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    • Signaling pathways that control the redox

    2021-09-24

    Signaling pathways that control the redox stress response in P. brasiliensis are poorly known, but in other dimorphic fungi, the involvement of MAPK is an important factor in this process [[13], [14], [15]]. For example, when alveolar macrophages are challenged with Aspergillus or Cryptococcus conidia, the macrophages are able to produce RNS levels as high as 50 μM, and the fungi are efficiently phagocytosed [16]. However, even when the host is able to produce high concentrations of NO, the fungi still possess mechanisms to adapt to nitrosative stress and cause disease. This is because fungi have an effective enzymatic repertoire to counteract the RNS produced by the host, which functions as a virulence factor [17,18]. Currently, different research groups have sought to study the mechanisms that regulate the response of fungi to oxidative stress [[19], [20], [21]], but despite the importance of RNS in the death of the fungi, as well as the signaling process, little progress has been made even though this field has become a topic of interest [18,22]. In this study, we highlight the cross-talk between Ras GTPase and Hog1 MAPK activation in the context of nitrosative stress in the human pathogenic fungus P. brasiliensis.
    Materials and methods
    Results We have previously shown that different levels of NO (sub-toxic or toxic concentrations) can induce distinct responses within P. brasiliensis [5]. Initially, we investigated the intracellular NO levels in P. brasiliensis yeast treated with 0.25 and 1 mM NO2 for 5 h at 37 °C (in mYPD pH 5.6). In both conditions tested, we detected the presence of intracellular NO, and the peak of the 1 mM NO showed 4 times more NO than the control (Fig. 1A). To better understand this type of stimulus in P. brasiliensis, logarithmically growing yeast bosentan were cultured in RPMI media for 24 h and subsequently treated with different concentrations bosentan of NO (0.25 or 1 mM) in vitro for 5 h at 37 °C in pH 5.6 in the presence or absence of the NO scavenger cPTIO (30 mM, Sigma-Aldrich, St. Louis, MO, USA). We observed a decrease in cellular viability in yeast cells preincubated for 5 h with higher concentrations of NO (1 mM) (Fig. 1B). Yeast cells preincubated with low concentrations of NO (0.25 mM) responded with significant cell proliferation (3.2 ± 0.29 × 105 CFU) compared to unstimulated controls (1.8 ± 0.16 × 105 CFU) (Fig. 1B). These data confirm our previous results [5]. However, the treatment of P. brasiliensis yeast cells with low levels of NO (0.25 mM) in the presence of the NO scavenger cPTIO (30 μM) inhibited fungal proliferation, while the treatment of yeasts cells with 1 mM NO in the presence of cPTIO inhibited cellular death (Fig. 1B). These data demonstrate the involvement of NO in both modulating cell proliferation (at sub-toxic concentrations) and cell death (at toxic concentrations). To confirm the participation of sub-toxic concentrations of NO in the process of cell proliferation, we evaluated the expression of the PCNA (proliferating cell nuclear antigen) gene, a marker of cell proliferation [32]. Thus, mRNA samples were prepared from yeast treated with low concentrations of NO in the presence or absence of the cPTIO, and RT-qPCR was performed. We observed a strong increase in the PCNA mRNA levels of P. brasiliensis after treatment with 0.25 mM NO. On the other hand, the PCNA expression in P. brasiliensis yeast cells stimulated with low concentrations of NO and preincubated with cPTIO, was similar to that observed in the controls without stimuli (Fig. 1C). These data confirm our previous observations that NO and Ras are able to stimulate cell proliferation in P. brasiliensis. Ras GTPase proteins are activated by several stimuli in response to stress, including heat shock and fungal virulence [8,33]. Thus, we evaluated Ras activation in response to nitrosative stress. We used the RBD (Byr2)-GST probe (produced in a previous study [5]) to assess the ability of high concentrations (toxic) of NO to activate Ras. Ras activity was determined in P. brasiliensis yeasts exposed to 1, 2 or 4 mM NO for 60 min. We observed that high concentrations of NO induced Ras activation. Under these conditions, the peak activation was observed with 4 mM NO (Fig. 2A). Thus, to verify the kinetics of activation of Ras in nitrosative stress, a concentration of 4 mM of NO was used for increasing periods of time. Early Ras activation was observed after 15 min of stimulation with NO (Fig. 2B). Maximal Ras activation was observed after 60 min of incubation (Fig. 2B). In longer times (up to 5 h) the Ras activation was not observed (data not show). Additionally, the fungal protein extract was incubated with glutathione-Sepharose beads alone (negative control) and analyzed by Western blot, but no reaction was observed (data not shown). The protein loading of the samples used in the Ras activity assay observed by Coomassie blue staining of the SDS-PAGE gel were fairly homogeneous. Therefore, nitrosative stress induced by NO promoted guanine nucleotide exchanges in the critical cellular signaling Ras in P. brasiliensis.