Characterization of a heme-protein responsive to hypoxia in Paracoccidioides brasiliensis.

Oxygen is fundamental to the life of aerobic organisms and is not always available to Paracoccidioides cells. During the life cycle stages, reduced oxygen levels directly affect general metabolic processes and oxygen adaptation mechanisms may play a fundamental role on fungal ability to survive under such condition. Heme proteins can bind to oxygen and participate in important biological processes. Several fungi, including Paracoccidioides, express a heme-binding globin (fungoglobin - FglA) presumable to regulate fungal adaptation to hypoxia. However, the characterization of fungoglobin in Paracoccidioides spp. has not yet been performed. In this study, we predicted the structure of fungoglobin and determined its level of expression during hypoxic-mimetic conditions. Genomic screening revealed that the fungoglobin gene is conserved in all species of the Paracoccidioides genus. Molecular modeling showed biochemical and biophysical characteristics that support the hypothesis that FglA binds to the heme group and oxygen as well. The fungoglobin transcript and proteins are expressed at higher levels at the early treatment time, remaining elevated while oxygen is limited. A P. brasiliensis fglA knockdown strain depicted reduced growth in hypoxia indicating that this protein can be essential for growth at low oxygen. Biochemical analysis confirmed the binding of fungoglobin to heme. Initial analyzes were carried out to establish the relationship between FlglA and iron metabolism. The FglA transcript was up regulated in pulmonary infection, suggesting its potential role in the disease establishment. We believe that this study can contribute to the understanding of fungal biology and open new perspectives for scientific investigations.

Interaction of Isocitrate Lyase with Proteins Involved in the Energetic Metabolism in Paracoccidioides lutzii.

BACKGROUND: Systemic mycosis is a cause of death of immunocompromised subjects. The treatment directed to evade fungal pathogens shows severe limitations, such as time of drug exposure and side effects. The paracoccidioidomycosis (PCM) treatment depends on the severity of the infection and may last from months to years. METHODS: To analyze the main interactions of Paracoccidioides lutzii isocitrate lyase (ICL) regarding the energetic metabolism through affinity chromatography, we performed blue native PAGE and co-immunoprecipitation to identify ICL interactions. We also performed in silico analysis by homology, docking, hot-spot prediction and contact preference analysis to identify the conformation of ICL complexes. RESULTS: ICL interacted with 18 proteins in mycelium, 19 in mycelium-to-yeast transition, and 70 in yeast cells. Thirty complexes were predicted through docking and contact preference analysis. ICL has seven main regions of interaction with protein partners. CONCLUSIONS: ICL seems to interfere with energetic metabolism of P. lutzii, regulating aerobic and anaerobic metabolism as it interacts with proteins from glycolysis, gluconeogenesis, TCA and methylcitrate cycles, mainly through seven hot-spot residues.