Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.

Background: Paracoccidioides brasiliensis is a human pathogen with a broad distribution in Latin America. The fungus is thermally dimorphic with two distinct forms corresponding to completely different lifestyles. Upon elevation of the temperature to that of the mammalian body, the fungus adopts a yeast-like form that is exclusively associated with its pathogenic lifestyle. We describe expressed sequence tags (ESTs) analysis to assess the expression profile of the mycelium to yeast transition. To identify P. brasiliensis differentially expressed sequences during conversion we performed a large-scale comparative analysis between P. brasiliensis ESTs identified in the transition transcriptome and databases.

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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.

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.

Paracoccidioides brasiliensis is a fungal human pathogen with a wide distribution in Latin America. It causes paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. Although gene expression in P. brasiliensis had been studied, little is known about the genome sequences expressed by this species during the infection process. To better understand the infection process, 4934 expressed sequence tags (ESTs) derived from a non-normalized cDNA library from P. brasiliensis (isolate Pb01) yeast-phase cells recovered from the livers of infected mice were annotated and clustered to a UniGene (clusters containing sequences that represent a unique gene) set with 1602 members. A large-scale comparative analysis was performed between the UniGene sequences of P. brasiliensis yeast-phase cells recovered from infected mice and a database constructed with sequences of the yeast-phase and mycelium transcriptome (isolate Pb01) (https://dna.biomol.unb.br/Pb/), as well as with all public ESTs available at GenBank, including sequences of the P. brasiliensis yeast-phase transcriptome (isolate Pb18) (http:// www.ncbi.nlm.nih.gov/). The focus was on the overexpressed and novel genes. From the total, 3184 ESTs (64.53 %) were also present in the previously described transcriptome of yeast-form and mycelium cells obtained from in vitro cultures (https://dna.biomol.unb.br/Pb/) and of those, 1172 ESTs (23.75 % of the described sequences) represented transcripts overexpressed during the infection process. Comparative analysis identified 1750 ESTs (35.47 % of the total), comprising 649 UniGene sequences representing novel transcripts of P. brasiliensis, not previously described for this isolate or for other isolates in public databases. KEGG pathway mapping showed that the novel and overexpressed transcripts represented standard metabolic pathways, including glycolysis, amino acid biosynthesis, lipid and sterol metabolism. The unique and divergent representation of transcripts in the cDNA library of yeast cells recovered from infected mice suggests differential gene expression in response to the host milieu.Abbreviations: EST, expressed sequence tag; KEGG, Kyoto Encyclopedia of Gene and Genomes; sqRT-PCR, semiquantitative RT-PCR.3These authors contributed equally to this work.The GenBank/EMBL/DDBJ accession numbers for the ESTs of Paracoccidioides brasiliensis identified in this study are EST1487-EST6420.Two supplementary tables listing the overexpressed and novel genes identified during this study and supplementary material describing the EST dataset analysed are available with the online version of this paper. Microbiology

An efficient oxidative stress response is important to the fungal pathogen Paracoccidioides to survive within the human host. In this study, oxidative stress was mimicked by exposure of yeast cells to hydrogen peroxide (2 mM H2O2). To investigate the effect of H2O2 on the proteome of Paracoccidioides, we used a large scale 2-DE protein gel electrophoresis approach to analyze differentially expressed proteins/isoforms that were detected in early (2 h) and in late (6 h) oxidative stress treatments. All proteins/isoforms were grouped based on their functional categories that revealed a global activation of antioxidant enzymes, such as catalase, superoxide dismutase, cytochrome C peroxidase and thioredoxin. A view of the metabolic cell profile, as determined by proteomics, depicted a shift in the yeast cells metabolism as suggested by the activation of the pentose phosphate pathway, a great source of cellular reducing power in the form of NADPH. Additionally, in silico analyzes depicted 34 oxidoreductases proteins/isoforms putatively involved with defense against oxidative stress. Confirmatory assays of enzymatic activity, flow cytometry, transcript levels and NADPH measurements, produced data in agreement with proteomic analysis.

Diseases caused by fungi can occur in healthy people, but immunocompromised patients are the major risk group for invasive fungal infections. Cases of fungal resistance and the difficulty of treatment make fungal infections a public health problem. This review explores mechanisms used by fungi to promote fungal resistance, such as the mutation or overexpression of drug targets, efflux and degradation systems, and pleiotropic drug responses. Alternative novel drug targets have been investigated; these include metabolic routes used by fungi during infection, such as trehalose and amino acid metabolism and mitochondrial proteins. An overview of new antifungal agents, including nanostructured antifungals, as well as of repositioning approaches is discussed. Studies focusing on the development of vaccines against antifungal diseases have increased in recent years, as these strategies can be applied in combination with antifungal therapy to prevent posttreatment sequelae. Studies focused on the development of a pan-fungal vaccine and antifungal drugs can improve the treatment of immunocompromised patients and reduce treatment costs.