RESUMO
Aim: To access the metabolic changes caused by a chalcone derivative (LabMol-75) through a proteomic approach. Methods: Proteomic analysis was performed after 9 h of Paracoccidioides brasiliensis yeast (Pb18) cell incubation with the LabMol-75 at MIC. The proteomic findings were validated through in vitro and in silico assays. Results: Exposure to the compound led to the downregulation of proteins associated with glycolysis and gluconeogenesis, ß-oxidation, the citrate cycle and the electron transport chain. Conclusion: LabMol-75 caused an energetic imbalance in the fungus metabolism and deep oxidative stress. Additionally, the in silico molecular docking approach pointed to this molecule as a putative competitive inhibitor of DHPS.
Assuntos
Paracoccidioides , Paracoccidioidomicose , Paracoccidioides/metabolismo , Proteômica , Simulação de Acoplamento Molecular , Estresse Oxidativo , Oxirredução , Paracoccidioidomicose/microbiologiaRESUMO
The enzyme Homoserine dehydrogenase from Paracoccidioides brasiliensis (PbHSD), an interesting enzyme in the search for new antifungal drugs against paracoccidioidomycosis, was expressed by E. coli. Thirty milligrams of PbHSD with 94% of purity were obtained per liter of culture medium. The analysis by CD spectroscopy indicates a composition of 45.5 ± 7.3% of α-helices and 10.5 ± 7.0% ß-strands. Gel filtration chromatography indicates a homodimer as biological unity. Fluorescence emission spectroscopy has shown stability of PbHSD in the presence of urea until Cm of 4.13 ± 0.21 M, and a broad pH range in which there is no conformational change. The protein analysis by differential scanning calorimetry indicates high stability at room temperature, but low stability at high temperatures, suffering irreversible denaturation, with Tm = 58.65 ± 0.87 °C. Kinetic studies of PbHSD by molecular absorption spectroscopy in UV/Vis have shown an optimum pH between 9.35 and 9.50, with Michaelian behavior, presenting KM of 224 ± 15 µM and specific activity at optimum pH of 2.10 ± 0.07 µmol/min/mg for homoserine. Therefore, protein expression and purification were efficient, and the structural characterization has shown that PbHSD presents native conformation with enzymatic activity in kinetic assays.
Assuntos
Paracoccidioides , Paracoccidioides/genética , Paracoccidioides/metabolismo , Homosserina Desidrogenase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Espectrometria de FluorescênciaRESUMO
Aims: Considering the need to identify new compounds with antifungal action, the activity of five 3-phenacylideneoxindoles compounds was evaluated. Materials & methods: The compounds were synthesized, and their antifungal activity was elucidated through minimum inhibitory concentration tests and interaction assay with other antifungals. Potential targets of compounds were predicted in silico. Results: 3-phenacylideneoxindoles compounds inhibited fungal growth with minimum inhibitory concentration and minimum fungicidal concentration ranging from 3.05 to 12.26 µM. The compounds demonstrated high selectivity index and presented a synergistic effect with itraconazole. In silico prediction revealed the pentafunctional AROM polypeptide, enolase, superoxide dismutase, catalase and kinases as proteins targets of the compound 4a. Conclusion: The results demonstrate that 3-phenacylideneoxindoles is a potential new class of antifungal compounds for paracoccidioidomycosis treatment.
Patients affected by paracoccidioidomycosis (PCM) require long-term treatment, which commonly influences their adherence. In addition, only three drugs are in clinical use, which indicates the relevance of research in identifying new drugs for treating PCM. Thus, five drugs were tested in the laboratory to verify whether they could prevent the growth of the fungus without being toxic to humans. In addition, whether these compounds in combination with drugs used to treat PCM could be even more potent was evaluated. All compounds tested efficiently inhibited the growth of Paracoccidioides, the fungus that causes PCM. One drug was identified that, combined with itraconazole, decreased the required dose of both the discovered compound and itraconazole needed to inhibit fungal growth. Using computational tools, this work suggests how the new drug could act against the fungus. The results demonstrate a potential new treatment option, but more studies are needed to confirm the safety of these drugs.
Assuntos
Antifúngicos , Oxindóis , Paracoccidioides , Paracoccidioidomicose , Antifúngicos/farmacologia , Antifúngicos/química , Itraconazol/farmacologia , Testes de Sensibilidade Microbiana , Oxindóis/química , Oxindóis/farmacologia , Paracoccidioides/metabolismo , Paracoccidioidomicose/tratamento farmacológicoRESUMO
Paracoccidioidomycosis is a systemic mycosis endemic in Latin America, and one of the etiological agents of the disease is Paracoccidioides brasiliensis. Currently, available treatments present adversities, such as duration, side effects, and drug interactions. In search of new therapy possibilities, this study evaluates drugs approved for use against the homoserine dehydrogenase enzyme, by an in silico approach, which performs an important biosynthesis phase for the fungus and is not present in the human body. The three-dimensional structure of the homoserine dehydrogenase enzyme from Paracoccidioides brasiliensis was obtained by homology modeling. The model was validated, and simulations were performed for virtual screening of molecules of drugs approved from the Drugs-libs database by the MTiOpenScreen web server. Molecular dynamics in three replicas were used for four drugs with better results, and in two more molecules as a control, the HS9 with inhibition against enzyme and HON which shows inhibition against mold structure. Based on the results of molecular dynamics and the comparison of binding free energy, the drug that obtained the best result was Bemcentinib. In comparison with the controls, it presented a highly relevant affinity with - 44.63 kcal/mol, in addition to good structural stability and occupation of the active site. Therefore, Bemcentinib is a promising molecule for the inhibition of PbHSD protein (homoserine dehydrogenase of Paracoccidioides brasiliensis) and a therapeutic option to be investigated.
Assuntos
Paracoccidioides , Humanos , Paracoccidioides/metabolismo , Homosserina Desidrogenase , Reposicionamento de Medicamentos , Antifúngicos/farmacologiaRESUMO
Paracoccidioides spp. are the etiological agent of paracoccidioidomycosis, a disease that causes skin lesions and affect the lungs and other organs. The current management of the disease is long and has several side effects that often lead the patient to give up the treatment, sequelae and even death. The search for new forms of treatment that minimize these drawbacks is very important. Thus, natural compounds are targets of great interest. Curcumin is one of the main components of the tubers of Curcuma longa, presenting medicinal effects well described in the literature, including the antifungal effect on Paracocidioides brasiliensis. Nevertheless, the mechanisms related to the antifungal effect of such compound are still unknown, so the objective of the present research is to understand what changes occur in the metabolism of P. brasiliensis after exposure to curcumin and to identify the main targets of the compound. Proteomic analysis as based on nanoUPLC-MS analysis and the functional classification of the identified proteins. The main metabolic processes that were being regulated were biologically validated through assays such as fluorescence microscopy, EPR and phagocytosis. Proteomic analysis revealed that curcumin regulates several metabolic processes of the fungus, including important pathways for energy production, such as the glycolytic pathway, beta oxidation and the glyoxylate cycle. Protein synthesis was down-regulated in fungi exposed to curcumin. The electron transport chain and the tricarboxylic acid cycle were also down-regulated, indicating that both the mitochondrial membrane and the mitochondrial activity were compromised. Plasma membrane and cell wall structure were altered following exposure to the compound. The fungus' ability to survive the phagocytosis process by alveolar macrophages was reduced. Thus, curcumin interferes with several metabolic pathways in the fungus that causes paracoccidioidomycosis. BIOLOGICAL SIGNIFICANCE: The challenges presented by the current treatment of paracoccidioidomycosis often contributing to patients' withdrawal from treatment, leading to sequelae or even death. Thus, the search for new treatment options against this disease is growing. The discovery that curcumin is active against Paracoccidioides was previously reported by our study group. Here, we clarify how the compound acts on the fungus causing its growth inhibition and decreased viability. Understanding the mechanisms of action of curcumin on P. brasiliensis elucidates how we can seek new alternatives and which metabolic pathways and molecular targets we should focus on in this incessant search to bring the patient a treatment with fewer adverse effects.
Assuntos
Curcumina , Paracoccidioides , Paracoccidioidomicose , Antifúngicos/farmacologia , Curcumina/farmacologia , Humanos , Paracoccidioides/metabolismo , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/microbiologia , ProteômicaRESUMO
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.
Assuntos
Hipóxia/metabolismo , Paracoccidioides/metabolismo , Proteoma/metabolismo , Proteômica , Parede Celular/metabolismo , Ergosterol/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Glicólise , Humanos , Peróxido de Hidrogênio/metabolismo , Nitrogênio/metabolismo , Paracoccidioides/genética , Paracoccidioides/patogenicidade , VirulênciaRESUMO
During pathogen interaction with the host, several mechanisms are used to favor or inhibit the infectious process; one is called nutritional immunity, characterized by restriction of micronutrients to pathogens. Several studies on fungi of the Paracoccidioides complex, have demonstrated that these pathogens remodel their metabolic pathways to overcome the hostile condition imposed by the host. However, molecular mechanisms that control the regulation of those metabolic changes are not fully understood. Therefore, this work characterizes the expression profile of miRNAs during iron deprivation and describes metabolic pathways putatively regulated by those molecules. Through analysis of RNAseq, 45 miRNAs were identified and eight presented alterations in the expression profile during iron deprivation. Among the differentially regulated miRNAs, five were more abundant in yeast cells during iron deprivation and interestingly, the analyses of genes potentially regulated by those five miRNAs, pointed to metabolic pathways as oxidative phosphorylation, altered in response to iron deprivation. In addition, miRNAs with more abundance in iron presence, have as target genes encoding transcriptional factors related to iron homeostasis and uptake. Therefore, we suggest that miRNAs produced by Paracoccidioides brasiliensis may contribute to the adaptive responses of this fungus in iron starvation environment.
Assuntos
Regulação Fúngica da Expressão Gênica , Ferro/metabolismo , MicroRNAs/metabolismo , Paracoccidioides/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Homeostase , Humanos , MicroRNAs/genética , Paracoccidioides/metabolismo , Paracoccidioidomicose/microbiologia , RNA Fúngico/genética , RNA Fúngico/metabolismoRESUMO
Oxygen is fundamental to the life of aerobic organisms and is not always available to Paracoccidioides cells. During the life cycle stages, reduced oxygen levels directly affect general metabolic processes and oxygen adaptation mechanisms may play a fundamental role on fungal ability to survive under such condition. Heme proteins can bind to oxygen and participate in important biological processes. Several fungi, including Paracoccidioides, express a heme-binding globin (fungoglobin - FglA) presumable to regulate fungal adaptation to hypoxia. However, the characterization of fungoglobin in Paracoccidioides spp. has not yet been performed. In this study, we predicted the structure of fungoglobin and determined its level of expression during hypoxic-mimetic conditions. Genomic screening revealed that the fungoglobin gene is conserved in all species of the Paracoccidioides genus. Molecular modeling showed biochemical and biophysical characteristics that support the hypothesis that FglA binds to the heme group and oxygen as well. The fungoglobin transcript and proteins are expressed at higher levels at the early treatment time, remaining elevated while oxygen is limited. A P. brasiliensis fglA knockdown strain depicted reduced growth in hypoxia indicating that this protein can be essential for growth at low oxygen. Biochemical analysis confirmed the binding of fungoglobin to heme. Initial analyzes were carried out to establish the relationship between FlglA and iron metabolism. The FglA transcript was up regulated in pulmonary infection, suggesting its potential role in the disease establishment. We believe that this study can contribute to the understanding of fungal biology and open new perspectives for scientific investigations.
Assuntos
Proteínas Fúngicas/genética , Heme/genética , Hemeproteínas/genética , Paracoccidioides/genética , Aerobiose/genética , Hipóxia Celular/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Heme/metabolismo , Hemeproteínas/metabolismo , Oxigênio/metabolismo , Paracoccidioides/metabolismoRESUMO
Studies on the effects of components derived from the human pathogenic fungi Paracoccidioides brasiliensis have identified paracoccin (PCN), as a bifunctional protein with lectin (GlcNAc-binding) and enzymatic (chitinase) activities, able to induce modulation of host immune response. Endogenous PCN acts as a fungal virulence factor, whereas exogenous purified PCN, administered to the host, confers protective immunity in a murine model of paracoccidioidomycosis. The immunomodulation induced by purified-PCN injection has characterized it as an agent applicable in the therapy and vaccine against paracoccidioidomycosis. This section describes methods for PCN purification and validation of its lectin and enzymatic activities. It includes detailed protocols to obtain homogeneous PCN from P. brasiliensis yeasts, as well as to purify recombinant PCN from transformed heterologous microorganisms.
Assuntos
Acetilglucosamina/metabolismo , Proteínas Fúngicas/administração & dosagem , Lectinas/administração & dosagem , Paracoccidioides/patogenicidade , Paracoccidioidomicose/prevenção & controle , Animais , Quitinases/metabolismo , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Lectinas/genética , Lectinas/isolamento & purificação , Lectinas/metabolismo , Camundongos , Paracoccidioides/imunologia , Paracoccidioides/metabolismo , Paracoccidioidomicose/imunologia , Ligação Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Paracoccidiodomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. The disease requires long and complicated treatment. The aim of this review is to address the fungal virulence factors that could be the target of the development of new drugs for PCM treatment. Virulence factors favoring the process of fungal infection and pathogenicity are considered as a microbial attribute associated with host susceptibility. P. brasiliensis has some known virulence factors which are 43 kDa glycoprotein (gp 43) which is an important fungal antigen, 70 kDa glycoprotein (gp 70), the carbohydrates constituting the fungal cell wall α-1,3, glucan and ß-1,3-glucan, cell adhesion molecules and the presence of melanin pigments. The discovery and development of drugs that interact with these factors, such as inhibitors of ß-1,3-glucan, reduced synthesis of gp 43, inhibitors of melanin production, is of great importance for the treatment of PCM. The study of virulence factors favors the understanding of pathogen-host relationships, aiming to evaluate the possibility of developing new therapeutic targets and mechanisms that these molecules play in the infectious process, favoring the design of a more specific treatment for this disease.
Assuntos
Paracoccidioides , Paracoccidioidomicose , Fatores de Virulência/metabolismo , Animais , Antifúngicos/uso terapêutico , Parede Celular/metabolismo , América Central/epidemiologia , Proteínas Fúngicas/metabolismo , Glucanos/metabolismo , Glicoproteínas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Melaninas/metabolismo , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/isolamento & purificação , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidade , Paracoccidioidomicose/epidemiologia , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/patologia , Paracoccidioidomicose/terapia , Prevalência , América do Sul/epidemiologiaRESUMO
The genus Paracoccidioides consist of dimorphic fungi geographically limited to the subtropical regions of Latin America, which are responsible for causing deep systemic mycosis in humans. However, the molecular mechanisms by which Paracoccidioides spp. causes the disease remain poorly understood. Paracoccidioides spp. harbor genes that encode proteins involved in host cell interaction and mitochondrial function, which together are required for pathogenicity and mediate virulence. Previously, we identified TufM (previously known as EF-Tu) in Paracoccidioides brasiliensis (PbTufM) and suggested that it may be involved in the pathogenicity of this fungus. In this study, we examined the effects of downregulating PbTUFM using a silenced strain with a 55% reduction in PbTUFM expression obtained by antisense-RNA (aRNA) technology. Silencing PbTUFM yielded phenotypic differences, such as altered translation elongation, respiratory defects, increased sensitivity of yeast cells to reactive oxygen stress, survival after macrophage phagocytosis, and reduced interaction with pneumocytes. These results were associated with reduced virulence in Galleria mellonella and murine infection models, emphasizing the importance of PbTufM in the full virulence of P. brasiliensis and its potential as a target for antifungal agents against paracoccidioidomycosis.
Assuntos
Comunicação Celular , Interações Hospedeiro-Patógeno , Paracoccidioides/patogenicidade , Paracoccidioidomicose/microbiologia , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Virulência/metabolismo , Virulência , Animais , Regulação para Baixo , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioides/metabolismo , Paracoccidioidomicose/metabolismo , FagocitoseRESUMO
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi of the genus Paracoccidioides spp., which mainly affects workers in rural regions of Latin America. Although the antifungal agents currently available for the treatment of PCM are effective in controlling the disease, many months are needed for healing, making the side effects and drug interactions relevant. In addition, conventional treatments are not able to control the sequelae left by PCM, even after the cure, justifying the search for new therapeutic options against PCM. In this context, the enzyme homoserine dehydrogenase of P. brasiliensis (PbHSD) was used to screen a library of natural products from the Zinc database using three different docking programs, i.e. Autodock, Molegro, and CLC Drugdiscovery Workbench. Three molecules (Zinc codes 2123137, 15967722, and 20611644) were better ranked than the homoserine substrate (HSE) and were used for in vitro trials of the minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MCF). All three molecules presented a fungicidal profile with MICs/MCFs of 8, 32, and 128 µg mL-1, respectively. The two most promising molecules presented satisfactory results with wide therapeutic ranges in the cytotoxicity assays. Molecular dynamics simulations of PbHSD indicated that the ligands remained bound to the protein by a common mechanism throughout the simulation. The molecule with the lowest MIC value presented the highest number of contacts with the protein. The results presented in this work suggest that the molecule Zinc2123137 may be considered as a hit in the development of new therapeutic options for PCM.
Assuntos
Antifúngicos/farmacologia , Homosserina Desidrogenase/antagonistas & inibidores , Paracoccidioides/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HeLa , Humanos , Ligantes , Testes de Sensibilidade Microbiana/métodos , Simulação de Dinâmica Molecular , Células VeroRESUMO
Paracoccidioidomycosis (PCM) is a neglected human systemic disease caused by species of the genus Paracoccidioides. The disease attacks the host's lungs and may disseminate to many other organs. Treatment involves amphotericin B, sulfadiazine, trimethoprim-sulfamethoxazole, itraconazole, ketoconazole, or fluconazole. The treatment duration is usually long, from 6 months to 2 years, and many adverse effects may occur in relation to the treatment; co-morbidities and poor treatment adherence have been noted. Therefore, the discovery of more effective and less toxic drugs is needed. Thiosemicarbazide (TSC) and a camphene derivative of thiosemicarbazide (TSC-C) were able to inhibit P. brasiliensis growth at a low dosage and were not toxic to fibroblast cells. In order to investigate the mode of action of those compounds, we used a chemoproteomic approach to determine which fungal proteins were bound to each of these compounds. The compounds were able to inhibit the activities of the enzyme formamidase and interfered in P. brasiliensis dimorphism. In comparison with the transcriptomic and proteomic data previously obtained by our group, we determined that TSC and TSC-C were multitarget compounds that exerted effects on the electron-transport chain and cell cycle regulation, increased ROS formation, inhibited proteasomes and peptidases, modulated glycolysis, lipid, protein and carbohydrate metabolisms, and caused suppressed the mycelium to yeast transition.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Proteínas Fúngicas/metabolismo , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/metabolismo , Proteômica , Semicarbazidas/química , Semicarbazidas/farmacologia , Amidoidrolases/metabolismo , Animais , Antifúngicos/isolamento & purificação , Células 3T3 BALB , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Ativação Enzimática/efeitos dos fármacos , Proteínas Fúngicas/antagonistas & inibidores , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Ligação Proteica , Proteômica/métodos , Semicarbazidas/isolamento & purificaçãoRESUMO
The sensitivity of the double agar gel immunodiffusion test is about 90% in patients with untreated paracoccidioidomycosis (PCM), but it is much lower in cases of relapse. In addition, serum from patients with PCM caused by Paracoccidioides lutzii, frequent in the Midwest region of Brazil, do not react with the classical antigen obtained from Pb B-339. These findings showed the need for alternative diagnostic methods, such as biological markers through proteomics. The aim of this study was to identify biomarkers for the safe identification of PCM relapse and specific proteins that could distinguish infections caused by Paracoccidioides brasiliensis from those produced by Paracoccidioides lutzii. Proteomic analysis was performed in serum from 9 patients with PCM caused by P. brasiliensis, with and without relapse, from 4 patients with PCM produced by P. lutzii, and from 3 healthy controls. The comparative evaluation of the 29 identified plasma proteins suggested that the presence of the immunoglobulin (Ig) alpha-2 chain C region and the absence of Ig heavy chain V-III TIL indicate infection by P. lutzii. In addition, the absence of complement factor B protein might be a predictor of relapse. The evaluation of these proteins in a higher number of patients should be carried out in order to validate these findings.
Assuntos
Biomarcadores/sangue , Paracoccidioides/metabolismo , Paracoccidioidomicose/diagnóstico , Proteômica , Adolescente , Adulto , Idoso de 80 Anos ou mais , Anticorpos Antifúngicos/química , Anticorpos Antifúngicos/imunologia , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Feminino , Proteínas Fúngicas/análise , Proteínas Fúngicas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Paracoccidioides/isolamento & purificação , Paracoccidioidomicose/microbiologia , Recidiva , Risco , Espectrometria de Massas em TandemRESUMO
Paracoccidioidomycosis is an important systemic mycosis caused by thermodimorphic fungi of the Paracoccidioides genus. During the infective process, the cell wall acts at the interface between the fungus and the host. In this way, the cell wall has a key role in growth, environment sensing and interaction, as well as morphogenesis of the fungus. Since the cell wall is absent in mammals, it may present molecules that are described as target sites for new antifungal drugs. Despite its importance, up to now few studies have been conducted employing proteomics in for the identification of cell wall proteins in Paracoccidioides spp. Here, a detailed proteomic approach, including cell wall-fractionation coupled to NanoUPLC-MSE, was used to study and compare the cell wall fractions from Paracoccidioides lutzii mycelia and yeast cells. The analyzed samples consisted of cell wall proteins extracted by hot SDS followed by extraction by mild alkali. In summary, 512 proteins constituting different cell wall fractions were identified, including 7 predicted GPI-dependent cell wall proteins that are potentially involved in cell wall metabolism. Adhesins previously described in Paracoccidioides spp. such as enolase, glyceraldehyde-3-phosphate dehydrogenase were identified. Comparing the proteins in mycelium and yeast cells, we detected some that are common to both fungal phases, such as Ecm33, and some specific proteins, as glucanase Crf1. All of those proteins were described in the metabolism of cell wall. Our study provides an important elucidation of cell wall composition of fractions in Paracoccidioides, opening a way to understand the fungus cell wall architecture.
Assuntos
Parede Celular/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Micélio/genética , Paracoccidioides/genética , Bicarbonatos/química , Parede Celular/química , Cromatografia Líquida , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Ontologia Genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/isolamento & purificação , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Extração Líquido-Líquido/métodos , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/isolamento & purificação , Fosfopiruvato Hidratase/metabolismo , Proteômica/métodos , Dodecilsulfato de Sódio/química , Espectrometria de Massas em TandemRESUMO
Paracoccidioides brasiliensis yeast was reported to express paracoccin, a GlcNAc-binding protein that displays N-acetyl-ß-d-glucosaminidase (NAGase) activity. Highly specific anti-paracoccin antibodies have been previously used to examine the localization of paracoccin in yeast and inhibit its growth in vitro. In the present study, anti-paracoccin antibodies were used to characterize, by scanning confocal microscopy, the distribution of paracoccin in P. brasiliensis hyphae, transition forms from hyphae to yeast, and mature yeast. In the mycelial phase, paracoccin was detected mainly in the hyphae tips, where it demonstrated a punctate distribution, and was associated with the cell wall. During the first 48 hours after a temperature shift from 26°C to 37°C, paracoccin expression in the differentiating hyphae was mainly detected in the budding regions, i.e. lateral protrusions, and inside the new daughter cells. There was an increased number of chlamydoconidia that expressed a high concentration of paracoccin on their surfaces and/or in their interiors 72-96 hours after the temperature shift. After 120 hours, yeast cells were the predominant form and their cytoplasm stained extensively for paracoccin, whereas Wheat Germ Agglutinin (WGA) staining was predominant on their exterior walls. After 10 days at 37°C, the interior of both mother and daughter yeast cells, as well as the budding regions, stained intensely for paracoccin. The comparison of mRNA-expression in the different fungal forms showed that PCN transcripts, although detected in all evaluated morphological forms, were higher in hypha and yeast-to-hypha transition forms. In conclusion, the pattern of paracoccin distribution in all P. brasiliensis morphotypes supports prevalent beliefs that it plays important roles in fungal growth and dimorphic transformation.
Assuntos
Proteínas Fúngicas/metabolismo , Paracoccidioides/metabolismo , Paracoccidioides/crescimento & desenvolvimento , Aglutininas do Germe de Trigo/metabolismoRESUMO
This work evaluated new potential inhibitors of the enzyme homoserine dehydrogenase (HSD) of Paracoccidioides brasiliensis, one of the etiological agents of paracoccidioidomycosis. The tertiary structure of the protein bonded to the analogue NAD, and l-homoserine was modeled by homology. The model with the best output was subjected to gradient minimization, redocking, and molecular dynamics simulation. Virtual screening simulations with 187,841 molecules purchasable from the Zinc database were performed. After the screenings, 14 molecules were selected and analyzed by the use of absorption, distribution, metabolism, excretion, and toxicity criteria, resulting in four compounds for in vitro assays. The molecules HS1 and HS2 were promising, exhibiting MICs of 64 and 32 µg · ml-1, respectively, for the Pb18 isolate of P. brasilensis, 64 µg · ml-1 for two isolates of P. lutzii, and also synergy with itraconazole. The application of these molecules to human-pathogenic fungi confirmed that the HSD enzyme may be used as a target for the development of drugs with specific action against paracoccidioidomycosis; moreover, these compounds may serve as leads in the design of new antifungals.
Assuntos
Antifúngicos/farmacologia , Homosserina Desidrogenase/metabolismo , Paracoccidioides/efeitos dos fármacos , Paracoccidioidomicose/tratamento farmacológico , Linhagem Celular Tumoral , Células HeLa , Humanos , Itraconazol/farmacologia , Testes de Sensibilidade Microbiana/métodos , Paracoccidioides/metabolismo , Paracoccidioidomicose/metabolismoRESUMO
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the dimorphic fungi Paracoccidioides spp. The duration of antifungal treatment ranges from months to years and relapses may nevertheless occur despite protracted therapy. Thus, there remains an urgent need for new therapeutic options. Miltefosine (MLT), an analogue of alkylphospholipids, has antifungal activity against species of yeast and filamentous fungi. The aim of this study was to evaluate the antifungal effects of MLT on the yeast forms of Paracoccidioides brasiliensis and Paracoccidioides lutzii. MLT demonstrated inhibitory activity from 0.12 to 1 µg/mL, which was similar to amphotericin B or the combination trimethoprim/sulfamethoxazole but was not more effective than itraconazole. The fungicidal activity of MLT occurred at concentrations ≥1 µg/mL. Ultrastructural alterations were observed following exposure of the fungus to a subinhibitory concentration of MLT, such as cytoplasmic membrane alteration, mitochondrial swelling, electron-lucent vacuole accumulation and increasing melanosome-like structures. Melanin production by yeasts following MLT exposure was confirmed by labelling with antibodies to melanin. In addition, the combination of a subinhibitory concentration of MLT and tricyclazole, an inhibitor of DHN-melanin biosynthesis, drastically reduced yeast viability. In conclusion, MLT had a fungicidal effect against both Paracoccidioides spp., and a subinhibitory concentration impacted melanogenesis. These findings suggest that additional investigations should be pursued to establish a role for MLT in the treatment of PCM.
Assuntos
Antifúngicos/farmacologia , Melaninas/biossíntese , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/metabolismo , Fosforilcolina/análogos & derivados , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Cães , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Organelas/efeitos dos fármacos , Organelas/ultraestrutura , Paracoccidioides/isolamento & purificação , Paracoccidioides/ultraestrutura , Fosforilcolina/farmacologiaRESUMO
The genus Paracoccidioides is composed of thermal dimorphic fungi, causative agents of paracoccidioidomycosis, one of the most frequent systemic mycoses in Latin America. Mitochondria have sophisticated machinery for ATP production, which involves metabolic pathways such as citric acid and glyoxylate cycles, electron transport chain and oxidative phosphorylation. In addition, this organelle performs a variety of functions in the cell, working as an exceptional metabolic signalling centre that contributes to cellular stress responses, as autophagy and apoptosis in eukaryotic organisms. The aim of this work was to perform a descriptive proteomic analysis of mitochondria in Paracoccidioides lutzii yeast cells. After mitochondria fractionation, samples enriched in mitochondrial proteins were digested with trypsin and analysed using a NanoUPLC-MSE system (Waters Corporation, Manchester, UK). Ours results revealed that the established protocol for purification of mitochondria was very effective for P. lutzii, and 298 proteins were identified as primarily mitochondrial, in our analysis. To our knowledge, this is the first compilation of mitochondrial proteins from P. lutzii, to date. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Mitocôndrias/metabolismo , Paracoccidioides/genética , Paracoccidioides/metabolismo , Proteoma/genética , Proteômica/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Metabolismo dos Lipídeos , Mitocôndrias/genética , Estresse Oxidativo/fisiologiaRESUMO
Paracoccidioidomycosis is an endemic disease in Latin America, caused by thermo dimorphic fungi of the genus Paracoccidioides. Although previous proteome analyses of Paracoccidioides spp. have been carried out, the nuclear subproteome of this pathogen has not been described. In this way, we aimed to characterize the nuclear proteome of Paracoccidioides species, in the yeast form. For that, yeast cells were disrupted and submitted to cell fractionation. The purity of the nuclear fraction was confirmed by fluorescence and electron microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 867 proteins. In order to support our enrichment method for nuclear proteins, bioinformatics analysis were applied that allowed the identification of 281 proteins with nuclear localization. The analysis revealed proteins related to DNA maintenance, gene expression, synthesis and processing of messenger and ribosomal RNAs, likewise proteins of nuclear-cytoplasmic traffic. It was also possible to detect some proteins that are poorly expressed, like transcription factors involved in important roles such as resistance to abiotic stress, sporulation, cellular growth and DNA and chromatin maintenance. This is the first descriptive nuclear proteome of Paracoccidioides spp. that can be useful as an important platform base for fungi-specific nuclear processes.