Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.

The genus Paracoccidioides comprises human thermal dimorphic fungi, which cause paracoccidioidomycosis (PCM), an important mycosis in Latin America. Adaptation to environmental conditions is key to fungal survival during human host infection. The adaptability of carbon metabolism is a vital fitness attribute during pathogenesis. The fungal pathogen Paracoccidioides spp. is exposed to numerous adverse conditions, such as nutrient deprivation, in the human host. In this study, a comprehensive response of Paracoccidioides, Pb01, under carbon starvation was investigated using high-resolution transcriptomic (RNAseq) and proteomic (NanoUPLC-MS^E) approaches. A total of 1,063 transcripts and 421 proteins were differentially regulated, providing a global view of metabolic reprogramming during carbon starvation. The main changes were those related to cells shifting to gluconeogenesis and ethanol production, supported by the degradation of amino acids and fatty acids and by the modulation of the glyoxylate and tricarboxylic cycles. This proposed carbon flow hypothesis was supported by gene and protein expression profiles assessed using qRT-PCR and western blot analysis, respectively, as well as using enzymatic, cell dry weight and fungus-macrophage interaction assays. The carbon source provides a survival advantage to Paracoccidioides inside macrophages. For a complete understanding of the physiological processes in an organism, the integration of approaches addressing different levels of regulation is important. To the best of our knowledge, this report presents the first description of the responses of Paracoccidioides spp. to host-like conditions using large-scale expression approaches. The alternative metabolic pathways that could be adopted by the organism during carbon starvation can be important for a better understanding of the fungal adaptation to the host, because systems for detecting and responding to carbon sources play a major role in adaptation and persistence in the host niche. The species of the Paracoccidioides genus, a neglected human pathogen, represent the causative agents of paracoccidioidomycosis (PCM), one of the most frequent systemic mycoses in Latin America. Despite being phagocytosed, the fungus conidia differentiate into the parasitic yeast form that subverts the normally harsh intraphagosomal environment and survives and replicates into murine and human macrophages. It has been suggested that alternative carbon metabolism plays a role in the survival and virulence of Paracoccidioides spp. within host cells. We used large-scale transcriptome and proteome approaches to better characterize the responses of Paracoccidioides, Pb01, yeast parasitic cells, to carbon starvation. We aimed to identify important molecules used by the fungus to adapt to these hostile conditions. The shift to a starvation mode, including gluconeogenesis and ethanol increases, activation of fatty acids, and amino acid degradation are the strategies used by the pathogen to persist under this stress. Our study provides a detailed map of Paracoccidioides spp. responses under carbon starvation conditions and contributes to further investigations of the importance of alternative carbon adaptation during fungus pathogenesis.

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-MS^E 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. Fungal infections contribute substantially to human morbidity and mortality. During infectious processes, fungi have evolved mechanisms to obtain iron from high-affinity iron-binding proteins. In the current study, we demonstrated that hemoglobin is the preferential host iron source for the thermodimorphic fungus Paracoccidioides spp. To acquire hemoglobin, the fungus presents hemolytic activity and the ability to internalize protoporphyrin rings. A putative hemoglobin receptor, Rbt5, was demonstrated to be GPI-anchored at the yeast cell surface. Rbt5 was able to bind to hemin, protoporphyrin and hemoglobin in vitro. When rbt5 expression was inhibited, the survival of Paracoccidioides sp. inside macrophages and the fungal burden in mouse spleen diminished, which indicated that Rbt5 could participate in the establishment of the fungus inside the host. Drugs or vaccines could be developed against Paracoccidioides spp. Rbt5 to disturb iron uptake of this micronutrient and, thus, the proliferation of the fungus. Moreover, this protein could be used in routes to introduce antifungal agents into fungal cells.

Paracoccidoides brasiliensis and Paracoccidioides lutzii, the etiologic agents of paracoccidioidomycosis, cause disease in healthy and immunocompromised persons in Latin America. We developed a method for harvesting P. brasiliensis yeast cells from infected murine lung to facilitate in vivo transcriptional and proteomic profiling. P. brasiliensis harvested at 6 h post-infection were analyzed using RNAseq and LC-MS^E. In vivo yeast cells had 594 differentially expressed transcripts and 350 differentially expressed proteins. Integration of transcriptional and proteomic data indicated that early in infection (6 h), P. brasiliensis yeast cells underwent a shift in metabolism from glycolysis to β-oxidation, upregulated detoxifying enzymes to defend against oxidative stress, and repressed cell wall biosynthesis. Bioinformatics and functional analyses also demonstrated that a serine proteinase was upregulated and secreted in vivo. To our knowledge this is the first study depicting transcriptional and proteomic data of P. brasiliensis yeast cells upon 6 h post-infection of mouse lung.

Paracoccidioides, a complex of several phylogenetic species, is the causative agent of paracoccidioidomycosis. The ability of pathogenic fungi to develop a multifaceted response to the wide variety of stressors found in the host environment is important for virulence and pathogenesis. Extracellular proteins represent key mediators of the host-parasite interaction. To analyze the expression profile of the proteins secreted by Paracoccidioides, Pb01 mycelia and yeast cells, we used a proteomics approach combining two-dimensional electrophoresis with matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF MS/MS). From three biological replicates, 356 and 388 spots were detected, in mycelium and yeast cell secretomes, respectively. In this study, 160 non-redundant proteins/isoforms were indentified, including 30 and 24 proteins preferentially secreted in mycelia and yeast cells, respectively. In silico analyses revealed that 65% of the identified proteins/isoforms were secreted primarily via non-conventional pathways. We also investigated the influence of protein export inhibition in the phagocytosis of Paracoccidioides by macrophages. The addition of Brefeldin A to the culture medium significantly decreased the production of secreted proteins by both Paracoccidioides and internalized yeast cells by macrophages. In contrast, the addition of concentrated culture supernatant to the co-cultivation significantly increased the number of internalized yeast cells by macrophages. Importantly, the proteins detected in the fungal secretome were also identified within macrophages. These results indicate that Paracoccidioides extracellular proteins are important for the fungal interaction with the host.

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.