Exposure of Paracoccidioides brasiliensis to Mebendazole Leads to Inhibition of Fungal Energy Production.

Paracoccidioidomycosis (PCM) is a fungal disease caused by organisms of the genus Paracoccidioides spp. The treatment of the disease is lengthy and includes several adverse effects. Various methodologies focus on the search for new treatments against fungal disease, including the repositioning of drugs. Our group showed the fungicidal effect of mebendazole in P. brasiliensis cells. Thus, understanding the effect of exposing fungal cells to mebendazole is significant for further studies in order to demonstrate it as a potential drug for the treatment of PCM. A proteomic analysis of P. brasiliensis exposed to mebendazole was carried out. Analyses showed that exposure strongly affected the pathways related to energy production, such as glycolysis, fermentation, and the electron transport chain. The quantification of adenosine triphosphate (ATP) and mitochondrial activity demonstrated that the drug alters the electron chain, resulting in an increase in oxidative stress. Enzymes such as superoxide dismutase (SOD) and cytochrome c oxidase (Cyt C) were repressed in cells exposed to mebendazole. The concentration of ethanol produced by the cells under treatment demonstrated that the attempt to produce energy through fermentation is also arrested. Thus, the drug inhibits fungal growth through changes in energy metabolism, making it a promising compound for use in the treatment of PCM.

Iron Deprivation Modulates the Exoproteome in Paracoccidioides brasiliensis.

Fungi of the Paracoccidioides genus are the etiological agents of the systemic mycosis paracoccidioidomycosis and, when in the host, they find a challenging environment that is scarce in nutrients and micronutrients, such as Fe, which is indispensable for the survival of the pathogen. Previous studies have shown that fungi of this genus, in response to Fe deprivation, are able to synthesize and capture siderophores (Fe3+ chelators), use Fe-containing host proteins as a source of the metal, and use a non-canonical reductive pathway for Fe3+ assimilation. Despite all of these findings, there are still gaps that need to be filled in the pathogen response to metal deprivation. To contribute to the knowledge related to this subject, we obtained the exoproteome of Paracoccidioides brasiliensis (Pb18) undergoing Fe deprivation and by nanoUPLC-MSE. One hundred forty-one proteins were identified, and out of these, 64 proteins were predicted to be secreted. We also identified the regulation of several virulence factors. Among the results, we highlight Cyb5 as a secreted molecule of Paracoccidioides in the exoproteome obtained during Fe deprivation. Cyb5 is described as necessary for the Fe deprivation response of Saccharomyces cerevisiae and Aspergillus fumigatus. Experimental data and molecular modeling indicated that Cyb5 can bind to Fe ions in vitro, suggesting that it can be relevant in the arsenal of molecules related to iron homeostasis in P. brasiliensis.

Interactome of Glyceraldehyde-3-Phosphate Dehydrogenase Points to the Existence of Metabolons in Paracoccidioides lutzii.

Paracoccidioides is a dimorphic fungus, the causative agent of paracoccidioidomycosis. The disease is endemic within Latin America and prevalent in Brazil. The treatment is based on azoles, sulfonamides and amphotericin B. The seeking for new treatment approaches is a real necessity for neglected infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential glycolytic enzyme, well known for its multitude of functions within cells, therefore categorized as a moonlight protein. To our knowledge, this is the first approach performed on the Paracoccidioides genus regarding the description of PPIs having GAPDH as a target. Here, we show an overview of experimental GAPDH interactome in different phases of Paracoccidioides lutzii and an in silico analysis of 18 proteins partners. GAPDH interacted with 207 proteins in P. lutzii. Several proteins bound to GAPDH in mycelium, transition and yeast phases are common to important pathways such as glycolysis and TCA. We performed a co-immunoprecipitation assay to validate the complex formed by GAPDH with triose phosphate isomerase, enolase, isocitrate lyase and 2-methylcitrate synthase. We found GAPDH participating in complexes with proteins of specific pathways, indicating the existence of a glycolytic and a TCA metabolon in P. lutzii. GAPDH interacted with several proteins that undergoes regulation by nitrosylation. In addition, we modeled the GAPDH 3-D structure, performed molecular dynamics and molecular docking in order to identify the interacting interface between GAPDH and the interacting proteins. Despite the large number of interacting proteins, GAPDH has only four main regions of contact with interacting proteins, reflecting its ancestrality and conservation over evolution.