Ras pathways are involved in the

Ras pathways are involved in the regulation of virulence in Cryptococcus neoformans [7] and Candida albicans [8,9]. To verify the importance of Ras in the survival response of the fungus in the host, a pathogenicity test of P. brasiliensis was performed before and after treatment with the Ras inhibitor (FPT inhibitor III). BALB/c mice were inoculated intratracheally with yeast Riboflavin preincubated in the presence or absence (control) of the Ras inhibitor. In this experiment, all the infected animals were euthanized after 30 days of infection; the lungs were isolated and macerated, and the material was loaded onto supplemented BHI plates. After 10 days, the plates were analyzed and the CFUs counted (Fig. 2C). Animals infected with yeast cells plus inhibitor showed significant reductions in the fungal cell numbers in their lungs, as demonstrated by the reduced CFUs/g of tissue recovered from this group compared to that recovered from the positive control (untreated infected group) (p < 0.05) (Fig. 2C). Therefore, these data indicate that Ras GTPase is required for the pathogenicity of P. brasiliensis.
Discussion Phagocytic cells generate potent ROS and RNS, which are toxic to most fungal pathogens [37]. However, some fungi have an efficient response that detoxifies these chemicals and repairs the molecular damage that they cause. This response helps fungal pathogens to survive their initial contact with the host immune system, which is predicted to be an important mechanism of virulence and is crucial for establishing disease [22,38]. NO homeostasis, based on the balance between NO synthesis and degradation, is important for regulating its physiological functions, since an excess of NO causes nitrosative stress due to the high reactivity of NO and NO-derived compounds [39]. In general, NO has toxic effects at higher concentrations. The cytotoxic effects caused by excessive levels of NO occur through various mechanisms, such as an increase in metal toxicity [40] and the promotion of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-targeted proteolysis by metacaspase through the S-nitrosylation of GAPDH [41,42]. On the other hand, fungi contain several NO detoxification systems, including NO dioxygenase (NOD) and S-nitrosoglutathione reductase (GSNOR) [43]. In this study, we examined the response of P. brasiliensis yeast cells to nitrosative stress. In our attempt to understand the biological pathways that may be targets for NO, since this free radical shows signaling properties [44], we found that the Ras GTPase and Hog1 pathways play central roles in the responses to nitrosative stress. Several studies have reported the strong involvement of the Ras protein in response to stress, differentiation and fungal growth after different stimuli [33,45,46], including oxidative stress [47], but the relationship of Ras activation to the nitrosative stress response in fungi has not yet been demonstrated. In mammalian cells, RNS are known to be involved in Ras-dependent cell proliferation [48,49]. Previous data from our group demonstrated that low levels of NO induce cell proliferation of P. brasiliensis in a Ras-dependent manner [5]. We developed a specific probe to detect Ras activation (Ras-GTP form) under stimulus with sub-toxic concentrations of NO, and this activation may induce the PKA pathway. Using the same probe, we demonstrated that a high concentration of NO induces the activation of Ras and consequently the expression of different genes involved in the redox response. Ras can be activated by NO due to S-nitrosylation [5,35,[49], [50], [51], [52], [53]]. S-nitrosylation is a reversible posttranslational modification derived from the interaction of NO with the thiol group of specific cysteines [54]. Redox regulation of Ras GTPases occurs in a redox-active cysteine (X) present in a conserved NKXD motif [55]. In P. brasiliensis, there is a putative site of S-nitrosylation in Ras1, Cys123 [5]. Several studies have shown that NO reacts with Ras through Cys 118 (Cys nitrosylable in mammalian cells) to promote nucleotide exchange and, consequently, Ras activation [[56], [57], [58]]. Thus, NO can increase Ras downstream signaling through the mitogen-activated kinase pathway [35]. The ability of Ras to induce different pathways is widely known. Previously, we showed that Ras participates in cell proliferation stimulated by low concentrations of NO [5], but in this study, we observed that high concentrations are also able to activate Ras. In addition, we detected that Ras was S-nitrosylated in both conditions, but the levels of Ras S-nitrosylation observed in this study were higher. These phenomena, apparently contradictory, can be explained by the dual role that NO exerts in cell signaling [59,60]. NO is a gaseous messenger that affects various biological functions, either at low concentrations as a signal transducer in many physiological processes or at high concentrations as a cytotoxic defense mechanism against pathogens that can also have a role in signaling processes [47]. In this sense, we propose that the Ras-dependent response to nitrosative stress occurs concomitantly with the inhibition of the PKA pathway, as hypothesized by Brown et al. [22], thus releasing the pathway to trigger the stress response genes.