Departamento de Microbiologia, Imunologia e Parasitologia-Universidade Federal de São Paulo, São Paulo, Brazil.
PLoS One. 2013 Jul 29;8(7):e69590. doi: 10.1371/journal.pone.0069590. Print 2013.
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.
巴西副球孢子菌是引起副球孢子菌病（PCM）的病原体，在吸入空气中的分生孢子后，它应该能够适应感染宿主内的剧烈环境变化，并转变为致病性酵母。具有抗氧化功能的蛋白质可以保护真菌细胞免受吞噬细胞产生的活性氧（ROS）和活性氮（RNS）物质的侵害，因此可以作为潜在的毒力因子。Ras GTPases 参与了多种生物的应激反应、细胞形态和分化。处于激活状态的 Ras 与效应蛋白相互作用，可引发激酶级联反应。在较低等的真核生物中，Byr2 激酶是 Ras 的一个靶点。本研究探讨了 Ras 在巴西副球孢子菌体外受到 ROS 或 RNS 刺激后的作用。我们已经证明，低浓度的 H2O2（0.1 mM）或 NO2（0.1-0.25 µM）刺激巴西副球孢子菌酵母细胞增殖，但当酵母细胞预先用法呢基转移酶抑制剂孵育时，就不会观察到这种现象。我们构建了一个表达质粒，其中包含与 GST 融合的 Byr2 Ras 结合域（RBD）（RBD-Byr2-GST），以检测 Ras 的活性形式。在低浓度的 H2O2 或 NO2 刺激后，在真菌提取物中观察到 Ras 的活性形式。此外，NO2 诱导 Ras 水平的 S-亚硝基化迅速增加。这种 Ras 的翻译后修饰替代方式，可能在残基 Cys123 上，将导致 GDP 与 GTP 交换，继而导致 P. brasiliensis 中的 GTPase 激活。总之，低浓度的 H2O2 或 NO2 通过 Ras 激活刺激巴西副球孢子菌增殖。
