Background: The pathogenic fungus Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM). This is a pulmonary mycosis acquired by inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival.
Paracoccidioides spp., a dimorphic pathogenic fungus, is the etiologic agent of paracoccidioidomycosis (PCM). PCM is an endemic disease that affects at least 10 million people in Latin America, causing severe public health problems. The drugs used against pathogenic fungi have various side effects and limited efficacy; therefore, there is an inevitable and urgent medical need for the development of new antifungal drugs. In the present study, we evaluated the transcriptional profile of Paracoccidioides lutzii exposed to argentilactone, a constituent of the essential oil of Hyptis ovalifolia. A total of 1,058 genes were identified, of which 208 were up-regulated and 850 were down-regulated. Cell rescue, defense and virulence, with a total of 26 genes, was a functional category with a large number of genes induced, including heat shock protein 90 (hsp90), cytochrome c peroxidase (ccp), the hemoglobin ligand RBT5 (rbt5) and superoxide dismutase (sod). Quantitative real-time PCR revealed an increase in the expression level of all of those genes. An enzymatic assay showed a significant increase in SOD activity. The reduced growth of Pbhsp90-aRNA, Pbccp-aRNA, Pbsod-aRNA and Pbrbt5-aRNA isolates in the presence of argentilactone indicates the importance of these genes in the response of Paracoccidioides spp. to argentilactone. The response of the P. lutzii cell wall to argentilactone treatment was also evaluated. The results showed that argentilactone caused a decrease in the levels of polymers in the cell wall. These results suggest that argentilactone is a potential candidate for antifungal therapy.
BackgroundItraconazole is currently used to treat paracoccidioidomycosis. The mechanism of action of azoles has been elucidated in some fungi, although little is known regarding its mechanism of action in Paracoccidioides spp. The present work focused on identification of regulated transcripts using representational difference analysis of Paracoccidioides spp. yeast cells treated with itraconazole for 1 and 2 h.ResultsParacoccidioides Pb01 genes up-regulated by itraconazole included genes involved in cellular transport, metabolism/energy, transcription, cell rescue, defense and virulence. ERG11, ERG6, ERG3, ERG5 and ERG25 were up-regulated at multiple time points. In vivo infection experiments in mice corroborated the in vitro results. Ergosterol levels and distribution were evaluated in Paracoccidioides Pb18 yeast cells, and the results demonstrate that both factors were changed in the fungus treated with itraconazole.ConclusionTo our knowledge, this is the first transcriptional analysis of Paracoccidioides spp. exposed to a triazole drug. Here acetyl seems to be intensively produced from different metabolic pathways to produce ergosterol by the action of ergosterol synthesis related enzymes, which were also affected in other fungi. Among the genes affected, we identified genes in common with other fungi, as well as genes unique to Paracoccidioides Pb01. Those genes could be considered target to new drugs. Voltage-gated Ca2+ alpha subunit (CAV), Tetracycline resistance protein (TETA) and Hemolisyn-iii channel protein (HLYiii) were found only here and a probably involvement with resistence to itraconazole could be investigated in the future. However our findings do not permit inference to current clinical practice.
Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species (PbMLS). The three-dimensional (3D) structure of PbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with PbMLS, it was possible to identify -carboline moiety as a standard key structure. The compounds with -carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of PbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of -carboline moiety, was crucial for their stability in the binding pocket of PbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four -carboline alkaloid compounds showed the ability to inhibit recombinant PbMLS (PbMLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and PbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated. Several plants, human-pathogenic fungi, and bacteria utilize the glyoxylate cycle during host infection (1). The unique enzymes of this cycle are isocitrate lyase and malate synthase. This pathway has not been observed in mammals; therefore, this cycle has been identified as a potential target for the discovery of new drugs. Novel active molecules should shorten the duration of chemotherapy, prevent the development of resistance, and eliminate latent disease.In the human-pathogenic fungi from the genus Paracoccidioides, the malate synthase (PbMLS) participates in the glyoxylate pathway, which enables the fungus to assimilate two-carbon compounds, and in the allantoin degradation pathway associated with purine metabolism, which allows the fungus to use nitrogen compounds (2). PbMLS is localized in peroxisomes and on the cell surface and is secreted. In addition, PbMLS plays a role as an adhesin, mediating the adhesion and internalization of the fungus to host cells (3). PbMLS is regulated during the transition from mycelium to yeast (4) and in oxidative stress (5). However, an inhibitor to PbMLS has not yet been investigated.In recent years, virtual screening has become an accepted tool in drug discovery that has successfully been applied in a number of therapeutic programs, particularly at the lead discovery stage, during which high-throughput molecular docking can play an important r...
BackgroundThe fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands.ResultsThe search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software.ConclusionThese observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal transduction. These data suggest that PbMLS could play different roles in the fungal cell.
Aim: To perform the proteomic profile of Paracoccidioides lutzii after treatment with the compound camphene thiosemicarbazide (TSC-C) in order to study its mode of action. Methods: Proteomic analysis was carried out after cells were incubated with TSC-C in a subinhibitory concentration. Validation of the proteomic results comprised the azocasein assay, western blot and determination of the susceptibility of a mutant to the compound. Results: Proteins related to metabolism, energy and protein fate were regulated after treatment. In addition, TSC-C reduces the proteolytic activity of the protein extract similarly to different types of protease inhibitors. Conclusion: TSC-C showed encouraging antifungal activity, working as a protease inhibitor and downregulating important pathways impairing the ability of the fungi cells to produce important precursors.
As part of our continuing chemical and biological analyses of Rubiaceae species from Cerrado, we isolated novel alkaloids 1 and 2, along with known compounds epicatechin, ursolic acid, and oleanolic acid, from Galianthe ramosa. Alkaloid 2 inhibited malate synthase from the pathogenic fungus Paracoccidioides spp. This enzyme is considered an important molecular target because it is not found in humans. Molecular docking simulations were used to describe the interactions between the alkaloids and malate synthase.
Protein glycosylation is one of the most studied post-translational modifications and has received considerable attention for its critical role in the cell biology of eukaryotic cells. The genus Trichoderma has been extensively studied in the biocontrol of soil-borne fungal phytopathogens. The aim of this study was to identify the proteins secreted from Trichoderma harzianum after interacting with the cell walls of two phytopathogens, Sclerotinia sclerotiorum and Fusarium oxysporum. This study used N-glycoprotein enrichment with a concanavalin A (Con A) affinity column, staining detection differential SDS-PAGE, sequencing by mass spectrometric, and protein identification by comparison with the NCBI database to detect the protein expression of the two resulting secretome samples. The majority of the proteins found in both enriched secretomes belonged to a specific class of carbohydrate-active enzymes (CAZymes), within which glycosyl hydrolases (GHs), glycosyltransferases (GTs), and auxiliary activities (AAs) were identified. In this study was described two proteins that have not been previously reported in the secretomes of Trichoderma, a glycosyltransferase (six-harpin) and a galactose oxidase, belonging to the class of auxiliary activities (AA), classified as an AA subfamily AA5-2.The expression pattern of gene encoding to 17 identified proteins, evaluated by real-time quantitative PCR (RT-qPCR), showed significant difference of expression of some GHs and proteases, suggesting a specific gene expression regulation by T. harzianum in presence of different cell walls of two phytopathogens.
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