RESUMEN
Gallic acid is a powerful antioxidant with multiple therapeutic applications, usually obtained from the acidic hydrolysis of tannins produced by many plants. As this process generates a considerable amount of toxic waste, the use of tannases or tannase-producing microorganisms has become a greener alternative over the last years. However, their high costs still impose some barriers for industrial scalability, requiring solutions that could be both greener and cost-effective. Since Pseudomonas putida KT2440 is a powerful degrader of gallic acid, its metabolism offers pathways that can be engineered to produce it from cheap and renewable carbon sources, such as the crude glycerol generated in biodiesel units. In this study, a synthetic operon with the heterologous genes aroG4, quiC and pobA* was developed and expressed in P. putida, based on an in silico analysis of possible metabolic routes, resulting in no production. Then, the sequences pcaHG and galTAPR were deleted from the genome of this strain to avoid the degradation of gallic acid and its main intermediate, the protocatechuic acid. This mutant was transformed with the vector containing the synthetic operon and was finally able to convert glycerol into gallic acid. Production assays in shaker showed a final concentration of 346.7 ± 0.004 mg L-1 gallic acid after 72 h.
Asunto(s)
Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Glicerol/metabolismo , Ácido Gálico/metabolismoRESUMEN
PURPOSE: During the juvenile stage, such areas as the hippocampus and corpus callosum (CC) are still immature and sensitive to stress exposure. The present study investigated whether two different types of stressors in the juvenile stage of life have a long-lasting impact on behavior and biological outcomes in adult rats. METHODS: Male juvenile rats were exposed to restraint or predator stress on postnatal day 25 (P25) for 3 days. Thirty-two days later (P60-74), behavioral and biological analyses were conducted. The behavioral analysis included measures of anxiety-like behavior and recognition memory. The biological analysis investigated gross cerebral morphology, based on volume analysis of the CC and hippocampus, perirhinal cortex thickness, and dendritic spine density. RESULTS: Neither restraint stress nor predator stress affected anxiety-like behavior or object recognition memory in adulthood. Body weight and adrenal gland weight were unaffected by both types of stress. Overall, volumetric measures of the CC and hippocampus were not significant, with no changes in perirhinal cortex thickness. Spine density in the medial prefrontal cortex also was unaffected, but a decrease in dendritic spine density was found in the hippocampus in response to restraint stress and an increase to predator stress. CONCLUSION: Short-term and daily restraint and predator stress during the juvenile stage had no long-lasting effects on anxiety-like behavior, object memory, volume of the CC or hippocampus, or perirhinal cortex thickness, but a decrease in dendritic spine density was found in the hippocampus. These findings suggest that different types of stressors have different impacts on microstructures in the brain without affecting behavior or the gross morphology of stress-sensitive brain areas.
Asunto(s)
Espinas Dendríticas , Corteza Prefrontal , Ratas , Animales , Masculino , Espinas Dendríticas/fisiología , Encéfalo , Hipocampo , Ansiedad , Estrés PsicológicoRESUMEN
Nosocomial infections by fungi are important causes of morbidity and mortality, and the adhesion capacity of yeast on abiotic and biotic surfaces has been considered an important step in this process. Als3 proteins are widely studied for their ability to allow Candida albicans to bind to various surfaces. The objective of the present study was to verify, with more details, the action of F2768-0318 in relation to its antifungal activity as well as its ability to act on C. albicans virulence factors related to adhesion and biofilm formation in vitro and in vivo by inhibiting the Als3 protein. F2768-0318 was assessed in tests of biofilm formation and adhesion on abiotic surfaces (polystyrene plates) and adherence on biotic surfaces, including human endocervical (HeLa) cells, human umbilical vein endothelial cells (HUVECs), and fresh buccal epithelial cells (BEC). Our results showed F2768-0318 was useful in reducing the adhesion and biofilm formation of C. albicans on abiotic surfaces, indicating the possibility of treating hospital materials and preventing biofilm formation on these types of equipment. Further studies are still needed, including optimization of the molecule to allow this molecule to be effective on other types of surfaces, such as human cells.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Animales , Antifúngicos/uso terapéutico , Candida albicans/crecimiento & desarrollo , Candida albicans/fisiología , Candidemia/tratamiento farmacológico , Candidemia/microbiología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Células Epiteliales/fisiología , Femenino , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Pruebas de ToxicidadRESUMEN
BACKGROUND: Brazil is one of the highest tuberculosis (TB) burden countries of the world. Cutaneous tuberculosis (CTB) is a rare form of extrapulmonary manifestation of tuberculosis. This study aimed to describe the clinico-evolutive, laboratory and therapeutic aspects of CTB cases among patients from a cohort with TB in Rio de Janeiro, Brazil. METHODS: Cases of diagnosed CTB with microbiologic confirmation or clinical response to anti-tuberculous treatment associated with positive smear or histopathological findings between the years 2000 and 2016 were selected. RESULTS: Seventy-five patients with CTB were included, most were women (58.7%) with a median age of 42 years. CTB diagnosis was based on culture in only 42.7% of the cases. Scrofuloderma represented 50.7% of the cases, followed by erythema induratum of Bazin (EIB) (18.7%), tuberculous gumma (13.3%), lupus vulgaris (8%), TB verrucosa cutis (4%), orificial TB (2.7%) and associated forms (2.7%). Other TB presentations were pulmonary (22.7%), mammary (6.6%) and osteoarticular (4%). All patients who completed the treatment (97.3%) had their lesions healed. Only two patients (2.6%) needed to change the therapy due to adverse reactions. Fifty percent of EIB patients presented recurrence. CONCLUSIONS: These data highlight the diversity of CTB presentations and the importance of the skin to assist in early identification and treatment of TB. More studies are necessary to improve the knowledge on EIB for a better approach towards these patients, mainly in cases of recurrence.
Asunto(s)
Antituberculosos/uso terapéutico , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Cutánea/epidemiología , Tuberculosis Osteoarticular/epidemiología , Tuberculosis Pulmonar/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Brasil/epidemiología , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Piel/microbiología , Piel/patología , Resultado del Tratamiento , Tuberculosis Cutánea/tratamiento farmacológico , Tuberculosis Cutánea/microbiología , Tuberculosis Cutánea/patología , Tuberculosis Osteoarticular/tratamiento farmacológico , Tuberculosis Osteoarticular/microbiología , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/microbiología , Adulto JovenRESUMEN
The order Chaetothyriales (Pezizomycotina, Ascomycetes) harbours obligatorily melanised fungi and includes numerous etiologic agents of chromoblastomycosis, phaeohyphomycosis and other diseases of vertebrate hosts. Diseases range from mild cutaneous to fatal cerebral or disseminated infections and affect humans and cold-blooded animals globally. In addition, Chaetothyriales comprise species with aquatic, rock-inhabiting, ant-associated, and mycoparasitic life-styles, as well as species that tolerate toxic compounds, suggesting a high degree of versatile extremotolerance. To understand their biology and divergent niche occupation, we sequenced and annotated a set of 23 genomes of main the human opportunists within the Chaetothyriales as well as related environmental species. Our analyses included fungi with diverse life-styles, namely opportunistic pathogens and closely related saprobes, to identify genomic adaptations related to pathogenesis. Furthermore, ecological preferences of Chaetothyriales were analysed, in conjuncture with the order-level phylogeny based on conserved ribosomal genes. General characteristics, phylogenomic relationships, transposable elements, sex-related genes, protein family evolution, genes related to protein degradation (MEROPS), carbohydrate-active enzymes (CAZymes), melanin synthesis and secondary metabolism were investigated and compared between species. Genome assemblies varied from 25.81 Mb (Capronia coronata) to 43.03 Mb (Cladophialophora immunda). The bantiana-clade contained the highest number of predicted genes (12â817 on average) as well as larger genomes. We found a low content of mobile elements, with DNA transposons from Tc1/Mariner superfamily being the most abundant across analysed species. Additionally, we identified a reduction of carbohydrate degrading enzymes, specifically many of the Glycosyl Hydrolase (GH) class, while most of the Pectin Lyase (PL) genes were lost in etiological agents of chromoblastomycosis and phaeohyphomycosis. An expansion was found in protein degrading peptidase enzyme families S12 (serine-type D-Ala-D-Ala carboxypeptidases) and M38 (isoaspartyl dipeptidases). Based on genomic information, a wide range of abilities of melanin biosynthesis was revealed; genes related to metabolically distinct DHN, DOPA and pyomelanin pathways were identified. The MAT (MAting Type) locus and other sex-related genes were recognized in all 23 black fungi. Members of the asexual genera Fonsecaea and Cladophialophora appear to be heterothallic with a single copy of either MAT-1-1 or MAT-1-2 in each individual. All Capronia species are homothallic as both MAT1-1 and MAT1-2 genes were found in each single genome. The genomic synteny of the MAT-locus flanking genes (SLA2-APN2-COX13) is not conserved in black fungi as is commonly observed in Eurotiomycetes, indicating a unique genomic context for MAT in those species. The heterokaryon (het) genes expansion associated with the low selective pressure at the MAT-locus suggests that a parasexual cycle may play an important role in generating diversity among those fungi.
RESUMEN
Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.
Asunto(s)
Hibridación de Ácido Nucleico , Oligonucleótidos/química , Factor de Crecimiento Epidérmico/química , Receptores ErbB/química , Citometría de Flujo , Colorantes Fluorescentes/química , Unión ProteicaRESUMEN
The development of biocompatible polymeric nanoparticles has become an important strategy for optimizing the therapeutic efficacy of many classical drugs, as it may expand their activities, reduce their toxicity, increase their bioactivity and improve biodistribution. In this study, nanoparticles of Amphotericin B entrapped within poly (lactic-co-glycolic) acid and incorporated with dimercaptosuccinic acid (NANO-D-AMB) as a target molecule were evaluated for their physic-chemical characteristics, pharmacokinetics, biocompatibility and antifungal activity. We found high plasma concentrations of Amphotericin B upon treatment with NANO-D-AMB and a high uptake of nanoparticles in the lungs, liver and spleen. NANO-D-AMB exhibited antifungal efficacy against Paracoccidioides brasiliensis and induced much lower cytotoxicity levels compared to D-AMB formulation in vivo and in vitro. Together, these results confirm that NANO-D-AMB improves Amphotericin B delivery and suggest this delivery system as a potential alternative to the use of Amphotericin B sodium deoxycholate.
Asunto(s)
Anfotericina B/química , Anfotericina B/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Ácido Desoxicólico/química , Ácido Desoxicólico/farmacología , Portadores de Fármacos/química , Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , Anfotericina B/efectos adversos , Anfotericina B/uso terapéutico , Animales , Antifúngicos/efectos adversos , Antifúngicos/uso terapéutico , Ácido Desoxicólico/efectos adversos , Ácido Desoxicólico/uso terapéutico , Portadores de Fármacos/farmacocinética , Combinación de Medicamentos , Liberación de Fármacos , Ácido Láctico/farmacocinética , Ensayo de Materiales , Ratones , Paracoccidioides/efectos de los fármacos , Paracoccidioides/fisiología , Paracoccidioidomicosis/tratamiento farmacológico , Ácido Poliglicólico/farmacocinética , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Seguridad , Succímero/química , Distribución TisularRESUMEN
Biotechnology and nanotechnology are fields of science that can be applied together to solve a variety of biological issues. In the case of human health, biotechnology attempts to improve advances on the therapy against several diseases. Therapeutic peptides and proteins are promissory molecules for developing new medicines. Gene transfection and RNA interference have been considered important approaches for modern therapy to treat cancer and viral infections. However, because of their instability, these molecules alone cannot be used for in vivo application, since they are easily degraded or presenting a poor efficiency. Nanotechnology can contribute by the development of nanostructured delivery systems to increase the stability and potency of these molecules. Studies involving polymeric and magnetic nanoparticles, dendrimers, and carbon nanotubes have demonstrated a possibility to use these systems as vectors instead of the conventional viral ones, which present adverse effects, such as recombination and immunogenicity. This review presents some possibilities and strategies to efficiently delivery peptides, proteins, gene and RNA interference using nanotechnology approach.
Asunto(s)
Biotecnología/métodos , Sistemas de Liberación de Medicamentos/métodos , Nanotecnología/métodos , Animales , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/instrumentación , Estabilidad de Medicamentos , Humanos , Nanotecnología/instrumentaciónRESUMEN
Vaccines play an essential role in keeping humans healthy. Innovative approaches to their use include the utilization of plasmid DNA encoding sequences to express foreign antigens. DNAhsp65 from Mycobacterium leprae is suitable for this purpose due to its ability to elicit a powerful immune response. Controlled release systems represent a promising approach to delivering vaccines. In this work, we used liposomes or PLGA systems to deliver DNAhsp65 to treat the pulmonary fungal infection Paracoccidioidomycosis. Both formulations modulated a protective immune response and reduced the pulmonary fungal burden even in the groups receiving less than four times the amount of the DNAhps65 entrapped within the nanoparticles. Although both systems had the same effective therapeutic results, the advantage of the liposome formulation was that it was administered intranasally, which may be more easily accepted by patients. These systems are a great alternative to be considered as adjuvant vaccine therapy for systemic mycosis.
Asunto(s)
Biotecnología/métodos , Vacunas Fúngicas/administración & dosificación , Técnicas de Transferencia de Gen , Nanotecnología/métodos , Paracoccidioidomicosis/inmunología , Paracoccidioidomicosis/prevención & control , Vacunas de ADN/administración & dosificación , Animales , Proteínas Bacterianas/metabolismo , Proliferación Celular , Chaperonina 60/metabolismo , Citocinas/metabolismo , Vacunas Fúngicas/inmunología , Inmunidad Humoral/inmunología , Inmunoglobulina G/sangre , Ácido Láctico/química , Liposomas/química , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Mycobacterium leprae/metabolismo , Óxido Nítrico/metabolismo , Paracoccidioides/fisiología , Paracoccidioidomicosis/sangre , Paracoccidioidomicosis/microbiología , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Bazo/metabolismo , Vacunas de ADN/inmunologíaAsunto(s)
Osteomielitis/microbiología , Sporothrix/aislamiento & purificación , Esporotricosis/microbiología , Antifúngicos/uso terapéutico , Drenaje , Femenino , Humanos , Osteomielitis/diagnóstico , Osteomielitis/terapia , Esporotricosis/complicaciones , Esporotricosis/diagnóstico , Esporotricosis/terapia , Resultado del TratamientoRESUMEN
Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a facultative intracellular human pathogen that can persist within macrophage phagolysosomes, indicating that the fungus has evolved defense mechanisms in order to survive under nutritionally poor environments. The analysis of P. brasiliensis transcriptome revealed several virulence factor orthologs of other microorganisms, including the glyoxylate cycle genes. This cycle allows the utilization of two-carbon (C2) compounds as carbon source in gluconeogenesis. Semiquantitative RT-PCR analyses revealed that these genes were upregulated when P. brasiliensis was recovered from murine macrophages, without any additional in vitro growth. The induction of this cycle, in response to macrophage microenvironments, was shown to be coordinated with the upregulation of the gluconeogenic phosphoenolpyruvate carboxykinase gene. In addition, assays employing RNA extracted from P. brasiliensis grown in a medium with acetate instead of glucose also showed increased levels of glyoxylate cycle transcripts. Our main results suggest that P. brasiliensis uses the glyoxylate cycle as an important adaptive metabolic pathway.
Asunto(s)
Glioxilatos/metabolismo , Macrófagos/microbiología , Paracoccidioides/fisiología , Paracoccidioidomicosis/metabolismo , Animales , ADN de Hongos/análisis , Regulación Fúngica de la Expresión Génica , Macrófagos/fisiología , Ratones , Paracoccidioides/genética , Paracoccidioidomicosis/inmunología , ARN de Hongos/genética , ARN de Hongos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Introdução: o condiloma acuminado é provocado pelo Papilomavírus humano (HPV), do qual existem mais de 100 tipos distintos. Acredita-se que seja um dos facilitadores da transmissão sexual do HIV, sendo por isso importante o seu diagnóstico precoce e tratamento. Comumente utilizados em saúde pública, o ácido tricloroacético (TCA 90 por cento) e a eletrocauterização são métodos dolorosos e que necessitam de formação médica especializada. A solução de podofilina 25 por cento é bastante utilizada, porém a preparação é extremamente instável com curto período de durabilidade. Objetivo: os autores estudaram o uso domiciliar da podofilotoxina creme a 0,15 por cento, principal substância ativa da resina de podofilina, que inibe a metáfase celular, sendo uma medicação antiviral, que pode ser auto-aplicada. É considerada como forma de tratamento praticamente indolor quando comparada aos demais métodos. Métodos: os pacientes foram orientados a aplicarem o medicamento duas vezes ao dia, três vezes por semana por um máximo de quatro semanas (ciclos), com avaliação do número de lesões por consulta após cada ciclo. Resultados: a análise estatística provou ser significativa a diminuição do número de lesões ao longo do tratamento. O número de lesões da primeira consulta comparado respectivamente com o número após o primeiro ciclo e após o quarto ciclo mostraram p-valor=0,001134 e p-valor=0,000699. Ao final do quarto ciclo, observou-se cura em 72 por cento, melhora em 15 por cento e inalterado em 13 por cento dos pacientes. Conclusão: a auto-aplicação de podofilotoxina creme a 0,15 por cento para o tratamento de condiloma acuminado mostrou-se como um dos métodos mais eficazes para tratamento do HPV.
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Condiloma Acuminado , Papillomaviridae , Enfermedades de Transmisión SexualRESUMEN
The human fungal pathogen Paracoccidioides brasiliensis is an ascomycete that displays a temperature-dependent dimorphic transition, appearing as a mycelium at 22 degrees C and as a yeast at 37 degrees C, this latter being the virulent form. We report on the in silico search made of the P. brasiliensis transcriptome-expressed sequence tag database for components of signaling pathways previously known to be involved in morphogenesis and virulence in other species of fungi, including Saccharomyces cerevisiae, Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Using this approach, it was possible to identify several protein cascades in P. brasiliensis, such as i) mitogen-activated protein kinase signaling for cell integrity, cell wall construction, pheromone/mating, and osmo-regulation, ii) the cAMP/PKA system, which regulates fungal development and virulence, iii) the Ras protein, which allows cross-talking between cascades, iv) calcium-calmodulin-calcineurin, which controls cell survival under oxidative stress, high temperature, and membrane/cell wall perturbation, and v) the target of rapamycin pathway, controlling cell growth and proliferation. The ways in which P. brasiliensis responds to the environment and modulates the expression of genes required for its survival and virulence can be inferred through comparison with other fungi for which this type of data is already available.
Asunto(s)
Etiquetas de Secuencia Expresada , Proteínas Fúngicas/metabolismo , Paracoccidioides/fisiología , Transducción de Señal/genética , Transcripción Genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hongos/citología , Hongos/metabolismo , Hongos/patogenicidad , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ósmosis/fisiología , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidad , Feromonas/metabolismo , Alineación de Secuencia , Transducción de Señal/fisiología , Proteínas ras/metabolismoRESUMEN
The rise in antifungal resistance, observed as a result of the increasing numbers of immunocompromised patients, has made the discovery of new targets for drug therapy imperative. The description of the Paracoccidioides brasiliensis transcriptome has allowed us to find alternatives to refine current therapy against paracoccidioidomycosis. We used comparative analysis of expressed sequence tags to find promising drug targets that have been addressed in other pathogens. We divided the analysis into six different categories, based on the involvement of the targeted mechanisms in the cell: i) cell wall construction, ii) plasma membrane composition, iii) cellular machinery, iv) cellular metabolism, v) signaling pathways, and vi) other essential processes. Through this approach, it has been possible to infer strategies to develop alternative drugs against this pathogen.
Asunto(s)
Humanos , Antifúngicos/farmacología , Diseño de Fármacos , Etiquetas de Secuencia Expresada , Paracoccidioides/genética , Transcripción Genética/genética , Paracoccidioides/efectos de los fármacos , Paracoccidioides/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/enzimología , Pared Celular/metabolismoRESUMEN
Survival of pathogenic fungi inside human hosts depends on evasion from the host immune system and adaptation to the host environment. Among different insults that Paracoccidioides brasiliensis has to handle are reactive oxygen and nitrogen species produced by the human host cells, and by its own metabolism. Knowing how the parasite deals with reactive species is important to understand how it establishes infection and survives within humans. The initiative to describe the P. brasiliensis transcriptome fostered new approaches to study oxidative stress response in this organism. By examining genes related to oxidative stress response, one can evaluate the parasite's ability to face this condition and infer about possible ways to overcome this ability. We report the results of a search of the P. brasiliensis assembled expressed sequence tag database for homologous sequences involved in oxidative stress response. We described several genes coding proteins involved in antioxidant defense, for example, catalase and superoxide dismutase isoenzymes, peroxiredoxin, cytochrome c peroxidase, glutathione synthesis enzymes, thioredoxin, and the transcription factors Yap1 and Skn7. The transcriptome analysis of P. brasiliensis reveals a pathogen that has many resources to combat reactive species. Besides characterizing the antioxidant defense system in P. brasiliensis, we also compared the ways in which different fungi respond to oxidative damage, and we identified the basic features of this response.
Asunto(s)
Humanos , Antioxidantes/fisiología , Estrés Oxidativo/fisiología , Factores de Transcripción/fisiología , Paracoccidioides/fisiología , Especies Reactivas de Oxígeno/metabolismo , Etiquetas de Secuencia Expresada/metabolismo , Estallido Respiratorio/fisiología , Factores de Transcripción/genética , Macrófagos/inmunología , Paracoccidioides/genéticaRESUMEN
The human fungal pathogen Paracoccidioides brasiliensis is an ascomycete that displays a temperature-dependent dimorphic transition, appearing as a mycelium at 22 degrees C and as a yeast at 37 degrees C, this latter being the virulent form. We report on the in silico search made of the P. brasiliensis transcriptome-expressed sequence tag database for components of signaling pathways previously known to be involved in morphogenesis and virulence in other species of fungi, including Saccharomyces cerevisiae, Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Using this approach, it was possible to identify several protein cascades in P. brasiliensis, such as i) mitogen-activated protein kinase signaling for cell integrity, cell wall construction, pheromone/mating, and osmo-regulation, ii) the cAMP/PKA system, which regulates fungal development and virulence, iii) the Ras protein, which allows cross-talking between cascades, iv) calcium-calmodulin-calcineurin, which controls cell survival under oxidative stress, high temperature, and membrane/cell wall perturbation, and v) the target of rapamycin pathway, controlling cell growth and proliferation. The ways in which P. brasiliensis responds to the environment and modulates the expression of genes required for its survival and virulence can be inferred through comparison with other fungi for which this type of data is already available.
Asunto(s)
Humanos , Etiquetas de Secuencia Expresada , Paracoccidioides/fisiología , Proteínas Fúngicas/metabolismo , Transcripción Genética , Transducción de Señal/genética , Alineación de Secuencia , Feromonas/metabolismo , Hongos/citología , Hongos/metabolismo , Hongos/patogenicidad , Ósmosis/fisiología , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas ras/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Annotation of the transcriptome of the dimorphic fungus Paracoccidioides brasiliensis has set the grounds for a global understanding of its metabolism in both mycelium and yeast forms. This fungus is able to use the main carbohydrate sources, including starch, and it can store reduced carbons in the form of glycogen and trehalose; these provide energy reserves that are relevant for metabolic adaptation, protection against stress and infectivity mechanisms. The glyoxylate cycle, which is also involved in pathogenicity, is present in this fungus. Classical pathways of lipid biosynthesis and degradation, including those of ketone body and sterol production, are well represented in the database of P. brasiliensis. It is able to synthesize de novo all nucleotides and amino acids, with the sole exception of asparagine, which was confirmed by the fungus growth in minimal medium. Sulfur metabolism, as well as the accessory synthetic pathways of vitamins and co-factors, are likely to exist in this fungus.
Asunto(s)
Etiquetas de Secuencia Expresada/metabolismo , Paracoccidioides/metabolismo , Regulación Fúngica de la Expresión Génica , Transcripción Genética , Aminoácidos/metabolismo , Azufre/metabolismo , Fosforilación , Metabolismo de los Hidratos de Carbono , Paracoccidioides/genética , Pirimidinas/metabolismo , Purinas/metabolismo , Ácidos Grasos/metabolismoRESUMEN
Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, an endemic mycosis of Latin America. This fungus presents a dimorphic character; it grows as a mycelium at room temperature, but it is isolated as yeast from infected individuals. It is believed that the transition from mycelium to yeast is important for the infective process. The Functional and Differential Genome of Paracoccidioides brasiliensis Project--PbGenome Project was developed to study the infection process by analyzing expressed sequence tags--ESTs, isolated from both mycelial and yeast forms. The PbGenome Project was executed by a consortium that included 70 researchers (professors and students) from two sequencing laboratories of the midwest region of Brazil; this project produced 25,741 ESTs, 19,718 of which with sufficient quality to be analyzed. We describe the computational procedures used to receive process, analyze these ESTs, and help with their functional annotations; we also detail the services that were used for sequence data exploration. Various programs were compared for filtering and grouping the sequences, and they were adapted to a user-friendly interface. This system made the analysis of the differential transcriptome of P. brasiliensis possible.
Asunto(s)
Biología Computacional/métodos , Etiquetas de Secuencia Expresada , Genoma Fúngico/genética , Paracoccidioides/genética , Transcripción Genética/genética , Brasil , Interfaz Usuario-Computador , Regulación Fúngica de la Expresión Génica/genéticaRESUMEN
Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus which is found as mycelia (M) at 26 degrees C and as yeasts (Y) at 37 degrees C, or after the invasion of host tissues. Although the dimorphic transition in P. brasiliensis and other dimorphic fungi is an essential step in the establishment of infection, the molecular events regulating this process are yet poorly understood. Since the differential gene expression is a well-known mechanism which plays a central role in the dimorphic transition as well as in other biological process, in this work we describe the identification and characterization of two differentially expressed P. brasiliensis hydrophobin cDNAs (Pbhyd1 and Pbhyd2). Hydrophobins are small hydrophobic proteins related to a variety of important functions in fungal biology, including cell growth, development, infection, and virulence. These two hydrophobin genes are present as single copy in P. brasiliensis genome and Northern blot analysis revealed that both mRNAs are mycelium-specific and highly accumulated during the first 24 h of M to Y transition.