Vacuoles and peroxisomes are involved in Aspergillus fumigatus gliotoxin production and self-protection.

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. Peroxisomes are also required for proper GT production and self-defense. The Mitogen-Activated Protein (MAP) 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. Our work emphasizes the importance of dynamic compartmentalization of cellular events for GT production and self-defense.

Secondary Metabolites Produced during Aspergillus fumigatus and Pseudomonas aeruginosa Biofilm Formation.

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.

Pathological and mycological characterization of pulmonary Aspergillus fumigatus infection producing gliotoxin in a captive African grey parrot (Psittacus erithacus)

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)

Gliotoxin Aggravates Experimental Autoimmune Encephalomyelitis by Triggering Neuroinflammation.

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.

Curcumin decreases astrocytic reaction after gliotoxic injury in the rat brainstem / Curcumina reduz a reação astrocitária após injúria gliotóxica no tronco encefálico de ratos

ABSTRACT Recent studies have demonstrated that curcumin (Cur) has antioxidant, anti-inflammatory and anti-fibrotic effects. Ethidium bromide (EB) injections into the central nervous system (CNS) are known to induce local oligodendroglial and astrocytic loss, resulting in primary demyelination and neuroinflammation. Peripheral astrogliosis is seen around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP). This investigation aimed to evaluate the effect of Cur administration on astrocytic response following gliotoxic injury. Wistar rats were injected with EB into the cisterna pontis and treated, or not, with Cur (100 mg/kg/day, intraperitoneal route) during the experimental period. Brainstem sections were collected at 15, 21 and 31 days after EB injection and processed for GFAP immunohistochemical staining. Astrocytic reactivity was measured in a computerized system for image analysis. In Cur-treated rats, the GFAP-stained area around the lesion was significantly smaller in all periods after EB injection compared to untreated animals, showing that Cur reduces glial scar development following injury.

RESUMO Estudos recentes têm demonstrado que a curcumina (Cur) possui efeitos antioxidantes, anti-inflamatórios e antifibróticos. Sabe-se que a injeção de brometo de etídio (EB) no sistema nervoso central induz a perda oligodendroglial e astrocitária, resultando em desmielinização primária e neuroinflamação. Astrogliose periférica é observada ao redor da lesão com aumento da imunorreatividade à proteína glial fibrilar ácida (GFAP). A presente investigação objetivou avaliar o efeito da Cur sobre a resposta astrocitária após injúria gliotóxica. Ratos Wistar foram injetados com EB na cisterna basal e tratados ou não com Cur (100 mg/kg/dia, via intraperitoneal) durante o período experimental. Amostras do tronco encefálico foram coletadas aos 15, 21 e 31 dias pós-injeção de EB e processadas para estudo imuno-histoquímico para a GFAP. A reatividade astrocitária foi medida em um sistema computadorizado para análise de imagem. Nos ratos tratados com Cur, a área marcada para GFAP foi significantemente menor em todos os períodos pós-injeção de EB, indicando que a Cur reduz o desenvolvimento da cicatriz glial após injúria.

Aspergillosis, a Natural Infection in Poultry: Mycological and Molecular Characterization and Determination of Gliotoxin in Aspergillus fumigatus Isolates.

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.

Propentofylline reduces glial scar development following gliotoxic damage in the rat brainstem / Propentofilina reduz o desenvolvimento da cicatriz glial após dano gliotóxico no tronco encefálico de ratos

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.

Ecophysiology of environmental Aspergillus fumigatus and comparison with clinical strains on gliotoxin production and elastase activity.

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.

Systematic Global Analysis of Genes Encoding Protein Phosphatases in Aspergillus fumigatus.

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.

Bioprospection of cytotoxic compounds in fungal strains recovered from sediments of the Brazilian coast.

The cytotoxic activities of extracts (50 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 1.06 µg/ml, resp.).

Comparison of toxicogenic and immunosuppressive capacity of Aspergillus fumigatus strains isolated from clinical and corn silage samples.

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.

Natural co-occurrence of fungi and mycotoxins in poultry feeds from Entre Ríos, Argentina.

A total of 120 pelleted poultry feed samples from Entre Ríos Province, Argentina, were evaluated. The aims were to investigate (1) the presence of relevant toxigenic fungi, as well as to determine the ability to produce aflatoxins (AFs) by Aspergillus section Flavi isolated strains; and (2) the natural co-occurrence of AFs, fumonisins (FBs), gliotoxin, diacetoxyscirpenol (DAS), HT-2 and T-2 toxin by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Total fungal counts were below the established value (1 × 104 CFU g⁻¹). Aspergillus flavus and A. parasiticus were the only aflatoxigenic species isolated. Co-occurrence of fumonisin B1 (FB1), HT-2 and T-2 toxin was detected in 100% of the feeds, with mean levels from 4502 to 5813; 6.7 to 21.6 and 19.6 to 30.3 µg kg⁻¹, respectively. A large number of starter samples were co-contaminated with aflatoxin B1 (AFB1), FB1, HT-2 and T-2 toxins. Gliotoxin and DAS were not found in this survey.

Prevalência de aspergilose broncopulmonar alérgica em pacientes com fibrose cística na Bahia, Brasil / Prevalence of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis in the state of Bahia, Brazil

OBJETIVO: Determinar a prevalência de aspergilose broncopulmonar alérgica (ABPA) em pacientes com fibrose cística acompanhados em um centro de referência da Bahia. MÉTODOS: Estudo transversal, com coleta prospectiva de dados, realizado no Centro de Referência de Fibrose Cística da Bahia do Hospital Especializado Octavio Mangabeira. Foram incluídos no estudo 74 pacientes que tinham diagnóstico de fibrose cística, com idade acima de 6 anos e tratados entre 9 de dezembro de 2003 e 7 de março de 2005. Foram analisadas as seguintes variáveis: gênero, idade, capacidade vital forçada, volume expiratório forçado no primeiro segundo, resposta a prova farmacodinâmica, achados em radiografia torácica e de seios de face, presença de sibilância, culturas para Aspergillus spp., imunoglobulina E (IgE) total, IgE específica para Aspergillus fumigatus e teste cutâneo de leitura imediata para aspergilina. RESULTADOS: Dos 74 pacientes, 2 foram diagnosticados com ABPA. Níveis de IgE total > 1.000 UI/mL foram observados em 17 pacientes (23%), teste cutâneo de leitura imediata para A. fumigatus positivos em 19 (25,7%) e sibilância em 60 (81,1%). CONCLUSÕES: A taxa de prevalência de ABPA foi de 2,7%. As altas taxas de IgE total, de teste cutâneo imediato para A. fumigatus positivos e de sibilância sugerem que estes pacientes devam ser acompanhados cuidadosamente por haver a possibilidade do desenvolvimento de ABPA.

OBJECTIVE: To determine the prevalence of allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis treated at a referral center in the state of Bahia, Brazil. METHODS: A cross-sectional study, with prospective data collection, carried out at the Cystic Fibrosis Referral Center of Bahia of the Octavio Mangabeira Specialized Hospital. We evaluated 74 patients diagnosed with cystic fibrosis, older than six years of age, treated between December 9, 2003 and March 7, 2005. We analyzed the following variables: gender, age, forced vital capacity, forced expiratory volume in one second, pharmacodynamic response, chest X-ray findings, facial sinus X-ray findings, wheezing, cultures for Aspergillus spp., total immunoglobulin E (IgE), specific IgE for Aspergillus fumigatus and immediate skin test reactivity to A.fumigatus antigen. RESULTS: Of the 74 patients, 2 were diagnosed with ABPA. We found total IgE levels > 1,000 IU/mL in 17 (23%), positive immediate skin reactivity to A. fumigatus antigen in 19 (25.7%) and wheezing in 60 (81.1%). CONCLUSIONS: The prevalence of ABPA was 2.7%. The high levels of total IgE, high incidence of wheezing and high rate of immediate skin test reactivity to A. fumigatus antigen suggest that these patients should be carefully monitored due to their propensity to develop ABPA.