Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms.

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Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides brasiliensis. Currently, the treatment approach involves the use of antifungal drugs and requires years of medical therapy, which can induce nephrotoxicity and lead to resistance in yeast strains. Photodynamic inactivation (PDI) is a new therapy capable of killing microorganisms via the combination of a nontoxic dye with visible light to generate toxic reactive oxygen species (ROS). We investigated the phototoxic effect of 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin (TMPyP), a cationic porphyrin, on the survival of P. brasiliensis following exposure to light. Phototoxicity was found to depend on both the fluence and concentration of the photosensitizer (PS). Although the biological effects of PDI are known, the molecular mechanisms underlying the resultant damage to cells are poorly defined. Therefore, we evaluated the molecular response to PDI-induced oxidative stress by gene transcription analysis. We selected genes associated with the high-osmolarity glycerol (HOG)-mitogen-activated protein kinase (MAPK) pathway and antioxidant enzymes. The genes analyzed were all overexpressed after PDI treatment, suggesting that the oxidative stress generated in our experimental conditions induces antioxidant activity. In addition to PDI-induced gene expression, there was high cell mortality, suggesting that the antioxidant response was not sufficient to avoid fungal mortality.

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