events in many fungi. Ras, in its activated form (Ras-GTP), interacts with effector proteins and can initiate a kinase cascade. In lower eukaryotes, Byr2 kinase represents a Ras target. In present study, we investigated the role of Ras GTPase in P. brasiliensis after in vitro stimulus with nitric oxide (NO). 

Methods: We constructed an expression plasmid containing the Byr2 Ras-binding domain (RBD) fused with GST (RBD-GST probe, which detects the Ras active form). Immunobloting assays were performed to detect the Ras and Hog-1 activation. Ras and Hog-1 interaction were performed by immunoprecipitation assays.

Results: We observed that low concentrations of NO induce cell proliferation in P. brasiliensis, while high concentrations promoted decrease in fungal viability. Additionally, we observe that both events (cell proliferation and death) are reversed in the presence of a Nitric Oxide scavenger (carboxy-PTIO). We reported by immunobloting that after stimulation with NO, the Ras active form was observed in fungal extracts. High NO concentrations induces the S-nitrosylation of Ras in P. brasiliensis and, in this conditions, Ras modulates the expression of antioxidant genes in response to nitrosative stress. We also investigated whether Hog-1 would be activated in response to nitrosative stress. Under these conditions we observed that Hog-1 is phosphorylated after NO treatment. In addition, when we used the RBD-GST probe and blotted with anti-Hog-1 antibody we observed that Ras-GTP can interact with MAPK Hog-1 after stimulation with high concentrations of NO. 

Conclusion: Finally, our data indicate that Ras GTPase and Hog-1 pathways may cross-talk in response to nitrosative stress in P. brasiliensis. S-nitrosylation of Ras probably activates this GTPase, which initiates a signaling cascade involving Ras-Byr2-By1-Hog. These responses could help the fungus to survive after their initial contacts with the host immune system, which is crucial for disease establishment.


Ras-MAPK pathway participates of the nitrosative stress response in pathogenic fungus Paracoccidioides brasiliensis