Cross-talk between the Ras GTPase and the Hog1 survival pathways in response to nitrosative stress in Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a temperature-dependent dimorphic fungus that cause paracoccidioidomycosis (PCM), the major systemic mycosis in Latin America. The capacity to evade the innate immune response of the host is due to P. brasiliensis ability to respond and to survive the nitrosative stress caused by phagocytic cells. However, the regulation of signal transduction pathways associated to nitrosative stress response are poorly understood. Ras GTPase play an important role in the various cellular events in many fungi. Ras, in its activated form (Ras-GTP), interacts with effector proteins and can initiate a kinase cascade. In this report, we investigated the role of Ras GTPase in P. brasiliensis after in vitro stimulus with nitric oxide (NO). We observed that low concentrations of NO induced cell proliferation in P. brasiliensis, while high concentrations promoted decrease in fungal viability, and both events were reversed in the presence of a NO scavenger. We observed that high levels of NO induced Ras activation and its S-nitrosylation. Additionally, we showed that Ras modulated the expression of antioxidant genes in response to nitrosative stress. We find that the Hog1 MAP kinase contributed to nitrosative stress response in P. brasiliensis in a Ras-dependent manner. Taken together, our data demonstrate the relationship between Ras-GTPase and Hog1 MAPK pathway allowing for the P. brasiliensis adaptation to nitrosative stress.

Secreted aspartyl proteinase (PbSap) contributes to the virulence of Paracoccidioides brasiliensis infection.

Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and is caused by fungi from the Paracoccidioides genus. Virulence factors are important fungal characteristics that support the development of disease. Aspartyl proteases (Saps) are virulence factors in many human fungal pathogens that play an important role in the host invasion process. We report here that immunization with recombinant Sap from Paracoccidioides brasiliensis (rPbSap) imparted a protective effect in an experimental PCM model. The rPbSap-immunized mice had decreased fungal loads, and their lung parenchyma were notably preserved. An aspartyl protease inhibitor (pepstatin A) significantly decreased pulmonary injury and reduced fungal loads in the lung. Additionally, we observed that pepstatin A enhanced the fungicidal and phagocytic profile of macrophages against P. brasiliensis. Furthermore, PbSAP expression was highly altered by environmental conditions, including thermal stress, dimorphism switching and low pH. Hence, our data suggest that PbSap is an important virulence regulator in P. brasiliensis.