RESUMEN
Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. The Mitogen-Activated Protein kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. The sensor histidine kinase SlnASln1 is important for modulation of MpkA phosphorylation. Our work emphasizes the importance of MpkA and compartmentalization of cellular events for GT production and self-defense.
Asunto(s)
Aspergilosis , Gliotoxina , Humanos , Aspergillus fumigatus/metabolismo , Gliotoxina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Aspergilosis/microbiologíaRESUMEN
In cystic fibrosis (CF), mucus plaques are formed in the patient's lungs, creating a hypoxic condition and a propitious environment for colonization and persistence of many microorganisms. There is clinical evidence showing that Aspergillus fumigatus can cocolonize CF patients with Pseudomonas aeruginosa, which has been associated with lung function decline. P. aeruginosa produces several compounds with inhibitory and antibiofilm effects against A. fumigatus in vitro; however, little is known about the fungal compounds produced in counterattack. Here, we annotated fungal and bacterial secondary metabolites (SM) produced in mixed biofilms under normoxia and hypoxia conditions. We detected nine SM produced by P. aeruginosa. Phenazines and different analogs of pyoverdin were the main compounds produced by P. aeruginosa, and their secretion levels were increased by the fungal presence. The roles of the two operons responsible for phenazine production (phzA1 and phzA2) were also investigated, and mutants lacking one of those operons were able to produce partial sets of phenazines. We detected a total of 20 SM secreted by A. fumigatus either in monoculture or in coculture with P. aeruginosa. All these compounds were secreted during biofilm formation in either normoxia or hypoxia. However, only eight compounds (demethoxyfumitremorgin C, fumitremorgin, ferrichrome, ferricrocin, triacetylfusigen, gliotoxin, gliotoxin E, and pyripyropene A) were detected during biofilm formation by the coculture of A. fumigatus and P. aeruginosa under normoxia and hypoxia conditions. Overall, we showed how diverse SM secretion is during A. fumigatus and P. aeruginosa mixed culture and how this can affect biofilm formation in normoxia and hypoxia. IMPORTANCE The interaction between Pseudomonas aeruginosa and Aspergillus fumigatus has been well characterized in vitro. In this scenario, the bacterium exerts a strong inhibitory effect against the fungus. However, little is known about the metabolites produced by the fungus to counterattack the bacteria. Our work aimed to annotate secondary metabolites (SM) secreted during coculture between P. aeruginosa and A. fumigatus during biofilm formation in both normoxia and hypoxia. The bacterium produces several different types of phenazines and pyoverdins in response to presence of the fungus. In contrast, we were able to annotate 29 metabolites produced during A. fumigatus biofilm formation, but only 8 compounds were detected during biofilm formation by the coculture of A. fumigatus and P. aeruginosa upon either normoxia or hypoxia. In conclusion, we detected many SM secreted during A. fumigatus and P. aeruginosa biofilm formation. This analysis provides several opportunities to understand the interactions between these two species.
Asunto(s)
Fibrosis Quística , Gliotoxina , Aspergillus fumigatus , Biopelículas , Humanos , Hipoxia , Fenazinas/metabolismo , Fenazinas/farmacología , Pseudomonas aeruginosa/metabolismoRESUMEN
Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.
Asunto(s)
Aspergillus/crecimiento & desarrollo , Gliotoxina/farmacología , Metiltransferasas/genética , Factores de Transcripción/genética , Aspergillus/efectos de los fármacos , Aspergillus/genética , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/genética , Aspergillus nidulans/crecimiento & desarrollo , Aspergillus oryzae/efectos de los fármacos , Aspergillus oryzae/genética , Aspergillus oryzae/crecimiento & desarrollo , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Gliotoxina/biosíntesis , RNA-SeqRESUMEN
Aspergillosis, the main causative agent of which is Aspergillus fumigatus, causes mortality in all types of birds. Gliotoxin (GT), one of the multiple virulence factors of A. fumigatus, has a variety of immunosuppressive effects. The corpse of an African grey parrot (Psittacus erithacus) was sent for necropsy and diagnostic rule-out. The lungs were enlarged, firm, and had dark-red coloration, on the parietal faces of both lungs, some semi-circular caseous necrosis areas were observed. The caudal thoracic and abdominal air sacs were thickened and contained a fibrin-heterophilic exudate. Histopathologically, a necrotic and granulomatous bronchopneumonia was observed with intralesional hyphae with characteristics compatible with Aspergillus sp. that were positive with Grocott´s staining. Fibrinous and heterophilic airsacculitis was found in the air sacs. A. fumigatus was isolated from lungs, characterized using serial microcultures, and confirmed using polymerase chain reaction. In addition, GT production was detected in vitro from the culture filtrate in which the isolate was grown; the organic extract was analysed via thin-layer chromatography. This is the first detection of GT in a case of pulmonary aspergillosis in a parrot, which could help to understand the pathogenesis of the disease in psittacines.(AU)
Asunto(s)
Animales , Loros/microbiología , Aspergilosis Pulmonar/microbiología , Aspergilosis Pulmonar/patología , Inmunosupresores/análisis , Aspergillus fumigatus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Cromatografía , Gliotoxina/análisisRESUMEN
Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.
Asunto(s)
Aspergilosis , Aspergillus fumigatus/patogenicidad , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Gliotoxina/biosíntesis , Factores de Transcripción/metabolismo , Animales , Aspergilosis/metabolismo , Aspergilosis/microbiología , Aspergillus fumigatus/metabolismo , Ratones , Estrés Oxidativo/fisiología , Virulencia/fisiologíaRESUMEN
Gliotoxin (GTX) is the major and the most potent mycotoxin that is secreted by Aspergillus fumigatus, which is capable of injuring and killing microglial cells, astrocytes, and oligodendrocytes. During the last years, studies with patients and experimental models of multiple sclerosis (MS), which is an autoimmune disease of the central nervous system (CNS), suggested that fungal infections are among the possible initiators or aggravators of this pathology. The deleterious effect can occur through a direct interaction of the fungus with the CNS or by the toxin release from a non-neurological site. In the present work, we investigated the effect of GTX on experimental autoimmune encephalomyelitis (EAE) development. Female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein and then intraperitoneally injected with three doses of GTX (1 mg/kg b.w., each) on days 4, 7, and 10. GTX aggravated clinical symptoms of the disease in a dose-dependent way and this outcome was concomitant with an increased neuroinflammation. CNS analyses revealed that GTX locally increased the relative expression of inflammatory genes and the cytokine production. Our results indicate that GTX administered in a non-neuronal site was able to increase neuroinflammation in EAE. Other mycotoxins could also be deleterious to many neurological diseases by similar mechanisms.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Gliotoxina/toxicidad , Animales , Aspergillus fumigatus , Citocinas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Ratones Endogámicos C57BL , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
Aspergillosis affects all types of birds; it causes the loss of specimens with high ecologic value and also leads to significant economic losses within the poultry industry. The main etiologic agent is Aspergillus fumigatus , a filamentary fungus with multiple virulence factors, such as gliotoxin (GT), which is an immunosuppressive epipolythiodioxopiperazine molecule. Necropsy was performed on 73 poultry from different provenances, all of which presented with a respiratory semiology compatible with aspergillosis. A mycological culture was performed on the injured lungs of diseased birds, as was chloroform extraction of the GT, a thin-layer chromatography analysis (TLC), and a histopathology analysis with hematoxylin-eosin and Grocott stainings. The A. fumigatus identification was confirmed by PCR, where the ITS 1 5.1-5.8S-ITS 2 fragment of the rDNA complex was amplified. The in vitro GT production was studied by TLC in the recovered isolates from A. fumigatus . Seven isolates of A. fumigatus were obtained and in six of them, GT-like compounds were detected. In a lung sample, a compound with the same retention time (RF) as the reference GT was detected; whereas RF compounds different from the GT standard were observed in three lung samples.
Asunto(s)
Aspergilosis/veterinaria , Aspergillus fumigatus/aislamiento & purificación , Aspergillus fumigatus/metabolismo , Gliotoxina/metabolismo , Enfermedades de las Aves de Corral/microbiología , Animales , Aspergilosis/microbiología , Aspergilosis/patología , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/genética , Pollos , Pulmón/patología , Pulmón/virología , Enfermedades de las Aves de Corral/patologíaRESUMEN
OBJECTIVE: This study aimed to evaluate the effect of propentofylline administration on astrocytic response following gliotoxic injury. METHOD: Wistar rats were injected with ethidium bromide into the cisterna pontis and treated or not with propentofylline (12.5mg/kg/day, intraperitoneal) during the experimental period. Brainstem sections were collected from 15 to 31 days after gliotoxic injection and processed for GFAP immunohistochemistry. RESULTS AND CONCLUSION: Results demonstrate that propentofylline decreased astrocytic activation until the 21st day, suggesting that this drug may have a role in reducing glial scar development following injury.
Asunto(s)
Astrocitos/efectos de los fármacos , Tronco Encefálico/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Xantinas/farmacología , Animales , Astrocitos/metabolismo , Tronco Encefálico/metabolismo , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/prevención & control , Modelos Animales de Enfermedad , Etidio/toxicidad , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/efectos de los fármacos , Gliotoxina/toxicidad , Inmunohistoquímica , Masculino , Ratas Wistar , Reproducibilidad de los Resultados , Factores de Tiempo , Resultado del TratamientoRESUMEN
ABSTRACT Propentofylline is a xanthine derivative that depresses activation of glial cells, whose responses contribute to neural tissue damage during inflammation. Ethidium bromide injection into the central nervous system induces local oligodendroglial and astrocytic loss, resulting in primary demyelination, neuroinflammation and blood-brain barrier disruption. Surviving astrocytes present a vigorous reaction around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP). Objective This study aimed to evaluate the effect of propentofylline administration on astrocytic response following gliotoxic injury. Method Wistar rats were injected with ethidium bromide into the cisterna pontis and treated or not with propentofylline (12.5mg/kg/day, intraperitoneal) during the experimental period. Brainstem sections were collected from 15 to 31 days after gliotoxic injection and processed for GFAP immunohistochemistry. Results and Conclusion Results demonstrate that propentofylline decreased astrocytic activation until the 21st day, suggesting that this drug may have a role in reducing glial scar development following injury.
RESUMO A propentofilina é uma xantina que deprime a ativação das células gliais, cujas respostas contribuem para o dano neural durante inflamação. A injeção de brometo de etídio no sistema nervoso central induz a perda oligodendroglial e astrocitária, resultando em desmielinização, neuroinflamação e ruptura da barreira hematoencefálica. Os astrócitos sobreviventes apresentam vigorosa reação ao redor da lesão com aumento da imunorreatividade à proteína glial fibrilar ácida (GFAP). Objetivo Este estudo objetivou avaliar o efeito da propentofilina sobre a resposta astrocitária após injúria gliotóxica. Método Ratos Wistar foram injetados com brometo de etídio na cisterna basal e tratados ou não com propentofilina (12.5mg/kg/dia, intraperitoneal). Amostras do tronco encefálico foram coletadas dos 15 aos 31 dias pós-injeção do gliotóxico e processadas para estudo ultraestrutural e imuno-histoquímico para GFAP. Resultados e Conclusão Os resultados demonstram que a propentofilina reduziu a ativação astrocitária até o 21o dia, sugerindo que essa droga pode atuar na redução da cicatriz glial após injúria.
Asunto(s)
Animales , Masculino , Xantinas/farmacología , Tronco Encefálico/efectos de los fármacos , Astrocitos/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Factores de Tiempo , Tronco Encefálico/metabolismo , Inmunohistoquímica , Astrocitos/metabolismo , Reproducibilidad de los Resultados , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/prevención & control , Resultado del Tratamiento , Ratas Wistar , Modelos Animales de Enfermedad , Etidio/toxicidad , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/efectos de los fármacos , Gliotoxina/toxicidadRESUMEN
Proteasomal proteolysis is required for a wide range of cellular processes, including protein quality control, cell cycle progression, cell death and metabolic adaptation to environment changes or stress responses. Proteasome inhibitors are useful compounds for determining the roles of proteasome in eukaryotic cells. Here, we investigated the effects of gliotoxin, a proteasome inhibitor, on the cell growth, replication, ultrastructure, DNA integrity and proteasomal proteolytic activity of the protist parasite Tritrichomonas foetus. The effect of gliotoxin on the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, was investigated. Gliotoxin inhibited the culture growth, arrested cell cycle, and provoked a trichomonacidal effect in a dose-dependent manner. Parasites treated with gliotoxin displayed features typical of cell death, such as membrane blebbing, concentric membrane whorls containing remnants of organelles, intense cytosolic and nuclear vacuolisation, chromatin condensation, DNA fragmentation, cytoplasmic disintegration and plasma membrane disruption. The proteasomal peptidase activity was inhibited by gliotoxin in a dose-dependent manner. Gliotoxin treatment also induced an irreversible EFF transformation in a dose/time-dependent manner. We compared morphological characteristics between gliotoxin- and cold-induced EFF parasites. Our results suggest that gliotoxin could induce EFF transformation by a mechanism distinct from that provoked by cold temperature. This study further contributes to a better understanding of the role of proteasome system in cell cycle, cell death and EFF transformation in T. foetus.
Asunto(s)
Gliotoxina/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Proteolisis/efectos de los fármacos , Tritrichomonas foetus/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Tritrichomonas foetus/efectos de los fármacosRESUMEN
UNLABELLED: The aim of this manuscript was to study the influence of water activity (aW ) and pH in the ecophysiological behaviour of Aspergillus fumigatus strains at human body temperature. In addition, gliotoxin production and enzymatic ability among environmental (n = 2) and clinical (n = 5) strains were compared. Ecophysiological study of environmental strains was performed on agar silage incubated at 37°C, studying the interaction at eight aW levels (0·8, 0·85, 0·9, 0·92, 0·94, 0·96, 0·98 and 0·99) and eight pH levels (3·5, 4, 4·5, 5, 6, 7, 7·5 and 8). Considering the influence of the assumed lung conditions on growth of A. fumigatus (aW 0·98/0·99 and pH of 7/7·5), the optimal condition for the development of A. fumigatus RC031 was at aW 0·99 at pH 7. At aW 0·98/0·99 and pH of 7/7·5, the highest growth rate and the lowest lag phase was reported, whereas there were no significant differences at aW 0·98/0·99 and pH 7/7·5 interactions on growth of A. fumigatus RC032. Gliotoxin production of A. fumigatus strains was evaluated. The gliotoxin production was similar in clinical and environmental strains. Elastin activity was studied in solid medium, highest elastase activity index was found for clinical strain A. fumigatus RC0676, followed by the environmental strain A. fumigatus RC031. Opportunistic environmental strains can be considered as pathogenic in some cases when rural workers are exposed constantly to handling silage. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus fumigatus is one of the main opportunist pathogen agents causing invasive aspergillosis. Rural workers present a constant exposition to A. fumigatus spores caused by feed-borne manipulation. In this study, environmental A. fumigatus strains were able to grow and produce gliotoxin onto the studied conditions including the lung ones. Environmental and clinical strains were physiologically similar and could be an important putative infection source in rural workers.
Asunto(s)
Aspergillus fumigatus/enzimología , Aspergillus fumigatus/metabolismo , Elastina/metabolismo , Gliotoxina/biosíntesis , Ensilaje/microbiología , Aspergilosis/microbiología , Aspergilosis/patología , Aspergillus fumigatus/fisiología , HumanosRESUMEN
Aspergillus fumigatus is a fungal pathogen that causes several invasive and noninvasive diseases named aspergillosis. This disease is generally regarded as multifactorial, considering that several pathogenicity determinants are present during the establishment of this illness. It is necessary to obtain an increased knowledge of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes. Protein phosphatases are essential to several signal transduction pathways. We identified 32 phosphatase catalytic subunit-encoding genes in A. fumigatus, of which we were able to construct 24 viable deletion mutants. The role of nine phosphatase mutants in the HOG (high osmolarity glycerol response) pathway was evaluated by measuring phosphorylation of the p38 MAPK (SakA) and expression of osmo-dependent genes. We were also able to identify 11 phosphatases involved in iron assimilation, six that are related to gliotoxin resistance, and three implicated in gliotoxin production. These results present the creation of a fundamental resource for the study of signaling in A. fumigatus and its implications in the regulation of pathogenicity determinants and virulence in this important pathogen.
Asunto(s)
Aspergillus fumigatus/enzimología , Proteínas Fúngicas/genética , Fosfoproteínas Fosfatasas/genética , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Gliotoxina/análisis , Gliotoxina/metabolismo , Mutación , Fenotipo , Fosfoproteínas Fosfatasas/clasificación , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Filogenia , Sideróforos/análisis , Transducción de Señal , Espectrometría de Masas en Tándem , Virulencia/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The cytotoxic activities of extracts (50â µg/ml) from 48 fungal strains, recovered from sediments of Pecém's offshore port terminal (Northeast coast of Brazil), against HCT-116 colon cancer cell lines were investigated. The most promising extract was obtained from strain BRF082, identified as Dichotomomyces cejpii by phylogenetic analyses of partial RPB2 gene sequence. Thus, it was selected for bioassay-guided isolation of the cytotoxic compounds. Large-scale fermentation of BRF082 in potato dextrose broth, followed by chromatographic purification of the bioactive fractions from the liquid medium, yielded gliotoxin (4) and its derivatives acetylgliotoxin G (3), bis(dethio)bis(methylsulfanyl)gliotoxin (1), acetylgliotoxin (5), 6-acetylbis(dethio)bis(methylsulfanyl)gliotoxin (2), besides the quinazolinone alkaloid fiscalin B. All isolated compounds were tested for their cytotoxicities against the tumor cell lines HCT-116, revealing 4 and 3 as the most cytotoxic ones (IC50 0.41 and 1.06â µg/ml, resp.).
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Productos Biológicos/química , Productos Biológicos/farmacología , Hongos/química , Sedimentos Geológicos/microbiología , Antineoplásicos/aislamiento & purificación , Productos Biológicos/aislamiento & purificación , Brasil , Neoplasias del Colon/tratamiento farmacológico , Hongos/genética , Gliotoxina/análogos & derivados , Gliotoxina/química , Gliotoxina/aislamiento & purificación , Gliotoxina/farmacología , Células HCT116 , Humanos , Indoles/química , Indoles/aislamiento & purificación , Indoles/farmacología , Filogenia , Quinazolinas/química , Quinazolinas/aislamiento & purificación , Quinazolinas/farmacologíaRESUMEN
Environmental conditions play a key role in fungal development. During the silage production process, humidity, oxygen availability and pH vary among lactic-fermentation phases and among different silage sections. The aim of this work was to study the physiological behaviour of gliotoxicogenic Aspergillus fumigatus strains isolated from maize silage under simulated natural physicochemical conditions - different water activities (a(W)), temperatures (Tº), pH and oxygen pressure - on the growth parameters (growth rate and lag phase) and gliotoxin production. The silage was made with the harvested whole maize plant that was chopped and used for trench-type silo fabrication. Water activity and pH of the silage samples were determined. Total fungal counts were performed on Dichloran Rose Bengal Chloramphenicol agar and Dichloran 18% Glycerol agar. The morphological identification of A. fumigatus was performed with different culture media and at different growth temperature to observe microscopic and macroscopic characteristics. Gliotoxin production by A. fumigatus was determined by HPLC. All strains isolated were morphologically identified as A. fumigatus. Two A. fumigatus strains isolated from the silage samples were selected for the ecophysiological study (A. fumigatus sensu stricto RC031 and RC032). The results of this investigation showed that the fungus grows in the simulated natural physicochemical conditions of corn silage and produces gliotoxin. The study of the physiological behaviour of gliotoxigenic A. fumigatus under simulated environmental conditions allowed its behaviour to be predicted in silage and this will in future enable appropriate control strategies to be developed to prevent the spread of this fungus and toxin production that leads to impairment and reduced quality of silage.
Asunto(s)
Aspergillus fumigatus/química , Aspergillus fumigatus/aislamiento & purificación , Gliotoxina/análisis , Ensilaje/microbiología , Zea mays/microbiología , Aspergillus fumigatus/metabolismo , Química Física , Gliotoxina/biosíntesis , Zea mays/metabolismoRESUMEN
AIMS: To compare clinical and environmental Aspergillus fumigatus strains on their toxicogenic and immunosuppressive capacity. METHODS AND RESULTS: A total of 51 strains of A. fumigatus isolated from clinical and corn silage samples were assayed. All A. fumigatus strains were assayed for gliotoxin production, therefore strains with different gliotoxin capacities and isolated from different sources were selected and assayed for their effects on bovine macrophages and lymphocytes. Spore diffusates (SDs) obtained from all A. fumigatus strains were able to inhibit macrophage phagocytosys, regardless of their gliotoxin production capacity. However, most but not all strains were able to inhibit bactericidal activity. SDs from all A. fumigatus strains reduced lymphocytes viability. The heat treatment was not always able to inhibit the negative effect on immune cells. CONCLUSIONS: There was no difference between clinical and environmental isolates in their toxicogenic and immunosuppressive capacity. Gliotoxin would not be responsible for the immunosuppressive activity observed by the assayed A. fumigatus strains. However, gliotoxin could be present in the SD, together with some other substances. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained suggest that any environmental strain of A. fumigatus is a putative infectious strain. Prevention measures should be applied to control environmental Aspergillus conidia.
Asunto(s)
Aspergillus fumigatus/patogenicidad , Animales , Aspergillus fumigatus/aislamiento & purificación , Aspergillus fumigatus/metabolismo , Bovinos , Citotoxinas/toxicidad , Gliotoxina/biosíntesis , Humanos , Inmunosupresores/metabolismo , Inmunosupresores/farmacología , Linfocitos/efectos de los fármacos , Macrófagos/fisiología , Ensilaje/microbiología , Esporas Fúngicas , Zea maysRESUMEN
Using a gene disruption strategy, we generated mutants in the gliP locus of the plant-beneficial fungus Trichoderma virens that were no longer capable of producing gliotoxin. Phenotypic assays demonstrated that the gliP-disrupted mutants grew faster, were more sensitive to oxidative stress and exhibited a sparse colony edge compared with the WT strain. In a plate confrontation assay, the mutants deficient in gliotoxin production were ineffective as mycoparasites against the oomycete, Pythium ultimum, and the necrotrophic fungal pathogen, Sclerotinia sclerotiorum, but retained mycoparasitic ability against Rhizoctonia solani. Biocontrol assays in soil showed that the mutants were incapable of protecting cotton seedlings from attack by P. ultimum, against which the WT strain was highly effective. The mutants, however, were as effective as the WT strain in protecting cotton seedlings against R. solani. Loss of gliotoxin production also resulted in a reduced ability of the mutants to attack the sclerotia of S. sclerotiorum compared with the WT. The addition of exogenous gliotoxin to the sclerotia colonized by the mutants partially restored their degradative abilities. Interestingly, as in Aspergillus fumigatus, an opportunistic human pathogen, gliotoxin was found to be involved in pathogenicity of T. virens against larvae of the wax moth, Galleria mellonella. The loss of gliotoxin production in T. virens was restored by complementation with the gliP gene from A. fumigatus. We have, thus, demonstrated that the putative gliP cluster of T. virens is responsible for the biosynthesis of gliotoxin, and gliotoxin is involved in mycoparasitism and biocontrol properties of this plant-beneficial fungus.
Asunto(s)
Gliotoxina/metabolismo , Gossypium/microbiología , Enfermedades de las Plantas/microbiología , Simbiosis , Trichoderma/fisiología , Animales , Ascomicetos/crecimiento & desarrollo , Lepidópteros/microbiología , Interacciones Microbianas , Mutagénesis Insercional , Estrés Oxidativo , Control Biológico de Vectores , Pythium/crecimiento & desarrollo , Rhizoctonia/crecimiento & desarrollo , Plantones/microbiología , Microbiología del Suelo , Análisis de Supervivencia , Trichoderma/crecimiento & desarrollo , Trichoderma/metabolismo , VirulenciaRESUMEN
Invasive aspergillosis is an opportunistic infection that is mainly caused by Aspergillus fumigatus, which is known to produce several secondary metabolites, including gliotoxin, the most abundant metabolite produced during hyphal growth. The diagnosis of invasive aspergillosis is often made late in the infection because of the lack of reliable and feasible diagnostic techniques; therefore, early detection is critical to begin treatment and avoid more serious complications. The present work reports the development and validation of an HPLC-MS/MS method for the detection of gliotoxin in the serum of patients with suspected aspergillosis. Chromatographic separation was achieved using an XBridge C18 column (150 × 2.1 mm id; 5 mm particle size) maintained at 25 °C with the corresponding guard column (XBridge C18, 10 × 2.1 mm id, 5 mm particle size). The mobile phase was composed of a gradient of water and acetonitrile/water (95:5 v/v), both containing 1 mM ammonium formate with a flow rate of 0.45 mL min(-1). Data from the validation studies demonstrate that this new method is highly sensitive, selective, linear, precise, accurate and free from matrix interference. The developed method was successfully applied to samples from patients suspected of having aspergillosis. Therefore, the developed method has considerable potential as a diagnostic technique for aspergillosis.
Asunto(s)
Aspergilosis/diagnóstico , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodos , Gliotoxina/sangre , Gliotoxina/química , Humanos , Extracción Líquido-Líquido , Quercetina/química , Estándares de Referencia , Reproducibilidad de los Resultados , Factores de Riesgo , Solventes/químicaRESUMEN
UNLABELLED: Aspergillus fumigatus, a well-known human and animal pathogen causing aspergillosis, has been historically identified by morphological and microscopic features. However, recent studies have shown that species identification on the basis of morphology alone is problematic. The aim of this work was to confirm the taxonomic state at specie level of a set of clinical (human and animal) and animal environment A. fumigatus strains identified by morphological criteria applying a PCR-RFLP assay by an in silico and in situ analysis with three restriction enzymes. The A. fumigatus gliotoxin-producing ability was also determined. Previous to the in situ PCR-RFLP analysis, an in silico assay with BccI, MspI and Sau3AI restriction enzymes was carried out. After that, these enzymes were used for in situ assay. All A. fumigatus strains isolated from corn silage, human aspergillosis and bovine mastitis and high per cent of the strains isolated from cereals, animal feedstuff and sorghum silage were able to produce high gliotoxin levels. Also, all these strains identified by morphological criteria as A. fumigatus, regardless of its isolation source, had band patterns according to A. fumigatus sensu stricto by PCR-RFLP markers. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus fumigatus is a well-known human and animal pathogen causing aspergillosis. In this study, clinical (human and animal) and animal environment strains were able to produce high gliotoxin levels and had band profiles according to A. fumigatus sensu stricto by PCR-RFLP markers. The results obtained here suggest that strains involved in human and animal aspergillosis could come from the animal environment in which A. fumigatus is frequently found. Its presence in animal environments could affect animal health and productivity; in addition, there are risks of contamination for rural workers during handling and storage of animal feedstuffs.
Asunto(s)
Aspergilosis/microbiología , Aspergilosis/veterinaria , Aspergillus fumigatus/clasificación , Grano Comestible/microbiología , Gliotoxina/metabolismo , Mastitis Bovina/microbiología , Ensilaje/microbiología , Animales , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Aspergillus fumigatus/metabolismo , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Brasil , Bovinos , Femenino , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The potential association between hygienic conditions in the environment of lactating cows and the presence of gliotoxinogenic Aspergillus fumigatus strains was studied. Milk samples (individual cow's milk [ICM], bulk tank milk [BTM]) from 44 dairy farms were sampled. In ICM samples, eight different species of Aspergillus were identified. A. flavus and A. fumigatus were predominant, with 37.8% and 26.1% relative densities, respectively. A. fumigatus strains were isolated from 61.4% of the BTM samples, and 34% of these strains were able to produce gliotoxin. Principal component analysis was used to associate the presence of A. fumigatus with some hygienic and sanitary practices. A significant and positive correlation was observed between dry cow therapy and forestripping. The presence of A. fumigatus gliotoxin producers in milk was associated with high somatic cells count (SCC) samples. Good hygienic and sanitary practices were associated with absence of A. fumigatus and relatively low SCCs of <250,000 cells/ml. In general, a high percentage of dairy farms were positive for A. fumigatus in BTM samples. This is the first work that indicates the positive effects of adequate hygienic and sanitary practices in dairy herds on the control of A. fumigatus and related species. By reducing the frequency of Aspergillus spp. in the dairy environment, the risk of farm handlers' exposure and the risk of intramammary fungal infections would also be reduced.
Asunto(s)
Aspergillus fumigatus/aislamiento & purificación , Aspergillus fumigatus/metabolismo , Microbiología Ambiental , Gliotoxina/metabolismo , Leche/microbiología , Animales , Animales Domésticos , Bovinos , Leche/citologíaRESUMEN
AIMS: The aim of this study was to determine total fungal counts and the relative density of Aspergillus fumigatus and related species in silage samples intended for bovines before and after fermentation as well as to monitor the natural occurrence of gliotoxin in silage samples (pre- and postfermentation). METHODS AND METHODS: The survey was performed in farms located in São Paulo and Rio de Janeiro States in Brazil. In addition, the ability of A. fumigatus strains and related species strains to produce gliotoxin was also evaluated. A total of 300 samples were taken, immediately after opening of the silo (3-5 months) and during the ensiling period. Fungal counts were done by the surface-spread method. Gliotoxin production ability of isolates and natural contamination were determined by HPLC. RESULTS: All postfermented samples had a total number of moulds exceeding 1 × 10(4) CFU g(-1), with Aspergillus sp. as the most prevalent genus. Frequency of strains, among A. fumigatus and related species, was able to produce gliotoxin was similar in pre- and postfermented samples, except for sorghum, which showed differences between both kinds of samples. The highest toxin levels were produced by strains isolated from postfermented samples. More than 50% of the samples showed gliotoxin contamination levels that exceeded concentrations known to induce immunosuppressive and apoptotic effects in cells. CONCLUSIONS: The present data suggest that care should be taken because gliotoxin contamination in feedstuffs could affect productivity and also present a health risk for herds. SIGNIFICANCE AND IMPACT OF THE STUDY: Gliotoxin was found at quite important concentrations levels in pre- and postfermented substrates and its presence could therefore probably affect the productivity and health of herds. Current conservation and management practices do not avoid contamination with A. fumigatus on silage. Therefore, farm workers should be adequately protected during its handling.