Paracoccidioides brasiliensis and P. lutzii are the causative agents of paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America. These fungi are considered facultative intracellular pathogens, able to survive and replicate inside macrophages. After infection, the survival depends on its adaptability to host conditions, such as nitrosative stress caused by nitric oxide (NO) produced by the immune cells. Currently, NO is viewed as a remarkably important signaling molecule, involved in regulating stress responses. Protein S-nitrosylation, characterized by covalent attachment of nitroso group to cysteine thiol, has emerged as a central mechanism of NO-dependent cellular regulation. We have previously reported that low NO concentrations leads to Ras S-nitrosylation and induces cell proliferation in P. brasiliensis. However, others targets of NO in this fungus are unknown. In present study, using a combination of the biotin switch assay and label-free LC-MS/MS analysis, we revealed the S-nitroso-proteome profile of P. brasiliensis isolate 18 (Pb18) under different NO concentration. 

Methods: Immunobloting and enzymatic assays were performed to assess protein S-nitrosylated profiles and redox profile respectively. In order to determine S-nitrosylated proteins, first it was performed biotinylation assay followed by protein reduction, alkylation, digestion and peptide desalting. Next, samples were injected in Orbitrap Velos mass spectrometry connected to an Easy-nLCII. The MS raw files were processed and searched using MaxQuant software. All validated proteins were submitted to Blast2GO algorithm. 

Results: Immunoblotting results showed distinct band patterns among samples treated with different NO concentrations were observed. S-nytrosilation levels increased when cells were treated with 0.25 µM NO, suggesting the participation of this post-translational modifications in the fungus growth induced by low concentrations of RNS. Changes in the fungus redox state according with NO concentrations were also observed by enzymatic assays and evaluation of the GSH (reduced glutathione) and GSSG (oxidized glutathione) levels. Using a mass spectrometry-based approach, we mapped 335 S-nitrosylated proteins, where 48 and 6 were exclusively found on the groups treated with 0.25 and 10 µM of NO2 respectively. With this approach, we observed that proteins treated with NO low concentrations presented a proliferative response pattern, with several proteins involved with cellular cycle regulation and growth and would represent, as last analysis, molecules with an important role to fungi virulence. On the other hand, proteins stimulated with NO high concentrations exhibited a survival response pattern.

Conclusions: It has been demonstrated that the distinct redox-dependent events (cell proliferation or survival) were promoted by different NO concentrations, which may help us understand RNS antifungal properties and identify potential molecular targets to future development of new drugs



Differential modulation of S-nitrosoproteome of Paracoccidioides brasiliensis by Nitric Oxide: change in S-nitrosylation levels and fungal redox status.