RESUMO
BACKGROUND: The thermodimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis. Although poorly studied, paracoccin (PCN) from Paracoccidioides brasiliensis has been shown to harbor lectinic, enzymatic, and immunomodulatory properties that affect disease development. METHODS: Mutants of P. brasiliensis overexpressing PCN (ov-PCN) were constructed by Agrobacterium tumefaciens-mediated transformation. ov-PCN strains were analyzed and inoculated intranasally or intravenously to mice. Fungal burden, lung pathology, and survival were monitored to evaluate virulence. Electron microscopy was used to evaluate the size of chito-oligomer particles released by ov-PCN or wild-type strains to growth media. RESULTS: ov-PCN strains revealed no differences in cell growth and viability, although PCN overexpression favored cell separation, chitin processing that results in the release of smaller chito-oligomer particles, and enhanced virulence. Our data show that PCN triggers a critical effect in the cell wall biogenesis through the chitinase activity resulting from overexpression of PCN. As such, PCN overexpression aggravates the disease caused by P. brasiliensis. CONCLUSIONS: Our data are consistent with a model in which PCN modulates the cell wall architecture via its chitinase activity. These findings highlight the potential for exploiting PCN function in future therapeutic approaches.
Assuntos
Parede Celular/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/fisiologia , Lectinas/fisiologia , Paracoccidioides/patogenicidade , Animais , Citocinas/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioidomicose/imunologia , Fagocitose , VirulênciaRESUMO
Autophagy is a mechanism responsible for intracellular degradation and recycling of macromolecules and organelles, essential for cell survival in adverse conditions. More than 40 autophagy-related (ATG) genes have been identified and characterized in fungi, among them ATG4 and ATG8. ATG4 encodes a cysteine protease (Atg4) that plays an important role in autophagy by initially processing Atg8 at its C-terminus region. Atg8 is a ubiquitin-like protein essential for the synthesis of the double-layer membrane that constitutes the autophagosome vesicle, responsible for delivering the cargo from the cytoplasm to the vacuole lumen. The contributions of Atg-related proteins in the pathogenic yeast in the genus Cryptococcus remain to be explored, to elucidate the molecular basis of the autophagy pathway. In this context, we aimed to investigate the role of autophagy-related proteins 4 and 8 (Atg4 and Atg8) during autophagy induction and their contribution with non-autophagic events in C. neoformans. We found that Atg4 and Atg8 are conserved proteins and that they interact physically with each other. ATG gene deletions resulted in cells sensitive to nitrogen starvation. ATG4 gene disruption affects Atg8 degradation and its translocation to the vacuole lumen, after autophagy induction. Both atg4 and atg8 mutants are more resistant to oxidative stress, have an impaired growth in the presence of the cell wall-perturbing agent Congo Red, and are sensitive to the proteasome inhibitor bortezomib (BTZ). By that, we conclude that in C. neoformans the autophagy-related proteins Atg4 and Atg8 play an important role in the autophagy pathway; which are required for autophagy regulation, maintenance of amino acid levels and cell adaptation to stressful conditions.
Assuntos
Família da Proteína 8 Relacionada à Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/fisiologia , Cryptococcus neoformans/fisiologia , Proteínas Fúngicas/fisiologia , Aminoácidos/metabolismo , Autofagia/genética , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Bortezomib/farmacologia , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/genética , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Genes Fúngicos , Teste de Complementação Genética , Humanos , Técnicas In Vitro , Mutação , Nitrogênio/metabolismo , Estresse Oxidativo/genética , Inibidores de Proteassoma/farmacologia , Processamento de Proteína Pós-Traducional , Vacúolos/metabolismoRESUMO
Paracoccidioides brasiliensis is a temperature-dependent dimorphic fungus that cause paracoccidioidomycosis (PCM), the major systemic mycosis in Latin America. The capacity to evade the innate immune response of the host is due to P. brasiliensis ability to respond and to survive the nitrosative stress caused by phagocytic cells. However, the regulation of signal transduction pathways associated to nitrosative stress response are poorly understood. Ras GTPase play an important role in the various cellular events in many fungi. Ras, in its activated form (Ras-GTP), interacts with effector proteins and can initiate a kinase cascade. In this report, we investigated the role of Ras GTPase in P. brasiliensis after in vitro stimulus with nitric oxide (NO). We observed that low concentrations of NO induced cell proliferation in P. brasiliensis, while high concentrations promoted decrease in fungal viability, and both events were reversed in the presence of a NO scavenger. We observed that high levels of NO induced Ras activation and its S-nitrosylation. Additionally, we showed that Ras modulated the expression of antioxidant genes in response to nitrosative stress. We find that the Hog1 MAP kinase contributed to nitrosative stress response in P. brasiliensis in a Ras-dependent manner. Taken together, our data demonstrate the relationship between Ras-GTPase and Hog1 MAPK pathway allowing for the P. brasiliensis adaptation to nitrosative stress.
Assuntos
Proteínas Fúngicas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Estresse Nitrosativo/fisiologia , Paracoccidioides/fisiologia , Proteínas ras/fisiologia , Sequência de Aminoácidos , Animais , Morte Celular/fisiologia , Proliferação de Células/fisiologia , Expressão Gênica/fisiologia , Masculino , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico/química , Óxido Nítrico/farmacologia , Processamento de Proteína Pós-TraducionalRESUMO
Penicillium roqueforti is used in the production of several kinds of ripened blue-veined cheeses. In addition, this fungus produces interesting secondary metabolites such as roquefortine C, andrastin A and mycophenolic acid. To date, there is scarce information concerning the regulation of the production of these secondary metabolites. Recently, the gene named pcz1 (Penicillium C6 zinc domain protein 1) was described in P. roqueforti, which encodes for a Zn(II)2Cys6 protein that controls growth and developmental processes in this fungus. However, its effect on secondary metabolism is currently unknown. In this work, we have analyzed how the overexpression and down-regulation of pcz1 affect the production of roquefortine C, andrastin A and mycophenolic acid in P. roqueforti. The three metabolites were drastically reduced in the pcz1 down-regulated strains. However, when pcz1 was overexpressed, only mycophenolic acid was overproduced while, on the contrary, levels of roquefortine C and andrastin A were diminished. Importantly, these results match the expression pattern of key genes involved in the biosynthesis of these metabolites. Taken together, our results suggest that Pcz1 plays a key role in regulating secondary metabolism in the fungus Penicillium roqueforti.
Assuntos
Proteínas Fúngicas/fisiologia , Fungos/genética , Fungos/metabolismo , Penicillium/genética , Penicillium/metabolismo , Metabolismo Secundário/genética , Androstadienos/metabolismo , Queijo/microbiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Indóis/metabolismo , Ácido Micofenólico/metabolismo , Piperazinas/metabolismoRESUMO
Here, we report that the Neurospora crassa FLB-3 protein, the ortholog of the Aspergillus nidulans FlbC transcription factor, is required for developmental control. Deletion of flb-3 leads to changes in hyphae morphology and affects sexual and asexual development. We identified, as putative FLB-3 targets, the N. crassa aba-1, wet-1 and vos-1 genes, orthologs of the ones involved in A. nidulans asexual development and that work downstream of FlbC (abaA, wetA and vosA). In N. crassa, these three genes require FLB-3 for proper expression; however, they appear not to be required for normal development, as demonstrated by gene expression analyses during vegetative growth and asexual development. Moreover, mutant strains in the three genes conidiate well and produce viable conidia. We also determined FLB-3 DNA-binding preferences via protein-binding microarrays (PBMs) and demonstrated by chromatin immunoprecipitation (ChIP) that FLB-3 binds the aba-1, wet-1 and vos-1 promoters. Our data support an important role for FLB-3 in N. crassa development and highlight differences between the regulatory pathways controlled by this transcription factor in different fungal species.
Assuntos
Proteínas Fúngicas/fisiologia , Neurospora crassa/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Proteínas Fúngicas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Hifas/genética , Hifas/crescimento & desenvolvimento , Neurospora crassa/genética , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Fatores de Transcrição/genéticaRESUMO
The Mitogen-Activated Protein Kinase (MAPK) signaling pathways constitute one of the most important and evolutionarily conserved mechanisms for the perception of extracellular information in all the eukaryotic organisms. The MAPK pathways are involved in the transfer to the cell of the information perceived from extracellular stimuli, with the final outcome of activation of different transcription factors that regulate gene expression in response to them. In all species of fungi, the MAPK pathways have important roles in their physiology and development; e.g. cell cycle control, mating, morphogenesis, response to different stresses, resistance to UV radiation and to temperature changes, cell wall assembly and integrity, degradation of cellular organelles, virulence, cell-cell signaling, fungus-plant interaction, and response to damage-associated molecular patterns (DAMPs). Considering the importance of the phylogenetically conserved MAPK pathways in fungi, an updated review of the knowledge on them is discussed in this article. This information reveals their importance, their distribution in fungal species evolutionarily distant and with different lifestyles, their organization and function, and the interactions occurring between different MAPK pathways, and with other signaling pathways, for the regulation of the most complex cellular processes.
Assuntos
Proteínas Fúngicas/fisiologia , Fungos/enzimologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Sequência de Aminoácidos , Sequência Conservada , Fungos/genética , Fungos/crescimento & desenvolvimento , Fungos/patogenicidade , Proteínas de Ligação ao GTP/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Modelos Biológicos , Morfogênese/fisiologia , Filogenia , Receptores Acoplados a Proteínas G/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Virulência/fisiologiaRESUMO
Adhesion is the first step for Candida species to form biofilms on medical devices implanted in the human host. Both the physicochemical nature of the biomaterial and cell wall proteins (CWP) of the pathogen play a determinant role in the process. While it is true that some CWP have been identified in vitro, little is known about the CWP of pathogenic species of Candida involved in adhesion. On this background, we considered it important to investigate the potential role of CWP of C. albicans, C. glabrata, C. krusei and C. parapsilosis in adhesion to different medical devices. Our results indicate that the four species strongly adher to polyvinyl chloride (PVC) devices, followed by polyurethane and finally by silicone. It was interesting to identify fructose-bisphosphate aldolase (Fba1) and enolase 1 (Eno1) as the CWP involved in adhesion of C. albicans, C. glabrata and C. krusei to PVC devices whereas phosphoglycerate kinase (Pgk) and Eno1 allow C. parapsilosis to adher to silicone-made implants. Results presented here suggest that these CWP participate in the initial event of adhesion and are probably followed by other proteins that covalently bind to the biomaterial thus providing conditions for biofilm formation and eventually the onset of infection.
Assuntos
Candida/fisiologia , Adesão Celular , Parede Celular/química , Equipamentos e Provisões/microbiologia , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/fisiologia , Antifúngicos/farmacologia , Materiais Biocompatíveis/química , Biofilmes/crescimento & desenvolvimento , Candida/efeitos dos fármacos , Candida/enzimologia , Candida/metabolismo , Adesão Celular/efeitos dos fármacos , Parede Celular/enzimologia , Parede Celular/metabolismo , Frutose-Bifosfato Aldolase/isolamento & purificação , Frutose-Bifosfato Aldolase/fisiologia , Proteínas Fúngicas/fisiologia , Humanos , Peróxido de Hidrogênio/farmacologia , Fosfoglicerato Quinase , Fosfopiruvato Hidratase/isolamento & purificação , Fosfopiruvato Hidratase/fisiologia , Poliuretanos/química , Cloreto de Polivinila/química , Silicones/químicaRESUMO
Fungi represent a large group of biodiverse microorganisms with potential applications ranging from industrial fields to the treatment for human diseases. A large number of pharmacologically active compounds including terpenoids, polysaccharides and proteins have been derived from these microorganisms. Fungal Immunomodulatory Proteins (FIPs) are a group of active compounds that are being considered for the treatment of asthma, allergy, autoimmune diseases and cancer. Here, we discuss the discovery, heterologous production bioactive mechanisms of action and their potential use in biomedicine.
Assuntos
Adjuvantes Imunológicos , Proteínas Fúngicas/fisiologia , Genes FúngicosRESUMO
Aspergillus fumigatus, the main etiologic agent causing invasive aspergillosis, can induce an inflammatory response and a prothrombotic phenotype upon contact with human umbilical vein endothelial cells (HUVECs). However, the fungal molecules involved in this endothelial response remain unknown. A. fumigatus hyphae produce an extracellular matrix composed of galactomannan, galactosaminogalactan and α-(1,3)-glucan. In this study, we investigated the consequences of UGM1 gene deletion in A. fumigatus, which produces a mutant with increased galactosaminogalactan production. The ∆ugm1 mutant exhibited an HUVEC-hyperadhesive phenotype and induced increased endothelial TNF-α secretion and tissue factor mRNA overexpression in this "semi-professional" immune host cell. Using a shotgun proteomics approach, we show that the A. fumigatus ∆ugm1 strain can modulate the levels of proteins in important endothelial pathways related to the inflammatory response mediated by TNF-α and to stress response pathways. Furthermore, a purified galactosaminogalactan fraction was also able to induce TNF-α secretion and the coincident HUVEC pathways regulated by the ∆ugm1 mutant, which overexpresses this component, as demonstrated by fluorescence microscopy. This work contributes new data regarding endothelial mechanisms in response to A. fumigatus infection. SIGNIFICANCE: Invasive aspergillosis is the main opportunistic fungal infection described in neutropenic hematologic patients. One important clinical aspect of this invasive fungal infection is vascular thrombosis, which could be related, at least in part, to the activation of endothelial cells, as shown in previous reports from our group. It is known that direct contact between the A. fumigatus hyphal cell wall and the HUVEC cell surface is necessary to induce an endothelial prothrombotic phenotype and secretion of pro-inflammatory cytokines, though the cell surface components of this angioinvasive fungus that trigger this endothelial response are unknown. The present work employs a discovery-driven proteomics approach to reveal the role of one important cell wall polysaccharide of A. fumigatus, galactosaminogalactan, in the HUVEC interaction and the consequent mechanisms of endothelial activation. This is the first report of the overall panel of proteins related to the HUVEC response to a specific and purified cell wall component of the angioinvasive fungus A. fumigatus.
Assuntos
Aspergillus fumigatus/química , Células Endoteliais da Veia Umbilical Humana/química , Células Endoteliais da Veia Umbilical Humana/microbiologia , Hifas/química , Inflamação , Estresse Fisiológico , Aspergillus fumigatus/genética , Células Endoteliais/metabolismo , Proteínas Fúngicas/fisiologia , Deleção de Genes , Interações Hospedeiro-Patógeno , Humanos , Polissacarídeos/biossíntese , Trombose/etiologia , Trombose/microbiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
AIM: This study aims to understand which Candida orthopsilosis protein aids fungus adaptation upon its switching from planktonic growth to biofilm. MATERIALS & METHODS: Ion mobility separation within mass spectrometry analysis combination were used. RESULTS: Proteins mapped for different biosynthetic pathways showed that selective ribosome autophagy might occur in biofilms. Glucose, used as a carbon source in the glycolytic flux, changed to glycogen and trehalose. CONCLUSION: Candida orthopsilosis expresses proteins that combine a variety of mechanisms to provide yeasts with the means to adjust the catalytic properties of enzymes. Adjustment of the enzymes helps modulate the biosynthesis/degradation rates of the available nutrients, in order to control and coordinate the metabolic pathways that enable cells to express an adequate response to nutrient availability.