iTRAQ-based proteomic analysis of Paracoccidioides brasiliensis in response to hypoxia.

Aerobic organisms require oxygen for energy. In the course of the infection, adaptation to hypoxia is crucial for survival of human pathogenic fungi. Members of the Paracoccidioides complex face decreased oxygen tensions during the life cycle stages. In Paracoccidioides brasiliensis proteomic responses to hypoxia have not been investigated and the regulation of the adaptive process is still unknown, and this approach allowed the identification of 216 differentially expressed proteins in hypoxia using iTRAQ-labelling. Data suggest that P. brasiliensis reprograms its metabolism when submitted to hypoxia. The fungus reduces its basal metabolism and general transport proteins. Energy and general metabolism were more representative and up regulated. Glucose is apparently directed towards glycolysis or the production of cell wall polymers. Plasma membrane/cell wall are modulated by increasing ergosterol and glucan, respectively. In addition, molecules such as ethanol and acetate are produced by this fungus indicating that alternative carbon sources probably are activated to obtain energy. Also, detoxification mechanisms are activated. The results were compared with label free proteomics data from Paracoccidioides lutzii. Biochemical pathways involved with acetyl-CoA, pyruvate and ergosterol synthesis were up-regulated in both fungi. On the other hand, proteins from TCA, transcription, protein fate/degradation, cellular transport, signal transduction and cell defense/virulence processes presented different profiles between species. Particularly, proteins related to methylcitrate cycle and those involved with acetate and ethanol synthesis were increased in P. brasiliensis proteome, whereas GABA shunt were accumulated only in P. lutzii. The results emphasize metabolic adaptation processes for distinct Paracoccidioides species.

Comparative proteomics in the three major human pathogenic species of the genus Sporothrix.

Sporotrichosis is a subcutaneous mycosis of humans and other mammals, caused by dimorphic species of the genus Sporothrix. In Brazil, human disease is broadly linked to transmission by infected cats and is mainly caused by Sporothrix brasiliensis, Sporothrix schenckii and Sporothrix globosa. In this study, we used a nanoscale liquid chromatography coupled with tandem mass spectrometry approach to provide the yeast proteomic profiles of S. brasiliensis, S. schenckii and S. globosa. From a total of 247 identified proteins, 137 were found as differentially expressed. Functional classification revealed that most are related to carbohydrate and amino acid metabolism as well as stress response. Our data indicate that S. brasiliensis metabolism is distinct of that of S. schenckii and S. globosa, mainly regarding amino acid metabolism and cell wall remodeling, which are induced in the former. Enzymes belonging to glycolytic pathway are, on the other hand, up-regulated in S. schenckii and S. globosa. These findings may explain the previously described more virulent character of S. brasiliensis. Besides complementing genomic comparisons already published, this first comparative proteomic study provided information that indicates new aspects of Sporothrix species metabolism as well as offers information that may be useful in the development of prospective functional studies.

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.

Characterization of the Paracoccidioides Hypoxia Response Reveals New Insights into Pathogenesis Mechanisms of This Important Human Pathogenic Fungus.

BACKGROUND: Hypoxic microenvironments are generated during fungal infection. It has been described that to survive in the human host, fungi must also tolerate and overcome in vivo microenvironmental stress conditions including low oxygen tension; however nothing is known how Paracoccidioides species respond to hypoxia. The genus Paracoccidioides comprises human thermal dimorphic fungi and are causative agents of paracoccidioidomycosis (PCM), an important mycosis in Latin America. METHODOLOGY/PRINCIPAL FINDINGS: In this work, a detailed hypoxia characterization was performed in Paracoccidioides. Using NanoUPLC-MSE proteomic approach, we obtained a total of 288 proteins differentially regulated in 12 and 24 h of hypoxia, providing a global view of metabolic changes during this stress. In addition, a functional characterization of the homologue to the most important molecule involved in hypoxia responses in other fungi, the SREBP (sterol regulatory element binding protein) was performed. We observed that Paracoccidioides species have a functional homologue of SREBP, named here as SrbA, detected by using a heterologous genetic approach in the srbA null mutant in Aspergillus fumigatus. Paracoccidioides srbA (PbsrbA), in addition to involvement in hypoxia, is probable involved in iron adaptation and azole drug resistance responses. CONCLUSIONS/SIGNIFICANCE: In this study, the hypoxia was characterized in Paracoccidioides. The first results can be important for a better understanding of the fungal adaptation to the host and improve the arsenal of molecules for the development of alternative treatment options in future, since molecules related to fungal adaptation to low oxygen levels are important to virulence and pathogenesis in human pathogenic fungi.

Paracoccidioides spp. ferrous and ferric iron assimilation pathways.

Iron is an essential micronutrient for almost all organisms, including fungi. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA). Traditionally, the RIA pathway consists of ferric reductases (Fres), ferroxidase (Fet3) and a high-affinity iron permease (Ftr1). Paracoccidioides spp. genomes do not present an Ftr1 homolog. However, this fungus expresses zinc regulated transporter homologs (Zrts), members of the ZIP family of membrane transporters that are able in some organisms to transport zinc and iron. A 2,3,5-triphenyltetrazolium chloride (TTC)-overlay assay indicates that both Pb01 and Pb18 express a ferric reductase activity; however, (59)Fe uptake assays indicate that only in Pb18 is this activity coupled to a reductase-dependent iron uptake pathway. In addition, Zrts are up-regulated in iron deprivation, as indicated by RNAseq and qRT-PCR using Pb01 transcripts. RNAseq strategy also demonstrated that transcripts related to siderophore uptake and biosynthesis are up-regulated in iron-deprived condition. The data suggest that the fungus could use both a non-classical RIA, comprising ferric reductases and Fe/Zn permeases (Zrts), and siderophore uptake pathways under iron-limited conditions. The study of iron metabolism reveals novel surface molecules that could function as accessible targets for drugs to block iron uptake and, consequently, inhibit pathogen's proliferation.

Transcriptional and proteomic responses to carbon starvation in Paracoccidioides.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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-MSE) 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. CONCLUSIONS/SIGNIFICANCE: 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.