RESUMEN
Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America. It is caused by the temperature-dependent dimorphic fungus Paracoccidioides brasiliensis. The P. brasiliensis cell wall is a dynamic outer structure, composed of a network of glycoproteins and polysaccharides, such as chitin, glucan and N-glycosylated proteins. These glycoproteins can interact with the host to affect infection rates, and are known to perform other functions. We inhibited N-linked glycosylation using tunicamycin (TM), and then evaluated the expression of P. brasiliensis genes related to cell wall remodeling. Our results suggest that cell wall synthesis related genes, such as ß-1,3-glucanosyltransferase (PbGEL3), 1,3-ß-D-glucan synthase (PbFKS1), and α-1,4-amylase (PbAMY), as well as cell wall degrading related genes, such as N-acetyl-ß-D-glucosaminidase (PbNAG1), α-1,3-glucanase (PbAGN), and ß-1,3-glucanase (PbBGN1 and PbBGN2), have their expression increased by the N-glycosylation inhibition, as detected by qRT-PCR. The observed increases in gene expression levels reveal possible compensatory mechanisms for diminished enzyme activity due to the lack of glycosylation caused by TM.
RESUMEN
The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N-glycosylation of proteins such as N-acetyl-ß-D-glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)-mediated inhibition of N-linked glycosylation on α- and ß-(1,3)-glucanases and on α-(1,4)-amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 µg/ml TM to the fungal cultures did not interfere with either α- or ß-(1,3)-glucanase production and secretion. Moreover, incubation with TM did not alter α- and ß-(1,3)-glucanase activity in yeast and mycelium cell extracts. In contrast, α-(1,4)-amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N-glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N-glycans were essential for α-(1,4)-amylase activity involved in the production of malto-oligosaccharides that act as primer molecules for the biosynthesis of α-(1,3)-glucan. Our results suggest that reduced fungal α-(1,4)-amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells.
Asunto(s)
Antiinfecciosos/farmacología , Glucano 1,3-beta-Glucosidasa/metabolismo , Glicósido Hidrolasas/metabolismo , Glicosilación/efectos de los fármacos , Paracoccidioides/crecimiento & desarrollo , Tunicamicina/farmacología , alfa-Amilasas/metabolismo , Paracoccidioides/citología , Paracoccidioides/efectos de los fármacos , Paracoccidioides/enzimologíaRESUMEN
The genus Paracoccidioides comprises a complex of phylogenetic species of dimorphic pathogenic fungi, the etiologic agents of paracoccidioidomycosis (PCM), a disease confined to Latin America and of marked relevance in its endemic areas due to its high frequency and severity. The members of the Paracoccidioides genus are distributed in distinct phylogenetic species (S1, PS2, PS3 and 01-like) that potentially differ in their biochemical and molecular characteristics. In this work, we performed the proteomic characterization of different members of the genus Paracoccidioides. We compared the proteomic profiles of Pb01 (01-like), Pb2 (PS2), Pb339 (S1) and PbEPM83 (PS3) using 2D electrophoresis and mass spectrometry. The proteins/isoforms were selected based on the staining intensity of the spots as determined by image analysis. The proteins/isoforms were in-gel digested and identified by peptide mass fingerprinting and ion fragmentation. A total of 714 spots were detected, of which 343 were analyzed. From these spots, 301 represented differentially expressed proteins/isoforms among the four analyzed isolates, as determined by ANOVA. After applying the FDR correction, a total of 267 spots were determined to be differentially expressed. From the total, 193 proteins/isoforms were identified by PMF and confirmed by ion fragmentation. Comparing the expression profiles of the isolates, the proteins/isoforms that were related to glycolysis/gluconeogenesis and to alcohol fermentation were more abundant in Pb01 than in other representatives of the genus Paracoccidioides, indicating ahigher use of anaerobic pathways for energy production. Those enzymes related to the oxidative stress response were more abundant in Pb01, Pb2 and Pb339, indicating a better response to ROS in these members of the Paracoccidioides complex. The enzymes of the pentose phosphate pathway were abundant in Pb2. Antigenic proteins, such as GP43 and a 27-kDa antigenic protein, were less abundant in Pb01 and Pb2. The proteomic profile indicates metabolic differences among the analyzed members of the Paracoccidioides genus.
Asunto(s)
Proteínas Fúngicas/análisis , Paracoccidioidomicosis/genética , Paracoccidioidomicosis/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Respuesta al Choque Térmico/genética , Estrés Oxidativo/genética , Paracoccidioides/clasificación , Paracoccidioides/genética , Mapeo Peptídico , Filogenia , ProteomaRESUMEN
Paracoccidioides spp. are endemic fungi from Latin America that cause Paracoccidioidomycosis, a systemic disease. These fungi present systems for high-affinity metal uptake, storage, and mobilization, which counteract host nutritional immunity and mitigate the toxic effects of metals. Regarding Cu mobilization, the metallochaperone Atx1 is regulated according to Cu bioavailability in Paracoccidioides spp., contributing to metal homeostasis. However, additional information in the literature on PbAtx1 is scarce. Therefore, in the present work, we aimed to study the PbAtx1 protein-protein interaction networks. Heterologous expressed PbAtx1 was used in a pull-down assay with Paracoccidioides brasiliensis cytoplasmic extract. Nineteen proteins that interacted with PbAtx1 were identified by HPLC-MSE. Among them, a relevant finding was a Cytochrome b5 (PbCyb5), regulated by Fe bioavailability in Aspergillus fumigatus and highly secreted by P. brasiliensis in Fe deprivation. We validated the interaction between PbAtx1-PbCyb5 through molecular modeling and far-Western analyses. It is known that there is a relationship between Fe homeostasis and Cu homeostasis in organisms. In this sense, would PbAtx1-PbCyb5 interaction be a new metal-sensor system? Would it be supported by the presence/absence of metals? We intend to answer those questions in future works to contribute to the understanding of the strategies employed by Paracoccidioides spp. to overcome host defenses.
RESUMEN
The fungal pathogen Paracoccidioides lutzii causes systemic mycosis Paracoccidioidomycosis (PCM), which presents a broad distribution in Latin America. Upon infection, the fungus undergoes a morphological transition to yeast cells and provokes an inflammatory granulomatous reaction with a high number of neutrophils in the lungs. In this work, we employed proteomic analysis to investigate the in vitro response of the fungus to the interaction with human neutrophils. Proteomic profiling of P. lutzii yeast cells harvested at 2 and 4 h post interaction with human polymorphonuclear cells allowed the identification of 505 proteins differentially accumulated. The data indicated that P. lutzii yeast cells underwent a shift in metabolism from glycolysis to Beta oxidation, increasing enzymes of the glyoxylate cycle and upregulating enzymes related to the detoxification of oxidative and heat shock stress. To our knowledge, this is the first study employing proteomic analysis in the investigation of the response of a member of the Paracoccidioides genus to the interaction with neutrophils.
RESUMEN
Fungi of the Paracoccidioides genus are the etiological agents of the systemic mycosis paracoccidioidomycosis and, when in the host, they find a challenging environment that is scarce in nutrients and micronutrients, such as Fe, which is indispensable for the survival of the pathogen. Previous studies have shown that fungi of this genus, in response to Fe deprivation, are able to synthesize and capture siderophores (Fe3+ chelators), use Fe-containing host proteins as a source of the metal, and use a non-canonical reductive pathway for Fe3+ assimilation. Despite all of these findings, there are still gaps that need to be filled in the pathogen response to metal deprivation. To contribute to the knowledge related to this subject, we obtained the exoproteome of Paracoccidioides brasiliensis (Pb18) undergoing Fe deprivation and by nanoUPLC-MSE. One hundred forty-one proteins were identified, and out of these, 64 proteins were predicted to be secreted. We also identified the regulation of several virulence factors. Among the results, we highlight Cyb5 as a secreted molecule of Paracoccidioides in the exoproteome obtained during Fe deprivation. Cyb5 is described as necessary for the Fe deprivation response of Saccharomyces cerevisiae and Aspergillus fumigatus. Experimental data and molecular modeling indicated that Cyb5 can bind to Fe ions in vitro, suggesting that it can be relevant in the arsenal of molecules related to iron homeostasis in P. brasiliensis.
Asunto(s)
Paracoccidioides , Paracoccidioidomicosis , Aspergillus fumigatus/metabolismo , Hierro/metabolismo , Saccharomyces cerevisiae/metabolismo , Sideróforos/metabolismoRESUMEN
Members of the Paracoccidioides complex are human pathogens that infect different anatomic sites in the host. The ability of Paracoccidioides spp. to infect host niches is putatively supported by a wide range of virulence factors, as well as fitness attributes that may comprise the transition from mycelia/conidia to yeast cells, response to deprivation of micronutrients in the host, expression of adhesins on the cell surface, response to oxidative and nitrosative stresses, as well as the secretion of hydrolytic enzymes in the host tissue. Our understanding of how those molecules can contribute to the infection establishment has been increasing significantly, through the utilization of several models, including in vitro, ex vivo and in vivo infection in animal models. In this review we present an update of our understanding on the strategies used by the pathogen to establish infection. Our results were obtained through a comparative proteomic analysis of Paracoccidioides spp. in models of infection.
Asunto(s)
Proteínas Fúngicas/metabolismo , Interacciones Huésped-Patógeno , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidad , Paracoccidioidomicosis/microbiología , Factores de Virulencia/metabolismo , Animales , Humanos , Ratones , Estrés Nitrosativo , Estrés Oxidativo , Proteómica/métodos , Esporas Fúngicas/metabolismoRESUMEN
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.