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Sample records for aspergillus fumigatus infection

  1. Targeting zinc homeostasis to combat Aspergillus fumigatus infections

    PubMed Central

    Vicentefranqueira, Rocío; Amich, Jorge; Laskaris, Paris; Ibrahim-Granet, Oumaima; Latgé, Jean P.; Toledo, Héctor; Leal, Fernando; Calera, José A.

    2015-01-01

    Aspergillus fumigatus is able to invade and grow in the lungs of immunosuppressed individuals and causes invasive pulmonary aspergillosis. The concentration of free zinc in living tissues is much lower than that required for optimal fungal growth in vitro because most of it is tightly bound to proteins. To obtain efficiently zinc from a living host A. fumigatus uses the zinc transporters ZrfA, ZrfB, and ZrfC. The ZafA transcriptional regulator induces the expression of all these transporters and is essential for virulence. Thus, ZafA could be targeted therapeutically to inhibit fungal growth. The ZrfC transporter plays the major role in zinc acquisition from the host whereas ZrfA and ZrfB rather have a supplementary role to that of ZrfC. In addition, only ZrfC enables A. fumigatus to overcome the inhibitory effect of calprotectin, which is an antimicrobial Zn/Mn-chelating protein synthesized and released by neutrophils within the fungal abscesses of immunosuppressed non-leucopenic animals. Hence, fungal survival in these animals would be undermined upon blocking therapeutically the function of ZrfC. Therefore, both ZafA and ZrfC have emerged as promising targets for the discovery of new antifungals to treat Aspergillus infections. PMID:25774155

  2. Targeting zinc homeostasis to combat Aspergillus fumigatus infections.

    PubMed

    Vicentefranqueira, Rocío; Amich, Jorge; Laskaris, Paris; Ibrahim-Granet, Oumaima; Latgé, Jean P; Toledo, Héctor; Leal, Fernando; Calera, José A

    2015-01-01

    Aspergillus fumigatus is able to invade and grow in the lungs of immunosuppressed individuals and causes invasive pulmonary aspergillosis. The concentration of free zinc in living tissues is much lower than that required for optimal fungal growth in vitro because most of it is tightly bound to proteins. To obtain efficiently zinc from a living host A. fumigatus uses the zinc transporters ZrfA, ZrfB, and ZrfC. The ZafA transcriptional regulator induces the expression of all these transporters and is essential for virulence. Thus, ZafA could be targeted therapeutically to inhibit fungal growth. The ZrfC transporter plays the major role in zinc acquisition from the host whereas ZrfA and ZrfB rather have a supplementary role to that of ZrfC. In addition, only ZrfC enables A. fumigatus to overcome the inhibitory effect of calprotectin, which is an antimicrobial Zn/Mn-chelating protein synthesized and released by neutrophils within the fungal abscesses of immunosuppressed non-leucopenic animals. Hence, fungal survival in these animals would be undermined upon blocking therapeutically the function of ZrfC. Therefore, both ZafA and ZrfC have emerged as promising targets for the discovery of new antifungals to treat Aspergillus infections. PMID:25774155

  3. Aspergillus fumigatus in Poultry

    PubMed Central

    Arné, Pascal; Thierry, Simon; Wang, Dongying; Deville, Manjula; Le Loc'h, Guillaume; Desoutter, Anaïs; Féménia, Françoise; Nieguitsila, Adélaïde; Huang, Weiyi; Chermette, René; Guillot, Jacques

    2011-01-01

    Aspergillus fumigatus remains a major respiratory pathogen in birds. In poultry, infection by A. fumigatus may induce significant economic losses particularly in turkey production. A. fumigatus develops and sporulates easily in poor quality bedding or contaminated feedstuffs in indoor farm environments. Inadequate ventilation and dusty conditions increase the risk of bird exposure to aerosolized spores. Acute cases are seen in young animals following inhalation of spores, causing high morbidity and mortality. The chronic form affects older birds and looks more sporadic. The respiratory tract is the primary site of A. fumigatus development leading to severe respiratory distress and associated granulomatous airsacculitis and pneumonia. Treatments for infected poultry are nonexistent; therefore, prevention is the only way to protect poultry. Development of avian models of aspergillosis may improve our understanding of its pathogenesis, which remains poorly understood. PMID:21826144

  4. Metabolomics of Aspergillus fumigatus.

    PubMed

    Frisvad, Jens C; Rank, Christian; Nielsen, Kristian F; Larsen, Thomas O

    2009-01-01

    Aspergillus fumigatus is the most important species in Aspergillus causing infective lung diseases. This species has been reported to produce a large number of extrolites, including secondary metabolites, acids, and proteins such as hydrophobins and extracellular enzymes. At least 226 potentially bioactive secondary metabolites have been reported from A. fumigatus that can be ordered into 24 biosynthetic families. Of these families we have detected representatives from the following families of secondary metabolites: fumigatins, fumigaclavines, fumiquinazolines, trypacidin and monomethylsulochrin, fumagillins, gliotoxins, pseurotins, chloroanthraquinones, fumitremorgins, verruculogen, helvolic acids, and pyripyropenes by HPLC with diode array detection and mass spectrometric detection. There is still doubt whether A. fumigatus can produce tryptoquivalins, but all isolates produce the related fumiquinazolines. We also tentatively detected sphingofungins in A. fumigatus Af293 and in an isolate of A. lentulus. The sphingofungins may have a similar role as the toxic fumonisins, found in A. niger. A further number of mycotoxins, including ochratoxin A, and other secondary metabolites have been reported from A. fumigatus, but in those cases either the fungus or its metabolite appear to be misidentified. PMID:18763205

  5. Pharmacodynamics of Isavuconazole in an Aspergillus fumigatus Mouse Infection Model

    PubMed Central

    Brüggemann, Roger J. M.; Meis, Jacques F.; Melchers, Willem J. G.; Verweij, Paul E.

    2015-01-01

    Azole resistance is an emerging problem in Aspergillus fumigatus which translates into treatment failure. Alternative treatments with new azoles may improve therapeutic outcome in invasive aspergillosis (IA) even for strains with decreased susceptibility to current azoles. The in vivo efficacy of 0.25, 1, 4, 16, 64, 128, 256, and 512 mg/kg of body weight/day prodrug isavuconazonium sulfate (BAL8557) (isavuconazole [ISA]-equivalent doses of 0.12, 0.48, 1.92, 7.68, 30.7, 61.4, 122.9, and 245.8 mg/kg/day, respectively) administered by oral gavage was assessed in an immunocompetent murine model of IA against four clinical A. fumigatus isolates: a wild-type isolate (ISA MICEUCAST, 0.5 mg/liter) and three azole-resistant isolates harboring substitutions in the cyp51A gene: G54W (ISA MICEUCAST, 0.5 mg/liter), M220I (ISA MICEUCAST, 4 mg/liter), and TR34/L98H (ISA MICEUCAST, 8 mg/liter). The maximum effect (100% survival) was reached at a prodrug isavuconazonium sulfate dose of 64 mg/kg for the wild-type isolate, 128 mg/kg for the G54W mutant, and 256 mg/kg two times per day (q12) for the M220I mutant. A maximum response was not achieved with the TR34/L98H isolates with the highest dose of prodrug isavuconazonium sulfate (256 mg/kg q12). For a survival rate of 50%, the effective AUC0–24/MICEUCAST ratio for ISA total drug was 24.73 (95% confidence interval, 22.50 to 27.18). The efficacy of isavuconazole depended on both the drug exposure and the isavuconazole MIC of the isolates. The quantitative relationship between exposure and effect (AUC0–24/MIC) can be used to optimize the treatment of human infections by A. fumigatus, including strains with decreased susceptibility. PMID:25753636

  6. Dynamic Immune Cell Recruitment After Murine Pulmonary Aspergillus fumigatus Infection under Different Immunosuppressive Regimens

    PubMed Central

    Kalleda, Natarajaswamy; Amich, Jorge; Arslan, Berkan; Poreddy, Spoorthi; Mattenheimer, Katharina; Mokhtari, Zeinab; Einsele, Hermann; Brock, Matthias; Heinze, Katrin Gertrud; Beilhack, Andreas

    2016-01-01

    Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. However, in healthy individuals pulmonary host defense mechanisms efficiently eliminate the fungus. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. Host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control invasive fungal disease. In different immunocompromised murine models, myeloid, notably neutrophils, and macrophages, but not lymphoid cells were strongly recruited to the lungs upon infection. Other myeloid cells, particularly dendritic cells and monocytes, were only recruited to lungs of corticosteroid treated mice, which developed a strong pulmonary inflammation after infection. Lymphoid cells, particularly CD4+ or CD8+ T-cells and NK cells were highly reduced upon immunosuppression and not recruited after A. fumigatus infection. Moreover, adoptive CD11b+ myeloid cell transfer rescued cyclophosphamide immunosuppressed mice from lethal A. fumigatus infection but not cortisone and cyclophosphamide immunosuppressed mice. Our findings illustrate that CD11b+ myeloid cells are critical for anti-A. fumigatus defense under cyclophosphamide immunosuppressed conditions. PMID:27468286

  7. Dynamic Immune Cell Recruitment After Murine Pulmonary Aspergillus fumigatus Infection under Different Immunosuppressive Regimens.

    PubMed

    Kalleda, Natarajaswamy; Amich, Jorge; Arslan, Berkan; Poreddy, Spoorthi; Mattenheimer, Katharina; Mokhtari, Zeinab; Einsele, Hermann; Brock, Matthias; Heinze, Katrin Gertrud; Beilhack, Andreas

    2016-01-01

    Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. However, in healthy individuals pulmonary host defense mechanisms efficiently eliminate the fungus. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. Host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control invasive fungal disease. In different immunocompromised murine models, myeloid, notably neutrophils, and macrophages, but not lymphoid cells were strongly recruited to the lungs upon infection. Other myeloid cells, particularly dendritic cells and monocytes, were only recruited to lungs of corticosteroid treated mice, which developed a strong pulmonary inflammation after infection. Lymphoid cells, particularly CD4(+) or CD8(+) T-cells and NK cells were highly reduced upon immunosuppression and not recruited after A. fumigatus infection. Moreover, adoptive CD11b(+) myeloid cell transfer rescued cyclophosphamide immunosuppressed mice from lethal A. fumigatus infection but not cortisone and cyclophosphamide immunosuppressed mice. Our findings illustrate that CD11b(+) myeloid cells are critical for anti-A. fumigatus defense under cyclophosphamide immunosuppressed conditions. PMID:27468286

  8. Aspergillus fumigatus and Aspergillosis

    PubMed Central

    Latgé, Jean-Paul

    1999-01-01

    Aspergillus fumigatus is one of the most ubiquitous of the airborne saprophytic fungi. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes, are the only infections observed in such hosts. Thus, A. fumigatus was considered for years to be a weak pathogen. With increases in the number of immunosuppressed patients, however, there has been a dramatic increase in severe and usually fatal invasive aspergillosis, now the most common mold infection worldwide. In this review, the focus is on the biology of A. fumigatus and the diseases it causes. Included are discussions of (i) genomic and molecular characterization of the organism, (ii) clinical and laboratory methods available for the diagnosis of aspergillosis in immunocompetent and immunocompromised hosts, (iii) identification of host and fungal factors that play a role in the establishment of the fungus in vivo, and (iv) problems associated with antifungal therapy. PMID:10194462

  9. FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection.

    PubMed

    Kerr, Sheena C; Fischer, Gregory J; Sinha, Meenal; McCabe, Orla; Palmer, Jonathan M; Choera, Tsokyi; Lim, Fang Yun; Wimmerova, Michaela; Carrington, Stephen D; Yuan, Shaopeng; Lowell, Clifford A; Oscarson, Stefan; Keller, Nancy P; Fahy, John V

    2016-04-01

    The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia. PMID:27058347

  10. FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection

    PubMed Central

    Sinha, Meenal; McCabe, Orla; Palmer, Jonathan M.; Choera, Tsokyi; Yun Lim, Fang; Wimmerova, Michaela; Carrington, Stephen D.; Yuan, Shaopeng; Lowell, Clifford A.; Oscarson, Stefan; Keller, Nancy P.; Fahy, John V.

    2016-01-01

    The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia. PMID:27058347

  11. Infection-Mediated Priming of Phagocytes Protects against Lethal Secondary Aspergillus fumigatus Challenge

    PubMed Central

    Savers, Amélie; Rasid, Orhan; Parlato, Marianna; Brock, Matthias; Jouvion, Gregory; Ryffel, Bernhard; Cavaillon, Jean-Marc; Eberl, Gerard; Ibrahim-Granet, Oumaïma

    2016-01-01

    Phagocytes restrict the germination of Aspergillus fumigatus conidia and prevent the establishment of invasive pulmonary aspergillosis in immunecompetent mice. Here we report that immunecompetent mice recovering from a primary A. fumigatus challenge are protected against a secondary lethal challenge. Using RAGγc knock-out mice we show that this protection is independent of T, B and NK cells. In protected mice, lung phagocytes are recruited more rapidly and are more efficient in conidial phagocytosis and killing. Protection was also associated with an enhanced expression of CXCR2 and Dectin-1 on bone marrow phagocytes. We also show that protective lung cytokine and chemokine responses are induced more rapidly and with enhanced dynamics in protected mice. Our findings support the hypothesis that following a first encounter with a non-lethal dose of A. fumigatus conidia, the innate immune system is primed and can mediate protection against a secondary lethal infection. PMID:27078879

  12. Infection-Mediated Priming of Phagocytes Protects against Lethal Secondary Aspergillus fumigatus Challenge.

    PubMed

    Savers, Amélie; Rasid, Orhan; Parlato, Marianna; Brock, Matthias; Jouvion, Gregory; Ryffel, Bernhard; Cavaillon, Jean-Marc; Eberl, Gerard; Ibrahim-Granet, Oumaïma

    2016-01-01

    Phagocytes restrict the germination of Aspergillus fumigatus conidia and prevent the establishment of invasive pulmonary aspergillosis in immunecompetent mice. Here we report that immunecompetent mice recovering from a primary A. fumigatus challenge are protected against a secondary lethal challenge. Using RAGγc knock-out mice we show that this protection is independent of T, B and NK cells. In protected mice, lung phagocytes are recruited more rapidly and are more efficient in conidial phagocytosis and killing. Protection was also associated with an enhanced expression of CXCR2 and Dectin-1 on bone marrow phagocytes. We also show that protective lung cytokine and chemokine responses are induced more rapidly and with enhanced dynamics in protected mice. Our findings support the hypothesis that following a first encounter with a non-lethal dose of A. fumigatus conidia, the innate immune system is primed and can mediate protection against a secondary lethal infection. PMID:27078879

  13. Concurrent pulmonary Aspergillus fumigatus and mucor infection in a cardiac transplant recipient: a case report.

    PubMed

    Webb, B J; Blair, J E; Kusne, S; Scott, R L; Steidley, D E; Arabia, F A; Vikram, H R

    2013-03-01

    Invasive fungal infections are a significant complication of solid organ transplantation. Here we report the first case of concurrent invasive pulmonary fungal infection caused by Aspergillus fumigatus and Mucor species in a heart transplant recipient. Polymicrobial mold infection is rare but should be considered in solid organ transplant recipients who fail to respond to initial antifungal therapy targeting a single organism. It is also of interest that in addition to potent immunosuppression and prolonged voriconazole therapy, possible airway fungal colonization following hurricane Katrina cleaning efforts might have contributed to this dual invasive mold infection. PMID:23267784

  14. ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo.

    PubMed

    Rolle, Anna-Maria; Hasenberg, Mike; Thornton, Christopher R; Solouk-Saran, Djamschid; Männ, Linda; Weski, Juliane; Maurer, Andreas; Fischer, Eliane; Spycher, Philipp R; Schibli, Roger; Boschetti, Frederic; Stegemann-Koniszewski, Sabine; Bruder, Dunja; Severin, Gregory W; Autenrieth, Stella E; Krappmann, Sven; Davies, Genna; Pichler, Bernd J; Gunzer, Matthias; Wiehr, Stefan

    2016-02-23

    Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [(64)Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [(64)Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [(18)F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation. PMID:26787852

  15. ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo

    PubMed Central

    Rolle, Anna-Maria; Hasenberg, Mike; Thornton, Christopher R.; Solouk-Saran, Djamschid; Männ, Linda; Weski, Juliane; Maurer, Andreas; Fischer, Eliane; Spycher, Philipp R.; Schibli, Roger; Boschetti, Frederic; Stegemann-Koniszewski, Sabine; Bruder, Dunja; Severin, Gregory W.; Autenrieth, Stella E.; Krappmann, Sven; Davies, Genna; Pichler, Bernd J.; Gunzer, Matthias; Wiehr, Stefan

    2016-01-01

    Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [64Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [64Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [18F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation. PMID:26787852

  16. The Volatome of Aspergillus fumigatus

    PubMed Central

    Calvo, A. M.; Latgé, J. P.

    2014-01-01

    Early detection of invasive aspergillosis is absolutely required for efficient therapy of this fungal infection. The identification of fungal volatiles in patient breath can be an alternative for the detection of Aspergillus fumigatus that still remains problematic. In this work, we investigated the production of volatile organic compounds (VOCs) by A. fumigatus in vitro, and we show that volatile production depends on the nutritional environment. A. fumigatus produces a multiplicity of VOCs, predominantly terpenes and related compounds. The production of sesquiterpenoid compounds was found to be strongly induced by increased iron concentrations and certain drugs, i.e., pravastatin. Terpenes that were always detectable in large amounts were α-pinene, camphene, and limonene, as well as sesquiterpenes, identified as α-bergamotene and β-trans-bergamotene. Other substance classes that were found to be present in the volatome, such as 1-octen-3-ol, 3-octanone, and pyrazines, were found only under specific growth conditions. Drugs that interfere with the terpene biosynthesis pathway influenced the composition of the fungal volatome, and most notably, a block of sesquiterpene biosynthesis by the bisphosphonate alendronate fundamentally changed the VOC composition. Using deletion mutants, we also show that a terpene cyclase and a putative kaurene synthase are essential for the synthesis of volatile terpenes by A. fumigatus. The present analysis of in vitro volatile production by A. fumigatus suggests that VOCs may be used in the diagnosis of infections caused by this fungus. PMID:24906414

  17. Deciphering the Counterplay of Aspergillus fumigatus Infection and Host Inflammation by Evolutionary Games on Graphs.

    PubMed

    Pollmächer, Johannes; Timme, Sandra; Schuster, Stefan; Brakhage, Axel A; Zipfel, Peter F; Figge, Marc Thilo

    2016-01-01

    Microbial invaders are ubiquitously present and pose the constant risk of infections that are opposed by various defence mechanisms of the human immune system. A tight regulation of the immune response ensures clearance of microbial invaders and concomitantly limits host damage that is crucial for host viability. To investigate the counterplay of infection and inflammation, we simulated the invasion of the human-pathogenic fungus Aspergillus fumigatus in lung alveoli by evolutionary games on graphs. The layered structure of the innate immune system is represented by a sequence of games in the virtual model. We show that the inflammatory cascade of the immune response is essential for microbial clearance and that the inflammation level correlates with the infection-dose. At low infection-doses, corresponding to daily inhalation of conidia, the resident alveolar macrophages may be sufficient to clear infections, however, at higher infection-doses their primary task shifts towards recruitment of neutrophils to infection sites. PMID:27291424

  18. Deciphering the Counterplay of Aspergillus fumigatus Infection and Host Inflammation by Evolutionary Games on Graphs

    NASA Astrophysics Data System (ADS)

    Pollmächer, Johannes; Timme, Sandra; Schuster, Stefan; Brakhage, Axel A.; Zipfel, Peter F.; Figge, Marc Thilo

    2016-06-01

    Microbial invaders are ubiquitously present and pose the constant risk of infections that are opposed by various defence mechanisms of the human immune system. A tight regulation of the immune response ensures clearance of microbial invaders and concomitantly limits host damage that is crucial for host viability. To investigate the counterplay of infection and inflammation, we simulated the invasion of the human-pathogenic fungus Aspergillus fumigatus in lung alveoli by evolutionary games on graphs. The layered structure of the innate immune system is represented by a sequence of games in the virtual model. We show that the inflammatory cascade of the immune response is essential for microbial clearance and that the inflammation level correlates with the infection-dose. At low infection-doses, corresponding to daily inhalation of conidia, the resident alveolar macrophages may be sufficient to clear infections, however, at higher infection-doses their primary task shifts towards recruitment of neutrophils to infection sites.

  19. Deciphering the Counterplay of Aspergillus fumigatus Infection and Host Inflammation by Evolutionary Games on Graphs

    PubMed Central

    Pollmächer, Johannes; Timme, Sandra; Schuster, Stefan; Brakhage, Axel A.; Zipfel, Peter F.; Figge, Marc Thilo

    2016-01-01

    Microbial invaders are ubiquitously present and pose the constant risk of infections that are opposed by various defence mechanisms of the human immune system. A tight regulation of the immune response ensures clearance of microbial invaders and concomitantly limits host damage that is crucial for host viability. To investigate the counterplay of infection and inflammation, we simulated the invasion of the human-pathogenic fungus Aspergillus fumigatus in lung alveoli by evolutionary games on graphs. The layered structure of the innate immune system is represented by a sequence of games in the virtual model. We show that the inflammatory cascade of the immune response is essential for microbial clearance and that the inflammation level correlates with the infection-dose. At low infection-doses, corresponding to daily inhalation of conidia, the resident alveolar macrophages may be sufficient to clear infections, however, at higher infection-doses their primary task shifts towards recruitment of neutrophils to infection sites. PMID:27291424

  20. Indoleamine 2,3-Dioxygenase Is Involved in the Inflammation Response of Corneal Epithelial Cells to Aspergillus fumigatus Infections

    PubMed Central

    Jiang, Nan; Zhao, Guiqiu; Lin, Jing; Hu, Liting; Che, Chengye; Li, Cui; Wang, Qian; Xu, Qiang; Peng, Xudong

    2015-01-01

    Indoleamine 2,3-dioxygenase (IDO), which is mainly expressed in activated dendritic cells, is known as a regulator of immune responses. However, the role of IDO in immune responses against fungal corneal infection has not been investigated. To evaluate the regulatory mechanisms of IDO in fungal inflammation, we resorted to human corneal epithelial cells (HCECs), known as the first barrier of cornea against pathogenic microorganisms. We found that IDO was significantly up-regulated in corneal epithelium infected with Aspergillus fumigatus (A. fumigatus) and HCECs incubated with spores of A. fumigatus. Furthermore, IDO inhibitor (1-methyltryptophan, 1-MT) enhanced inflammatory cytokines IL-1β and IL-6 expression which were up-regulated by A. fumigatus spores infection. Dectin-1, as one of the important C-type lectin receptors, can identify β-glucan, and mediate fungal innate immune responses. In the present study, pre-treatment with curdlan, a Dectin-1 agonist, further enhanced IDO expression compared with A. fumigatus stimulation. While laminarin, the Dectin-1 specific inhibitor, partially inhibited IDO expression stimulated by A. fumigatus. Further studies demonstrated inhibition of IDO activity amplified the expressions of inflammatory cytokines IL-1β and IL-6 induced by activation of Dectin-1. These results suggested that IDO was involved in the immune responses of fungal keratitis. The activation of Dectin-1 may contribute to A. fumigatus spores-induced up-regulation of IDO. PMID:26361229

  1. Developmental regulators in Aspergillus fumigatus.

    PubMed

    Park, Hee-Soo; Yu, Jae-Hyuk

    2016-03-01

    The filamentous fungus Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing severe and usually fatal invasive aspergillosis in immunocompromised patients. This fungus produces a large number of small hydrophobic asexual spores called conidia as the primary means of reproduction, cell survival, propagation, and infectivity. The initiation, progression, and completion of asexual development (conidiation) is controlled by various regulators that govern expression of thousands of genes associated with formation of the asexual developmental structure conidiophore, and biogenesis of conidia. In this review, we summarize key regulators that directly or indirectly govern conidiation in this important pathogenic fungus. Better understanding these developmental regulators may provide insights into the improvement in controlling both beneficial and detrimental aspects of various Aspergillus species. PMID:26920882

  2. Toxicity and efficacy differences between liposomal amphotericin B formulations in uninfected and Aspergillus fumigatus infected mice.

    PubMed

    Olson, J A; Schwartz, J A; Hahka, D; Nguyen, N; Bunch, T; Jensen, G M; Adler-Moore, J P

    2015-02-01

    Because of the reduced toxicity associated with liposomal amphotericin B preparations, different amphotericin B liposome products have been made. In the present study, we compared the amphotericin B liposomal formulations, AmBisome(®) (AmBi) and Lambin(®) (Lbn), in uninfected and Aspergillus fumigatus infected mice, using several in vitro and in vivo toxicity and efficacy assays. The results showed that the formulations were significantly different, with Lbn 1.6-fold larger than AmBi. Lbn was also more toxic than AmBi based on the RBC potassium release assay and intravenous dosing in uninfected mice given a single 50 mg/kg dose (80% mortality for Lbn vs. 0% for AmBi). Renal tubular changes after intravenous daily dosing for 14 days were seen in uninfected mice given 5 mg/kg Lbn but not with AmBi. Survival following A. fumigatus challenge was 30% for 10 mg/kg Lbn and 60% for 10 mg/kg AmBi. When the BAL and lungs were collected 24 h after the second treatment, AmBi at 10 or 15 mg/kg or 15 mg/kg Lbn lowered the BAL fungal burden significantly vs. the controls (P ≤ 0.05), while there was no difference in lung fungal burden amongst the groups. In contrast, lung histopathology at this same early timepoint showed that AmBi was associated with markedly fewer fungal elements and less lung tissue damage than Lbn. In conclusion, given the differences in size, toxicity, and efficacy, AmBi and Lbn were not physically or functionally comparable, and these differences underscore the need for adequate testing when comparing amphotericin B liposome formulations. PMID:25550388

  3. In vivo efficacy of voriconazole and posaconazole therapy in a novel invertebrate model of Aspergillus fumigatus infection.

    PubMed

    Forastiero, A; Bernal-Martínez, L; Mellado, E; Cendejas, E; Gomez-Lopez, A

    2015-11-01

    The emergence of azole resistance in Aspergillus fumigatus is a clinically important issue in the management of invasive aspergillosis as it could limit therapeutic options. Accurate measurement of in vitro antifungal activity in terms of minimum inhibitory concentration (MIC) is considered of clinical relevance and often gives useful therapeutic information for physicians. However, the lack of in vitro-in vivo correlation is frequent and the observed in vitro phenotype does not always correlate with the in vivo response. In this regard, a wild-type strain and five A. fumigatus cyp51A mutated strains showing different azole susceptibility profiles were used to investigate whether the greater wax moth (Galleria mellonella) is an alternative model to assess the in vivo efficacy of voriconazole and posaconazole. Administration of both azoles improved the survival of larvae infected with susceptible strains. However, those larvae infected with resistant strains did not respond to treatment. The phenotype observed in vitro was found to correlate with the efficacy observed in vivo. Moreover, using this in vivo model, the pharmacodynamic target predicting therapeutic success (AUC(0-24)/MIC) was in the same range as previously described, allowing the use of the G. mellonella model to predict the azole susceptibility profile of A. fumigatus strains. PMID:26358971

  4. Development of monoclonal antibody-based galactomannoprotein antigen-capture ELISAs to detect Aspergillus fumigatus infection in the invasive aspergillosis rabbit models.

    PubMed

    Wang, Z-Y; Cai, J-P; Qiu, L-W; Hao, W; Pan, Y-X; Tung, E T K; Lau, C C Y; Woo, P C Y; Lau, S K P; Yuen, K-Y; Che, X-Y

    2012-11-01

    Aspergillus fumigatus is one of the most prominent opportunistic fungal pathogens in immunocompromised hosts. Early recognition of this infection along with prompt antifungal therapy may increase the survival rate. We expressed two potential bio-markers of A. fumigatus infection-galactomannoprotein Afmp1p and Afmp4p in Pichia pastoris. We generated 33 monoclonal antibodies (MAbs), 20 against recombinant Afmp1p (rAfmp1p) and the other 13 against recombinant Afmp4p (rAfmp4p). Subsequently, we developed two antigen-capture enzyme-linked immunosorbent assays (ELISAs) which employed MAbs as both the capture and the detection antibodies for rAfmp1p and rAfmp4p. The two antigen-capture ELISAs specifically detected Afmp1p/Afmp4p in cultures of A. fumigatus and had no cross-reaction with other tested pathogenic fungi, including Penicillium marneffei and other pathogenic Aspergillus species. The Afmp1p-captured ELISA would be positive even when the culture supernatant of A. fumigatus had been diluted to 128-fold of its original concentration. The two antigen ELISAs could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus-challenged animal models. We developed two antigen-capture ELISAs for the laboratory diagnosis of A. fumigatus infection. These two antigen-capture ELISAs may be useful in the clinical diagnosis of aspergillosis. PMID:22669560

  5. Detection of Aspergillus fumigatus-specific DNA, (1-3)-beta-D-glucan and galactomannan in serum and bronchoalveolar lavage specimens of experimentally infected rats.

    PubMed

    Khan, Z U; Ahmad, S; Theyyathel, A M

    2008-03-01

    The aim of this study was to detect Aspergillus fumigatus-specific DNA by nested PCR (nPCR) in serum and bronchoalveolar lavage (BAL) specimens of experimentally infected rats and compare the results with (1-3)-beta-D-glucan (BDG) and galactomannan (GM) detection. Sixty Wistar rats, immunosuppressed with an intraperitoneal injection of cyclophosphamide (70 mg kg(-1)) were infected with 1 x 10(6)A. fumigatus conidia. The rats were killed on days 1, 3, 5, 7 and 9 postinfection in groups of six each and their BAL, blood and lungs were cultured. The A. fumigatus-specific DNA, BDG and GM in serum and BAL were detected by nPCR, Fungitell kit and Aspergillus Platelia kit respectively. Base line values were obtained by using sera from six healthy rats. Except the lungs, blood and BAL specimens of all the infected rats were negative for A. fumigatus culture. The BDG, GM and nPCR positivity in serum specimens was 80%, 77% and 63% respectively. The sensitivity of GM and nPCR tests in BAL specimens was 77% and 70% respectively. The data suggest that BDG and GM appear early in the course of infection, and have similar kinetics (r = 0.483, P = 0.007). Hence, their combined detection could be useful in the early diagnosis of invasive aspergillosis. PMID:18254749

  6. Agent-based model of human alveoli predicts chemotactic signaling by epithelial cells during early Aspergillus fumigatus infection.

    PubMed

    Pollmächer, Johannes; Figge, Marc Thilo

    2014-01-01

    Aspergillus fumigatus is one of the most important human fungal pathogens, causing life-threatening diseases. Since humans inhale hundreds to thousands of fungal conidia every day, the lower respiratory tract is the primary site of infection. Current interaction networks of the innate immune response attribute fungal recognition and detection to alveolar macrophages, which are thought to be the first cells to get in contact with the fungus. At present, these networks are derived from in vitro or in situ assays, as the peculiar physiology of the human lung makes in vivo experiments, including imaging on the cell-level, hard to realize. We implemented a spatio-temporal agent-based model of a human alveolus in order to perform in silico experiments of a virtual infection scenario, for an alveolus infected with A. fumigatus under physiological conditions. The virtual analog captures the three-dimensional alveolar morphology consisting of the two major alveolar epithelial cell types and the pores of Kohn as well as the dynamic process of respiration. To the best of our knowledge this is the first agent-based model of a dynamic human alveolus in the presence of respiration. A key readout of our simulations is the first-passage-time of alveolar macrophages, which is the period of time that elapses until the first physical macrophage-conidium contact is established. We tested for random and chemotactic migration modes of alveolar macrophages and varied their corresponding parameter sets. The resulting first-passage-time distributions imply that randomly migrating macrophages fail to find the conidium before the start of germination, whereas guidance by chemotactic signals derived from the alveolar epithelial cell associated with the fungus enables a secure and successful discovery of the pathogen in time. PMID:25360787

  7. Deciphering chemokine properties by a hybrid agent-based model of Aspergillus fumigatus infection in human alveoli

    PubMed Central

    Pollmächer, Johannes; Figge, Marc Thilo

    2015-01-01

    The ubiquitous airborne fungal pathogen Aspergillus fumigatus is inhaled by humans every day. In the lung, it is able to quickly adapt to the humid environment and, if not removed within a time frame of 4–8 h, the pathogen may cause damage by germination and invasive growth. Applying a to-scale agent-based model of human alveoli to simulate early A. fumigatus infection under physiological conditions, we recently demonstrated that alveolar macrophages require chemotactic cues to accomplish the task of pathogen detection within the aforementioned time frame. The objective of this study is to specify our general prediction on the as yet unidentified chemokine by a quantitative analysis of its expected properties, such as the diffusion coefficient and the rates of secretion and degradation. To this end, the rule-based implementation of chemokine diffusion in the initial agent-based model is revised by numerically solving the spatio-temporal reaction-diffusion equation in the complex structure of the alveolus. In this hybrid agent-based model, alveolar macrophages are represented as migrating agents that are coupled to the interactive layer of diffusing molecule concentrations by the kinetics of chemokine receptor binding, internalization and re-expression. Performing simulations for more than a million virtual infection scenarios, we find that the ratio of secretion rate to the diffusion coefficient is the main indicator for the success of pathogen detection. Moreover, a subdivision of the parameter space into regimes of successful and unsuccessful parameter combination by this ratio is specific for values of the migration speed and the directional persistence time of alveolar macrophages, but depends only weakly on chemokine degradation rates. PMID:26074897

  8. Activity and Safety of Inhaled Itraconazole Nanosuspension in a Model Pulmonary Aspergillus fumigatus Infection in Inoculated Young Quails.

    PubMed

    Wlaź, Piotr; Knaga, Sebastian; Kasperek, Kornel; Wlaź, Aleksandra; Poleszak, Ewa; Jeżewska-Witkowska, Grażyna; Winiarczyk, Stanisław; Wyska, Elżbieta; Heinekamp, Thorsten; Rundfeldt, Chris

    2015-08-01

    Pulmonary aspergillosis is frequently reported in parrots, falcons, and other birds held in captivity. Inhalation is the main route of infection for Aspergillus fumigatus, resulting in both acute and chronic disease conditions. Itraconazole (ITRA) is an antifungal commonly used in birds, but its administration requires repeated oral dosing, and the safety margin is narrow. To investigate the efficacy of inhaled ITRA, six groups of ten young quails (Coturnix japonica) were inoculated intratracheally with 5 × 10(6) spores (3 groups) or 5 × 10(7) spores (3 groups). Animals were exposed to nebulized ITRA nanosuspension as 10 % suspension or 4 % suspension, once daily for 30 min, starting 2 h after inoculation for 6 days. Control groups were exposed to nebulized saline for the same period of time. Survival and clinical scores were evaluated, and animals were subjected to gross pathology. In control animals, aspergillosis resulted in systemic disease without pulmonary or air sac granulomas. Animals died from multiple organ failure. Inhalation of 10 % ITRA nanosuspension blocked lethality and prevented disease-related symptoms in the quails exposed to the low dose of spores, while the disease course in quails inoculated with the high-spore dose was retarded. Inhalation of 4 % ITRA nanosuspension was less effective. Both inhalations were well tolerated, and gross pathology did not reveal signs of local toxicity. The data indicate that inhaled administration of 10 % ITRA nanosuspension is capable of alleviating an acute A. fumigatus infection in quails. A lower ITRA concentration may be only active in chronic pulmonary aspergillosis. PMID:25790942

  9. Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis

    PubMed Central

    Dagenais, Taylor R. T.; Keller, Nancy P.

    2009-01-01

    Summary: Aspergillus species are globally ubiquitous saprophytes found in a variety of ecological niches. Almost 200 species of aspergilli have been identified, less than 20 of which are known to cause human disease. Among them, Aspergillus fumigatus is the most prevalent and is largely responsible for the increased incidence of invasive aspergillosis (IA) in the immunocompromised patient population. IA is a devastating illness, with mortality rates in some patient groups reaching as high as 90%. Studies identifying and assessing the roles of specific factors of A. fumigatus that contribute to the pathogenesis of IA have traditionally focused on single-gene deletion and mutant characterization. In combination with recent large-scale approaches analyzing global fungal responses to distinct environmental or host conditions, these studies have identified many factors that contribute to the overall pathogenic potential of A. fumigatus. Here, we provide an overview of the significant findings regarding A. fumigatus pathogenesis as it pertains to invasive disease. PMID:19597008

  10. 4-Ethylphenol metabolism by Aspergillus fumigatus

    SciTech Connect

    Jones, K.H.; Trudgill, P.W.; Hopper, D.J.

    1994-06-01

    Many industrial pollutants are phenolic, and the degradation these compounds is important in the carbon cycle. Aspergillus fumigatus ATCC 28282 can grow on p-cresol. However 4-Ethylphenol, the higher homolog of p-cresol, presents different possibilities for putative metabolic pathways. This study shows that A. fumigatus is able to grow and 4-ethylphenol and the pathway is described. 17 refs., 4 figs., 2 tabs.

  11. Targeting Iron Acquisition Blocks Infection with the Fungal Pathogens Aspergillus fumigatus and Fusarium oxysporum

    PubMed Central

    Leal, Sixto M.; Roy, Sanhita; Vareechon, Chairut; Carrion, Steven deJesus; Clark, Heather; Lopez-Berges, Manuel S.; diPietro, Antonio; Schrettl, Marcus; Beckmann, Nicola; Redl, Bernhard; Haas, Hubertus; Pearlman, Eric

    2013-01-01

    Filamentous fungi are an important cause of pulmonary and systemic morbidity and mortality, and also cause corneal blindness and visual impairment worldwide. Utilizing in vitro neutrophil killing assays and a model of fungal infection of the cornea, we demonstrated that Dectin-1 dependent IL-6 production regulates expression of iron chelators, heme and siderophore binding proteins and hepcidin in infected mice. In addition, we show that human neutrophils synthesize lipocalin-1, which sequesters fungal siderophores, and that topical lipocalin-1 or lactoferrin restricts fungal growth in vivo. Conversely, we show that exogenous iron or the xenosiderophore deferroxamine enhances fungal growth in infected mice. By examining mutant Aspergillus and Fusarium strains, we found that fungal transcriptional responses to low iron levels and extracellular siderophores are essential for fungal growth during infection. Further, we showed that targeting fungal iron acquisition or siderophore biosynthesis by topical application of iron chelators or statins reduces fungal growth in the cornea by 60% and that dual therapy with the iron chelator deferiprone and statins further restricts fungal growth by 75%. Together, these studies identify specific host iron-chelating and fungal iron-acquisition mediators that regulate fungal growth, and demonstrate that therapeutic inhibition of fungal iron acquisition can be utilized to treat topical fungal infections. PMID:23853581

  12. Evaluation of plasma (1-->3) beta-D-glucan concentrations in birds naturally and experimentally infected with aspergillus fumigatus.

    PubMed

    Burco, Julia D; Ziccardi, Michael H; Clemons, Karl V; Tell, Lisa A

    2012-03-01

    Avian aspergillosis, most often caused by Aspergillus fumigatus, is a common and devastating disease affecting a range of bird species. Early diagnosis is difficult and often unreliable. The current study evaluated the utility of measuring (1-->3)-beta-D-glucan (BG) concentrations in avian plasma samples to aid in the diagnosis of aspergillosis. We evaluated a commercially available BG assay (Fungitell, Beacon Diagnostics) using 178 plasma samples from naturally infected, experimentally infected, and aspergillosis-free birds. Although there was variation in BG concentration, as reflected by high standard deviations, seabirds with confirmed aspergillosis had the highest mean BG concentrations (M = 3098.7 pg/dl, SD = 5022.6, n = 22) followed by companion avian species and raptors with confirmed aspergillosis (M = 1033.8 pg/dl, SD = 1531.6, n = 19) and experimentally infected Japanese quail (Coturnix japonica; M = 1066.5 pg/dl, SD = 1348.2, n = 17). Variation in severity of disease, differences among species of birds with and without disease, and also different levels in environmental exposure likely contribute to the differences among avian groups. The overall sensitivity and specificity of the BG test for diagnosis of aspergillosis in birds was 60.0 and 92.7%, respectively, with an overall optimized avian cut-offvalue of > or = 461 pg/dl for positive disease. Our findings suggest that, although BG concentrations are highly variable between and within different avian groups, it could serve as a useful adjunctive diagnostic test for aspergillosis that is applicable to multiple avian species in some settings, particularly as a negative predictor of infection. PMID:22545545

  13. Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection.

    PubMed

    Clark, Heather L; Jhingran, Anupam; Sun, Yan; Vareechon, Chairut; de Jesus Carrion, Steven; Skaar, Eric P; Chazin, Walter J; Calera, José Antonio; Hohl, Tobias M; Pearlman, Eric

    2016-01-01

    Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein that possesses antimicrobial activity primarily because of its ability to chelate zinc and manganese. In the current study, we showed that neutrophils from calprotectin-deficient S100A9(-/-) mice have an impaired ability to inhibit Aspergillus fumigatus hyphal growth in vitro and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9(-/-) mice by injecting recombinant calprotectin. Furthermore, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both zinc and manganese binding are necessary for calprotectin's antihyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatus ∆zafA mutant, which demonstrates deficient zinc transport, exhibits impaired growth in infected corneas and following incubation with neutrophils or calprotectin in vitro as compared with wild-type. Collectively, these studies demonstrate a novel stage-specific susceptibility of A. fumigatus to zinc and manganese chelation by neutrophil-derived calprotectin. PMID:26582948

  14. Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

    PubMed Central

    Dinamarco, Taísa Magnani; Freitas, Fernanda Zanolli; Almeida, Ricardo S.; Brown, Neil Andrew; dos Reis, Thaila Fernanda; Ramalho, Leandra Naira Zambelli; Savoldi, Marcela; Goldman, Maria Helena S.; Bertolini, Maria Célia; Goldman, Gustavo Henrique

    2012-01-01

    Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5′-CACAGCCAC-3′ and 5′-CCCTGCCCC-3′ sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -B and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The ΔpmcA and ΔpmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the ΔcalA and ΔcrzA mutant strains. However, only the A. fumigatus ΔpmcA was avirulent in the murine model of invasive pulmonary aspergillosis. PMID:22649543

  15. International expert opinion on the management of infection caused by azole-resistant Aspergillus fumigatus.

    PubMed

    Verweij, Paul E; Ananda-Rajah, Michelle; Andes, David; Arendrup, Maiken C; Brüggemann, Roger J; Chowdhary, Anuradha; Cornely, Oliver A; Denning, David W; Groll, Andreas H; Izumikawa, Koichi; Kullberg, Bart Jan; Lagrou, Katrien; Maertens, Johan; Meis, Jacques F; Newton, Pippa; Page, Iain; Seyedmousavi, Seyedmojtaba; Sheppard, Donald C; Viscoli, Claudio; Warris, Adilia; Donnelly, J Peter

    2015-01-01

    An international expert panel was convened to deliberate the management of azole-resistant aspergillosis. In culture-positive cases, in vitro susceptibility testing should always be performed if antifungal therapy is intended. Different patterns of resistance are seen, with multi-azole and pan-azole resistance more common than resistance to a single triazole. In confirmed invasive pulmonary aspergillosis due to an azole-resistant Aspergillus, the experts recommended a switch from voriconazole to liposomal amphotericin B (L-AmB; Ambisome(®)). In regions with environmental resistance rates of ≥10%, a voriconazole-echinocandin combination or L-AmB were favoured as initial therapy. All experts recommended L-AmB as core therapy for central nervous system aspergillosis suspected to be due to an azole-resistant Aspergillus, and considered the addition of a second agent with the majority favouring flucytosine. Intravenous therapy with either micafungin or L-AmB given as either intermittent or continuous therapy was recommended for chronic pulmonary aspergillosis due to a pan-azole-resistant Aspergillus. Local and national surveillance with identification of clinical and environmental resistance patterns, rapid diagnostics, better quality clinical outcome data, and a greater understanding of the factors driving or minimising environmental resistance are areas where research is urgently needed, as well as the development of new oral agents outside the azole drug class. PMID:26282594

  16. Mycotic aneurysm of the thoracic aorta caused by Aspergillus fumigatus.

    PubMed

    Rose, H D; Stuart, J L

    1976-07-01

    A 54-year-old diabetic patient had unexplained fever and embolic occlusion of the splenic, right renal, right hypogastric, right superficial femoral, and left popliteal arteries. Aspergillus fumigatus was recovered from a femoral clot. An aortogram revealed a mycotic aneurysm of the thoracic aorta to be the source of the infected emboli. Surgical excision of the aneurysm and therapy with amphotericin B were unsuccessful. PMID:776547

  17. Secondary metabolite profiles and antifungal drug susceptibility of Aspergillus fumigatus and closely related species, Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans.

    PubMed

    Tamiya, Hiroyuki; Ochiai, Eri; Kikuchi, Kazuyo; Yahiro, Maki; Toyotome, Takahito; Watanabe, Akira; Yaguchi, Takashi; Kamei, Katsuhiko

    2015-05-01

    The incidence of Aspergillus infection has been increasing in the past few years. Also, new Aspergillus fumigatus-related species, namely Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans, were shown to infect humans. These fungi exhibit marked morphological similarities to A. fumigatus, albeit with different clinical courses and antifungal drug susceptibilities. The present study used liquid chromatography/time-of-flight mass spectrometry to identify the secondary metabolites secreted as virulence factors by these Aspergillus species and compared their antifungal susceptibility. The metabolite profiles varied widely among A. fumigatus, A. lentulus, A. udagawae, and A. viridinutans, producing 27, 13, 8, and 11 substances, respectively. Among the mycotoxins, fumifungin, fumiquinazoline A/B and D, fumitremorgin B, gliotoxin, sphingofungins, pseurotins, and verruculogen were only found in A. fumigatus, whereas auranthine was only found in A. lentulus. The amount of gliotoxin, one of the most abundant mycotoxins in A. fumigatus, was negligible in these related species. In addition, they had decreased susceptibility to antifungal agents such as itraconazole and voriconazole, even though metabolites that were shared in the isolates showing higher minimum inhibitory concentrations than epidemiological cutoff values were not detected. These strikingly different secondary metabolite profiles may lead to the development of more discriminative identification protocols for such closely related Aspergillus species as well as improved treatment outcomes. PMID:25737146

  18. Molecular sub-typing suggests that the environment of rehabilitation centers may be a potential source of Aspergillus fumigatus infecting rehabilitating seabirds.

    PubMed

    Burco, Julia D; Etienne, Kizee A; Massey, J Gregory; Ziccardi, Michael H; Balajee, S Arunmozhi

    2012-01-01

    Aspergillosis remains a major cause of infection-related avian mortality in birds that are debilitated and undergoing rehabilitation for release into the wild. This study was designed to understand the source of avian aspergillosis in seabirds undergoing rehabilitation at selected northern California aquatic bird rehabilitation centers. Air, surface and water sampling was performed between August 2007 and July 2008 in three such centers and selected natural seabird loafing sites. Average air Aspergillus fumigatus counts were at least nine times higher in samples obtained from the rehabilitation sites (M = 7.34, SD = 9.78 CFU/m(3)), when compared to those found at natural sites (M = 0.76, SD = 2.24 CFU/m(3)), t (205) = -5.99, P < 0.001. A total of 37 A. fumigatus isolates from birds with confirmed aspergillosis and 42 isolates from environmental samples were identified using both morphological and molecular methods, and subsequently sub-typed using an eight-locus microsatellite panel with the neighbor joining algorithm. Results of the study demonstrated the presence of five clonal groups, 13 genotypically related clusters, and 59 distinct genotypes. Six of the 13 genotypically related clusters contained matching genotypes between clinical isolates and local environmental isolates from the rehabilitation center in which these birds were housed. We present evidence that the environment of rehabilitation centers may be a source for A. fumigatus infection in rehabilitated seabirds. PMID:21756021

  19. Essential gene identification and drug target prioritization in Aspergillus fumigatus.

    PubMed

    Hu, Wenqi; Sillaots, Susan; Lemieux, Sebastien; Davison, John; Kauffman, Sarah; Breton, Anouk; Linteau, Annie; Xin, Chunlin; Bowman, Joel; Becker, Jeff; Jiang, Bo; Roemer, Terry

    2007-03-01

    Aspergillus fumigatus is the most prevalent airborne filamentous fungal pathogen in humans, causing severe and often fatal invasive infections in immunocompromised patients. Currently available antifungal drugs to treat invasive aspergillosis have limited modes of action, and few are safe and effective. To identify and prioritize antifungal drug targets, we have developed a conditional promoter replacement (CPR) strategy using the nitrogen-regulated A. fumigatus NiiA promoter (pNiiA). The gene essentiality for 35 A. fumigatus genes was directly demonstrated by this pNiiA-CPR strategy from a set of 54 genes representing broad biological functions whose orthologs are confirmed to be essential for growth in Candida albicans and Saccharomyces cerevisiae. Extending this approach, we show that the ERG11 gene family (ERG11A and ERG11B) is essential in A. fumigatus despite neither member being essential individually. In addition, we demonstrate the pNiiA-CPR strategy is suitable for in vivo phenotypic analyses, as a number of conditional mutants, including an ERG11 double mutant (erg11BDelta, pNiiA-ERG11A), failed to establish a terminal infection in an immunocompromised mouse model of systemic aspergillosis. Collectively, the pNiiA-CPR strategy enables a rapid and reliable means to directly identify, phenotypically characterize, and facilitate target-based whole cell assays to screen A. fumigatus essential genes for cognate antifungal inhibitors. PMID:17352532

  20. Fatal coinfection with Legionella pneumophila serogroup 8 and Aspergillus fumigatus.

    PubMed

    Guillouzouic, Aurélie; Bemer, Pascale; Gay-Andrieu, Françoise; Bretonnière, Cédric; Lepelletier, Didier; Mahé, Pierre-Joachim; Villers, Daniel; Jarraud, Sophie; Reynaud, Alain; Corvec, Stéphane

    2008-02-01

    Legionella pneumophila is an important cause of community-acquired and nosocomial pneumonia. We report on a patient who simultaneously developed L. pneumophila serogroup 8 pneumonia and Aspergillus fumigatus lung abscesses. Despite appropriate treatments, Aspergillus disease progressed with metastasis. Coinfections caused by L. pneumophila and A. fumigatus remain exceptional. In apparently immunocompetent patients, corticosteroid therapy is a key risk factor for aspergillosis. PMID:17945454

  1. A patient with allergic bronchopulmonary mycosis caused by Aspergillus fumigatus and Candida albicans.

    PubMed

    Wardhana; Datau, E A

    2012-10-01

    Allergic Bronchopulmonary Mycosis (ABPM) is an exagregated immunologic response to fungal colonization in the lower airways. It may cause by many kinds of fungal, but Aspergillus fumigatus is the most common cause of ABPM, although other Aspergillus and other fungal organisms, like Candida albicans, have been implicated. Aspergllus fumigatus and Candida albicans may be found as outdoor and indoor fungi, and cause the sensitization, elicitation of the disease pathology, and its clinical manifestations. Several diagnostic procedurs may be impicated to support the diagnosis of ABPM caused by Aspergillus fumigatus and Candida albicans. A case of allergic bronchopulmonary mycosis caused by Aspergillus fumigatus and Candida albicans in a 48 year old man was discussed. The patient was treated with antifungal, corticosteroids, and antibiotic for the secondary bacterial infection. The patient's condition is improved without any significant side effects. PMID:23314973

  2. Important factors mediates the adhesion of aspergillus fumigatus to alveolar epithelial cells with E-cadherin.

    PubMed

    Xu, Xiao-Yong; Chen, Fei; Sun, He; Chen, Chen; Zhao, Bei-Lei

    2016-01-01

    Aspergillus is widely distributed in the Earth's biosphere. It has strong adaptive capacity, and lives as saprophytic or parasitic life. This study aims to investigate the role of E-cadherin for adhesion of Aspergillus fumigatus blastospores in a human epithelial cell line (A549) and search the correlated molecule in aspergillus. A. fumigatus blastospores were incubated with the total protein of A549 to investigate the binding of E-cadherin and blastospores followed by an affinity purification procedure. After establishing the adhesion model, the adhesion of A. fumigatus blastospores by A549 cells was evaluated by down-regulating E-cadherin of A549 cells with small interfering RNA (siRNA). FVB mice constructed with E-cadherin down-regulation were infected with aspergillus fumigatus. Preliminary exploration of E-cadherin interacting protein on the surface of aspergillus fumigates by immunoprecipitation and mass spectrometry analysis. E-cadherin was adhered to the surface of A. fumigatus blastospore. Adhesion of the blastospores was reduced by blocking or down-regulating E-cadherin in A549 cells. E-cadherin showed limited significance in the process of mice against aspergillus fumigates. Mass spectrometry (MS) analysis indicated the following proteins AFUA_8G07080, AfA24A6.130c, XP_747789 can bind to E-cadherin. In conclusion, E-cadherin is a receptor for adhesion of A. fumigatus blastospores in epithelial cells. This may open a new approach to treat this fungal infection. PMID:27347350

  3. Important factors mediates the adhesion of aspergillus fumigatus to alveolar epithelial cells with E-cadherin

    PubMed Central

    Xu, Xiao-Yong; Chen, Fei; Sun, He; Chen, Chen; Zhao, Bei-Lei

    2016-01-01

    Aspergillus is widely distributed in the Earth’s biosphere. It has strong adaptive capacity, and lives as saprophytic or parasitic life. This study aims to investigate the role of E-cadherin for adhesion of Aspergillus fumigatus blastospores in a human epithelial cell line (A549) and search the correlated molecule in aspergillus. A. fumigatus blastospores were incubated with the total protein of A549 to investigate the binding of E-cadherin and blastospores followed by an affinity purification procedure. After establishing the adhesion model, the adhesion of A. fumigatus blastospores by A549 cells was evaluated by down-regulating E-cadherin of A549 cells with small interfering RNA (siRNA). FVB mice constructed with E-cadherin down-regulation were infected with aspergillus fumigatus. Preliminary exploration of E-cadherin interacting protein on the surface of aspergillus fumigates by immunoprecipitation and mass spectrometry analysis. E-cadherin was adhered to the surface of A. fumigatus blastospore. Adhesion of the blastospores was reduced by blocking or down-regulating E-cadherin in A549 cells. E-cadherin showed limited significance in the process of mice against aspergillus fumigates. Mass spectrometry (MS) analysis indicated the following proteins AFUA_8G07080, AfA24A6.130c, XP_747789 can bind to E-cadherin. In conclusion, E-cadherin is a receptor for adhesion of A. fumigatus blastospores in epithelial cells. This may open a new approach to treat this fungal infection. PMID:27347350

  4. Fungal siderophore metabolism with a focus on Aspergillus fumigatus

    PubMed Central

    2014-01-01

    Covering: up to 2014 Siderophores are chelators synthesized by microbes to sequester iron. This article summarizes the knowledge on the fungal siderophore metabolism with a focus on Aspergillus fumigatus. In recent years, A. fumigatus became a role model for fungal biosynthesis, uptake and degradation of siderophores as well as regulation of siderophore-mediated iron handling and the elucidation of siderophore functions. Siderophore functions comprise uptake, intracellular transport and storage of iron. This proved to be crucial not only for adaptation to iron starvation conditions but also for germination, asexual and sexual propagation, antioxidative defense, mutual interaction, microbial competition as well as virulence in plant and animal hosts. Recent studies also indicate the high potential of siderophores and its biosynthetic pathway to improve diagnosis and therapy of fungal infections. PMID:25140791

  5. Mycotoxins produced by Aspergillus fumigatus isolated from silage.

    PubMed

    Cole, R J; Kirksey, J W; Dorner, J W; Wilson, D M; Johnson, J; Bedell, D; Springer, J P; Chexal, K K; Clardy, J; Cox, R H

    1977-01-01

    Results are presented which show that Aspergillus fumigatus was one of the predominant fungi contaminating moldy silage. Growth of A. fumigatus on silage appeared to depend on a preliminary aerobic fermentation by other natural microflora in silage. The clavine alkaloid, fumigaclavine A, and a new clavine alkaloid designated fumigaclavine C were produced by A. fumigatus. The LD50 of fumigaclavine C was approximately 150 mg/kg oral dose in day-old cockerels. PMID:350117

  6. Degradation of melanin by Aspergillus fumigatus.

    PubMed Central

    Luther, J P; Lipke, H

    1980-01-01

    A strain of Aspergillus fumigatus from composted coffee and garden wastes utilized natural deproteinized insect, banana, hair, octopus, and synthetic tyrosine and dopa melanins as sole sources of carbon. With a sucrose supplement, degradation was essentially complete after 50 days in Czapek medium pH 6.5 at 30 degrees C. The catabolic rate differed for each substrate pigment, as did the molecular weight distribution of products accumulating in the medium. After incubation with L-[U-14C]melanin, over 50% was recovered in a dark fungal pigment, the remainder appearing as cell protein, chitin, lipid, CO2, and polar metabolites. When grown on melanin, the normally pale mycelia darkened with the production of a fungal allomelanin, with infrared spectrum and alkali fusion products differing from those of the substrate pigment. Isotope distribution in amino acids for A. fumigatus grown on labeled melanin supplemented with sucrose suggested separate pools for synthesis of cell proteins and melanoproteins. Deposition of allomelanin increased resistance of conidia, sterigma, and conidiophores to lytic carbohydrases as judged by scanning electron microscopy. Images PMID:6996615

  7. Azole Drug Import into the Pathogenic Fungus Aspergillus fumigatus

    PubMed Central

    Esquivel, Brooke D.; Smith, Adam R.; Zavrel, Martin

    2015-01-01

    The fungal pathogen Aspergillus fumigatus causes serious illness and often death when it invades tissues, especially in immunocompromised individuals. The azole class of drugs is the most commonly prescribed treatment for many fungal infections and acts on the ergosterol biosynthesis pathway. One common mechanism of acquired azole drug resistance in fungi is the prevention of drug accumulation to toxic levels in the cell. While drug efflux is a well-known resistance strategy, reduced azole import would be another strategy to maintain low intracellular azole levels. Recently, azole uptake in Candida albicans and other yeasts was analyzed using [3H]fluconazole. Defective drug import was suggested to be a potential mechanism of drug resistance in several pathogenic fungi, including Cryptococcus neoformans, Candida krusei, and Saccharomyces cerevisiae. We have adapted and developed an assay to measure azole accumulation in A. fumigatus using radioactively labeled azole drugs, based on previous work done with C. albicans. We used this assay to study the differences in azole uptake in A. fumigatus isolates under a variety of drug treatment conditions, with different morphologies and with a select mutant strain with deficiencies in the sterol uptake and biosynthesis pathway. We conclude that azole drugs are specifically selected and imported into the fungal cell by a pH- and ATP-independent facilitated diffusion mechanism, not by passive diffusion. This method of drug transport is likely to be conserved across most fungal species. PMID:25824209

  8. Genomic Islands in Pathogenic Filamentous Fungus Aspergillus fumigatus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present the genome sequences of a new clinical isolate, CEA10, of an important human pathogen, Aspergillus fumigatus, and two closely related, but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of CEA10 with the recently sequen...

  9. Prospective multicenter international surveillance of azole resistance in Aspergillus fumigatus.

    PubMed

    van der Linden, J W M; Arendrup, M C; Warris, A; Lagrou, K; Pelloux, H; Hauser, P M; Chryssanthou, E; Mellado, E; Kidd, S E; Tortorano, A M; Dannaoui, E; Gaustad, P; Baddley, J W; Uekötter, A; Lass-Flörl, C; Klimko, N; Moore, C B; Denning, D W; Pasqualotto, A C; Kibbler, C; Arikan-Akdagli, S; Andes, D; Meletiadis, J; Naumiuk, L; Nucci, M; Melchers, W J G; Verweij, P E

    2015-06-01

    To investigate azole resistance in clinical Aspergillus isolates, we conducted prospective multicenter international surveillance. A total of 3,788 Aspergillus isolates were screened in 22 centers from 19 countries. Azole-resistant A. fumigatus was more frequently found (3.2% prevalence) than previously acknowledged, causing resistant invasive and noninvasive aspergillosis and severely compromising clinical use of azoles. PMID:25988348

  10. Galactosaminogalactan, a New Immunosuppressive Polysaccharide of Aspergillus fumigatus

    PubMed Central

    Simenel, Catherine; Coddeville, Bernadette; van Vliet, Sandra J.; van Kooyk, Yvette; Bozza, Silvia; Moretti, Silvia; Schwarz, Flavio; Trichot, Coline; Aebi, Markus; Delepierre, Muriel; Elbim, Carole; Romani, Luigina; Latgé, Jean-Paul

    2011-01-01

    A new polysaccharide secreted by the human opportunistic fungal pathogen Aspergillus fumigatus has been characterized. Carbohydrate analysis using specific chemical degradations, mass spectrometry, 1H and 13C nuclear magnetic resonance showed that this polysaccharide is a linear heterogeneous galactosaminogalactan composed of α1-4 linked galactose and α1-4 linked N-acetylgalactosamine residues where both monosacharides are randomly distributed and where the percentage of galactose per chain varied from 15 to 60%. This polysaccharide is antigenic and is recognized by a majority of the human population irrespectively of the occurrence of an Aspergillus infection. GalNAc oligosaccharides are an essential epitope of the galactosaminogalactan that explains the universal antibody reaction due to cross reactivity with other antigenic molecules containing GalNAc stretches such as the N-glycans of Campylobacter jejuni. The galactosaminogalactan has no protective effect during Aspergillus infections. Most importantly, the polysaccharide promotes fungal development in immunocompetent mice due to its immunosuppressive activity associated with disminished neutrophil infiltrates. PMID:22102815

  11. Multiple mechanisms contribute to the development of clinically significant azole resistance in Aspergillus fumigatus

    PubMed Central

    Moye-Rowley, W. S.

    2015-01-01

    Infections caused by the filamentous fungus Aspergillus fumigatus are a significant clinical issue and represent the second most-common form of fungal infection. Azole drugs are effective against this pathogen but resistant isolates are being found more frequently. Infections associated with azole resistant A. fumigatus have a significantly increased mortality making understanding drug resistance in this organism a priority. The target of azole drugs is the lanosterol α-14 demethylase enzyme encoded by the cyp51A gene in A. fumigatus. Mutations in cyp51A have been described that give rise to azole resistance and been argued to be the primary, if not sole, contributor to azole resistance. Here, I discuss recent developments that indicate multiple mechanisms, including increased expression of ATP-binding cassette (ABC) transporter proteins, contribute to azole resistance. ABC transporters are well-established determinants of drug resistance in other fungal pathogens and seem likely to play a similar role in A. fumigatus. PMID:25713565

  12. Mediastinitis caused by Aspergillus fumigatus with ruptured aortic pseudoaneurysm in a heart transplant recipient: case study.

    PubMed

    Byl, B; Jacobs, F; Antoine, M; Depierreux, M; Serruys, E; Primo, G; Thys, J P

    1993-01-01

    The case of a heart transplant recipient with a ruptured aortic pseudoaneurysm caused by an Aspergillus fumigatus mediastinitis is reported. Contamination of surgical fields occurring by air seeding during surgery appears to be the most probable source of infection. Subtle infectious signs of the wound and subacute course are remarkable features of this case. PMID:8449757

  13. Metabolism of p-cresol by the fungus Aspergillus fumigatus

    SciTech Connect

    Jones, K.H.; Trudgill, P.W.; Hopper, D.J. )

    1993-04-01

    Cresols are phenolic compounds that are industrial pollutants. Degradation of p-cresol by several species of fungus has been reported. Aspergillus fumigatus ATCC 28282 metabolizes both phenylacetic and homogentisic acids. This study shows that A. fumigatus ATCC 28282 also is capable of growth on p-cresol as its sole carbon source. Two metabolic routes for p-cresol degradation are described in the paper, but the relative contributions of each pathway is not evaluated. 21 refs., 3 figs., 3 tabs.

  14. Fitness Studies of Azole-Resistant Strains of Aspergillus fumigatus.

    PubMed

    Valsecchi, Isabel; Mellado, Emilia; Beau, Rémi; Raj, Shriya; Latgé, Jean-Paul

    2015-12-01

    Isogenic bar-coded strains of Aspergillus fumigatus carrying the G54W or M220K mutation in Cyp51A were constructed. In vitro, the growth and conidiation capacities of the mutants were similar to those of the parental strain. Competition studies in the absence of azoles showed that there was no adverse fitness cost for the azole-resistant A. fumigatus strains in vitro or in vivo compared to the parental strain. PMID:26416854

  15. Fitness Studies of Azole-Resistant Strains of Aspergillus fumigatus

    PubMed Central

    Valsecchi, Isabel; Mellado, Emilia; Beau, Rémi; Raj, Shriya

    2015-01-01

    Isogenic bar-coded strains of Aspergillus fumigatus carrying the G54W or M220K mutation in Cyp51A were constructed. In vitro, the growth and conidiation capacities of the mutants were similar to those of the parental strain. Competition studies in the absence of azoles showed that there was no adverse fitness cost for the azole-resistant A. fumigatus strains in vitro or in vivo compared to the parental strain. PMID:26416854

  16. Aspergillus fumigatus-Related Species in Clinical Practice

    PubMed Central

    Lamoth, Frédéric

    2016-01-01

    Aspergillus fumigatus is the main etiologic agent of invasive aspergillosis (IA). Other Aspergillus species belonging to the section Fumigati (A. fumigatus complex) may occasionally be the cause of IA. These strains are often misidentified, as they cannot be distinguished from A. fumigatus by conventional morphological analysis and sequencing methods. This lack of recognition may have important consequences as these A. fumigatus-related species often display some level of intrinsic resistance to azoles and other antifungal drugs. A. lentulus, A. udagawae, A. viridinutans, and A. thermomutatus (Neosartorya pseudofischeri) have been associated with refractory cases of IA. Microbiologists should be able to suspect the presence of these cryptic species behind a putative A. fumigatus isolate on the basis of some simple characteristics, such as defect in sporulation and/or unusual antifungal susceptibility profile. However, definitive species identification requires specific sequencing analyses of the beta-tubulin or calmodulin genes, which are not available in most laboratories. Multiplex PCR assays or matrix-assisted laser desorption ionization – time-of-flight mass spectrometry (MALDI-TOF MS) gave promising results for rapid and accurate distinction between A. fumigatus and other Aspergillus spp. of the section Fumigati in clinical practice. Improved diagnostic procedures and antifungal susceptibility testing may be helpful for the early detection and management of these particular IA cases. PMID:27242710

  17. Update on antifungal drug resistance mechanisms of Aspergillus fumigatus.

    PubMed

    Chamilos, G; Kontoyiannis, D P

    2005-12-01

    Although the arsenal of agents with anti-Aspergillus activity has expanded over the last decade, mortality due to invasive aspergillosis (IA) remains unacceptably high. Aspergillus fumigatus still accounts for the majority of cases of IA; however less susceptible to antifungals non-fumigatus aspergilli began to emerge. Antifungal drug resistance of Aspergillus might partially account for treatment failures. Recent advances in our understanding of mechanisms of antifungal drug action in Aspergillus, along with the standardization of in vitro susceptibility testing methods, has brought resistance testing to the forefront of clinical mycology. In addition, molecular biology has started to shed light on the mechanisms of resistance of A. fumigatus to azoles and the echinocandins, while genome-based assays show promise for high-throughput screening for genotypic antifungal resistance. Several problems remain, however, in the study of this complex area. Large multicenter clinical studies--point prevalence or longitudinal--to capture the incidence and prevalence of antifungal resistance in A. fumigatus isolates are lacking. Correlation of in vitro susceptibility with clinical outcome and susceptibility breakpoints has not been established. In addition, the issue of cross-resistance between the newer triazoles is of concern. Furthermore, in vitro resistance testing for polyenes and echinocandins is difficult, and their mechanisms of resistance are largely unknown. This review examines challenges in the diagnosis, epidemiology, and mechanisms of antifungal drug resistance in A. fumigatus. PMID:16488654

  18. Human Neutrophils Use Different Mechanisms To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects.

    PubMed

    Gazendam, Roel P; van Hamme, John L; Tool, Anton T J; Hoogenboezem, Mark; van den Berg, J Merlijn; Prins, Jan M; Vitkov, Ljubomir; van de Veerdonk, Frank L; van den Berg, Timo K; Roos, Dirk; Kuijpers, Taco W

    2016-02-01

    Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcγ receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response. PMID:26718340

  19. Genetic control of asexual development in aspergillus fumigatus.

    PubMed

    Alkhayyat, Fahad; Chang Kim, Sun; Yu, Jae-Hyuk

    2015-01-01

    Aspergillus fumigatus is one of the most common fungi found in the environment. It is an opportunistic human pathogen causing invasive pulmonary aspergillosis with a high mortality rate in immunocompromised patients. Conidia, the asexual spores, serve as the main dispersal and infection agent allowing entrance of the fungus into the host through the respiratory tract. Therefore, understanding the asexual developmental process that gives rise to the conidia is of great interest to the scientific community and is currently the focus of an immense load of research being conducted. We have been studying the genetic basis that controls asexual development and gliotoxin biosynthesis in A. fumigatus. In this review, we discuss the genetic regulatory system that dictates conidiation in this important fungus by covering the roles of crucial genetic factors from the upstream heterotrimeric G-protein signaling components to the more specific downstream central activators of the conidiation pathway. In addition, other key asexual regulators including the velvet regulators, the Flb proteins and their associated regulatory factors are discussed. PMID:25596030

  20. Distinct Innate Immune Phagocyte Responses to Aspergillus fumigatus Conidia and Hyphae in Zebrafish Larvae

    PubMed Central

    Knox, Benjamin P.; Deng, Qing; Rood, Mary; Eickhoff, Jens C.

    2014-01-01

    Aspergillus fumigatus is the most common filamentous fungal pathogen of immunocompromised hosts, resulting in invasive aspergillosis (IA) and high mortality rates. Innate immunity is known to be the predominant host defense against A. fumigatus; however, innate phagocyte responses to A. fumigatus in an intact host and their contributions to host survival remain unclear. Here, we describe a larval zebrafish A. fumigatus infection model amenable to real-time imaging of host-fungal interactions in live animals. Following infection with A. fumigatus, innate phagocyte populations exhibit clear preferences for different fungal morphologies: macrophages rapidly phagocytose conidia and form aggregates around hyphae, while the neutrophil response is dependent upon the presence of hyphae. Depletion of macrophages rendered host larvae susceptible to invasive disease. Moreover, a zebrafish model of human leukocyte adhesion deficiency with impaired neutrophil function also resulted in invasive disease and impaired host survival. In contrast, macrophage-deficient but not neutrophil-deficient larvae exhibited attenuated disease following challenge with a less virulent (ΔlaeA) strain of A. fumigatus, which has defects in secondary metabolite production. Taking these results together, we have established a new vertebrate model for studying innate immune responses to A. fumigatus that reveals distinct roles for neutrophils and macrophages in mediating host defense against IA. PMID:24879123

  1. Human Neutrophils Are Primed by Chemoattractant Gradients for Blocking the Growth of Aspergillus fumigatus.

    PubMed

    Jones, Caroline N; Dimisko, Laurie; Forrest, Kevin; Judice, Kevin; Poznansky, Mark C; Markmann, James F; Vyas, Jatin M; Irimia, Daniel

    2016-02-01

    The contribution of human neutrophils to the protection against fungal infections by Aspergillus fumigatus is essential but not fully understood. Whereas healthy people can inhale spores of A. fumigatus without developing disease, neutropenic patients and those receiving immunosuppressive drugs have a higher incidence of invasive fungal infections. To study the role of neutrophils in protection against A. fumigatus infections, we developed an in vitro assay in which the interactions between human neutrophils and A. fumigatus were observed in real time, at single-cell resolution, in precisely controlled conditions. We measured the outcomes of neutrophil-fungus interactions and found that human neutrophils have a limited ability to migrate toward A. fumigatus and block the growth of A. fumigatus conidia (proportion with growth blocked, 69%). The blocking ability of human neutrophils increased to 85.1% when they were stimulated by uniform concentrations of fMLP and was enhanced further, to 99.4%, in the presence of chemoattractant gradients. Neutrophils from patients receiving immunosuppressive treatment after transplantation were less effective against the fungus than those from healthy donors, and broader heterogeneity exists between patients, compared with healthy individuals. Further studies using this microfluidic platform will help understand the relevance of innate immune deficiencies responsible for the higher risk of fungal infections in patients with immunosuppressive disease. PMID:26272935

  2. Chemosensitization prevents tolerance of Aspergillus fumigatus to antimycotic drugs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tolerance of human pathogenic fungi to antifungal drugs is an emerging medical problem. We show how strains of the causative agent of human aspergillosis, Aspergillus fumigatus, tolerant to cell wall-interfering antimycotic drugs become susceptible through chemosensitization by natural compounds. To...

  3. Expression of indoleamine 2,3-dioxygenase in a murine model of Aspergillus fumigatus keratitis

    PubMed Central

    Jiang, Nan; Zhao, Gui-Qiu; Lin, Jing; Hu, Li-Ting; Che, Cheng-Ye; Li, Cui; Wang, Qian; Xu, Qiang; Zhang, Jie; Peng, Xu-Dong

    2016-01-01

    AIM To observe the presence and expression of indoleamine 2,3-dioxygenase (IDO) during the corneal immunity to Aspergillus fumigatus (A. fumigatus) in the murine models. METHODS The murine model of fungal keratitis was established by smearing with colonies of A. fumigatus after scraping central epithelium of cornea and covering with contact lenses in C57BL/6 mice. The mice were randomly divided into control group, sham group and A. fumigatus keratitis group. The cornea was monitored daily using a slit lamp and recorded disease score after infection. Corneal lesion was detected by immunofluorescence staining. IDO mRNA and protein were also detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. RESULTS The disease score and slit lamp photography indicated that disease severity was consistent with corneal inflammation in the murine models, and the disease scores in A. fumigatus keratitis group were obviously higher than those in the sham group. By immunofluorescence staining, IDO was mainly localized in corneal epithelium and stroma in the murine corneal tissues with A. fumigatus keratitis. Compared with the sham group, IDO mRNA expression was significantly enhanced in corneal epithelium infected by A. fumigatus. Furthermore, IDO protein expression detected by Western blot was in accord with transcript levels of IDO mRNA measured by qRT-PCR. IDO protein expression was enhanced after A. fumigatus infection compared with the sham group. CONCLUSION IDO is detected in corneal epithelium and stroma locally, which indicates IDO takes part in the pathogenesis of A. fumigatus keratitis and plays a key role in immune regulation at the early stage. PMID:27162718

  4. JAK/STAT regulation of Aspergillus fumigatus corneal infections and IL-6/23-stimulated neutrophil, IL-17, elastase, and MMP9 activity.

    PubMed

    Taylor, Patricia R; Roy, Sanhita; Meszaros, Evan C; Sun, Yan; Howell, Scott J; Malemud, Charles J; Pearlman, Eric

    2016-07-01

    IL-6 and IL-23 (IL-6/23) induce IL-17A (IL-17) production by a subpopulation of murine and human neutrophils, resulting in autocrine IL-17 activation, enhanced production of reactive oxygen species, and increased fungal killing. As IL-6 and IL-23 receptors trigger JAK1, -3/STAT3 and JAK2/STAT3 phosphorylation, respectively, we examined the role of this pathway in a murine model of fungal keratitis and also examined neutrophil elastase and gelatinase (matrix metalloproteinase 9) activity by IL-6/23-stimulated human neutrophils in vitro. We found that STAT3 phosphorylation of neutrophils in Aspergillus fumigatus-infected corne as was inhibited by the JAK/STAT inhibitor Ruxolitinib, resulting in impaired fungal killing and decreased matrix metalloproteinase 9 activity. In vitro, we showed that fungal killing by IL-6/23-stimulated human peripheral blood neutrophils was impaired by JAK/STAT inhibitors Ruxolitinib and Stattic, and by the retinoic acid receptor-related orphan receptor γt inhibitor SR1001. This was also associated with decreased reactive oxygen species, IL-17A production, and retinoic acid receptor-related orphan receptor γt translocation to the nucleus. We also demonstrate that IL-6/23-activated neutrophils exhibit increased elastase and gelatinase (matrix metalloproteinase 9) activity, which is inhibited by Ruxolitinib and Stattic but not by SR1001. Taken together, these observations indicate that the regulation of activity of IL-17-producing neutrophils by JAK/STAT inhibitors impairs reactive oxygen species production and fungal killing activity but also blocks elastase and gelatinase activity that can cause tissue damage. PMID:27034404

  5. [Aspergillus fumigatus mediastinitis in an immunocompetent pediatric patient after heart surgery].

    PubMed

    Acuña, Mirta; Farfán, Felipe; Cofré, Fernanda; Benadof, Dona

    2016-02-01

    Postsurgical aspergillosis occurs primarily in immunocompetent patients whose main predisposing factor is the loss of skin and mucosal integrity during surgery. Local infection tends to be destructive and refractory to treatment and relapses are common. It is important to consider aspergillosis in the differential diagnosis of slowly progressive and destructive surgical site infections with negative bacterial cultures. We present the case of a child who developed Aspergillus fumigatus mediastinitis months after heart surgery. PMID:26965883

  6. Hide, Keep Quiet, and Keep Low: Properties That Make Aspergillus fumigatus a Successful Lung Pathogen

    PubMed Central

    Escobar, Natalia; Ordonez, Soledad R.; Wösten, Han A. B.; Haas, Pieter-Jan A.; de Cock, Hans; Haagsman, Henk P.

    2016-01-01

    Representatives of the genus Aspergillus are opportunistic fungal pathogens. Their conidia can reach the alveoli by inhalation and can give rise to infections in immunocompromised individuals. Aspergillus fumigatus is the causal agent of invasive aspergillosis in nearly 90% of the cases. It is not yet well-established what makes this fungus more pathogenic than other aspergilli such as A. niger. Here, we show that A. fumigatus and A. niger conidia adhere with similar efficiency to lung epithelial A549 cells but A. fumigatus conidia internalized 17% more efficiently. Conidia of both aspergilli were taken up in phagolysosomes 8 h after the challenge. These organelles only acidified in the case of A. niger, which is probably due to the type of melanin coating of the conidia. Viability of both types of conidia was not affected after uptake in the phagolysosomes. Germination of A. fumigatus and A. niger conidia in the presence of epithelial cells was delayed when compared to conidia in the medium. However, germination of A. niger conidia was still higher than that of A. fumigatus 10 h after exposure to A549 cells. Remarkably, A. fumigatus hyphae grew mainly parallel to the epithelium, while growth direction of A. niger hyphae was predominantly perpendicular to the plane of the cells. Neutrophils reduced germination and hyphal growth of A. niger, but not of A fumigatus, in presence of epithelial cells. Taken together, efficient internalization, delayed germination, and hyphal growth parallel to the epithelium gives a new insight into what could be the causes for the success of A. fumigatus compared to A. niger as an opportunistic pathogen in the lung. PMID:27092115

  7. Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy

    PubMed Central

    Reichhardt, Courtney; Ferreira, Jose A. G.; Joubert, Lydia-Marie; Clemons, Karl V.; Stevens, David A.

    2015-01-01

    Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The 13C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional 15N and 31P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions. PMID:26163318

  8. Isolation and toxigenicity of Aspergillus fumigatus from moldy silage.

    PubMed

    dos Santos, Valentina Melo; Dorner, Joe W; Carreira, Fátima

    2003-01-01

    Thirty-nine silage samples were collected from various silos on Terceira Island in the Azores. Samples were examined for the presence of total fungi, and isolates of Aspergillus fumigatus were analyzed for their ability to produce fumitremorgens B and C, fumigaclavines B and C, and gliotoxin. Thirty-four silage samples (87%) were contaminated with fungi, and A. fumigatus was isolated from 27 samples (69%). Samples that were taken from the surface of silos had significantly higher populations of both total fungi and A. fumigatus than did samples taken from the middle of silos. Analysis of 27 A. fumigatus isolates (one representing each positive sample) showed that 59.3% produced fumitremorgen B; 33.3% produced fumitremorgen C; 29.6% produced fumigaclavine B; 7.4% produced fumigaclavine C; and 11.1% produced gliotoxin. Fifty-two percent of the isolates produced multiple toxins, and 25.9% did not produce any of these toxins. Gliotoxin and fumigaclavine C were always produced in combination with other toxins. Because of the demonstrated potential of these A. fumigatus isolates to produce mycotoxins, it is important to properly construct and manage silos to prevent their contamination with A. fumigatus. PMID:12733634

  9. Aspergillus fumigatus mycovirus causes mild hypervirulent effect on pathogenicity when tested on Galleria mellonella.

    PubMed

    Özkan, Selin; Coutts, Robert H A

    2015-03-01

    Mycoviruses are a specific group of viruses that naturally infect and replicate in fungi. The importance of mycoviruses was revealed after their effects were identified not only in economically important fungi but also in the human pathogenic fungus Aspergillus fumigatus. The latter was shown recently to harbor at least three different types of mycoviruses including a chrysovirus, a partitivirus and an as yet uncharacterized virus. Assessment of virulence in the presence and absence of mycoviruses in A. fumigatus is pivotal to understanding its pathogenicity. Here, we have investigated, for the first time, the effects of mycoviruses on the pathogenicity of A. fumigatus as assessed using larvae of the greater wax moth Galleria mellonella. In order to observe the effects of mycoviruses on pathogenicity, G. mellonella were injected with virus-free and virus-infected isolates of A. fumigatus and post-infection survival times were analyzed along with the fungal burden. Neither chrysovirus nor partitivirus infection affected fungal pathogenicity when survival rates were assessed which, for the chrysovirus, agreed with a previous study on murine pathogenicity. However statistically significant differences were observed in survival rates and fungal burden in the presence of the uncharacterized A78 virus. Here we show, for the first time, the effects of a partitivirus and an uncharacterized A78 virus on the pathogenicity of A. fumigatus. PMID:25626171

  10. Disseminated Trichosporon mycotoxinivorans, Aspergillus fumigatus, and Scedosporium apiospermum Coinfection after Lung and Liver Transplantation in a Cystic Fibrosis Patient

    PubMed Central

    Letscher-Bru, Valérie; Pottecher, Julien; Lannes, Béatrice; Jeung, Mi Young; Degot, Tristan; Santelmo, Nicola; Sabou, Alina Marcela; Herbrecht, Raoul; Kessler, Romain

    2012-01-01

    Trichosporon mycotoxinivorans is a novel pathogen recently found in cystic fibrosis patients. We report the first case of a disseminated fatal infection with T. mycotoxinivorans associated with invasive Aspergillus fumigatus and Scedosporium apiospermum infection after lung and liver transplantation in a cystic fibrosis patient. PMID:23035187

  11. Hypoxia attenuates anti-Aspergillus fumigatus immune responses initiated by human dendritic cells.

    PubMed

    Fliesser, Mirjam; Wallstein, Marion; Kurzai, Oliver; Einsele, Hermann; Löffler, Jürgen

    2016-08-01

    Aspergillus fumigatus is an opportunistic mould that causes invasive pulmonary aspergillosis (IPA), a life-threatening infection in immunocompromised patients. During the course of IPA, localised areas of tissue hypoxia occur. Bacterial infection models revealed that hypoxic microenvironments modulate the function of host immune cells. However, the influence of hypoxia on anti-fungal immunity has been largely unknown. We evaluated the impact of hypoxia on the human anti-A. fumigatus immune response. Human monocyte-derived dendritic cells (DCs) were stimulated in vitro with germ tubes of A. fumigatus under normoxia or hypoxia (1% O2 ), followed by analysis of DC viability, maturation and cytokine release. While DC viability was unaffected, hypoxia attenuated cytokine release from DCs and maturation of DCs upon stimulation with A. fumigatus. These data suggest that hypoxia at the site of A. fumigatus infection inhibits full activation and function of human DCs. Thereby, this study identified hypoxia as a crucial immune-modulating factor in the human anti-fungal immune response that might influence the course and outcome of IPA in immunocompromised patients. PMID:27005862

  12. Activation of NF-κB and respiratory burst following Aspergillus fumigatus stimulation of macrophages.

    PubMed

    Sun, He; Xu, Xiao-yong; Tian, Xiao-li; Shao, Hong-tao; Wu, Xiao-dong; Wang, Quan; Su, Xin; Shi, Yi

    2014-01-01

    Dectin-2, a C-type lectin receptor (CLR), plays an essential role in mediating nuclear factor-kappa B (NF-κB) activation and anti-fungal immunity in response to Candida albicans infection. However, the molecular mechanisms and function of Dectin-2 signaling in response to infection by the pathogenic fungus Aspergillus fumigatus have not been characterized. In order to characterize Dectin-2 signaling in response to A. fumigatus infection, activation of Dectin-2 was analyzed at both transcriptional and translational levels. Spleen tyrosine kinase (Syk) phosphorylation, NF-κB activation and cytokine production downstream of Dectin-2 activation were also investigated. In addition, Dectin-2-Syk function and its ability to mediate reactive oxygen species (ROS) production and elimination of A. fumigatus conidia was examined. We demonstrate that Syk is involved in Dectin-2-induced IκBα (inhibitor of kappa B protein) phosphorylation and NF-κB activation following A. fumigatus stimulation in a time dependent manner. Silencing of Dectin-2 and Syk as well as Syk inhibition blocks NF-κB activation and cytokine secretion. Furthermore, the killing of A. fumigatus conidia and ROS production are significantly affected by Dectin-2 or Syk silencing as well as Syk inhibition. Swelling and germination of the fungus followed by hyphae formation and not the resting and heat-inactivated form of A. fumigatus mediate the activation of Dectin-2 signaling. In conclusion, Syk plays an essential role in IκBα kinase phosphorylation, NF-κB activation, and ROS production mediated by Dectin-2 activation in response to A. fumigatus infection. PMID:23886693

  13. Concurrent sensitization to Aspergillus Fumigatus in tropical pulmonary eosinophilia.

    PubMed

    Chhabra, Sunil K; Dash, Devi Jyoti

    2016-01-01

    Tropical pulmonary eosinophilia (TPE) is characterized by lung tissue and peripheral blood eosinophilia. Serum total IgE is also markedly increased in TPE. However, an association with asthma or other hypersensitivity conditions has not been described. During the diagnostic workup of three patients eventually confirmed to have TPE, hypersensitivity to the fungus, Aspergillus Fumigatus was found. However, there was no evidence of diseases of aspergillus hypersensitivity such as severe asthma with fungal sensitization (SAFS) and allergic bronchopulmonary aspergillosis (ABPA). This association however raises the possibility of a future risk of these potentially serious allergic respiratory manifestations. PMID:27374215

  14. The 18-kilodalton antigen secreted by Aspergillus fumigatus.

    PubMed Central

    Latgé, J P; Moutaouakil, M; Debeaupuis, J P; Bouchara, J P; Haynes, K; Prévost, M C

    1991-01-01

    One of the major antigens secreted in vitro by Aspergillus fumigatus is an 18-kDa basic protein which has been purified by cation-exchange chromatography. It is recognized by sera from aspergilloma patients. It is also the major circulating antigen found in urine of patients with invasive aspergillosis. Our results indicated that this antigen has potential for the diagnosis of both aspergilloma and invasive aspergillosis. Images PMID:1855978

  15. Voriconazole for the treatment of refractory Aspergillus fumigatus keratitis

    PubMed Central

    Mehta, Hitendra B; Garg, Prashant; Kodial, Harish

    2008-01-01

    We report a case of Aspergillus fumigatus keratitis in a 53-year-old, well-controlled diabetic female who did not respond to standard antifungal treatment. She was started on topical natamycin eye drops, but the infiltrate continued to progress. Topical amphotericin B and systemic ketoconazole was added, however, there was no response and the infiltrate increased further. She was then switched to topical and systemic voriconazole. Steady resolution of the infiltrate was noted within 2 weeks of therapy. PMID:18417831

  16. Isolate-Dependent Growth, Virulence, and Cell Wall Composition in the Human Pathogen Aspergillus fumigatus

    PubMed Central

    Amarsaikhan, Nansalmaa; O’Dea, Evan M.; Tsoggerel, Angar; Owegi, Henry; Gillenwater, Jordan; Templeton, Steven P.

    2014-01-01

    The ubiquitous fungal pathogen Aspergillus fumigatus is a mediator of allergic sensitization and invasive disease in susceptible individuals. The significant genetic and phenotypic variability between and among clinical and environmental isolates are important considerations in host-pathogen studies of A. fumigatus-mediated disease. We observed decreased radial growth, rate of germination, and ability to establish colony growth in a single environmental isolate of A. fumigatus, Af5517, when compared to other clinical and environmental isolates. Af5517 also exhibited increased hyphal diameter and cell wall β-glucan and chitin content, with chitin most significantly increased. Morbidity, mortality, lung fungal burden, and tissue pathology were decreased in neutropenic Af5517-infected mice when compared to the clinical isolate Af293. Our results support previous findings that suggest a correlation between in vitro growth rates and in vivo virulence, and we propose that changes in cell wall composition may contribute to this phenotype. PMID:24945802

  17. Bimodular Peptide Synthetase SidE Produces Fumarylalanine in the Human Pathogen Aspergillus fumigatus

    PubMed Central

    Steinchen, Wieland; Lackner, Gerald; Yasmin, Sabiha; Schrettl, Markus; Dahse, Hans-Martin

    2013-01-01

    The filamentous mold Aspergillus fumigatus causes invasive aspergillosis, a potentially life-threatening infectious disease, in humans. The sidE gene encodes a bimodular peptide synthetase and was shown previously to be strongly upregulated during initiation of murine lung infection. In this study, we characterized the two adenylation domains of SidE with the ATP-[32P]pyrophosphate exchange assay in vitro, which identified fumarate and l-alanine, respectively, as the preferred substrates. Using full-length holo-SidE, fumarylalanine (FA) formation was observed in vitro. Furthermore, FA was identified in A. fumigatus culture supernatants under inducing conditions, unless sidE was genetically inactivated. As FA is structurally related to established pharmaceutical products exerting immunomodulatory activity, this work may contribute to our understanding of the virulence of A. fumigatus. PMID:23974138

  18. Volatile Compounds Emitted by Pseudomonas aeruginosa Stimulate Growth of the Fungal Pathogen Aspergillus fumigatus

    PubMed Central

    Briard, Benoit; Heddergott, Christoph

    2016-01-01

    ABSTRACT Chronic lung infections with opportunistic bacterial and fungal pathogens are a major cause of morbidity and mortality especially in patients with cystic fibrosis. Pseudomonas aeruginosa is the most frequently colonizing bacterium in these patients, and it is often found in association with the filamentous fungus Aspergillus fumigatus. P. aeruginosa is known to inhibit the growth of A. fumigatus in situations of direct contact, suggesting the existence of interspecies communication that may influence disease outcome. Our study shows that the lung pathogens P. aeruginosa and A. fumigatus can interact at a distance via volatile-mediated communication and expands our understanding of interspecific signaling in microbial communities. PMID:26980832

  19. Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus

    PubMed Central

    Lambou, Karine; Pennati, Andrea; Valsecchi, Isabel; Tada, Rui; Sherman, Stephen; Sato, Hajime; Beau, Remi

    2013-01-01

    The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD+ to NADH. Analysis of the AfchoAΔ::HPH and AfbadAΔ::HPH single mutants and the AfchoAΔAfbadAΔ::HPH double mutant showed that AfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. AfChoAp and AfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom. PMID:23563483

  20. Inhaled corticosteroids and Aspergillus fumigatus isolation in cystic fibrosis.

    PubMed

    Noni, Maria; Katelari, Anna; Dimopoulos, George; Kourlaba, Georgia; Spoulou, Vana; Alexandrou-Athanassoulis, Helen; Doudounakis, Stavros-Eleftherios; Tzoumaka-Bakoula, Chryssa

    2014-10-01

    Aspergillus fumigatus isolation in cultures from respiratory specimens of patients with cystic fibrosis (CF) is quite common; however, the role of A. fumigatus as a pathogen and whether its presence is associated with progression of pulmonary disease remain unclear. We investigated the association between inhaled corticosteroids and the recovery of A. fumigatus by performing a retrospective cohort study of CF patients born between 1988 and 1996. The patients' medical records from their first visit to the CF Center until December 2010 were reviewed. Outcomes were the occurrence of A. fumigatus first isolation, chronic colonization, or the last visit at the CF Center. A number of possible confounders were included in the multivariate logistic regression analysis in order to identify an independent association between inhaled corticosteroids and colonization status. A total of 121 patients were included in the study. Thirty-nine patients (32.2%) had at least one positive culture and 14 (11.6%) developed chronic colonization. Multivariate logistic regression analysis was used to determine the independent effect of inhaled corticosteroids on the odds of first isolation (odds ratio [OR], 1.165; 95% confidence interval [CI], 1.015-1.337; P = 0.029) and chronic colonization (OR, 1.180; 95% CI, 1.029-1.353; P = 0.018). In conclusion, A. fumigatus first isolation and chronic colonization are associated with the duration of inhaled corticosteroid treatment. PMID:25056962

  1. Extensive proteomic remodeling is induced by eukaryotic translation elongation factor 1Bγ deletion in Aspergillus fumigatus.

    PubMed

    O'Keeffe, Grainne; Jöchl, Christoph; Kavanagh, Kevin; Doyle, Sean

    2013-11-01

    The opportunistic pathogen Aspergillus fumigatus is ubiquitous in the environment and predominantly infects immunocompromised patients. The functions of many genes remain unknown despite sequencing of the fungal genome. A putative translation elongation factor 1Bγ (eEF1Bγ, termed elfA; 750 bp) is expressed, and exhibits glutathione S-transferase activity, in A. fumigatus. Here, we demonstrate the role of ElfA in the oxidative stress response, as well as a possible involvement in translation and actin cytoskeleton organization, respectively. Comparative proteomics, in addition to phenotypic analysis, under basal and oxidative stress conditions, demonstrated a role for A. fumigatus elfA in the oxidative stress response. An elfA-deficient strain (A. fumigatus ΔelfA) was significantly more sensitive to the oxidants H2O2, diamide, and 4,4'-dipyridyl disulfide (DPS) than the wild-type. This was further supported with the identification of differentially expressed proteins of the oxidative stress response, including; mitochondrial peroxiredoxin Prx1, molecular chaperone Hsp70 and mitochondrial glycerol-3-phosphate dehydrogenase. Phenotypic analysis also revealed that A. fumigatus ΔelfA was significantly more tolerant to voriconazole than the wild-type. The differential expression of two aminoacyl-tRNA synthetases suggests a role for A. fumigatus elfA in translation, while the identification of actin-bundling protein Sac6 and vacuolar dynamin-like GTPase VpsA link A. fumigatus elfA to the actin cytoskeleton. Overall, this work highlights the diverse roles of A. fumigatus elfA, with respect to translation, oxidative stress and actin cytoskeleton organization. In addition to this, the strategy of combining targeted gene deletion with comparative proteomics for elucidating the role of proteins of unknown function is further revealed. PMID:24023013

  2. The Aspergillus fumigatus pkcAG579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model

    PubMed Central

    Rocha, Marina Campos; de Godoy, Krissia Franco; de Castro, Patrícia Alves; Hori, Juliana Issa; Bom, Vinícius Leite Pedro; Brown, Neil Andrew; da Cunha, Anderson Ferreira; Goldman, Gustavo Henrique; Malavazi, Iran

    2015-01-01

    Aspergillus fumigatus is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved Cell Wall Integrity (CWI) pathway. In A. fumigatus this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that pkcA is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential A. fumigatus pkcAG579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcAG579R mutation has a reduced activation of the downstream Mitogen-Activated Protein Kinase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcAG579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The pkcAG579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the A. fumigatus cell wall during infection. PMID:26295576

  3. The Aspergillus fumigatus pkcA G579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model.

    PubMed

    Rocha, Marina Campos; Godoy, Krissia Franco de; de Castro, Patrícia Alves; Hori, Juliana Issa; Bom, Vinícius Leite Pedro; Brown, Neil Andrew; Cunha, Anderson Ferreira da; Goldman, Gustavo Henrique; Malavazi, Iran

    2015-01-01

    Aspergillus fumigatus is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved Cell Wall Integrity (CWI) pathway. In A. fumigatus this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that pkcA is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential A. fumigatus pkcAG579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcAG579R mutation has a reduced activation of the downstream Mitogen-Activated Protein Kinase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcAG579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The pkcAG579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the A. fumigatus cell wall during infection. PMID:26295576

  4. Proteomic Profiling of Serological Responses to Aspergillus fumigatus Antigens in Patients with Invasive Aspergillosis.

    PubMed

    Teutschbein, Janka; Simon, Svenja; Lother, Jasmin; Springer, Jan; Hortschansky, Peter; Morton, C Oliver; Löffler, Jürgen; Einsele, Hermann; Conneally, Eibhlin; Rogers, Thomas R; Guthke, Reinhard; Brakhage, Axel A; Kniemeyer, Olaf

    2016-05-01

    Aspergillus fumigatus is the species that most commonly causes the opportunistic infection invasive aspergillosis (IA) in patients being treated for hematological malignancies. Little is known about the A. fumigatus proteins that trigger the production of Aspergillus-specific IgG antibodies during the course of IA. To characterize the serological response to A. fumigatus protein antigens, mycelial proteins were separated by 2-D gel electrophoresis. The gels were immunoblotted with sera from patients with probable and proven IA and control patients without IA. We identified 49 different fungal proteins, which gave a positive IgG antibody signal. Most of these antigens play a role in primary metabolism and stress responses. Overall, our analysis identified 18 novel protein antigens from A. fumigatus. To determine whether these antigens can be used as diagnostic or prognostic markers or exhibit a protective activity, we employed supervised machine learning with decision trees. We identified two candidates for further analysis, the protein antigens CpcB and Shm2. Heterologously produced Shm2 induced a strongly proinflammatory response in human peripheral blood mononuclear cells after in vitro stimulation. In contrast, CpcB did not activate the immune response of PBMCs. These findings could serve as the basis for the development of an immunotherapy of IA. PMID:26974881

  5. Histidine biosynthesis plays a crucial role in metal homeostasis and virulence of Aspergillus fumigatus

    PubMed Central

    Dietl, Anna-Maria; Amich, Jorge; Leal, Sixto; Beckmann, Nicola; Binder, Ulrike; Beilhack, Andreas; Pearlman, Eric; Haas, Hubertus

    2016-01-01

    Abstract Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing invasive fungal infections in immunosuppressed individuals. The histidine biosynthetic pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants, but is absent in mammals. Here we demonstrate that deletion of the gene encoding imidazoleglycerol-phosphate dehydratase (HisB) in A. fumigatus causes (i) histidine auxotrophy, (ii) decreased resistance to both starvation and excess of various heavy metals, including iron, copper and zinc, which play a pivotal role in antimicrobial host defense, (iii) attenuation of pathogenicity in 4 virulence models: murine pulmonary infection, murine systemic infection, murine corneal infection, and wax moth larvae. In agreement with the in vivo importance of histidine biosynthesis, the HisB inhibitor 3-amino-1,2,4-triazole reduced the virulence of the A. fumigatus wild type and histidine supplementation partially rescued virulence of the histidine-auxotrophic mutant in the wax moth model. Taken together, this study reveals limited histidine availability in diverse A. fumigatus host niches, a crucial role for histidine in metal homeostasis, and the histidine biosynthetic pathway as being an attractive target for development of novel antifungal therapy approaches. PMID:26854126

  6. Histidine biosynthesis plays a crucial role in metal homeostasis and virulence of Aspergillus fumigatus.

    PubMed

    Dietl, Anna-Maria; Amich, Jorge; Leal, Sixto; Beckmann, Nicola; Binder, Ulrike; Beilhack, Andreas; Pearlman, Eric; Haas, Hubertus

    2016-05-18

    Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing invasive fungal infections in immunosuppressed individuals. The histidine biosynthetic pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants, but is absent in mammals. Here we demonstrate that deletion of the gene encoding imidazoleglycerol-phosphate dehydratase (HisB) in A. fumigatus causes (i) histidine auxotrophy, (ii) decreased resistance to both starvation and excess of various heavy metals, including iron, copper and zinc, which play a pivotal role in antimicrobial host defense, (iii) attenuation of pathogenicity in 4 virulence models: murine pulmonary infection, murine systemic infection, murine corneal infection, and wax moth larvae. In agreement with the in vivo importance of histidine biosynthesis, the HisB inhibitor 3-amino-1,2,4-triazole reduced the virulence of the A. fumigatus wild type and histidine supplementation partially rescued virulence of the histidine-auxotrophic mutant in the wax moth model. Taken together, this study reveals limited histidine availability in diverse A. fumigatus host niches, a crucial role for histidine in metal homeostasis, and the histidine biosynthetic pathway as being an attractive target for development of novel antifungal therapy approaches. PMID:26854126

  7. Diversity and origins of Indian multi-triazole resistant strains of Aspergillus fumigatus.

    PubMed

    Chang, Howard; Ashu, Eta; Sharma, Cheshta; Kathuria, Shallu; Chowdhary, Anuradha; Xu, Jianping

    2016-07-01

    Aspergillus fumigatus is a widespread opportunistic fungal pathogen causing an alarmingly high mortality rate in immunocompromised patients. Nosocomial infections by drug-resistant A. fumigatus strains are of particular concern, and there is a pressing need to understand the origin, dispersal and long-term evolution of drug resistance in this organism. The objective of this study was to investigate the diversity and putative origins of triazole resistance of A. fumigatus from India. Eighty-nine isolates, including 51 multiple triazole resistant (MTR) isolates and 38 azole-susceptible isolates, were genotyped using multilocus sequence typing (MLST), mating typing and PCR fingerprinting. MLST resolved the 51 MTR isolates into three genotypes, two of which have susceptible counterparts, suggesting that MTR isolates originated multiple times in India. The multiple-origin hypothesis was further supported by the diversity of sequences at the triazole target gene CYP51A among the MTR isolates, and by PCR fingerprints. Interestingly, there is abundant evidence for mating and recombination in natural population of A. fumigatus in India, suggesting that sexual spread of TR34 /L98H, the dominant MTR allele, is possible. Our results call for greater attention to MTR in A. fumigatus and for better management of antifungal drug use. PMID:26931802

  8. A single aspergillus fumigatus intracranial abscess in an immunocompetent patient with parietal lobe tumorectomy

    PubMed Central

    2014-01-01

    Aspergillosis of the central nervous system is a rare fungal infection that is mainly reported in patients with immune deficiency, such as AIDS patients and organ transplant patients treated with immunosuppressive agents, and is uncommon among patients with intact immune function. We report here a rare case of intracranial aspergillosis in a patient who had previously undergone a parietal lobe tumorectomy. Aspergillus fumigatus was confirmed by histopathology, and susceptibility tests reported that this infection should respond to voriconazole. We believe the immunosuppression resulting from surgical trauma and glucocorticosteroid treatment may be contributing to the infection, and therefore management of these two factors may improve the prognosis. PMID:24906819

  9. Interaction of the pathogenic mold Aspergillus fumigatus with lung epithelial cells

    PubMed Central

    Osherov, Nir

    2012-01-01

    Aspergillus fumigatus is an opportunistic environmental mold that can cause severe allergic responses in atopic individuals and poses a life-threatening risk for severely immunocompromised patients. Infection is caused by inhalation of fungal spores (conidia) into the lungs. The initial point of contact between the fungus and the host is a monolayer of lung epithelial cells. Understanding how these cells react to fungal contact is crucial to elucidating the pathobiology of Aspergillus-related disease states. The experimental systems, both in vitro and in vivo, used to study these interactions, are described. Distinction is made between bronchial and alveolar epithelial cells. The experimental findings suggest that lung epithelial cells are more than just “innocent bystanders” or a purely physical barrier against infection. They can be better described as an active extension of our innate immune system, operating as a surveillance mechanism that can specifically identify fungal spores and activate an offensive response to block infection. This response includes the internalization of adherent conidia and the release of cytokines, antimicrobial peptides, and reactive oxygen species. In the case of allergy, lung epithelial cells can dampen an over-reactive immune response by releasing anti-inflammatory compounds such as kinurenine. This review summarizes our current knowledge regarding the interaction of A. fumigatus with lung epithelial cells. A better understanding of the interactions between A. fumigatus and lung epithelial cells has therapeutic implications, as stimulation or inhibition of the epithelial response may alter disease outcome. PMID:23055997

  10. Phagocytosis of Aspergillus fumigatus conidia by primary nasal epithelial cells in vitro

    PubMed Central

    Botterel, Françoise; Gross, Karine; Ibrahim-Granet, Oumaïma; Khoufache, Khaled; Escabasse, Virginie; Coste, André; Cordonnier, Catherine; Escudier, Estelle; Bretagne, Stéphane

    2008-01-01

    Background Invasive aspergillosis, which is mainly caused by the fungus Aspergillus fumigatus, is an increasing problem in immunocompromised patients. Infection occurs by inhalation of airborne conidia, which are first encountered by airway epithelial cells. Internalization of these conidia into the epithelial cells could serve as a portal of entry for this pathogenic fungus. Results We used an in vitro model of primary cultures of human nasal epithelial cells (HNEC) at an air-liquid interface. A. fumigatus conidia were compared to Penicillium chrysogenum conidia, a mould that is rarely responsible for invasive disease. Confocal microscopy, transmission electron microscopy, and anti-LAMP1 antibody labeling studies showed that conidia of both species were phagocytosed and trafficked into a late endosomal-lysosomal compartment as early as 4 h post-infection. In double immunolabeling experiments, the mean percentage of A. fumigatus conidia undergoing phagocytosis 4 h post-infection was 21.8 ± 4.5%. Using combined staining with a fluorescence brightener and propidium iodide, the mean rate of phagocytosis was 18.7 ± 9.3% and the killing rate 16.7 ± 7.5% for A. fumigatus after 8 h. The phagocytosis rate did not differ between the two fungal species for a given primary culture. No germination of the conidia was observed until 20 h of observation. Conclusion HNEC can phagocytose fungal conidia but killing of phagocytosed conidia is low, although the spores do not germinate. This phagocytosis does not seem to be specific to A. fumigatus. Other immune cells or mechanisms are required to kill A. fumigatus conidia and to avoid further invasion. PMID:18564423

  11. Pseudallescheria boydii with Aspergillus fumigatus and Aspergillus terreus in a Critically Ill Hematopoietic Stem Cell Recipient with ARDS.

    PubMed

    Lahmer, Tobias; Messer, Marlena; Ehmer, Ursula; Eser, Stefan; Beitz, Analena; Fekecs, Lisa; Schmid, Roland M; Huber, Wolfgang

    2016-04-01

    Pseudallescheria boydii is a fungal organism known to affect immunocompromised patients. This organism is known to cause, in severe cases, invasive infection of various organs such as the central nervous, cardiovascular, and respiratory systems. We report an unusual case of pulmonary P. boydii pneumonia in an immunocompromised critically ill patient with a co-infection of Aspergillus fumigatus and Aspergillus terreus with ARDS. This case highlights the importance of a high index of suspicion for superimposed fungal infections in patients who are critically ill and immunocompromised. Uncommon fungal pathogens should be considered in the differential diagnosis of respiratory failure, especially if diagnostic markers such as galactomannan (from BAL and serum) or 1,3-beta-D-glucan are elevated. Further diagnostic interventions are warranted when insufficient clinical improvement is observed to prevent treatment failure and adverse outcomes. PMID:26455910

  12. Genomic Islands in the Pathogenic Filamentous Fungus Aspergillus fumigatus

    PubMed Central

    Fedorova, Natalie D.; Khaldi, Nora; Joardar, Vinita S.; Maiti, Rama; Amedeo, Paolo; Anderson, Michael J.; Crabtree, Jonathan; Silva, Joana C.; Badger, Jonathan H.; Albarraq, Ahmed; Angiuoli, Sam; Bussey, Howard; Bowyer, Paul; Cotty, Peter J.; Dyer, Paul S.; Egan, Amy; Galens, Kevin; Fraser-Liggett, Claire M.; Haas, Brian J.; Inman, Jason M.; Kent, Richard; Lemieux, Sebastien; Malavazi, Iran; Orvis, Joshua; Roemer, Terry; Ronning, Catherine M.; Sundaram, Jaideep P.; Sutton, Granger; Turner, Geoff; Venter, J. Craig; White, Owen R.; Whitty, Brett R.; Youngman, Phil; Wolfe, Kenneth H.; Goldman, Gustavo H.; Wortman, Jennifer R.; Jiang, Bo; Denning, David W.; Nierman, William C.

    2008-01-01

    We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated “gene dumps” and, perhaps, simultaneously, as “gene factories”. PMID:18404212

  13. Aspergillus fumigatus Endophthalmitis with Necrotizing Scleritis following Pars Plana Vitrectomy

    PubMed Central

    Allen, Felicity; Graham, Elizabeth M.

    2016-01-01

    We present a case of Aspergillus fumigatus endophthalmitis complicated by necrotizing scleritis in a 68-year-old man with diet-controlled diabetes, after retinal detachment repair. He was initially treated with systemic steroids for surgically induced necrotizing scleritis following routine pars plana vitrectomy. An additional diagnosis of endophthalmitis was made when the patient developed a hypopyon. Repeat vitreous culture isolated Aspergillus fumigatus. Symptoms improved following antifungal treatment leaving the patient with scleromalacia and an advanced postoperative cataract. Fungal scleritis and endophthalmitis are rare complications of intraocular surgery with sight-threatening consequences, and, as this case demonstrates, may even occur concomitantly. The overlapping features of both conditions can make differentiating one from the other difficult. A fungal aetiology should be considered in cases of postoperative scleritis and endophthalmitis that are protracted and refractory to standard therapy. Even in cases of early diagnosis and treatment, visual outcomes in Aspergillus endophthalmitis and scleritis are variable and often disappointing, not infrequently necessitating enucleation of a painful blind eye. PMID:27379189

  14. Aspergillus fumigatus Endophthalmitis with Necrotizing Scleritis following Pars Plana Vitrectomy.

    PubMed

    Gruener, Anna M; Allen, Felicity; Stanford, Miles R; Graham, Elizabeth M

    2016-01-01

    We present a case of Aspergillus fumigatus endophthalmitis complicated by necrotizing scleritis in a 68-year-old man with diet-controlled diabetes, after retinal detachment repair. He was initially treated with systemic steroids for surgically induced necrotizing scleritis following routine pars plana vitrectomy. An additional diagnosis of endophthalmitis was made when the patient developed a hypopyon. Repeat vitreous culture isolated Aspergillus fumigatus. Symptoms improved following antifungal treatment leaving the patient with scleromalacia and an advanced postoperative cataract. Fungal scleritis and endophthalmitis are rare complications of intraocular surgery with sight-threatening consequences, and, as this case demonstrates, may even occur concomitantly. The overlapping features of both conditions can make differentiating one from the other difficult. A fungal aetiology should be considered in cases of postoperative scleritis and endophthalmitis that are protracted and refractory to standard therapy. Even in cases of early diagnosis and treatment, visual outcomes in Aspergillus endophthalmitis and scleritis are variable and often disappointing, not infrequently necessitating enucleation of a painful blind eye. PMID:27379189

  15. Immunoproteome of Aspergillus fumigatus Using Sera of Patients with Invasive Aspergillosis

    PubMed Central

    Virginio, Emylli D.; Kubitschek-Barreira, Paula H.; Batista, Marjorie Vieira; Schirmer, Marcelo R.; Abdelhay, Eliana; Shikanai-Yasuda, Maria A.; Lopes-Bezerra, Leila M.

    2014-01-01

    Invasive aspergillosis is a life-threatening lung or systemic infection caused by the opportunistic mold Aspergillus fumigatus. The disease affects mainly immunocompromised hosts, and patients with hematological malignances or who have been submitted to stem cell transplantation are at high risk. Despite the current use of Platelia™ Aspergillus as a diagnostic test, the early diagnosis of invasive aspergillosis remains a major challenge in improving the prognosis of the disease. In this study, we used an immunoproteomic approach to identify proteins that could be putative candidates for the early diagnosis of invasive aspergillosis. Antigenic proteins expressed in the first steps of A. fumigatus germination occurring in a human host were revealed using 2-D Western immunoblots with the serum of patients who had previously been classified as probable and proven for invasive aspergillosis. Forty antigenic proteins were identified using mass spectrometry (MS/MS). A BLAST analysis revealed that two of these proteins showed low homology with proteins of either the human host or etiological agents of other invasive fungal infections. To our knowledge, this is the first report describing specific antigenic proteins of A. fumigatus germlings that are recognized by sera of patients with confirmed invasive aspergillosis who were from two separate hospital units. PMID:25141105

  16. Oxidative burst and neutrophil elastase contribute to clearance of Aspergillus fumigatus pneumonia in mice.

    PubMed

    Prüfer, Steve; Weber, Michael; Stein, Pamela; Bosmann, Markus; Stassen, Michael; Kreft, Andreas; Schild, Hansjörg; Radsak, Markus P

    2014-02-01

    Polymorphonuclear neutrophils (PMN) are important for the control of invasive aspergillosis (IA), a major threat to immunocompromised individuals. For clearance of Aspergillus fumigatus infections, PMN employ their potent oxidative and non-oxidative mechanisms. To clarify the relative contribution of these mechanisms, we analyzed p47(phox-/-), gp91(phox-/-) and elastase (ELA) deficient mice (ELANE) after intratracheal infection with A. fumigatus. Infected p47(phox-/-) and gp91(phox-/-) mice died within 4 days and had a significant higher fungal burden in the lungs compared to wild-type controls. Interestingly, the survival of ELANE mice after infection was unimpaired suggesting that ELA is not essential here. Nevertheless, A. fumigatus clearance was delayed in ELANE mice indicating a partial contribution of ELA to fungal immunity. Comparing p47(phox-/-), gp91(phox-/-) or ELANE mice for PMN activation and recruitment to the lungs, we were unable to detect significant differences in vitro or in vivo among mutant or wild-type strains suggesting intact PMN functionality of basic effector mechanisms. Fungal killing in vitro by ELA deficient PMN was comparably reduced as in p47(phox-/-) and gp91(phox-/-) deficient PMN corroborating the importance of oxidative and non-oxidative PMN mechanisms for the control of fungal outgrowth. Taken together, this suggests that intact oxidative as well as non-oxidative PMN effector functions are highly relevant for the control of A. fumigatus infections in vitro and in vivo. While ELA contributes to clearance of A. fumigatus, the oxidative functions are essential for survival. PMID:24054721

  17. The Aspergillus fumigatus cell wall integrity signaling pathway: drug target, compensatory pathways, and virulence

    PubMed Central

    Valiante, Vito; Macheleidt, Juliane; Föge, Martin; Brakhage, Axel A.

    2015-01-01

    Aspergillus fumigatus is the most important airborne fungal pathogen, causing severe infections with invasive growth in immunocompromised patients. The fungal cell wall (CW) prevents the cell from lysing and protects the fungus against environmental stress conditions. Because it is absent in humans and because of its essentiality, the fungal CW is a promising target for antifungal drugs. Nowadays, compounds acting on the CW, i.e., echinocandin derivatives, are used to treat A. fumigatus infections. However, studies demonstrating the clinical effectiveness of echinocandins in comparison with antifungals currently recommended for first-line treatment of invasive aspergillosis are still lacking. Therefore, it is important to elucidate CW biosynthesis pathways and their signal transduction cascades, which potentially compensate the inhibition caused by CW- perturbing compounds. Like in other fungi, the central core of the cell wall integrity (CWI) signaling pathway in A. fumigatus is composed of three mitogen activated protein kinases. Deletion of these genes resulted in severely enhanced sensitivity of the mutants against CW-disturbing compounds and in drastic alterations of the fungal morphology. Additionally, several cross-talk interactions between the CWI pathways and other signaling pathways are emerging, raising the question about their role in the CW compensatory mechanisms. In this review we focused on recent advances in understanding the CWI signaling pathway in A. fumigatus and its role during drug stress response and virulence. PMID:25932027

  18. Zinc acquisition: a key aspect in Aspergillus fumigatus virulence.

    PubMed

    Amich, Jorge; Calera, José Antonio

    2014-12-01

    Zinc is an essential micronutrient required for the growth of all microorganisms. To grow in the lungs of a susceptible patient Aspergillus fumigatus must obtain zinc from the surrounding tissues. The concentration of Zn(2+) ions in living tissues is much lower than that required for optimal fungal growth in vitro because most of them are tightly bound to proteins at the physiological pH. However, A. fumigatus has several zinc transporters (ZrfA, ZrfB and ZrfC) that enable it to uptake zinc efficiently under the extreme zinc-limiting conditions provided by a susceptible host. The ZafA transcriptional regulator induces the expression of these transporters and is essential for virulence. ZrfC is required for fungal growth within the host tissues, whereas ZrfA and ZrfB play an accessory role. The zinc-scavenging capacity of ZrfC relies on its unusually long N-terminus. In addition, ZrfC also enables A. fumigatus to overcome the inhibitory effect of calprotectin, which is an antimicrobial Zn/Mn-chelating protein synthesized in high amounts by neutrophils, even in immunosuppressed non-leucopenic animals. In summary, the regulation of zinc homeostasis and zinc acquisition could be promising targets for the discovery and development of a new generation of antifungals for the treatment of invasive pulmonary aspergillosis. PMID:24947168

  19. Interactions of Aspergillus fumigatus Conidia with Airway Epithelial Cells: A Critical Review

    PubMed Central

    Croft, Carys A.; Culibrk, Luka; Moore, Margo M.; Tebbutt, Scott J.

    2016-01-01

    Aspergillus fumigatus is an environmental filamentous fungus that also acts as an opportunistic pathogen able to cause a variety of symptoms, from an allergic response to a life-threatening disseminated fungal infection. The infectious agents are inhaled conidia whose first point of contact is most likely to be an airway epithelial cell (AEC). The interaction between epithelial cells and conidia is multifaceted and complex, and has implications for later steps in pathogenesis. Increasing evidence has demonstrated a key role for the airway epithelium in the response to respiratory pathogens, particularly at early stages of infection; therefore, elucidating the early stages of interaction of conidia with AECs is essential to understand the establishment of infection in cohorts of at-risk patients. Here, we present a comprehensive review of the early interactions between A. fumigatus and AECs, including bronchial and alveolar epithelial cells. We describe mechanisms of adhesion, internalization of conidia by AECs, the immune response of AECs, as well as the role of fungal virulence factors, and patterns of fungal gene expression characteristic of early infection. A clear understanding of the mechanisms involved in the early establishment of infection by A. fumigatus could point to novel targets for therapy and prophylaxis. PMID:27092126

  20. Two α(1-3) Glucan Synthases with Different Functions in Aspergillus fumigatus

    PubMed Central

    Beauvais, A.; Maubon, D.; Park, S.; Morelle, W.; Tanguy, M.; Huerre, M.; Perlin, D. S.; Latgé, J. P.

    2005-01-01

    α(1-3) glucan is a main component of the Aspergillus fumigatus cell wall. In spite of its importance, synthesis of this amorphous polymer has not been investigated to date. Two genes in A. fumigatus, AGS1 and AGS2, are highly homologous to the AGS genes of Schizosaccharomyces pombe, which encode putative α(1-3) glucan synthases. The predicted Ags proteins of A. fumigatus have an estimated molecular mass of 270 kDa. AGS1 and AGS2 were disrupted in A. fumigatus. Both Δags mutants have similar altered hyphal morphologies and reduced conidiation levels. Only Δags1 presented a reduction in the α(1-3) glucan content of the cell wall. These results showed that Ags1p and Ags2p were functionally different. The cellular localization of the two proteins was in agreement with their different functions: Ags1p was localized at the periphery of the cell in connection with the cell wall, whereas Ags2p was intracellularly located. An original experimental model of invasive aspergillosis based on mixed infection and quantitative PCR was developed to analyze the virulence of A. fumigatus mutant and wild-type strains. Using this model, it was shown that the cell wall and morphogenesis defects of Δags1 and Δags2 were not associated with a reduction in virulence in either mutant. This result showed that a 50% reduction in the content of the cell wall α(1-3) glucan does not play a significant role in A. fumigatus pathogenicity. PMID:15746357

  1. Identification of Aspergillus fumigatus and Related Species by Nested PCR Targeting Ribosomal DNA Internal Transcribed Spacer Regions

    PubMed Central

    Zhao, Jun; Kong, Fanrong; Li, Ruoyu; Wang, Xiaohong; Wan, Zhe; Wang, Duanli

    2001-01-01

    Aspergillus fumigatus is the most common species that causes invasive aspergillosis. In order to identify A. fumigatus, partial ribosomal DNA (rDNA) from two to six strains of five different Aspergillus species was sequenced. By comparing sequence data from GenBank, we designed specific primer pairs targeting rDNA internal transcribed spacer (ITS) regions of A. fumigatus. A nested PCR method for identification of other A. fumigatus-related species was established by using the primers. To evaluate the specificities and sensitivities of those primers, 24 isolates of A. fumigatus and variants, 8 isolates of Aspergillus nidulans, 7 isolates of Aspergillus flavus and variants, 8 isolates of Aspergillus terreus, 9 isolates of Aspergillus niger, 1 isolate each of Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus versicolor, Aspergillus wangduanlii, Aspergillus qizutongii, Aspergillus beijingensis, and Exophiala dermatitidis, 4 isolates of Candida, 4 isolates of bacteria, and human DNA were used. The nested PCR method specifically identified the A. fumigatus isolates and closely related species and showed a high degree of sensitivity. Additionally, four A. fumigatus strains that were recently isolated from our clinic were correctly identified by this method. Our results demonstrate that these primers are useful for the identification of A. fumigatus and closely related species in culture and suggest further studies for the identification of Aspergillus fumigatus species in clinical specimens. PMID:11376067

  2. Integrative analysis of the heat shock response in Aspergillus fumigatus

    PubMed Central

    2010-01-01

    Background Aspergillus fumigatus is a thermotolerant human-pathogenic mold and the most common cause of invasive aspergillosis (IA) in immunocompromised patients. Its predominance is based on several factors most of which are still unknown. The thermotolerance of A. fumigatus is one of the traits which have been assigned to pathogenicity. It allows the fungus to grow at temperatures up to and above that of a fevered human host. To elucidate the mechanisms of heat resistance, we analyzed the change of the A. fumigatus proteome during a temperature shift from 30°C to 48°C by 2D-fluorescence difference gel electrophoresis (DIGE). To improve 2D gel image analysis results, protein spot quantitation was optimized by missing value imputation and normalization. Differentially regulated proteins were compared to previously published transcriptome data of A. fumigatus. The study was augmented by bioinformatical analysis of transcription factor binding sites (TFBSs) in the promoter region of genes whose corresponding proteins were differentially regulated upon heat shock. Results 91 differentially regulated protein spots, representing 64 different proteins, were identified by mass spectrometry (MS). They showed a continuous up-, down- or an oscillating regulation. Many of the identified proteins were involved in protein folding (chaperones), oxidative stress response, signal transduction, transcription, translation, carbohydrate and nitrogen metabolism. A correlation between alteration of transcript levels and corresponding proteins was detected for half of the differentially regulated proteins. Interestingly, some previously undescribed putative targets for the heat shock regulator Hsf1 were identified. This provides evidence for Hsf1-dependent regulation of mannitol biosynthesis, translation, cytoskeletal dynamics and cell division in A. fumigatus. Furthermore, computational analysis of promoters revealed putative binding sites for an AP-2alpha-like transcription factor

  3. Heat shock protein 90 (Hsp90): A novel antifungal target against Aspergillus fumigatus.

    PubMed

    Lamoth, Frédéric; Juvvadi, Praveen R; Steinbach, William J

    2016-01-01

    Invasive aspergillosis is a life-threatening and difficult to treat infection in immunosuppressed patients. The efficacy of current anti-Aspergillus therapies, targeting the cell wall or membrane, is limited by toxicity (polyenes), fungistatic activity and some level of basal resistance (echinocandins), or the emergence of acquired resistance (triazoles). The heat shock protein 90 (Hsp90) is a conserved molecular chaperone involved in the rapid development of antifungal resistance in the yeast Candida albicans. Few studies have addressed its role in filamentous fungi such as Aspergillus fumigatus, in which mechanisms of resistance may differ substantially. Hsp90 is at the center of a complex network involving calcineurin, lysine deacetylases (KDAC) and other client proteins, which orchestrate compensatory repair mechanisms of the cell wall in response to the stress induced by antifungals. In A. fumigatus, Hsp90 is a trigger for resistance to high concentrations of caspofungin, known as the paradoxical effect. Disrupting Hsp90 circuitry by different means (Hsp90 inhibitors, KDAC inhibitors and anti-calcineurin drugs) potentiates the antifungal activity of caspofungin, thus representing a promising novel antifungal approach. This review will discuss the specific features of A. fumigatus Hsp90 and the potential for antifungal strategies of invasive aspergillosis targeting this essential chaperone. PMID:25243616

  4. A modified recombineering protocol for the genetic manipulation of gene clusters in Aspergillus fumigatus.

    PubMed

    Alcazar-Fuoli, Laura; Cairns, Timothy; Lopez, Jordi F; Zonja, Bozo; Pérez, Sandra; Barceló, Damià; Igarashi, Yasuhiro; Bowyer, Paul; Bignell, Elaine

    2014-01-01

    Genomic analyses of fungal genome structure have revealed the presence of physically-linked groups of genes, termed gene clusters, where collective functionality of encoded gene products serves a common biosynthetic purpose. In multiple fungal pathogens of humans and plants gene clusters have been shown to encode pathways for biosynthesis of secondary metabolites including metabolites required for pathogenicity. In the major mould pathogen of humans Aspergillus fumigatus, multiple clusters of co-ordinately upregulated genes were identified as having heightened transcript abundances, relative to laboratory cultured equivalents, during the early stages of murine infection. The aim of this study was to develop and optimise a methodology for manipulation of gene cluster architecture, thereby providing the means to assess their relevance to fungal pathogenicity. To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes. By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates. We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge. PMID:25372385

  5. Fungistatic activity of all-trans retinoic acid against Aspergillus fumigatus and Candida albicans

    PubMed Central

    Campione, Elena; Gaziano, Roberta; Marino, Daniele; Orlandi, Augusto

    2016-01-01

    Purpose Fungal infections are a major complication in hematologic and neoplastic patients causing severe morbidity and mortality. Aspergillus fumigatus and Candida albicans are among the most invasive opportunistic pathogens in immunocompromised patients, and classic antifungal drugs are frequently unsuccessful in these patients. Recent reports hypothesize that the antifungal efficacy of all-trans retinoic acid (ATRA) is mainly related to its strong capacity to stimulate monocyte-mediated immunity, but no consideration was given to its potential direct fungistatic activity. Moreover, ATRA offers the opportunity for systemic therapy. Methods and results We investigated the efficacy of ATRA at different concentrations for its antifungal activity against opportunistic A. fumigatus and C. albicans obtained from clinical samples according to standard protocols. A fungistatic activity of ATRA on A. fumigatus and C. albicans at 0.5–1 mM concentration was documented up to 7 days. Conclusion This is the first evidence of a direct and strong fungistatic activity of ATRA against A. fumigatus and C. albicans. The potential adjuvant therapeutic application of ATRA might be useful in the treatment and/or prevention of systemic mycoses in immunocompromised patients. The discovery of a direct fungistatic activity, in association with its reported immunomodulatory properties, makes ATRA an excellent candidate for new combined antifungal strategies for systemic mycoses in immunocompromised and cancer patients. PMID:27199548

  6. The innate immune response to Aspergillus fumigatus at the alveolar surface.

    PubMed

    Margalit, Anatte; Kavanagh, Kevin

    2015-09-01

    Aspergillus fumigatus is an ubiquitous, saprophytic mould that forms and releases airborne conidia which are inhaled by humans on a daily basis. When the immune system is compromised (e.g. immunosuppressive therapy prior to organ transplantation) or there is pre-existing pulmonary malfunction (e.g. asthma, cystic fibrosis, TB lesions), A. fumigatus exploits weaknesses in the host defenses which can result in the development of saphrophytic, allergic or invasive aspergillosis. If not effectively eliminated by the innate immune response, conidia germinate and form invasive hyphae which can penetrate pulmonary tissues. The innate immune response to A. fumigatus is stage-specific and various components of the host's defenses are recruited to challenge the different cellular forms of the pathogen. In immunocompetent hosts, anatomical barriers (e.g. the mucociliary elevator) and professional phagocytes such as alveolar macrophages (AM) and neutrophils prevent the development of aspergillosis by inhibiting the growth of conidia and hyphae. The recognition of inhaled conidia by AM leads to the intracellular degradation of the spores and the secretion of proinflammatory mediators which recruit neutrophils to assist in fungal clearance. During the later stages of infection, dendritic cells activate a protective A. fumigatus-specific adaptive immune response which is driven by Th1 CD4(+) T cells. PMID:25934117

  7. Evolutionary Analysis of Sequence Divergence and Diversity of Duplicate Genes in Aspergillus fumigatus

    PubMed Central

    Yang, Ence; Hulse, Amanda M.; Cai, James J.

    2012-01-01

    Gene duplication as a major source of novel genetic material plays an important role in evolution. In this study, we focus on duplicate genes in Aspergillus fumigatus, a ubiquitous filamentous fungus causing life-threatening human infections. We characterize the extent and evolutionary patterns of the duplicate genes in the genome of A. fumigatus. Our results show that A. fumigatus contains a large amount of duplicate genes with pronounced sequence divergence between two copies, and approximately 10% of them diverge asymmetrically, i.e. two copies of a duplicate gene pair diverge at significantly different rates. We use a Bayesian approach of the McDonald-Kreitman test to infer distributions of selective coefficients γ(=2Nes) and find that (1) the values of γ for two copies of duplicate genes co-vary positively and (2) the average γ for the two copies differs between genes from different gene families. This analysis highlights the usefulness of combining divergence and diversity data in studying the evolution of duplicate genes. Taken together, our results provide further support and refinement to the theories of gene duplication. Through characterizing the duplicate genes in the genome of A. fumigatus, we establish a computational framework, including parameter settings and methods, for comparative study of genetic redundancy and gene duplication between different fungal species. PMID:23225993

  8. The molecular mechanism of azole resistance in Aspergillus fumigatus: from bedside to bench and back.

    PubMed

    Wei, Xiaolei; Zhang, Yuanwei; Lu, Ling

    2015-02-01

    The growing use of immunosuppressive therapies has resulted in a dramatic increased incidence of invasive fungal infections (IFIs) caused by Aspergillus fumigatus, a common pathogen, and is also associated with a high mortality rate. Azoles are the primary guideline-recommended therapy agents for first-line treatment and prevention of IFIs. However, increased azole usage in medicinal and agricultural settings has caused azole-resistant isolates to repeatedly emerge in the environment, resulting in a significant threat to human health. In this review, we present and summarize current research on the resistance mechanisms of azoles in A. fumigatus as well as efficient susceptibility testing methods. Moreover, we analyze and discuss the putative clinical (bedside) indication of these findings from bench work. PMID:25626363

  9. A Nonribosomal Peptide Synthetase-derived Iron(III) Complex from the Pathogenic Fungus Aspergillus fumigatus

    PubMed Central

    Yin, Wen-Bing; Baccile, Joshua A.; Bok, Jin Woo; Chen, Yiming; Keller, Nancy P.; Schroeder, Frank C.

    2013-01-01

    Small molecules (SMs) play central roles as virulence factors of pathogenic fungi and bacteria; however, genomic analyses suggest that the majority of microbial SMs have remained uncharacterized. Based on microarray analysis followed by comparative metabolomics of overexpression/knockout mutants we identified a tryptophan-derived iron(III)-complex, hexadehydroastechrome (HAS), as the major product of the cryptic has non-ribosomal peptide synthetase (NRPS) gene cluster in the human pathogen Aspergillus fumigatus. Activation of the has cluster created a highly virulent A. fumigatus strain that increased mortality of infected mice. Comparative metabolomics of different mutant strains allowed to propose a pathway for HAS biosynthesis and further revealed cross-talk with another NRPS pathway producing the anti-cancer fumitremorgins. PMID:23360537

  10. The contribution of Aspergillus fumigatus stress responses to virulence and antifungal resistance.

    PubMed

    Brown, Neil A; Goldman, Gustavo H

    2016-03-01

    Invasive aspergillosis has emerged as one of the most common life-threatening fungal disease of humans. The emergence of antifungal resistant pathogens represents a current and increasing threat to society. In turn, new strategies to combat fungal infection are urgently required. Fungal adaptations to stresses experienced within the human host are a prerequisite for the survival and virulence strategies of the pathogen. Here, we review the latest information on the signalling pathways in Aspergillus fumigatus that contribute to stress adaptations and virulence, while highlighting their potential as targets for the development of novel combinational antifungal therapies. PMID:26920884

  11. Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion.

    PubMed

    Zarember, Kol A; Sugui, Janyce A; Chang, Yun C; Kwon-Chung, Kyung J; Gallin, John I

    2007-05-15

    Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus. PMID:17475866

  12. Dataset of differentially regulated proteins in HUVECs challenged with wild type and UGM1 mutant Aspergillus fumigatus strains.

    PubMed

    Neves, Gabriela Westerlund Peixoto; Curty, Nathália; Kubitschek-Barreira, Paula Helena; Fontaine, Thierry; Souza, Gustavo Henrique Martins Ferreira; Cunha, Marcel Lyra; Goldman, Gustavo H; Beauvais, Anne; Latgé, Jean-Paul; Lopes-Bezerra, Leila M

    2016-12-01

    Invasive aspergillosis is the primary opportunistic invasive fungal infection described in neutropenic hematologic patients, caused by the angioinvasive pathogen Aspergillus fumigatus. The molecular mechanisms associated with A. fumigatus infection in the vascular endothelium are poorly understood. In this context, we used a high-throughput proteomic approach to unveil the proteins modulated in HUVECs after interaction with a wild type strain and the UGM1 mutant (Δugm1) of A. fumigatus. The proteomic analysis was also performed in HUVECs challenged with a galactosaminogalactan (GAG) purified from A. fumigatus cell wall. The dataset presented here correspond to all proteins identified that fit a 2-fold change criteria (log 2 ratio ≥ 1 or ≤ -1), disregarding the statistical validation cut off, in order to supplement the research article entitled "Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates the HUVEC proteins associated with inflammatory and stress responses" (G.W.P. Neves, N.A. Curty, P.H. Kubitschek-Barreira, T. Fontaine, G.H.M.F. Souza, M. Lyra Cunha, G.H. Goldman, A. Beauvais, J.P. Latgé, L.M. Lopes-Bezerra, 2016) [1]. The mass spectrometry proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002823. PMID:27622208

  13. Automated Image Analysis of the Host-Pathogen Interaction between Phagocytes and Aspergillus fumigatus

    PubMed Central

    Guthke, Reinhard; Brakhage, Axel A.; Figge, Marc Thilo

    2011-01-01

    Aspergillus fumigatus is a ubiquitous airborne fungus and opportunistic human pathogen. In immunocompromised hosts, the fungus can cause life-threatening diseases like invasive pulmonary aspergillosis. Since the incidence of fungal systemic infections drastically increased over the last years, it is a major goal to investigate the pathobiology of A. fumigatus and in particular the interactions of A. fumigatus conidia with immune cells. Many of these studies include the activity of immune effector cells, in particular of macrophages, when they are confronted with conidia of A. fumigus wild-type and mutant strains. Here, we report the development of an automated analysis of confocal laser scanning microscopy images from macrophages coincubated with different A. fumigatus strains. At present, microscopy images are often analysed manually, including cell counting and determination of interrelations between cells, which is very time consuming and error-prone. Automation of this process overcomes these disadvantages and standardises the analysis, which is a prerequisite for further systems biological studies including mathematical modeling of the infection process. For this purpose, the cells in our experimental setup were differentially stained and monitored by confocal laser scanning microscopy. To perform the image analysis in an automatic fashion, we developed a ruleset that is generally applicable to phagocytosis assays and in the present case was processed by the software Definiens Developer XD. As a result of a complete image analysis we obtained features such as size, shape, number of cells and cell-cell contacts. The analysis reported here, reveals that different mutants of A. fumigatus have a major influence on the ability of macrophages to adhere and to phagocytose the respective conidia. In particular, we observe that the phagocytosis ratio and the aggregation behaviour of pksP mutant compared to wild-type conidia are both significantly increased. PMID

  14. Aspergillus fumigatus Invasion Increases with Progressive Airway Ischemia

    PubMed Central

    Hsu, Joe L.; Khan, Mohammad A.; Sobel, Raymond A.; Jiang, Xinguo; Clemons, Karl V.; Nguyen, Tom T.; Stevens, David A.; Martinez, Marife; Nicolls, Mark R.

    2013-01-01

    Despite the prevalence of Aspergillus-related disease in immune suppressed lung transplant patients, little is known of the host-pathogen interaction. Because of the mould’s angiotropic nature and because of its capacity to thrive in hypoxic conditions, we hypothesized that the degree of Aspergillus invasion would increase with progressive rejection-mediated ischemia of the allograft. To study this relationship, we utilized a novel orthotopic tracheal transplant model of Aspergillus infection, in which it was possible to assess the effects of tissue hypoxia and ischemia on airway infectivity. Laser Doppler flowmetry and FITC-lectin were used to determine blood perfusion, and a fiber optic microsensor was used to measure airway tissue oxygen tension. Fungal burden and depth of invasion were graded using histopathology. We demonstrated a high efficacy (80%) for producing a localized fungal tracheal infection with the majority of infection occurring at the donor-recipient anastomosis; Aspergillus was more invasive in allogeneic compared to syngeneic groups. During the study period, the overall kinetics of both non-infected and infected allografts was similar, demonstrating a progressive loss of perfusion and oxygenation, which reached a nadir by days 10-12 post-transplantation. The extent of Aspergillus invasion directly correlated with the degree of graft hypoxia and ischemia. Compared to the midtrachea, the donor-recipient anastomotic site exhibited lower perfusion and more invasive disease; a finding consistent with clinical experience. For the first time, we identify ischemia as a putative risk factor for Aspergillus invasion. Therapeutic approaches focused on preserving vascular health may play an important role in limiting Aspergillus infections. PMID:24155924

  15. Genetic relatedness versus biological compatibility between Aspergillus fumigatus and related species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus section Fumigati contains twelve clinically relevant species. Among them, A. fumigatus is the most frequent agent of invasive aspergillosis followed by A. lentulus and A. viridinutans. Genealogical concordance and mating experiments were performed to examine the relationship between phyl...

  16. Phenotypic and genotypic analysis of variability in Aspergillus fumigatus.

    PubMed Central

    Rinyu, E; Varga, J; Ferenczy, L

    1995-01-01

    Sixty-one isolates and collection strains of Aspergillus fumigatus were compared for their phenotypic (morphological features and isoenzyme profiles) and genotypic (restriction enzyme-generated mitochondrial DNA and ribosomal DNA profiles and random amplified polymorphic DNA patterns) features. The examined strains exhibited highly variable colony morphologies and growth rates at different temperatures, but their micromorphologies and conidial diameters were characteristic of the species. Of the isoenzymes studied, the beta-arylesterase and phosphatase patterns were the most divergent, and the 61 strains could be classified into seven groups. The glucose 6-phosphate dehydrogenase and catalase isoenzyme patterns displayed only a limited variability, while the profiles of superoxide dismutase, lactate dehydrogenase, and glutamate dehydrogenase were highly conserved. The HaeIII-generated mitochondrial DNA patterns and SmaI-digested repetitive DNA and ribosomal DNA hybridization patterns of almost all strains were also invariable. The level of variation was much higher when random amplified polymorphic DNA analysis was applied. Although the patterns of the strains were very similar with most of the primers, the application of some primers made it possible to cluster the A. fumigatus isolates into several groups. The results indicate that the random amplified polymorphic DNA technique could be used more efficiently than isoenzyme analysis for typing A. fumigatus isolates. A good correlation was found between the dendrograms obtained from the isoenzyme and random amplified polymorphic DNA data, but the isoenzyme and amplified DNA patterns did not correlate with the pathogenicity, pigment production, or geographical origin of the strains.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8567884

  17. Accumulation of ergot alkaloids during conidiophore development in Aspergillus fumigatus.

    PubMed

    Mulinti, Prashanthi; Allen, Natalie A; Coyle, Christine M; Gravelat, Fabrice N; Sheppard, Donald C; Panaccione, Daniel G

    2014-01-01

    Production of ergot alkaloids in the opportunistic fungal pathogen Aspergillus fumigatus is restricted to conidiating cultures. These cultures typically accumulate several pathway intermediates at concentrations comparable to that of the pathway end product. We investigated the contribution of different cell types that constitute the multicellular conidiophore of A. fumigatus to the production of ergot alkaloid pathway intermediates versus the pathway end product, fumigaclavine C. A relatively minor share (11 %) of the ergot alkaloid yield on a molar basis was secreted into the medium, whereas the remainder was associated with the conidiating colonies. Entire conidiating cultures (containing hyphae, vesicle of conidiophore, phialides of conidiophore, and conidia) accumulated higher levels of the pathway intermediate festuclavine and lower levels of the pathway end product fumigaclavine C than did isolated, abscised conidia, indicating that conidiophores and/or hyphae have a quantitatively different ergot alkaloid profile compared to that of conidia. Differences in alkaloid accumulation among cell types also were indicated by studies with conidiophore development mutants. A ∆medA mutant, in which conidiophores are numerous but develop poorly, accumulated higher levels of pathway intermediates than did the wildtype or a complemented ∆medA mutant. A ∆stuA mutant, which grows mainly as hyphae and produces very few, abnormal conidiophores, produced no detectable ergot alkaloids. The data indicated heterogeneous spatial distribution of ergot alkaloid pathway intermediates versus pathway end product in conidiating cultures of A. fumigatus. This skewed distribution may reflect differences in abundance or activity of pathway enzymes among cell types of those conidiating cultures. PMID:23925951

  18. IgG antibodies to Aspergillus fumigatus in cystic fibrosis: a laboratory correlate of disease activity.

    PubMed Central

    Forsyth, K D; Hohmann, A W; Martin, A J; Bradley, J

    1988-01-01

    Serum was collected from 50 patients with cystic fibrosis, and IgG antibodies to Aspergillus fumigatus were measured by enzyme linked immunosorbent assay (ELISA). In addition, total IgE and Aspergillus specific IgE antibodies were measured in 41 of the 50. A close association was found between pulmonary function and clinical state, and IgG antibodies to Aspergillus. There was no association between pulmonary function or clinical state and IgE antibodies. It is postulated that in patients with cystic fibrosis, Aspergillus fumigatus may contribute to deterioration in pulmonary function by local pathogenicity, or by hypersensitivity mechanisms mediated by IgG. PMID:3046514

  19. Thorium biosorption by Aspergillus fumigatus, a filamentous fungal biomass.

    PubMed

    Bhainsa, Kuber C; D'Souza, Stanislaus F

    2009-06-15

    Thorium biosorption by Aspergillus fumigatus was carried out in a batch reactor to study the effect of initial pH and metal ion concentration, contact time, biomass dose and kinetics and equilibrium Th uptake. Thorium(IV) uptake by A. fumigatus was pH dependent (pH range, 2.0-6.0) and maximum sorption was observed at pH 4.0. The uptake was rapid and the biosorption process reached equilibrium within 2h of contact times at pH 2-4 and initial Th concentration of 50 and 100mg/L. The kinetics data fitted well to Lagergren's pseudo-second-order rate equation (r(2)>0.99). A maximum initial sorption rate of 71.94 (mg/g min) and second-order rate constant of 7.82 x 10(-2) (g/mg min) were observed at pH 4.0, 50 mg Th/L. The observed maximum uptake of thorium was 370 mg Th/g at equilibrium. Biosorption process could be well described by Langmuir isotherm in comparison to Freundlich and Temkin isotherms. Sodium bicarbonate was the most efficient desorbing reagent with desorption efficiency of more than 99%. Environmental scanning electron micrograph (ESEM) showed that the surface of the biomass after desorption was intact. PMID:19036508

  20. Chemical and immunological analysis of the Aspergillus fumigatus cell wall.

    PubMed

    Hearn, V M; Sietsma, J H

    1994-04-01

    Hyphal-wall preparations of Aspergillus fumigatus have been analysed by sequential treatment with KOH, nitrous acid and again with KOH. By acidification of the alkali-soluble extract, a polyglucose was precipitated which showed an X-ray diffraction pattern similar to that of (1-->3)-alpha-glucan. The remainder of the alkali-soluble fraction was precipitated with ethanol; it contained all the mannose, galactose and protein of the wall and, in addition, 6.2% of the amino sugars. This wall-associated glycoprotein, following SDS-PAGE and immunoblotting, reacted with antisera raised against several mycelial extracts of A. fumigatus. Sera from patients with aspergilloma have antibodies which recognize components of this glycoprotein. The glycoprotein nature of these antigens was shown by their ability to bind Lens culinaris lectin. In addition, the antigen/antibody binding could be disrupted by exposure of antigen to periodate oxidation, hydrolysis with dilute acid or pretreatment with a large excess of an exo-beta-D-galactofuranosidase. The alkali-insoluble fraction consisted of a covalently linked glucan-chitin complex. Nitrous acid treatment, which specifically disrupts glycosidic linkages involving glucosamine, did not solubilize much material but changed the X-ray diffraction pattern from diffuse to a pattern showing the characteristic lines of crystalline (1-->3)-beta-glucan and chitin. Most of the glucan became alkali-soluble after this treatment, and the insoluble residue appeared to contain crystalline chitin. PMID:8012598

  1. Bioconversion of tea polyphenols to bioactive theabrownins by Aspergillus fumigatus.

    PubMed

    Wang, Qiuping; Gong, Jiashun; Chisti, Yusuf; Sirisansaneeyakul, Sarote

    2014-12-01

    Theabrownins (TB) are water-soluble phenolic compounds associated with the various health benefits of Pu-erh tea, a post-fermented Chinese dark tea. This work reports on the production of theabrownins from infusions of sun-dried green tea leaves using a pure culture of Aspergillus fumigatus isolated from a solid-state Pu-erh tea fermentation. A theabrownins yield of 158 g kg(-1) sun-dried green tea leaves was obtained in 6 days at 45 °C in an aerobic fermentation. In a 2 l fermenter, the yield of theabrownins was 151 g kg(-1) sun-dried green tea leaves in 48 h of aerobic culture (45 °C, 1 vvm aeration rate, 250 rpm agitation speed). Extracellular polyphenol oxidase and peroxidase of A. fumigatus contributed to this bioconversion. Repeated batch fermentation process was used for producing theabrownins but was less productive than the batch process. PMID:25214210

  2. Testing the efficacy of RNA interference constructs in Aspergillus fumigatus.

    PubMed

    Henry, Christine; Mouyna, Isabelle; Latgé, Jean-Paul

    2007-04-01

    We recently developed a silencing vector in Aspergillus fumigatus which carries a hygromycin resistance marker and a transcriptional unit for hairpin RNA expression under the control of the inducible glucoamylase promoter (pGla) (Mouyna et al. in FEMS Microbiol Lett 237:317-324, 2004). We showed previously that this vector can be used for the RNA interference application of two genes ALB1 and FKS1 of which reduced mRNA levels occurred for both, with phenotypic consequences resembling disruptions of genes involved in melanin (ALB1) and beta(1-3)glucan biosynthesis (FKS1). We reported here the silencing of KRE6 and CRH1, two other genes putatively involved in cell wall biosynthesis using a similar construction under the control of the constitutive promoter glyceraldehyde-3-phosphate dehydrogenase (pgpdA). Silencing of the expression of these two genes was obtained. Further analysis of the transformants showed however that (1) a 100% loss of expression was never achieved for all genes tested (2) the vector used for RNAi is lost or modified over successive transfers resulting in an inhibition of the silencing. These disadvantages of RNAi indicate that classical gene disruption by gene replacement remains the most efficient method for a molecular analysis of gene function in A. fumigatus. PMID:17273823

  3. In Vitro Biochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus

    PubMed Central

    Warrilow, Andrew G. S.; Parker, Josie E.; Price, Claire L.; Nes, W. David

    2015-01-01

    The incidence of triazole-resistant Aspergillus infections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol, purified recombinant Aspergillus fumigatus CPR1 (AfCPR1), and Escherichia coli membrane suspensions containing recombinant A. fumigatus CYP51 proteins, allowing in vitro screening of azole antifungals. Azole-CYP51 studies determining the 50% inhibitory concentration (IC50) showed that A. fumigatus CYP51B (Af51B IC50, 0.50 μM) was 34-fold more susceptible to inhibition by fluconazole than A. fumigatus CYP51A (Af51A IC50, 17 μM) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC50s of 0.16 to 0.38 μM). Af51A-G54W and Af51A-M220K enzymes were 11- and 15-fold less susceptible to inhibition by itraconazole and 30- and 8-fold less susceptible to inhibition by posaconazole than wild-type Af51A, confirming the azole-resistant phenotype of these two Af51A mutations. Susceptibility to voriconazole of Af51A-G54W and Af51A-M220K was only marginally lower than that of wild-type Af51A. Susceptibility of Af51A-L98H to inhibition by voriconazole, itraconazole, and posaconazole was only marginally lower (less than 2-fold) than that of wild-type Af51A. However, Af51A-L98H retained 5 to 8% residual activity in the presence of 32 μM triazole, which could confer azole resistance in A. fumigatus strains that harbor the Af51A-L98H mutation. The AfCPR1/Af51 assay system demonstrated the biochemical basis for the increased azole resistance of A. fumigatus strains harboring G54W, L98H, and M220K Af51A point mutations. PMID:26459890

  4. In Vitro Biochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus.

    PubMed

    Warrilow, Andrew G S; Parker, Josie E; Price, Claire L; Nes, W David; Kelly, Steven L; Kelly, Diane E

    2015-12-01

    The incidence of triazole-resistant Aspergillus infections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol, purified recombinant Aspergillus fumigatus CPR1 (AfCPR1), and Escherichia coli membrane suspensions containing recombinant A. fumigatus CYP51 proteins, allowing in vitro screening of azole antifungals. Azole-CYP51 studies determining the 50% inhibitory concentration (IC50) showed that A. fumigatus CYP51B (Af51B IC50, 0.50 μM) was 34-fold more susceptible to inhibition by fluconazole than A. fumigatus CYP51A (Af51A IC50, 17 μM) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC50s of 0.16 to 0.38 μM). Af51A-G54W and Af51A-M220K enzymes were 11- and 15-fold less susceptible to inhibition by itraconazole and 30- and 8-fold less susceptible to inhibition by posaconazole than wild-type Af51A, confirming the azole-resistant phenotype of these two Af51A mutations. Susceptibility to voriconazole of Af51A-G54W and Af51A-M220K was only marginally lower than that of wild-type Af51A. Susceptibility of Af51A-L98H to inhibition by voriconazole, itraconazole, and posaconazole was only marginally lower (less than 2-fold) than that of wild-type Af51A. However, Af51A-L98H retained 5 to 8% residual activity in the presence of 32 μM triazole, which could confer azole resistance in A. fumigatus strains that harbor the Af51A-L98H mutation. The AfCPR1/Af51 assay system demonstrated the biochemical basis for the increased azole resistance of A. fumigatus strains harboring G54W, L98H, and M220K Af51A point mutations. PMID:26459890

  5. Calcineurin Orchestrates Hyphal Growth, Septation, Drug Resistance and Pathogenesis of Aspergillus fumigatus: Where Do We Go from Here?

    PubMed

    Juvvadi, Praveen R; Steinbach, William J

    2015-01-01

    Studies on fungal pathogens belonging to the ascomycota phylum are critical given the ubiquity and frequency with which these fungi cause infections in humans. Among these species, Aspergillus fumigatus causes invasive aspergillosis, a leading cause of death in immunocompromised patients. Fundamental to A. fumigatus pathogenesis is hyphal growth. However, the precise mechanisms underlying hyphal growth and virulence are poorly understood. Over the past 10 years, our research towards the identification of molecular targets responsible for hyphal growth, drug resistance and virulence led to the elucidation of calcineurin as a key signaling molecule governing these processes. In this review, we summarize our salient findings on the significance of calcineurin for hyphal growth and septation in A. fumigatus and propose future perspectives on exploiting this pathway for designing new fungal-specific therapeutics. PMID:26694470

  6. [Azole resistance in Aspergillus fumigatus in the Netherlands--increase due to environmental fungicides?].

    PubMed

    Verweij, Paul E; van de Sande-Bruisma, Nienke; Kema, Gert H J; Melchers, Willem J G

    2012-01-01

    The mould Aspergillus fumigatus may develop mechanisms that confer resistance to itraconazole, voriconazole and posaconazole. In the Netherlands a dominant resistance mechanism referred to as TR/L98H is found. In A. fumigatus isolates recovered from clinical samples in Dutch hospitals the prevalence of azole resistance varied between 0.8% and 9.4%. The TR/L98H resistance mechanism probably develops in our environment, as azoles are frequently used for crop protection and material preservation. It is likely that breathing in the resistant spores of these strains from the environment leads to clinical infection. More research is needed to understand the environmental route of resistance development and to enable effective measures to prevent this occurring. Azole resistance is associated with treatment failure. Of 8 patients with azole-resistant invasive aspergillosis 7 died within 12 weeks of diagnosis. Alternative treatment regimens might include lipid-formulation of amphotericin B or a combination of voriconazole and an echinocandin, but there is little data available to support these choices. Physicians who treat patients with Aspergillus diseases should be aware of the possibility of azole resistance, also in azole-naïve patients. PMID:22748367

  7. Recent advances in the understanding of the Aspergillus fumigatus cell wall.

    PubMed

    Lee, Mark J; Sheppard, Donald C

    2016-03-01

    Over the past several decades, research on the synthesis and organization of the cell wall polysaccharides of Aspergillus fumigatus has expanded our knowledge of this important fungal structure. Besides protecting the fungus from environmental stresses and maintaining structural integrity of the organism, the cell wall is also the primary site for interaction with host tissues during infection. Cell wall polysaccharides are important ligands for the recognition of fungi by the innate immune system and they can mediate potent immunomodulatory effects. The synthesis of cell wall polysaccharides is a complicated process that requires coordinated regulation of many biosynthetic and metabolic pathways. Continuous synthesis and remodeling of the polysaccharides of the cell wall is essential for the survival of the fungus during development, reproduction, colonization and invasion. As these polysaccharides are absent from the human host, these biosynthetic pathways are attractive targets for antifungal development. In this review, we present recent advances in our understanding of Aspergillus fumigatus cell wall polysaccharides, including the emerging role of cell wall polysaccharides in the host-pathogen interaction. PMID:26920883

  8. RbdB, a Rhomboid Protease Critical for SREBP Activation and Virulence in Aspergillus fumigatus

    PubMed Central

    Dhingra, Sourabh; Kowlaski, Caitlin H.; Thammahong, Arsa; Beattie, Sarah R.; Bultman, Katherine M.

    2016-01-01

    ABSTRACT SREBP transcription factors play a critical role in fungal virulence; however, the mechanisms of sterol regulatory element binding protein (SREBP) activation in pathogenic fungi remains ill-defined. Screening of the Neurospora crassa whole-genome deletion collection for genes involved in hypoxia responses identified a gene for an uncharacterized rhomboid protease homolog, rbdB, required for growth under hypoxic conditions. Loss of rbdB in Aspergillus fumigatus also inhibited growth under hypoxic conditions. In addition, the A. fumigatus ΔrbdB strain also displayed phenotypes consistent with defective SREBP activity, including increased azole drug susceptibility, reduced siderophore production, and full loss of virulence. Expression of the basic helix-loop-helix (bHLH) DNA binding domain of the SREBP SrbA in ΔrbdB restored all of the phenotypes linking RdbB activity with SrbA function. Furthermore, the N-terminal domain of SrbA containing the bHLH DNA binding region was absent from ΔrbdB under inducing conditions, suggesting that RbdB regulates the protein levels of this important transcription factor. As SrbA controls clinically relevant aspects of fungal pathobiology in A. fumigatus, understanding the mechanisms of SrbA activation provides opportunities to target this pathway for therapeutic development. IMPORTANCE Aspergillus fumigatus causes life-threatening infections, and treatment options remain limited. Thus, there is an urgent need to find new therapeutic targets to treat this deadly disease. Previously, we have shown that SREBP transcription factors and their regulatory components are critical for the pathobiology of A. fumigatus. Here we identify a role for RbdB, a rhomboid protease, as an essential component of SREBP activity. Our results indicate that mutants lacking rbdB have growth defects under hypoxic conditions, are hypersusceptible to voriconazole, lack extracellular siderophore production, and fail to cause disease in a murine

  9. AbaA and WetA govern distinct stages of Aspergillus fumigatus development.

    PubMed

    Tao, Li; Yu, Jae-Hyuk

    2011-02-01

    The opportunistic human pathogen Aspergillus fumigatus produces a massive number of asexual spores (conidia) as the primary means of dispersal, survival, genome protection and infection of hosts. In this report, we investigate the functions of two developmental regulators, AfuAbaA and AfuWetA, in A. fumigatus. The AfuabaA gene is predicted to encode an ATTS/TEA DNA-binding domain protein and is activated by AfuBrlA during the middle stage of A. fumigatus asexual development (conidiation). The deletion of AfuabaA results in the formation of aberrant conidiophores exhibiting reiterated cylinder-like terminal cells lacking spores. Furthermore, the absence of AfuabaA causes delayed autolysis and cell death, whereas the overexpression of AfuabaA accelerates these processes, indicating an additional role for AfuAbaA. The AfuwetA gene is sequentially activated by AfuAbaA in the late phase of conidiation. The deletion of AfuwetA causes the formation of defective spore walls and a lack of trehalose biogenesis, leading to a rapid loss of spore viability and reduced tolerance to various stresses. This is the first report to demonstrate that WetA is essential for trehalose biogenesis in conidia. Moreover, the absence of AfuwetA causes delayed germ-tube formation and reduced hyphal branching, suggesting a role of AfuWetA in the early phase of fungal growth. A genetic model depicting the regulation of conidiation in A. fumigatus is proposed. PMID:20966095

  10. Identification of Aspergillus fumigatus multidrug transporter genes and their potential involvement in antifungal resistance.

    PubMed

    Meneau, Isabelle; Coste, Alix T; Sanglard, Dominique

    2016-08-01

    Aspergillus fumigatus can cause severe fatal invasive aspergillosis in immunocompromised patients but is also found in the environment. A. fumigatus infections can be treated with antifungals agents among which azole and echinocandins. Resistance to the class of azoles has been reported not only from patient samples but also from environmental samples. Azole resistance mechanisms involve for most isolates alterations at the site of the azole target (cyp51A); however, a substantial number of isolates can also exhibit non-cyp51A-mediated mechanisms.We aimed here to identify novel A. fumigatus genes involved in azole resistance. For this purpose, we designed a functional complementation system of A. fumigatus cDNAs expressed in a Saccharomyces cerevisiae isolate lacking the ATP Binding Cassette (ABC) transporter PDR5 and that was therefore more azole-susceptible than the parent wild type. Several genes were recovered including two distinct ABC transporters (atrF, atrI) and a Major Facilitator transporter (mdrA), from which atrI (Afu3g07300) and mdrA (Afu1g13800) were not yet described. atrI mediated resistance to itraconazole and voriconazole, while atrF only to voriconazole in S. cerevisiae Gene inactivation of each transporter in A. fumigatus indicated that the transporters were involved in the basal level of azole susceptibility. The expression of the transporters was addressed in clinical and environmental isolates with several azole resistance profiles. Our results show that atrI and mdrA tended to be expressed at higher levels than atrF in normal growth conditions. atrF was upregulated in 2/4 of azole-resistant environmental isolates and was the only gene with a significant association between transporter expression and azole resistance. In conclusion, this work showed the potential of complementation to identify functional transporters. The identified transporters were suggested to participate in azole resistance of A. fumigatus; however, this hypothesis will

  11. Plant-like biosynthesis of isoquinoline alkaloids in Aspergillus fumigatus.

    PubMed

    Baccile, Joshua A; Spraker, Joseph E; Le, Henry H; Brandenburger, Eileen; Gomez, Christian; Bok, Jin Woo; Macheleidt, Juliane; Brakhage, Axel A; Hoffmeister, Dirk; Keller, Nancy P; Schroeder, Frank C

    2016-06-01

    Natural product discovery efforts have focused primarily on microbial biosynthetic gene clusters (BGCs) containing large multimodular polyketide synthases and nonribosomal peptide synthetases; however, sequencing of fungal genomes has revealed a vast number of BGCs containing smaller NRPS-like genes of unknown biosynthetic function. Using comparative metabolomics, we show that a BGC in the human pathogen Aspergillus fumigatus named fsq, which contains an NRPS-like gene lacking a condensation domain, produces several new isoquinoline alkaloids known as the fumisoquins. These compounds derive from carbon-carbon bond formation between two amino acid-derived moieties followed by a sequence that is directly analogous to isoquinoline alkaloid biosynthesis in plants. Fumisoquin biosynthesis requires the N-methyltransferase FsqC and the FAD-dependent oxidase FsqB, which represent functional analogs of coclaurine N-methyltransferase and berberine bridge enzyme in plants. Our results show that BGCs containing incomplete NRPS modules may reveal new biosynthetic paradigms and suggest that plant-like isoquinoline biosynthesis occurs in diverse fungi. PMID:27065235

  12. The mtfA Transcription Factor Gene Controls Morphogenesis, Gliotoxin Production, and Virulence in the Opportunistic Human Pathogen Aspergillus fumigatus

    PubMed Central

    Smith, Timothy D.

    2014-01-01

    Aspergillus fumigatus is the leading causative agent of invasive aspergillosis (IA). The number of cases is on the rise, with mortality rates as high as 90% among immunocompromised patients. Molecular genetic studies in A. fumigatus could provide novel targets to potentially set the basis for antifungal therapies. In the current study, we investigated the role of the transcription factor gene mtfA in A. fumigatus. Our results revealed that mtfA plays a role in the growth and development of the fungus. Deletion or overexpression of mtfA leads to a slight reduction in colony growth, as well as a reduction in conidiation levels, in the overexpression strain compared to the wild-type strain. Furthermore, production of the secondary metabolite gliotoxin increased when mtfA was overexpressed, coinciding with an increase in the transcription levels of the gliotoxin genes gliZ and gliP with respect to the wild type. In addition, our study showed that mtfA is also necessary for normal protease activity in A. fumigatus; deletion of mtfA resulted in a reduction of protease activity compared to wild-type levels. Importantly, the absence of mtfA caused a decrease in virulence in the Galleria mellonella infection model, indicating that mtfA is necessary for A. fumigatus wild-type pathogenesis. PMID:24728192

  13. Levels of gram-negative bacteria, Aspergillus fumigatus, dust, and endotoxin at compost plants.

    PubMed Central

    Clark, C S; Rylander, R; Larsson, L

    1983-01-01

    Airborne gram-negative bacteria, endotoxins, dust, and Aspergillus fumigatus were measured in four compost plants in Sweden. At sites where material was processed, the number of airborne A. fumigatus exceeded 10(6)/m3, whereas the number of gram-negative bacteria was usually lower. Dust levels were moderate, and endotoxin levels were well below 0.5 micrograms/m3. Medical studies to evaluate the effects of this type of microbial exposure are recommended. PMID:6347061

  14. Genotypic characterization of sequential Aspergillus fumigatus isolates from patients with cystic fibrosis.

    PubMed Central

    Verweij, P E; Meis, J F; Sarfati, J; Hoogkamp-Korstanje, J A; Latgé, J P; Melchers, W J

    1996-01-01

    Twenty-three sequential Aspergillus fumigatus sputum isolates, which had been collected over a period of 2 years, from two patients with cystic fibrosis were genotyped by random amplified polymorphic DNA PCR and restriction fragment length polymorphism analysis. In patient B, one genotype was predominantly present in the sputum samples, while in the other patient up to nine different genotypes were identified. This study suggests that different patterns of colonization with A.fumigatus exist in patients with cystic fibrosis. PMID:8880528

  15. Pharmacodynamics of itraconazole against Aspergillus fumigatus in an in vitro model of the human alveolus: perspectives on the treatment of triazole-resistant infection and utility of airway administration.

    PubMed

    Al-Nakeeb, Zaid; Sudan, Ajay; Jeans, Adam R; Gregson, Lea; Goodwin, Joanne; Warn, Peter A; Felton, Timothy W; Howard, Susan J; Hope, William W

    2012-08-01

    Itraconazole is used for the prevention and treatment of infections caused by Aspergillus fumigatus. An understanding of the pharmacodynamics of itraconazole against wild-type and triazole-resistant strains provides a basis for innovative therapeutic strategies for treatment of infections. An in vitro model of the human alveolus was used to define the pharmacodynamics of itraconazole. Galactomannan was used as a biomarker. The effect of systemic and airway administration of itraconazole was assessed, as was a combination of itraconazole administered to the airway and systemically administered 5FC. Systemically administered itraconazole against the wild type induced a concentration-dependent decline in galactomannan in the alveolar and endothelial compartments. No exposure-response relationships were apparent for the L98H, M220T, or G138C mutant. The administration of itraconazole to the airway resulted in comparable exposure-response relationships to those observed with systemic therapy. This was achieved without detectable concentrations of drug within the endothelial compartment. The airway administration of itraconazole resulted in a definite but submaximal effect in the endothelial compartment against the L98H mutant. The administration of 5FC resulted in a concentration-dependent decline in galactomannan in both the alveolar and endothelial compartments. The combination of airway administration of itraconazole and systemically administered 5FC was additive. Systemic administration of itraconazole is ineffective against Cyp51 mutants. The airway administration of itraconazole is effective for the treatment of wild-type strains and appears to have some activity against the L98H mutants. Combination with other agents, such as 5FC, may enable the attainment of near-maximal antifungal activity. PMID:22615280

  16. Identification of the antiphagocytic trypacidin gene cluster in the human-pathogenic fungus Aspergillus fumigatus.

    PubMed

    Mattern, Derek J; Schoeler, Hanno; Weber, Jakob; Novohradská, Silvia; Kraibooj, Kaswara; Dahse, Hans-Martin; Hillmann, Falk; Valiante, Vito; Figge, Marc Thilo; Brakhage, Axel A

    2015-12-01

    The opportunistic human pathogen Aspergillus fumigatus produces numerous different natural products. The genetic basis for the biosynthesis of a number of known metabolites has remained unknown. The gene cluster encoding for the biosynthesis of the conidia-bound metabolite trypacidin is of particular interest because of its antiprotozoal activity and possible role in the infection process. Here, we show that the genes encoding the biosynthesis enzymes of trypacidin reside within an orphan gene cluster in A. fumigatus. Genome mining identified tynC as an uncharacterized polyketide synthase with high similarity to known enzymes, whose products are structurally related to trypacidin including endocrocin and fumicycline. Gene deletion of tynC resulted in the complete absence of trypacidin production, which was fully restored when the mutant strain was complemented with the wild-type gene. When confronted with macrophages, the tynC deletion mutant conidia were more frequently phagocytosed than those of the parental wild-type strain. This was also found for phagocytic amoebae of the species Dictyostelium discoideum, which showed increased phagocytosis of ΔtynC conidia. Both macrophages and amoebae were also sensitive to trypacidin. Therefore, our results suggest that the conidium-bound trypacidin could have a protective function against phagocytes both in the environment and during the infection process. PMID:26278536

  17. cyp51A-based mechanism of azole resistance in Aspergillus fumigatus: Illustration by a new 3D Structural Model of Aspergillus fumigatus CYP51A protein.

    PubMed

    Liu, Musang; Zheng, Nan; Li, Dongmei; Zheng, Hailin; Zhang, Lili; Ge, Hu; Liu, Weida

    2016-05-01

    Mutations of CYP51A protein (Cytochrome P450 14-α Sterol demethylase) play a central role in the azole resistance of Aspergillus fumigatus The available structural models of CYP51A protein ofA. fumigatus are built based on that of Homo sapiens and that of Mycobacterium tuberculosis, of which the amino acid homology is only 38% and 29% compared with CYP51A protein ofA. fumigatus, respectively. In the present study, we constructed a new 3D structural model ofA. fumigatus CYP51A protein based on a recently resolved crystal structure of the homologous protein in the fungus S. cerevisiae, which shares 50% amino acid homology with A. fumigatus CYP51A protein. Three azole molecules, itraconazole, voriconazole, and posaconazole, were docked to the wild-type and the mutant A. fumigatus CYP51A protein models, respectively, to illustrate the impact of cyp51A mutations to azole-resistance. We found the mutations that occurred at L98, M220, and Y431 positions would decrease the binding affinity of azoles to the CYP51A protein and therefore would reduce their inhibitory effects. Additionally, the mutations of L98 and G432 would reduce the stability of the protein, which might lead to conformational change of its binding pocket and eventually the resistance to azoles. PMID:26768370

  18. Bioluminescent Aspergillus fumigatus, a New Tool for Drug Efficiency Testing and In Vivo Monitoring of Invasive Aspergillosis▿

    PubMed Central

    Brock, Matthias; Jouvion, Grégory; Droin-Bergère, Sabrina; Dussurget, Olivier; Nicola, Marie-Anne; Ibrahim-Granet, Oumaïma

    2008-01-01

    Aspergillus fumigatus is the main cause of invasive aspergillosis in immunocompromised patients, and only a limited number of drugs for treatment are available. A screening method for new antifungal compounds is urgently required, preferably an approach suitable for in vitro and in vivo studies. Bioluminescence imaging is a powerful tool to study the temporal and spatial resolutions of the infection and the effectiveness of antifungal drugs. Here, we describe the construction of a bioluminescent A. fumigatus strain by fusing the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene from A. fumigatus with the luciferase gene from Photinus pyralis to control the expression of the bioluminescent reporter. A. fumigatus transformed with this construct revealed high bioluminescence under all tested growth conditions. Furthermore, light emission correlated with the number of conidia used for inoculation and with the biomass formed after different incubation times. The bioluminescent strains were suitable to study the effectiveness of antifungals in vitro by several independent methods, including the determination of light emission with a microplate reader and the direct visualization of light emission with an IVIS 100 system. Moreover, when glucocorticoid-treated immunosuppressed mice were infected with a bioluminescent strain, light emission was detected from infected lungs, allowing the visualization of the progression of invasive aspergillosis. Therefore, this new bioluminescence tool is suitable to study the in vitro effectiveness of drugs and the disease development, localization, and burden of fungi within tissues and may also provide a powerful tool to study the effectiveness of antifungals in vivo. PMID:18820063

  19. The Aspergillus fumigatus Dihydroxyacid Dehydratase Ilv3A/IlvC Is Required for Full Virulence

    PubMed Central

    Oliver, Jason D.; Kaye, Sarah J.; Tuckwell, Danny; Johns, Anna E.; Macdonald, Darel A.; Livermore, Joanne; Warn, Peter A.; Birch, Mike; Bromley, Michael J.

    2012-01-01

    Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC. PMID:23028460

  20. Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    PubMed Central

    2009-01-01

    primary culture of human respiratory cells exposed to A. fumigatus points to the biological significance of described phenomena. Conclusion Our findings provide evidence that respiratory epithelium might play an important role in the immune response during Aspergillus infection. Understanding the mechanisms of regulation of defensin expression may thus lead to new approaches that could enhance expression of antimicrobial peptides for potential therapeutic use during aspergillosis treatment. PMID:19208266

  1. Extrolites of Aspergillus fumigatus and Other Pathogenic Species in Aspergillus Section Fumigati

    PubMed Central

    Frisvad, Jens C.; Larsen, Thomas O.

    2016-01-01

    Aspergillus fumigatus is an important opportunistic human pathogen known for its production of a large array of extrolites. Up to 63 species have been described in Aspergillus section Fumigati, some of which have also been reliably reported to be pathogenic, including A. felis, A. fischeri, A. fumigatiaffinis, A. fumisynnematus, A. hiratsukae, A. laciniosus, A. lentulus, A. novofumigatus, A. parafelis, A. pseudofelis, A. pseudoviridinutans, A. spinosus, A. thermomutatus, and A. udagawae. These species share the production of hydrophobins, melanins, and siderophores and ability to grow well at 37°C, but they only share some small molecule extrolites, that could be important factors in pathogenicity. According to the literature gliotoxin and other exometabolites can be contributing factors to pathogenicity, but these exometabolites are apparently not produced by all pathogenic species. It is our hypothesis that species unable to produce some of these metabolites can produce proxy-exometabolites that may serve the same function. We tabulate all exometabolites reported from species in Aspergillus section Fumigati and by comparing the profile of those extrolites, suggest that those producing many different kinds of exometabolites are potential opportunistic pathogens. The exometabolite data also suggest that the profile of exometabolites are highly specific and can be used for identification of these closely related species. PMID:26779142

  2. New applications for known drugs: Human glycogen synthase kinase 3 inhibitors as modulators of Aspergillus fumigatus growth.

    PubMed

    Sebastián, Víctor; Manoli, Maria-Tsampika; Pérez, Daniel I; Gil, Carmen; Mellado, Emilia; Martínez, Ana; Espeso, Eduardo A; Campillo, Nuria E

    2016-06-30

    Invasive aspergillosis (IA) is one of the most severe forms of fungi infection. IA disease is mainly due to Aspergillus fumigatus, an air-borne opportunistic pathogen. Mortality rate caused by IA is still very high (50-95%), because of difficulty in early diagnostics and reduced antifungal treatment options, thus new and efficient drugs are necessary. The aim of this work is, using Aspergillus nidulans as non-pathogen model, to develop efficient drugs to treat IA. The recent discovered role of glycogen synthase kinase-3 homologue, GskA, in A. fumigatus human infection and our previous experience on human GSK-3 inhibitors focus our attention on this kinase as a target for the development of antifungal drugs. With the aim to identify effective inhibitors of colonial growth of A. fumigatus we use A. nidulans as an accurate model for in vivo and in silico studies. Several well-known human GSK-3β inhibitors were tested for inhibition of A. nidulans colony growth. Computational tools as docking studies and binding site prediction was used to explain the different biological profile of the tested inhibitors. Three of the five tested hGSK3β inhibitors are able to reduce completely the colonial growth by covalent bind to the enzyme. Therefore these compounds may be useful in different applications to eradicate IA. PMID:27131621

  3. The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence

    PubMed Central

    Bom, Vinícius Leite Pedro; de Castro, Patrícia Alves; Winkelströter, Lizziane K.; Marine, Marçal; Hori, Juliana I.; Ramalho, Leandra Naira Zambelli; dos Reis, Thaila Fernanda; Goldman, Maria Helena S.; Brown, Neil Andrew; Rajendran, Ranjith; Ramage, Gordon; Walker, Louise A.; Munro, Carol A.; Rocha, Marina Campos; Malavazi, Iran; Hagiwara, Daisuke

    2015-01-01

    Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ΔsitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased β-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitA strain is more sensitive to several metals and ions, such as MnCl2, CaCl2, and LiCl, but it is more resistant to ZnSO4. The ΔsitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway. PMID:25911225

  4. Acquired antifungal drug resistance in Aspergillus fumigatus: epidemiology and detection.

    PubMed

    Howard, Susan Julie; Arendrup, Maiken Cavling

    2011-04-01

    Voriconazole is the recommended agent for invasive aspergillosis, with lipid amphotericin B or caspofungin as second line treatment choices. Being the only agents available in oral formulation, azoles are used in chronic infections and often over longer time periods. In addition to being used in clinical medicine, azoles are employed extensively in agriculture. Azole-resistant Aspergillus has been isolated in azole naïve patients, in azole exposed patients and in the environment. The primary underlying mechanism of resistance is as a result of alterations in the cyp51A target gene, with a variety of mutations found in clinical isolates but just one identified in a environmental strain (a point mutation at codon 98 accompanied by a tandem repeat in the promoter region). Much less is currently known about echinocandin resistance in Aspergillus, in part because susceptibility testing is not routinely performed and because the methods suffer from technical difficulties and suboptimal reproducibility. Clinical breakthrough cases have been reported however, and resistance has been confirmed in vivo. In this paper we review the current knowledge on epidemiology, susceptibility testing and underlying mechanisms involved in azole and echinocandin resistance in Aspergillus. PMID:20795765

  5. Distinct Roles of Myosins in Aspergillus fumigatus Hyphal Growth and Pathogenesis.

    PubMed

    Renshaw, Hilary; Vargas-Muñiz, José M; Richards, Amber D; Asfaw, Yohannes G; Juvvadi, Praveen R; Steinbach, William J

    2016-05-01

    Myosins are a family of actin-based motor proteins found in many organisms and are categorized into classes based on their structures. Class II and V myosins are known to be important for critical cellular processes, including cytokinesis, endocytosis, exocytosis, and organelle trafficking, in the model fungi Saccharomyces cerevisiae and Aspergillus nidulans However, the roles of myosins in the growth and virulence of the pathogen Aspergillus fumigatus are unknown. We constructed single- and double-deletion strains of the class II and class V myosins in A. fumigatus and found that while the class II myosin (myoB) is dispensable for growth, the class V myosin (myoE) is required for proper hyphal extension; deletion of myoE resulted in hyperbranching and loss of hyphal polarity. Both myoB and myoE are necessary for proper septation, conidiation, and conidial germination, but only myoB is required for conidial viability. Infection with the ΔmyoE strain in the invertebrate Galleria mellonella model and also in a persistently immunosuppressed murine model of invasive aspergillosis resulted in hypovirulence, while analysis of bronchoalveolar lavage fluid revealed that tumor necrosis factor alpha (TNF-α) release and cellular infiltration were similar compared to those of the wild-type strain. The ΔmyoE strain showed fungal growth in the murine lung, while the ΔmyoB strain exhibited little fungal burden, most likely due to the reduced conidial viability. These results show, for the first time, the important role these cytoskeletal components play in the growth of and disease caused by a known pathogen, prompting future studies to understand their regulation and potential targeting for novel antifungal therapies. PMID:26953327

  6. Pituitary aspergillus infection.

    PubMed

    Moore, Lauren A; Erstine, Emily M; Prayson, Richard A

    2016-07-01

    Fungal infection should be considered in the differential diagnosis of a pituitary or sellar mass, albeit fungal infections involving the pituitary gland and sella are a rare occurrence. We report a case of Aspergillus infection involving the pituitary gland and sellar region discovered in a 74-year-old man. The patient had a history of hypertension, chronic renal disease, autoimmune hemolytic anemia and presented with right eye pain, headaches and worsening hemiparesis. Imaging studies revealed a right internal carotid artery occlusion and an acute right pontine stroke along with smaller infarcts in the right middle cerebral artery distribution. Clinically, the patient was thought to have vasculitis. An infectious etiology was not identified. He developed respiratory distress and died. At autopsy, necrotizing meningitis was discovered. A predominantly chronic inflammatory cell infiltrate consisting of benign-appearing lymphocytes, plasma cells and macrophages was accompanied by acute angle branching, angioinvasive hyphae which were highlighted on Gomori methenamine silver staining and were morphologically consistent with Aspergillus species. In previously reported cases of Aspergillus infection involving the pituitary or sella, most presented with headaches or impaired vision and were not immunocompromised. A transsphenoidal surgical approach is recommended in suspected cases in order to minimize the risk of dissemination of the infection. Some patients have responded well to antifungal medications once diagnosed. PMID:26896907

  7. Abundant respirable ergot alkaloids from the common airborne fungus Aspergillus fumigatus.

    PubMed

    Panaccione, Daniel G; Coyle, Christine M

    2005-06-01

    Ergot alkaloids are mycotoxins that interact with several monoamine receptors, negatively affecting cardiovascular, nervous, reproductive, and immune systems of exposed humans and animals. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, can produce ergot alkaloids in broth culture. The objectives of this study were to determine if A. fumigatus accumulates ergot alkaloids in a respirable form in or on its conidia, to quantify ergot alkaloids associated with conidia produced on several different substrates, and to measure relevant physical properties of the conidia. We found at least four ergot alkaloids, fumigaclavine C, festuclavine, fumigaclavine A, and fumigaclavine B (in order of abundance), associated with conidia of A. fumigatus. Under environmentally relevant conditions, the total mass of ergot alkaloids often constituted >1% of the mass of the conidium. Ergot alkaloids were extracted from conidia produced on all media tested, and the greatest quantities were observed when the fungus was cultured on latex paint or cultured maize seedlings. The values for physical properties of conidia likely to affect their respirability (i.e., diameter, mass, and specific gravity) were significantly lower for A. fumigatus than for Aspergillus nidulans, Aspergillus niger, and Stachybotrys chartarum. The demonstration of relatively high concentrations of ergot alkaloids associated with conidia of A. fumigatus presents opportunities for investigations of potential contributions of the toxins to adverse health effects associated with the fungus and to aspects of the biology of the fungus that contribute to its success. PMID:15933008

  8. Aspergillus fumigatus MedA governs adherence, host cell interactions and virulence

    PubMed Central

    Gravelat, Fabrice N.; Ejzykowicz, Daniele E.; Chiang, Lisa Y.; Chabot, Josée C.; Urb, Mirjam; Macdonald, K. Denyese; al-Bader, Nadia; Filler, Scott G.; Sheppard, Donald C.

    2010-01-01

    In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared to A. nidulans. The A. fumigatus ΔmedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The ΔmedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro-inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatus ΔmedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis. PMID:19889083

  9. Transcription Factor SomA Is Required for Adhesion, Development and Virulence of the Human Pathogen Aspergillus fumigatus

    PubMed Central

    Lin, Chi-Jan; Sasse, Christoph; Gerke, Jennifer; Valerius, Oliver; Irmer, Henriette; Frauendorf, Holm; Heinekamp, Thorsten; Straßburger, Maria; Tran, Van Tuan; Herzog, Britta; Braus-Stromeyer, Susanna A.; Braus, Gerhard H.

    2015-01-01

    The transcription factor Flo8/Som1 controls filamentous growth in Saccharomyces cerevisiae and virulence in the plant pathogen Magnaporthe oryzae. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS) domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from Aspergillus fumigatus rescued in yeast flo8 mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. A. fumigatus SomA acts similarly to yeast Flo8 on the promoter of FLO11 fused with reporter gene (LacZ) in S. cerevisiae. FLO11 expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the somA gene in A. fumigatus resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of somA using the inducible Tet-on system. A adherence assay with the conditional somA expression strain indicated that SomA is required for biofilm formation. A ptaB deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls A. fumigatus biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of A. fumigatus. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen A. fumigatus. PMID:26529322

  10. The Temporal Dynamics of Differential Gene Expression in Aspergillus fumigatus Interacting with Human Immature Dendritic Cells In Vitro

    PubMed Central

    Morton, Charles O.; Varga, John J.; Hornbach, Anke; Mezger, Markus; Sennefelder, Helga; Kneitz, Susanne; Kurzai, Oliver; Krappmann, Sven; Einsele, Hermann; Nierman, William C.; Rogers, Thomas R.; Loeffler, Juergen

    2011-01-01

    Dendritic cells (DC) are the most important antigen presenting cells and play a pivotal role in host immunity to infectious agents by acting as a bridge between the innate and adaptive immune systems. Monocyte-derived immature DCs (iDC) were infected with viable resting conidia of Aspergillus fumigatus (Af293) for 12 hours at an MOI of 5; cells were sampled every three hours. RNA was extracted from both organisms at each time point and hybridised to microarrays. iDC cell death increased at 6 h in the presence of A. fumigatus which coincided with fungal germ tube emergence; >80% of conidia were associated with iDC. Over the time course A. fumigatus differentially regulated 210 genes, FunCat analysis indicated significant up-regulation of genes involved in fermentation, drug transport, pathogenesis and response to oxidative stress. Genes related to cytotoxicity were differentially regulated but the gliotoxin biosynthesis genes were down regulated over the time course, while Aspf1 was up-regulated at 9 h and 12 h. There was an up-regulation of genes in the subtelomeric regions of the genome as the interaction progressed. The genes up-regulated by iDC in the presence of A. fumigatus indicated that they were producing a pro-inflammatory response which was consistent with previous transcriptome studies of iDC interacting with A. fumigatus germ tubes. This study shows that A. fumigatus adapts to phagocytosis by iDCs by utilising genes that allow it to survive the interaction rather than just up-regulation of specific virulence genes. PMID:21264256

  11. Draft Genome Sequences of Two Aspergillus fumigatus Strains, Isolated from the International Space Station.

    PubMed

    Singh, Nitin Kumar; Blachowicz, Adriana; Checinska, Aleksandra; Wang, Clay; Venkateswaran, Kasthuri

    2016-01-01

    Draft genome sequences of Aspergillus fumigatus strains (ISSFT-021 and IF1SW-F4), opportunistic pathogens isolated from the International Space Station (ISS), were assembled to facilitate investigations of the nature of the virulence characteristics of the ISS strains to other clinical strains isolated on Earth. PMID:27417828

  12. Effect of essential oil of Hyssopus officinalis on the lipid composition of Aspergillus fumigatus.

    PubMed

    Ghfir, B; Fonvieille, J L; Koulali, Y; Ecalle, R; Dargent, R

    1994-06-01

    Addition of the essential oil of Hyssopus officinalis to the culture medium of Aspergillus fumigatus induced alterations in both growth and lipid composition of this mould. Total lipids and sterols were reduced, whereas total phospholipids were increased. There were alterations in the proportions of fatty acids, neutral lipid and phospholipid fractions. PMID:7935731

  13. Draft Genome Sequences of Two Aspergillus fumigatus Strains, Isolated from the International Space Station

    PubMed Central

    Singh, Nitin Kumar; Blachowicz, Adriana; Checinska, Aleksandra; Wang, Clay

    2016-01-01

    Draft genome sequences of Aspergillus fumigatus strains (ISSFT-021 and IF1SW-F4), opportunistic pathogens isolated from the International Space Station (ISS), were assembled to facilitate investigations of the nature of the virulence characteristics of the ISS strains to other clinical strains isolated on Earth. PMID:27417828

  14. Dispersal of Aspergillus fumigatus from Sewage Sludge Compost Piles Subjected to Mechanical Agitation in Open Air

    PubMed Central

    Millner, Patricia D.; Bassett, David A.; Marsh, Paul B.

    1980-01-01

    Aerosolization of the thermophilous fungal opportunist Aspergillus fumigatus from mechanically agitated compost piles was examined at a pilot-scale sewage sludge composting facility and two other selected test sites. Aerosols of A. fumigatus downwind from stationary compost piles were insignificant in comparison with those downwind from agitated piles. These aerosols were generated by a front-end loader moving and dropping compost. Aerial concentrations of the fungus at distances downwind from the point of emission were used to determine an emission rate for A. fumigatus associated with the moving operations. The maximum emission rate, 4.6 × 106A. fumigatus particles per s, was used to calculate predicted concentrations in an unobstructed plume with restrictive, neutral, and dispersive atmospheric mixing conditions up to 1 km downwind from the emission source. PMID:16345563

  15. Exploration of Sulfur Assimilation of Aspergillus fumigatus Reveals Biosynthesis of Sulfur-Containing Amino Acids as a Virulence Determinant.

    PubMed

    Amich, Jorge; Dümig, Michaela; O'Keeffe, Gráinne; Binder, Jasmin; Doyle, Sean; Beilhack, Andreas; Krappmann, Sven

    2016-04-01

    Fungal infections are of major relevance due to the increased numbers of immunocompromised patients, frequently delayed diagnosis, and limited therapeutics. To date, the growth and nutritional requirements of fungi during infection, which are relevant for invasion of the host, are poorly understood. This is particularly true for invasive pulmonary aspergillosis, as so far, sources of (macro)elements that are exploited during infection have been identified to only a limited extent. Here, we have investigated sulfur (S) utilization by the human-pathogenic mold Aspergillus fumigatus during invasive growth. Our data reveal that inorganic S compounds or taurine is unlikely to serve as an S source during invasive pulmonary aspergillosis since a sulfate transporter mutant strain and a sulfite reductase mutant strain are fully virulent. In contrast, the S-containing amino acid cysteine is limiting for fungal growth, as proven by the reduced virulence of a cysteine auxotroph. Moreover, phenotypic characterization of this strain further revealed the robustness of the subordinate glutathione redox system. Interestingly, we demonstrate that methionine synthase is essential for A. fumigatus virulence, defining the biosynthetic route of this proteinogenic amino acid as a potential antifungal target. In conclusion, we provide novel insights into the nutritional requirements ofA. fumigatus during pathogenesis, a prerequisite to understanding and fighting infection. PMID:26787716

  16. Characterization of the Aspergillus fumigatus chitosanase CsnB and evaluation of its potential use in serological diagnostics.

    PubMed

    Beck, Julia; Broniszewska, Marzena; Schwienbacher, Monika; Ebel, Frank

    2014-07-01

    Aspergillus fumigatus is currently the major air-borne fungal pathogen as its asexual spores are distributed through the air. In severely immunocompromised patients, inhalation of these conidia can result in life-threatening infections. Invasive Aspergillosis, a major Aspergillus-associated disease, is associated with a high mortality reflecting short-comings in diagnostics and therapy. Current diagnostics largely rely on the serological detection of the galactomannan antigen. Detection of circulating antibodies is an alternative approach. In this study, we have characterized the chitosanase CsnB, a protein that was previously shown to be a major secreted A. fumigatus antigen and therefore a potential target for antibody-based diagnostics. To analyze the biological function of CsnB we have deleted the csnB gene and generated CsnB-specific antibodies. We found that A. fumigatus is able to grow on chitosan in a CsnB-dependent manner. During growth on chitosan elevated levels of CsnB are found in the supernatants indicating that chitosan triggers enhanced CsnB production. Unexpectedly we have found a similar activity for tartrate. Using recombinant proteins we analyzed antibody responses in patients at risk to develop invasive aspergillosis. We focussed this study on two antigens: CsnB and for comparison mitogillin, a secreted A. fumigatus ribotoxin. IgG responses were found to both proteins, but elevated antibody levels to CsnB and/or mitogillin showed no correlation to the results of the galactomannan antigen assay or clinical signs that are characteristic for fungal infections. PMID:24880790

  17. Prevalence and mechanism of triazole resistance in Aspergillus fumigatus in a referral chest hospital in Delhi, India and an update of the situation in Asia

    PubMed Central

    Chowdhary, Anuradha; Sharma, Cheshta; Kathuria, Shallu; Hagen, Ferry; Meis, Jacques F.

    2015-01-01

    Aspergillus fumigatus causes varied clinical syndromes ranging from colonization to deep infections. The mainstay of therapy of Aspergillus diseases is triazoles but several studies globally highlighted variable prevalence of triazole resistance, which hampers the management of aspergillosis. We studied the prevalence of resistance in clinical A. fumigatus isolates during 4 years in a referral Chest Hospital in Delhi, India and reviewed the scenario in Asia and the Middle East. Aspergillus species (n = 2117) were screened with selective plates for azole resistance. The isolates included 45.4% A. flavus, followed by 32.4% A. fumigatus, 15.6% Aspergillus species and 6.6% A. terreus. Azole resistance was found in only 12 (1.7%) A. fumigatus isolates. These triazole resistant A. fumigatus (TRAF) isolates were subjected to (a) calmodulin and β tubulin gene sequencing (b) in vitro antifungal susceptibility testing against triazoles using CLSI M38-A2 (c) sequencing of cyp51A gene and real-time PCR assay for detection of mutations and (d) microsatellite typing of the resistant isolates. TRAF harbored TR34/L98H mutation in 10 (83.3%) isolates with a pan-azole resistant phenotype. Among the remaining two TRAF isolates, one had G54E and the other had three non-synonymous point mutations. The majority of patients were diagnosed as invasive aspergillosis followed by allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis. The Indian TR34/L98H isolates had a unique genotype and were distinct from the Chinese, Middle East, and European TR34/L98H strains. This resistance mechanism has been linked to the use of fungicide azoles in agricultural practices in Europe as it has been mainly reported from azole naïve patients. Reports published from Asia demonstrate the same environmental resistance mechanism in A. fumigatus isolates from two highly populated countries in Asia, i.e., China and India and also from the neighboring Middle East. PMID:26005442

  18. Purification and characterization of factors produced by Aspergillus fumigatus which affect human ciliated respiratory epithelium.

    PubMed Central

    Amitani, R; Taylor, G; Elezis, E N; Llewellyn-Jones, C; Mitchell, J; Kuze, F; Cole, P J; Wilson, R

    1995-01-01

    The mechanisms by which Aspergillus fumigatus colonizes the respiratory mucosa are unknown. Culture filtrates of eight of nine clinical isolates of A. fumigatus slowed ciliary beat frequency and damaged human respiratory epithelium in vitro. These changes appeared to occur concurrently. Culture filtrates of two clinical isolates of Candida albicans had no effect on ciliated epithelium. We have purified and characterized cilioinhibitory factors of a clinical isolate of A. fumigatus. The cilioinhibitory activity was heat labile, reduced by dialysis, and partially extractable into chloroform. The activity was associated with both high- and low-molecular-weight factors, as determined by gel filtration on Sephadex G-50. A low-molecular-weight cilioinhibitory factor was further purified by reverse-phase high-performance liquid chromatography and shown by mass spectrometry to be gliotoxin, a known metabolite of A. fumigatus. Gliotoxin significantly slowed ciliary beat frequency in association with epithelial damage at concentrations above 0.2 microgram/ml; other Aspergillus toxins, i.e., fumagillin and helvolic acid, were also cilioinhibitory but at much higher concentrations. High-molecular-weight (> or = 35,000 and 25,000) cilioinhibitory materials had neither elastolytic nor proteolytic activity and remain to be identified. Thus, A. fumigatus produces a number of biologically active substances which slow ciliary beating and damage epithelium and which may influence colonization of the airways. PMID:7543879

  19. Role of prostaglandin D2/CRTH2 pathway on asthma exacerbation induced by Aspergillus fumigatus

    PubMed Central

    Liu, Haixia; Zheng, Mingrui; Qiao, Jianou; Dang, Yajie; Zhang, Pengyu; Jin, Xianqiao

    2014-01-01

    Aspergillus fumigatus is often associated in asthmatic patients with the exacerbation of asthma symptoms. The pathomechanism of this phenomenon has not been fully understood. Here, we evaluated the immunological mechanisms and the role of the prostaglandin D2/ Chemoattractant Receptor-Homologous Molecule Expressed on Th2 Cells (CRTH2) pathway in the development of Aspergillus-associated asthma exacerbation. We studied the effects of A. fumigatus on airway inflammation and bronchial hyper-responsiveness in a rat model of chronic asthma. Inhalation delivery of A. fumigatus conidia increased the airway eosinophilia and bronchial hyper-responsiveness in ovalbumin-sensitized, challenged rats. These changes were associated with prostaglandin D2 synthesis and CRTH2 expression in the lungs. Direct inflammation occurred in ovalbumin-sensitized, challenged animals, whereas pre-treatment with an antagonist against CRTH2 nearly completely eliminated the A. fumigatus-induced worsening of airway eosinophilia and bronchial hyper-responsiveness. Our data demonstrate that production of prostaglandin D2 followed by eosinophil recruitment into the airways via a CRTH2 receptor are the major pathogenic factors responsible for the A. fumigatus-induced enhancement of airway inflammation and responsiveness. PMID:24329550

  20. Role of prostaglandin D2 /CRTH2 pathway on asthma exacerbation induced by Aspergillus fumigatus.

    PubMed

    Liu, Haixia; Zheng, Mingrui; Qiao, Jianou; Dang, Yajie; Zhang, Pengyu; Jin, Xianqiao

    2014-05-01

    Aspergillus fumigatus is often associated in asthmatic patients with the exacerbation of asthma symptoms. The pathomechanism of this phenomenon has not been fully understood. Here, we evaluated the immunological mechanisms and the role of the prostaglandin D2 / Chemoattractant Receptor-Homologous Molecule Expressed on Th2 Cells (CRTH2) pathway in the development of Aspergillus-associated asthma exacerbation. We studied the effects of A. fumigatus on airway inflammation and bronchial hyper-responsiveness in a rat model of chronic asthma. Inhalation delivery of A. fumigatus conidia increased the airway eosinophilia and bronchial hyper-responsiveness in ovalbumin-sensitized, challenged rats. These changes were associated with prostaglandin D2 synthesis and CRTH2 expression in the lungs. Direct inflammation occurred in ovalbumin-sensitized, challenged animals, whereas pre-treatment with an antagonist against CRTH2 nearly completely eliminated the A. fumigatus-induced worsening of airway eosinophilia and bronchial hyper-responsiveness. Our data demonstrate that production of prostaglandin D2 followed by eosinophil recruitment into the airways via a CRTH2 receptor are the major pathogenic factors responsible for the A. fumigatus-induced enhancement of airway inflammation and responsiveness. PMID:24329550

  1. Differentiation between Isolates of Aspergillus fumigatus from Breeding Turkeys and Their Environment by Genotyping with Microsatellite Markers

    PubMed Central

    Lair-Fulleringer, Sybille; Guillot, Jacques; Desterke, Christophe; Seguin, Dominique; Warin, Stephan; Bezille, Arnaud; Chermette, René; Bretagne, Stéphane

    2003-01-01

    To elucidate the epidemiology of the different forms of avian aspergillosis, 114 Aspergillus fumigatus isolates from sacrificed turkeys and 134 A. fumigatus isolates from air samples were collected and genotyped by microsatellite polymorphism marker analysis. Air sampling confirmed the huge diversity of A. fumigatus populations. Whereas older animals harbored several combinations of genotypes, 1-day-old chicks carried a unique genotype, suggesting a unique source of contamination. PMID:12682192

  2. Septic arthritis due to tubercular and Aspergillus co-infection.

    PubMed

    Kumar, Mukesh; Thilak, Jai; Zahoor, Adnan; Jyothi, Arun

    2016-01-01

    Aspergillus septic arthritis is a rare and serious medical and surgical problem. It occurs mainly in immunocompromised patients. Aspergillus fumigatus is the most common causative organism followed by Aspergillus flavus. The most common site affected is knee followed by shoulder, ankle, wrist, hip and sacroiliac joint. Debridement and voriconazole are primary treatment of articular aspergilosis. To the best of our knowledge, there are no reported cases of co-infection of tuberculosis (TB) and Aspergillus infecting joints. We report a case of co-infection of TB and A. flavus of hip and knee of a 60-year-old male, with type 2 diabetes mellitus. He was treated with debridement, intravenous voriconazole, and antitubercular drugs. PMID:27293296

  3. Aspergillus oerlinghausenensis, a new mould species closely related to A. fumigatus.

    PubMed

    Houbraken, Jos; Weig, Michael; Groß, Uwe; Meijer, Martin; Bader, Oliver

    2016-02-01

    Two isolates belonging to Aspergillus section Fumigati were recovered from German soil on itraconazole containing agar media. Phylogenetic analysis and phenotypic characterization of both isolates show that they represent a novel species named Aspergillus oerlinghausenensis (holotype CBS H-22119(HT), ex-type CBS 139183(T) = IBT 33878 = DTO 316-A3). The species is phylogenetically related to A. fischeri and A. fumigatus. Aspergillus oerlinghausenensis can be differentiated from A. fischeri by its higher growth rate at 50°C. Furthermore, A. oerlinghausenensis is protoheterothallic as only the MAT1-1 idiomorph was detected, while A. fischeri is homothallic. The species differs from A. fumigatus by a weak sporulation on malt extract agar at 25°C, a floccose colony texture on Czapek yeast extract agar and malt extract agar and subglobose instead of subclavate vesicles. The cyp51A promoter region of A. oerlinghausenensis deviates from the previously reported cyp51A promoter regions in A. fumigatus and potentially presents a novel azole resistance conferring modification. Due to the close relationship of A. oerlinghausenensis with A. fischeri and A. fumigatus, this species is placed in a good position for comparative studies involving these species. PMID:26667219

  4. DNA typing of epidemiologically-related isolates of Aspergillus fumigatus.

    PubMed Central

    Birch, M.; Nolard, N.; Shankland, G. S.; Denning, D. W.

    1995-01-01

    Invasive aspergillosis is often nosocomially acquired and carries a high mortality. Molecular typing methods to discriminate isolates have now been developed. Using simple restriction endonuclease (Sal1 and Xho1) digestion of total genomic DNA, we have typed 25 epidemiologically-related isolates of A. fumigatus from six hospital episodes of invasive aspergillosis. Eight DNA types were found and in each case the DNA type matched precisely the epidemiological data. Thus DNA typing of A. fumigatus can provide the means to match isolates from linked sources and distinguish isolates from diverse origins. Images Fig. 1 PMID:7867735

  5. The Aspergillus fumigatus Septins Play Pleiotropic Roles in Septation, Conidiation, and Cell Wall Stress, but are Dispensable for Virulence

    PubMed Central

    Vargas-Muñiz, José M.; Renshaw, Hilary; Richards, Amber D.; Lamoth, Frédéric; Soderblom, Erik J.; Moseley, M. Arthur; Juvvadi, Praveen R.; Steinbach, William J.

    2015-01-01

    Septins are a conserved family of GTPases that regulate important cellular processes such as cell wall integrity, and septation in fungi. The requirement of septins for virulence has been demonstrated in the human pathogenic yeasts Candida albicans and Cryptococcus neoformans, as well as the plant pathogen Magnaporthe oryzae. Aspergillus spp. contains five genes encoding for septins (aspA-E). While the importance of septins AspA, AspB, AspC, and AspE for growth and conidiation has been elucidated in the filamentous fungal model Aspergillus nidulans, nothing is known on the role of septins in growth and virulence in the human pathogen Aspergillus fumigatus. Here we deleted all five A. fumigatus septins, and generated certain double and triple septin deletion strains. Phenotypic analyses revealed that while all the septins are dispensable in normal growth conditions, AspA, AspB, AspC and AspE are required for regular septation. Furthermore, deletion of only the core septin genes significantly reduced conidiation. Concomitant with the absence of an electron-dense outer conidial wall, the ΔaspB strain was also sensitive to anti-cell wall agents. Infection with the ΔaspB strain in a Galleria mellonella model of invasive aspergillosis showed hypervirulence, but no virulence difference was noted when compared to the wild-type strain in a murine model of invasive aspergillosis. Although the deletion of aspB resulted in increased release of TNF-α from the macrophages, no significant inflammation differences in lung histology was noted between the ΔaspB strain and the wild-type strain. Taken together, these results point to the importance of septins in A. fumigatus growth, but not virulence in a murine model. PMID:26051489

  6. The RodA hydrophobin on Aspergillus fumigatus spores masks Dectin-1 and Dectin-2 dependent responses and enhances fungal survival in vivo

    PubMed Central

    de Jesus Carrion, Steven; Leal, Sixto M.; Ghannoum, Mahmoud A.; Pearlman, Eric

    2014-01-01

    Aspergillus and Fusarium species are important causes of fungal infections worldwide. Airborne spores (conidia) of these filamentous fungi express a surface protein that confers hydrophobicity (hydrophobin), and which covers cell wall components that would otherwise induce a host immune cell response. Using a mutant Aspergillus fumigatus strain that does not express the RodA hydrophobin (ΔrodA), and Aspergillus and Fusarium conidia from clinical isolates that were treated with hydrofluoric acid (HF, which removes the A. fumigatus RodA protein), we observed increased surface exposure of β1,3-glucan and α-mannose on Aspergillus and Fusarium conidia. We also found that ΔrodA and HF treated conidia stimulate significantly higher NF-κB p65 nuclear translocation and cytokine production by macrophages from C57BL/6, but not from Dectin-1−/− or Dectin-2−/− mice. Using a murine model of A. fumigatus corneal infection, we found that ΔrodA conidia exhibited increased cytokine production, neutrophil infiltration, and more rapid fungal clearance from C57BL/6 corneas compared with the parent G10 strain, which was dependent on Dectin-1 and Dectin-2. Together, these findings identify the hydrophobin RodA as a virulence factor that masks Dectin-1 and Dectin-2 recognition of conidia, resulting in impaired neutrophil recruitment to the cornea and increased fungal survival and clinical disease. PMID:23926321

  7. Culture condition-dependent metabolite profiling of Aspergillus fumigatus with antifungal activity.

    PubMed

    Kang, Daejung; Son, Gun Hee; Park, Hye Min; Kim, Jiyoung; Choi, Jung Nam; Kim, Hyang Yeon; Lee, Sarah; Hong, Seung-Beom; Lee, Choong Hwan

    2013-03-01

    Three sections of Aspergillus (five species, 21 strains) were classified according to culture medium-dependent and time-dependent secondary metabolite profile-based chemotaxonomy. Secondary metabolites were analysed by liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS-MS) and multivariate statistical methods. From the Aspergillus sections that were cultured on malt extract agar (MEA) and Czapek yeast extract agar (CYA) for 7, 12, and 16 d, Aspergillus sections Fumigati (A. fumigatus), Nigri (A. niger), and Flavi (A. flavus, A. oryzae, and A. sojae) clustered separately on the basis of the results of the secondary metabolite analyses at 16 d regardless of culture medium. Based on orthogonal projection to latent structures discriminant analysis by partial least squares discriminant analysis (PLS-DA), we identified the secondary metabolites that helped differentiate sections between A. fumigatus and Aspergillus section Flavi to be gliotoxin G, fumigatin oxide, fumigatin, pseurotin A or D, fumiquinazoline D, fumagillin, helvolic acid, 1,2-dihydrohelvolic acid, and 5,8-dihydroxy-9,12-octadecadienoic acid (5,8-diHODE). Among these compounds, fumagillin, helvolic acid, and 1,2-dihydrohelvolic acid of A. fumigatus showed antifungal activities against Malassezia furfur, which is lipophilic yeast that causes epidermal skin disorders. PMID:23537878

  8. Screening-based discovery of Aspergillus fumigatus plant-type chitinase inhibitors.

    PubMed

    Lockhart, Deborah E A; Schuettelkopf, Alexander; Blair, David E; van Aalten, Daan M F

    2014-08-25

    A limited therapeutic arsenal against increasing clinical disease due to Aspergillus spp. necessitates urgent characterisation of new antifungal targets. Here we describe the discovery of novel, low micromolar chemical inhibitors of Aspergillus fumigatus family 18 plant-type chitinase A1 (AfChiA1) by high-throughput screening (HTS). Analysis of the binding mode by X-ray crystallography confirmed competitive inhibition and kinetic studies revealed two compounds with selectivity towards fungal plant-type chitinases. These inhibitors provide new chemical tools to probe the effects of chitinase inhibition on A. fumigatus growth and virulence, presenting attractive starting points for the development of further potent drug-like molecules. PMID:25063338

  9. Can airborne fungal allergens pass through an air-conditioning system. [Aspergillus fumigatus

    SciTech Connect

    Elixmann, J.H. ); Linskens, H.F.; Schata, M.; Jorde, W. )

    1989-01-01

    Fungal spores, an important fraction of aeroplankton particles, can be filtered in an air-conditioning system, resulting in a drastic reduction of the spore count in the air-conditioned rooms. Nevertheless, using the EISA inhibition test against Aspergillus fumigatus, it was found that air samples from air-conditioned rooms show inhibition of the serum activity of a highly sensitized patient. There is evidence that airborne allergens can pass both coarse and fine filters of an air-conditioning system.

  10. Secondary metabolites from Aspergillus fumigatus, an endophytic fungus from the liverwort Heteroscyphus tener (Steph.) Schiffn.

    PubMed

    Xie, Fei; Li, Xiao-Bin; Zhou, Jin-Chuan; Xu, Qing-Qing; Wang, Xiao-Ning; Yuan, Hui-Qing; Lou, Hong-Xiang

    2015-09-01

    Three new metabolites, asperfumigatin (1), isochaetominine (10), and 8'-O-methylasterric acid (21), together with nineteen known compounds, were obtained from the culture of Aspergillus fumigatus, an endophytic fungus from the Chinese liverwort Heteroscyphus tener (Steph.) Schiffn. Their structures were established by extensive analysis of the spectroscopic data. The absolute configurations of 1 and 10 were determined by analysis of their respective CD spectra. Cytotoxicity of these isolates against four human cancer cell lines was also determined. PMID:26363876

  11. Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

    PubMed Central

    Fuller, Kevin K.; Chen, Shan

    2015-01-01

    Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen. PMID:26318395

  12. Investigation of Aspergillus fumigatus biofilm formation by various “omics” approaches

    PubMed Central

    Muszkieta, Laetitia; Beauvais, Anne; Pähtz, Vera; Gibbons, John G.; Anton Leberre, Véronique; Beau, Rémi; Shibuya, Kazutoshi; Rokas, Antonis; Francois, Jean M.; Kniemeyer, Olaf; Brakhage, Axel A.; Latgé, Jean P.

    2013-01-01

    In the lung, Aspergillus fumigatus usually forms a dense colony of filaments embedded in a polymeric extracellular matrix called biofilm (BF). This extracellular matrix embeds and glues hyphae together and protects the fungus from an outside hostile environment. This extracellular matrix is absent in fungal colonies grown under classical liquid shake conditions (PL), which were historically used to understand A. fumigatus pathobiology. Recent works have shown that the fungus in this aerial grown BF-like state exhibits reduced susceptibility to antifungal drugs and undergoes major metabolic changes that are thought to be associated to virulence. These differences in pathological and physiological characteristics between BF and liquid shake conditions suggest that the PL condition is a poor in vitro disease model. In the laboratory, A. fumigatus mycelium embedded by the extracellular matrix can be produced in vitro in aerial condition using an agar-based medium. To provide a global and accurate understanding of A. fumigatus in vitro BF growth, we utilized microarray, RNA-sequencing, and proteomic analysis to compare the global gene and protein expression profiles of A. fumigatus grown under BF and PL conditions. In this review, we will present the different signatures obtained with these three “omics” methods. We will discuss the advantages and limitations of each method and their complementarity. PMID:23407341

  13. Molecular Characterization of the Putative Transcription Factor SebA Involved in Virulence in Aspergillus fumigatus

    PubMed Central

    Dinamarco, Taísa Magnani; Almeida, Ricardo S.; Alves de Castro, Patrícia; Brown, Neil Andrew; dos Reis, Thaila Fernanda; Zambelli Ramalho, Leandra Naira; Savoldi, Marcela; Goldman, Maria Helena S.

    2012-01-01

    Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for A. fumigatus virulence and survival in the mammalian host. This study characterized the A. fumigatus SebA transcription factor, which is the putative homologue of the factor encoded by Trichoderma atroviride seb1. The ΔsebA mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl2, and poor nutritional conditions, while viability associated with sebA was also affected by heat shock exposure. Accordingly, SebA::GFP (SebA::green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the ΔsebA mutant. The A. fumigatus ΔsebA strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the ΔsebA mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain. A. fumigatus SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses. PMID:22345349

  14. Molecular characterization of the putative transcription factor SebA involved in virulence in Aspergillus fumigatus.

    PubMed

    Dinamarco, Taísa Magnani; Almeida, Ricardo S; de Castro, Patrícia Alves; Brown, Neil Andrew; dos Reis, Thaila Fernanda; Ramalho, Leandra Naira Zambelli; Savoldi, Marcela; Goldman, Maria Helena S; Goldman, Gustavo Henrique

    2012-04-01

    Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for A. fumigatus virulence and survival in the mammalian host. This study characterized the A. fumigatus SebA transcription factor, which is the putative homologue of the factor encoded by Trichoderma atroviride seb1. The ΔsebA mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl2, and poor nutritional conditions, while viability associated with sebA was also affected by heat shock exposure. Accordingly, SebA::GFP (SebA::green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the ΔsebA mutant. The A. fumigatus ΔsebA strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the ΔsebA mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain. A. fumigatus SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses. PMID:22345349

  15. Identification of fibrinogen-binding proteins of Aspergillus fumigatus using proteomic approach.

    PubMed

    Upadhyay, Santosh Kumar; Gautam, Poonam; Pandit, Hrishikesh; Singh, Yogendra; Basir, Seemi Farhat; Madan, Taruna

    2012-03-01

    Aspergillus fumigatus, the main etiological agent for various forms of human aspergillosis, gets access to the respiratory system of human host by inhalation of airborne conidia. These conidia possibly adhere to extracellular matrix (ECM) proteins. Among the ECM proteins involved in adherence, fibrinogen is thought to be crucial. Here, we studied whether A. fumigatus three-week culture filtrate (3wcf) proteins promote binding of A. fumigatus to ECM proteins and promote fungal growth. We observed that incubation of ECM with 3wcf proteins led to dose- and time-dependent increase in adherence of conidia to the ECM. In order to identify the catalogue of fibrinogen-binding A. fumigatus proteins, we carried out fibrinogen affinity blotting using two-dimensional gel electrophoresed 3wcf proteins. A total of 15 fibrinogen-binding protein spots corresponding to 7 unique proteins were identified in 3wcf using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF). Among these, 4 proteins, namely, beta-glucosidase, alpha-mannosidase, pectate lyase A and oryzin precursor were predicted to have cell wall or extracellular localization, whereas amidase family protein and two hypothetical proteins did not display the signal sequence. This study reports seven novel fibrinogen-binding proteins of A. fumigatus, some of which could be further explored for targeting the adhesion phenomenon as antifungal strategy. PMID:21870122

  16. Dectin-1 agonist curdlan modulates innate immunity to Aspergillus fumigatus in human corneal epithelial cells

    PubMed Central

    Zhu, Cheng-Cheng; Zhao, Gui-Qiu; Lin, Jing; Hu, Li-Ting; Xu, Qiang; Peng, Xu-Dong; Wang, Xue; Qiu, Sheng

    2015-01-01

    AIM To explore the immunomodulatory effects of curdlan on innate immune responses against Aspergillus fumigatus (A. fumigatus) in cultured human corneal epithelial cells (HCECs), and whether C-type lectin receptor Dectin-1 mediates the immunomodulatory effects of curdlan. METHODS The HCECs were stimulated by curdlan in different concentrations (50, 100, 200, 400 µg/mL) for various time. Then HCECs pretreated with or without laminarin (Dectin-1 blocker, 0.3 mg/mL) and curdlan were stimulated by A. fumigatus hyphae. The mRNA and protein production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The protein level of Dectin-1 was measured by Western blot. RESULTS Curdlan stimulated mRNA expression of TNF-α and IL-6 in a dose and time dependent manner in HCECs. Curdlan pretreatment before A. fumigatus hyphae stimulation significantly enhanced the expression of TNF-α and IL-6 at mRNA and protein levels compared with A. fumigatus hyphae stimulation group (P<0.05). Both curdlan and A. fumigatus hyphae up-regulated Dectin-1 protein expression in HCECs, and Dectin-1 expression was elevated to 1.5- to 2-fold by curdlan pretreatment followed hyphae stimulation. The Dectin-1 blocker laminarin suppressed the mRNA expression and protein production of TNF-α and IL-6 induced by curdlan and hyphae (P<0.05). CONCLUSION These findings demonstrated that curdlan pretreatment enhanced the inflammatory response induced by A. fumigatus hyphae in HCECs. Dectin-1 is essential for the immunomodulatory effects of curdlan. Curdlan may have high clinical application values in fungal keratitis treatment. PMID:26309863

  17. Expression and secretion of Aspergillus fumigatus proteases are regulated in response to different protein substrates

    PubMed Central

    Farnell, Edward; Rousseau, Karine; Thornton, David J.; Bowyer, Paul; Herrick, Sarah E.

    2012-01-01

    The ubiquitous filamentous fungus Aspergillus fumigatus secretes a number of allergens with protease activity and has been linked to a variety of allergic conditions such as Severe Asthma with Fungal Sensitization (SAFS) and Allergic Bronchopulmonary Aspergillosis (ABPA). However, it is unclear which allergen proteases are being secreted during fungal invasion and whether the local biological environment regulates their expression. Understanding the dynamic expression of allergen proteases during growth of A. fumigatus may lead to further characterisation of the pathogenesis of these disorders as well as improved standardisation in the commercial production of these allergens. Secretion of proteases during germination and early growth of A. fumigatus was investigated in response to various complex protein sources (pig lung homogenate, mucin or casein). Protease inhibitor studies demonstrated that A. fumigatus (AF293 strain) secretes predominately serine proteases during growth in pig lung based medium and mainly metalloproteases during growth in casein based medium but suppressed protease secretion in unmodified Vogel's minimal medium and secreted both types in mucin based medium. Analysis of gene transcription and protein identification by mass spectrometry showed that the matrix metalloprotease, Mep/Asp f 5 and the serine protease, Alp1/Asp f 13, were upregulated and secreted during growth in pig lung medium, whereas Alp1 was predominately expressed and secreted in mucin based medium. In casein medium, the matrix metalloprotease, Lap1, was also upregulated and secreted in addition to Mep and Alp1. These findings suggest that A. fumigatus is able to detect different complex proteins available as substrates in its environment and regulate protease secretion accordingly. There is a requirement for the standardisation of A. fumigatus allergen extracts used both in clinical diagnosis of A. fumigatus allergy and in research studies. PMID:22954343

  18. Disruption of a Nonribosomal Peptide Synthetase in Aspergillus fumigatus Eliminates Gliotoxin Production

    PubMed Central

    Cramer, Robert A.; Gamcsik, Michael P.; Brooking, Rhea M.; Najvar, Laura K.; Kirkpatrick, William R.; Patterson, Thomas F.; Balibar, Carl J.; Graybill, John R.; Perfect, John R.; Abraham, Soman N.; Steinbach, William J.

    2006-01-01

    The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in ΔgliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the ΔgliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the ΔgliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection. PMID:16757745

  19. Aspergillus fumigatus mitochondrial electron transport chain mediates oxidative stress homeostasis, hypoxia responses, and fungal pathogenesis

    PubMed Central

    Grahl, Nora; Dinamarco, Taisa Magnani; Willger, Sven D.; Goldman, Gustavo H.; Cramer, Robert A.

    2012-01-01

    Summary We previously observed that hypoxia is an important component of host microenvironments during pulmonary fungal infections. However, mechanisms of fungal growth in these in vivo hypoxic conditions are poorly understood. Here, we report that mitochondrial respiration is active in hypoxia (1% oxygen) and critical for fungal pathogenesis. We generated Aspergillus fumigatus alternative oxidase (aoxA) and cytochrome C (cycA) null mutants and assessed their ability to tolerate hypoxia, macrophage killing, and virulence. In contrast to ΔaoxA, ΔcycA was found to be significantly impaired in conidia germination, growth in normoxia and hypoxia, and displayed attenuated virulence. Intriguingly, loss of cycA results in increased levels of AoxA activity, which results in increased resistance to oxidative stress, macrophage killing, and long-term persistence in murine lungs. Thus, our results demonstrate a previously unidentified role for fungal mitochondrial respiration in the pathogenesis of aspergillosis, and lay the foundation for future research into its role in hypoxia signaling and adaptation. PMID:22443190

  20. Binding of pulmonary surfactant proteins A and D to Aspergillus fumigatus conidia enhances phagocytosis and killing by human neutrophils and alveolar macrophages.

    PubMed Central

    Madan, T; Eggleton, P; Kishore, U; Strong, P; Aggrawal, S S; Sarma, P U; Reid, K B

    1997-01-01

    To determine whether the lung surfactant proteins A (SP-A) and D (SP-D) are involved in the initial protective immunity against opportunistic pulmonary fungal infections caused by Aspergillus fumigatus, we performed a series of in vitro functional studies to see if SP-A and SP-D enhanced binding, phagocytosis, activation, and killing of A. fumigatus conidia by human alveolar macrophages and circulating neutrophils. Both SP-A and SP-D bound to carbohydrate structures on A. fumigatus conidia in a calcium-dependent manner. SP-A and SP-D were also chemoattractant and significantly enhanced agglutination and binding of conidia to alveolar macrophages and neutrophils. Furthermore, in the presence of SP-A and SP-D, the phagocytosis, oxidative burst, and killing of A. fumigatus conidia by neutrophils were significantly increased. These findings indicate that SP-A and SP-D may have an important immunological role in the early antifungal defense responses in the lung, through inhibiting infectivity of conidia by agglutination and by enhancing uptake and killing of A. fumigatus by phagocytic cells. PMID:9234771

  1. An alternative host model of a mixed fungal infection by azole susceptible and resistant Aspergillus spp strains.

    PubMed

    Alcazar-Fuoli, L; Buitrago, Mj; Gomez-Lopez, A; Mellado, E

    2015-01-01

    Aspergillus fumigatus is the most common mold involved in human infections. However, the number of non-fumigatus species able to cause disease is continuously increasing. Among them, Aspergillus lentulus is reported in hematological and cystic fibrosis patients and in those treated with corticosteroids. A. lentulus differs from A. fumigatus in some clinically relevant aspects such as virulence and antifungal susceptibility, showing high MICs to most antifungals. Previous studies proved that A. lentulus was pathogenic in immunocompromised mice, although the course of the infection was delayed compared to A. fumigatus. These differences could explain why A. lentulus is mostly found in mixed infections with A. fumigatus challenging the diagnosis and treatment. We used the alternative model host Galleria mellonella to compare virulence, host interaction, fungal burden and antifungal response when larvae were infected with A. fumigatus or A. lentulus alone, and with a mixture of both species. A. lentulus was pathogenic in G. mellonella but infected larvae did not respond to therapeutic doses of voriconazole. We were able to simultaneously detect A. fumigatus and A. lentulus by a multiplex Nested Real Time PCR (MN-PCR). Comparative analysis of larvae histological sections showed melanization of both species but presented a different pattern of immune response by haemocytes. Analysis of fungal burden and histology showed that A. lentulus survived in the G. mellonella despite the antifungal treatment in single and mixed infections. We conclude that the simultaneous presence of antifungal susceptible and resistant Aspergillus species would likely complicate the management of these infections. PMID:26065322

  2. Regulation of Sulphur Assimilation Is Essential for Virulence and Affects Iron Homeostasis of the Human-Pathogenic Mould Aspergillus fumigatus

    PubMed Central

    Amich, Jorge; Schafferer, Lukas; Haas, Hubertus; Krappmann, Sven

    2013-01-01

    Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen. PMID:24009505

  3. Nonribosomal peptide synthetase genes pesL and pes1 are essential for Fumigaclavine C production in Aspergillus fumigatus.

    PubMed

    O'Hanlon, Karen A; Gallagher, Lorna; Schrettl, Markus; Jöchl, Christoph; Kavanagh, Kevin; Larsen, Thomas O; Doyle, Sean

    2012-05-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H(2)O(2), and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P < 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for A. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both A. fumigatus wild-type and ΔpesL strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis in vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature. PMID:22344643

  4. Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

    PubMed Central

    O'Hanlon, Karen A.; Gallagher, Lorna; Schrettl, Markus; Jöchl, Christoph; Kavanagh, Kevin; Larsen, Thomas O.

    2012-01-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P < 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for A. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both A. fumigatus wild-type and ΔpesL strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis in vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature. PMID:22344643

  5. Dsc orthologs are required for hypoxia adaptation, triazole drug responses, and fungal virulence in Aspergillus fumigatus.

    PubMed

    Willger, Sven D; Cornish, E Jean; Chung, Dawoon; Fleming, Brittany A; Lehmann, Margaret M; Puttikamonkul, Srisombat; Cramer, Robert A

    2012-12-01

    Hypoxia is an environmental stress encountered by Aspergillus fumigatus during invasive pulmonary aspergillosis (IPA). The ability of this mold to adapt to hypoxia is important for fungal virulence and genetically regulated in part by the sterol regulatory element binding protein (SREBP) SrbA. SrbA is required for fungal growth in the murine lung and to ultimately cause lethal disease in murine models of IPA. Here we identified and partially characterized four genes (dscA, dscB, dscC, and dscD, here referred to as dscA-D) with previously unknown functions in A. fumigatus that are orthologs of the Schizosaccharomyces pombe genes dsc1, dsc2, dsc3, and dsc4 (dsc1-4), which encode a Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. A. fumigatus null dscA-D mutants displayed remarkable defects in hypoxic growth and increased susceptibility to triazole antifungal drugs. Consistent with the confirmed role of these genes in S. pombe, both ΔdscA and ΔdscC resulted in reduced cleavage of the SrbA precursor protein in A. fumigatus. Inoculation of corticosteroid immunosuppressed mice with ΔdscA and ΔdscC strains revealed that these genes are critical for A. fumigatus virulence. Reintroduction of SrbA amino acids 1 to 425, encompassing the N terminus DNA binding domain, into the ΔdscA strain was able to partially restore virulence, further supporting a mechanistic link between DscA and SrbA function. Thus, we have shown for the first time the importance of a previously uncharacterized group of genes in A. fumigatus that mediate hypoxia adaptation, fungal virulence, and triazole drug susceptibility and that are likely linked to regulation of SrbA function. PMID:23104569

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

    PubMed Central

    Winkelströter, Lizziane K.; Dolan, Stephen K.; Fernanda dos Reis, Thaila; Bom, Vinícius Leite Pedro; Alves de Castro, Patrícia; Hagiwara, Daisuke; Alowni, Raneem; Jones, Gary W.; Doyle, Sean; Brown, Neil Andrew; Goldman, Gustavo H.

    2015-01-01

    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. PMID:25943523

  7. Pulmonary immune responses to Aspergillus fumigatus in an immunocompetent mouse model of repeated exposures.

    PubMed

    Buskirk, Amanda D; Templeton, Steven P; Nayak, Ajay P; Hettick, Justin M; Law, Brandon F; Green, Brett J; Beezhold, Donald H

    2014-01-01

    Aspergillus fumigatus is a filamentous fungus that produces abundant pigmented conidia. Several fungal components have been identified as virulence factors, including melanin; however, the impact of these factors in a repeated exposure model resembling natural environmental exposures remains unknown. This study examined the role of fungal melanin in the stimulation of pulmonary immune responses using immunocompetent BALB/c mice in a multiple exposure model. It compared conidia from wild-type A. fumigatus to two melanin mutants of the same strain, Δarp2 (tan) or Δalb1 (white). Mass spectrometry-based analysis of conidial extracts demonstrated that there was little difference in the protein fingerprint profiles between the three strains. Field emission scanning electron microscopy demonstrated that the immunologically inert Rodlet A layer remained intact in melanin-deficient conidia. Thus, the primary difference between the strains was the extent of melanization. Histopathology indicated that each A. fumigatus strain induced lung inflammation, regardless of the extent of melanization. In mice exposed to Δalb1 conidia, an increase in airway eosinophils and a decrease in neutrophils and CD8(+) IL-17(+) (Tc17) cells were observed. Additionally, it was shown that melanin mutant conidia were more rapidly cleared from the lungs than wild-type conidia. These data suggest that the presence of fungal melanin may modulate the pulmonary immune response in a mouse model of repeated exposures to A. fumigatus conidia. PMID:23919459

  8. High osmolarity glycerol response PtcB phosphatase is important for Aspergillus fumigatus virulence.

    PubMed

    Winkelströter, Lizziane K; Bom, Vinícius Leite Pedro; de Castro, Patrícia Alves; Ramalho, Leandra Naira Zambelli; Goldman, Maria Helena S; Brown, Neil Andrew; Rajendran, Ranjith; Ramage, Gordon; Bovier, Elodie; Dos Reis, Thaila Fernanda; Savoldi, Marcela; Hagiwara, Daisuke; Goldman, Gustavo H

    2015-04-01

    Aspergillus fumigatus is a fungal pathogen that is capable of adapting to different host niches and to avoid host defenses. An enhanced understanding of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes is essential for the development of improved disease control strategies. Protein phosphatases are central to numerous signal transduction pathways. To comprehend the functions of protein phosphatases in A. fumigatus, 32 phosphatase catalytic subunit encoding genes were identified. We have recognized PtcB as one of the phosphatases involved in the high osmolarity glycerol response (HOG) pathway. The ΔptcB mutant has both increased phosphorylation of the p38 MAPK (SakA) and expression of osmo-dependent genes. The ΔptcB strain was more sensitive to cell wall damaging agents, had increased chitin and β-1,3-glucan, and impaired biofilm formation. The ΔptcB strain was avirulent in a murine model of invasive pulmonary aspergillosis. These results stress the importance of the HOG pathway in the regulation of pathogenicity determinants and virulence in A. fumigatus. PMID:25597841

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

    PubMed

    Winkelströter, Lizziane K; Dolan, Stephen K; Fernanda Dos Reis, Thaila; Bom, Vinícius Leite Pedro; Alves de Castro, Patrícia; Hagiwara, Daisuke; Alowni, Raneem; Jones, Gary W; Doyle, Sean; Brown, Neil Andrew; Goldman, Gustavo H

    2015-07-01

    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. PMID:25943523

  10. Pulmonary immune responses to Aspergillus fumigatus in an immunocompetent mouse model of repeated exposures

    PubMed Central

    Buskirk, Amanda D.; Templeton, Steven P.; Nayak, Ajay P.; Hettick, Justin M.; Law, Brandon F.; Green, Brett J.; Beezhold, Donald H.

    2015-01-01

    Aspergillus fumigatus is a filamentous fungus that produces abundant pigmented conidia. Several fungal components have been identified as virulence factors, including melanin; however, the impact of these factors in a repeated exposure model resembling natural environmental exposures remains unknown. This study examined the role of fungal melanin in the stimulation of pulmonary immune responses using immunocompetent BALB/c mice in a multiple exposure model. It compared conidia from wild-type A. fumigatus to two melanin mutants of the same strain, Δarp2 (tan) or Δalb1 (white). Mass spectrometry-based analysis of conidial extracts demonstrated that there was little difference in the protein fingerprint profiles between the three strains. Field emission scanning electron microscopy demonstrated that the immunologically inert Rodlet A layer remained intact in melanin-deficient conidia. Thus, the primary difference between the strains was the extent of melanization. Histopathology indicated that each A. fumigatus strain induced lung inflammation, regardless of the extent of melanization. In mice exposed to Δalb1 conidia, an increase in airway eosinophils and a decrease in neutrophils and CD8+ IL-17+ (Tc17) cells were observed. Additionally, it was shown that melanin mutant conidia were more rapidly cleared from the lungs than wild-type conidia. These data suggest that the presence of fungal melanin may modulate the pulmonary immune response in a mouse model of repeated exposures to A. fumigatus conidia. PMID:23919459

  11. The allergenicity of Aspergillus fumigatus conidia is influenced by growth temperature.

    PubMed

    Low, Swee Yang; Dannemiller, Karen; Yao, Maosheng; Yamamoto, Naomichi; Peccia, Jordan

    2011-07-01

    Common indoor and outdoor environmental fungi such as Aspergillus fumigatus produce asexual spores containing a collection of proteins that can bind IgE antibodies and trigger allergic reactions. We characterized the impact of sporulation temperature on the IgE-binding capacity (allergenicity) of A. fumigatus and explored the links between variable allergenicity and temperature-dependant expression of genes encoding these allergenic proteins. A 12-fold increase in A. fumigatus allergenicity per spore was observed when sporulation temperatures were decreased from 32°C to 17°C. Per spore protein mass and Asp f 1 allergen mass also followed this trend. Functional gene expression analysis of A. fumigatus sporulating cultures by real-time reverse-transcription PCR and gene expression microarrays revealed that a greater number of genes encoding known, major allergens are more highly expressed at lower sporulation temperatures. The results of this study indicate that environmental conditions at growth significantly influence the allergenicity of this common mould through the differential production of allergenic proteins, and highlight the importance of in vivo or in vitro allergenicity measurements for understanding environmental exposure to airborne allergenic fungi. PMID:21724168

  12. Verruculogen production in airborne and clinical isolates of Aspergillus fumigatus Fres.

    PubMed

    Kosalec, Ivan; Klarić, Maja Segvić; Pepeljnjak, Stjepan

    2005-12-01

    Among airborne aspergilli sampled in outdoor air of the Zagreb area (2002/2003), Aspergillus niger (v. Teigh.) and A. fumigatus (Fres.) were the most abundant species (20-30%), with low mean annual concentrations (0.21-1.04 CFU m-3). Higher concentrations of A. fumigatus were observed in autumn and winter (0.5-1.05 CFU m-3) than in spring and summer (0-0.4 CFU m-3). On the other hand, A. fumigatus was found to be the most frequent isolate from upper and/or lower respiratory tracts of imunocompromised patients in many studies. This species produces several mycotoxins, including the tremorgenic mycotoxin verruculogen that can be found in spores and during myceliar growth. Verruculogen production ability was tested on 30 airborne and 33 clinical isolates of A. fumigatus. In both groups, high percentage of verruculogen-producing strains was noticed (84% of airborne and 91% of clinical isolates). Verruculogen production was not significantly different in the groups of airborne isolates (0.34+/-0.16 mg mL-1), and clinical isolates (0.26+/-0.19 mg mL-1). PMID:16375825

  13. cyp51A gene silencing using RNA interference in azole-resistant Aspergillus fumigatus.

    PubMed

    Mousavi, Bita; Hedayati, Mohammad T; Teimoori-Toolabi, Ladan; Guillot, Jacques; Alizadeh, Ahad; Badali, Hamid

    2015-12-01

    An increasing number of reports have described the emergence of acquired resistance of Aspergillus fumigatus to azole compounds. The primary mechanism of resistance in clinical isolates is the mutation of the azole drug target enzyme, which is encoded by the cyp51A gene. The aim of this study was to evaluate the impact of silencing the cyp51A gene in azole-resistant A. fumigatus isolates. A 21-nucleotide small-interfering RNA (siRNA) was designed based on the cDNA sequence of the A. fumigatus cyp51A gene. After silencing the cyp51A gene in germinated conidia (15, 20, 25 and 50 nM), azole-resistant A. fumigatus was cultured on broth media and gene expression was analysed by measuring the cyp51A mRNA level using RT-PCR assay. Hyphae were successfully transfected by siRNA and expression of the cyp51A gene was significantly reduced by siRNA at the concentration of 50 nM (P ≤ 0.05). In addition, at this siRNA concentration, the minimum inhibitory concentration of itraconazole for the treated cells was decreased, compared with that for untreated control cells, from 16 to 4 μg/ml. PMID:26448519

  14. IgE Sensitization to Aspergillus fumigatus Is Associated with Reduced Lung Function in Asthma

    PubMed Central

    Fairs, Abbie; Agbetile, Joshua; Hargadon, Beverley; Bourne, Michelle; Monteiro, William R.; Brightling, Christopher E.; Bradding, Peter; Green, Ruth H.; Mutalithas, Kugathasan; Desai, Dhananjay; Pavord, Ian D.; Wardlaw, Andrew J.; Pashley, Catherine H.

    2010-01-01

    Rationale: The importance of Aspergillus fumigatus sensitization and colonization of the airways in patients with asthma is unclear. Objectives: To define the relationship between the clinical and laboratory features of A. fumigatus–associated asthma. Methods: We studied 79 patients with asthma (89% classed as GINA 4 or 5) classified into 3 groups according to A. fumigatus sensitization: (1) IgE-sensitized (immediate cutaneous reactivity > 3 mm and/or IgE > 0.35 kU/L); (2) IgG-only–sensitized (IgG > 40 mg/L); and (3) nonsensitized. These were compared with 14 healthy control subjects. Sputum culture was focused toward detection of A. fumigatus and compared with clinical assessment data. Measurements and Main Results: A. fumigatus was cultured from 63% of IgE-sensitized patients with asthma (n = 40), 39% of IgG-only–sensitized patients with asthma (n = 13), 31% of nonsensitized patients with asthma (n = 26) and 7% of healthy control subjects (n = 14). Patients sensitized to A. fumigatus compared with nonsensitized patients with asthma had lower lung function (postbronchodilator FEV1 % predicted, mean [SEM]: 68 [±5]% versus 88 [±5]%; P < 0.05), more bronchiectasis (68% versus 35%; P < 0.05), and more sputum neutrophils (median [interquartile range]: 80.9 [50.1–94.1]% versus 49.5 [21.2–71.4]%; P < 0.01). In a multilinear regression model, A. fumigatus–IgE sensitization and sputum neutrophil differential cell count were important predictors of lung function (P = 0.016), supported by culture of A. fumigatus (P = 0.046) and eosinophil differential cell count (P = 0.024). Conclusions: A. fumigatus detection in sputum is associated with A. fumigatus–IgE sensitization, neutrophilic airway inflammation, and reduced lung function. This supports the concept that development of fixed airflow obstruction in asthma is consequent upon the damaging effects of airway colonization with A. fumigatus. PMID:20639442

  15. Antifungal Activity of Selenium Nanoparticles Synthesized by Bacillus species Msh-1 Against Aspergillus fumigatus and Candida albicans

    PubMed Central

    Shakibaie, Mojtaba; Salari Mohazab, Naser; Ayatollahi Mousavi, Seyyed Amin

    2015-01-01

    Background: Fungal infections affect various parts of the body and can be difficult to treat. Aspergillus infection causes a spectrum of diverse diseases particularly in lung according to host immunity. The two major entities are invasive pulmonary aspergillosis and chronic pulmonary aspergillosis. Candida infections can be superficial or invasive. Superficial infections often affect the skin or mucous membranes. However, invasive fungal infections are often life-threatening. Advances in nanotechnology have opened new horizons in nanomedicine, allowing the synthesis of nanoparticles that can be assembled into complex architectures. Novel studies and technologies are devoted to understanding the mechanisms of disease for the design of new drugs. Objectives: In the present study, the antifungal activity of biogenic selenium nanoparticles (Se NPs) against Aspergillus fumigatus and Candida albicans was investigated. Materials and Methods: Se-reducing bacteria previously identified as Bacillus sp. MSh-1 were used for the intracellular biosynthesis of elemental Se NPs. The shape, size, and purity of the extracted NPs were determined with various instrumental techniques. The nanoparticles antifungal characterization mainly derives from the following pathways: (i) to generate sustained flux of nano-ions from the compounds that deposited on special substrates or imbedded in colloidal or semisolid matrices. (ii) To transport active those ions to sensitive targets on plasma membrane of fungi. Results: The results of energy-dispersive X-ray demonstrated that the purified NPs consisted of only Se. In addition, transmission electron micrographs showed that 120- to 140-nm spherical Se NPs were the most common. An antifungal assay was performed with a standard Clinical and Laboratory Standards Institute broth microdilution method. Minimum inhibitory concentration (MIC) measurements of the antifungal activity of the Se NPs against C. albicans (70 μg/mL) and A. fumigatus (100

  16. Mycotoxins of Aspergillus fumigatus in pure culture and in native bioaerosols from compost facilities.

    PubMed

    Fischer, G; Müller, T; Ostrowski, R; Dott, W

    1999-04-01

    Exposure to secondary metabolites of airborne fungi in waste handling facilities is discussed in regard to potential toxic impacts on human health. The relevance of mycotoxins has been intensely studied in connection with contamination of food and feed. Toxic secondary metabolites are expected to be present in airborne spores, but exposure to mycotoxins in bioaerosols has not been studied sufficiently. Aspergillus fumigatus is one of the most frequent species in the air of compost plants. A wide range of secondary metabolites was found in pure cultures of freshly isolated strains of A. fumigatus. Tryptoquivaline, a compound with tremorgenic properties, and trypacidin, for which no toxic properties are described, were found in native bioaerosols in a compost facility. The highly toxic metabolites gliotoxin and verruculogen were not found in the bioaerosols. PMID:10101846

  17. Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

    PubMed Central

    Richardson, M D; Stubbins, J M; Warnock, D W

    1982-01-01

    A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen 1 in both forms of ELISA, but two failed to react with antigen 2 in the standard ELISA and three failed to react with this antigen in the rapid method. Thirteen AGDD-negative sera were also negative in both forms of ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of allergic aspergillosis. PMID:6813358

  18. Convergent synthesis of isomeric heterosaccharides related to the fragments of galactomannan from Aspergillus fumigatus.

    PubMed

    Argunov, D A; Krylov, V B; Nifantiev, N E

    2015-03-21

    Aspergillus fumigatus is a very common fungus with high pathogenic potential for immunosuppressed hospital patients. A. fumigatus galactomannan, being the part of its cell wall, is considered as a promising candidate for vaccine and diagnostic test-systems. In this article we report the convergent synthesis of pentasaccharide fragments of the galactomannan containing the β-(1→5)-linked galactofuranoside chain attached to O-3 or O-6 of a spacer-armed mannopyranoside residue. The synthesis of selectively protected galactofuranoside precursors has been performed using recently developed pyranoside-into-furanoside (PIF) rearrangement. For assembling the target galactomannan structures the [1 + 2 + 2]-scheme was applied. This strategy was shown to be highly efficient and can easily be extended to the synthesis of longer fragments of thegalactomannan. PMID:25643073

  19. The hypoxia-induced dehydrogenase HorA is required for coenzyme Q10 biosynthesis, azole sensitivity and virulence of Aspergillus fumigatus.

    PubMed

    Kroll, Kristin; Shekhova, Elena; Mattern, Derek J; Thywissen, Andreas; Jacobsen, Ilse D; Strassburger, Maria; Heinekamp, Thorsten; Shelest, Ekaterina; Brakhage, Axel A; Kniemeyer, Olaf

    2016-07-01

    Aspergillus fumigatus is the predominant airborne pathogenic fungus causing invasive aspergillosis in immunocompromised patients. During infection A. fumigatus has to adapt to oxygen-limiting conditions in inflammatory or necrotic tissue. Previously, we identified a mitochondrial protein to be highly up-regulated during hypoxic adaptation. Here, this protein was found to represent the novel oxidoreductase HorA. In Saccharomyces cerevisiae a homologue was shown to play a role in biosynthesis of coenzyme Q. Consistently, reduced coenzyme Q content in the generated ΔhorA mutant indicated a respective function in A. fumigatus. Since coenzyme Q is involved in cellular respiration and maintaining cellular redox homeostasis, the strain ΔhorA displayed an impaired response to both oxidative and reductive stress, a delay in germination and an accumulation of NADH. Moreover, an increased resistance against antifungal drugs was observed. All phenotypes were completely reversed by the addition of the synthetic electron carrier menadione. The deletion strain ΔhorA showed significantly attenuated virulence in two murine infection models of invasive pulmonary aspergillosis. Therefore, the biosynthesis of coenzyme Q and, particularly, the fungal-specific protein HorA play a crucial role in virulence of A. fumigatus. Due to its absence in mammals, HorA might represent a novel therapeutic target against fungal infections. PMID:26991818

  20. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae.

    PubMed

    Galagan, James E; Calvo, Sarah E; Cuomo, Christina; Ma, Li-Jun; Wortman, Jennifer R; Batzoglou, Serafim; Lee, Su-In; Baştürkmen, Meray; Spevak, Christina C; Clutterbuck, John; Kapitonov, Vladimir; Jurka, Jerzy; Scazzocchio, Claudio; Farman, Mark; Butler, Jonathan; Purcell, Seth; Harris, Steve; Braus, Gerhard H; Draht, Oliver; Busch, Silke; D'Enfert, Christophe; Bouchier, Christiane; Goldman, Gustavo H; Bell-Pedersen, Deborah; Griffiths-Jones, Sam; Doonan, John H; Yu, Jaehyuk; Vienken, Kay; Pain, Arnab; Freitag, Michael; Selker, Eric U; Archer, David B; Peñalva, Miguel A; Oakley, Berl R; Momany, Michelle; Tanaka, Toshihiro; Kumagai, Toshitaka; Asai, Kiyoshi; Machida, Masayuki; Nierman, William C; Denning, David W; Caddick, Mark; Hynes, Michael; Paoletti, Mathieu; Fischer, Reinhard; Miller, Bruce; Dyer, Paul; Sachs, Matthew S; Osmani, Stephen A; Birren, Bruce W

    2005-12-22

    The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation. PMID:16372000

  1. Genetic relatedness versus biological compatibility between Aspergillus fumigatus and related species.

    PubMed

    Sugui, Janyce A; Peterson, Stephen W; Figat, Abigail; Hansen, Bryan; Samson, Robert A; Mellado, Emilia; Cuenca-Estrella, Manuel; Kwon-Chung, Kyung J

    2014-10-01

    Aspergillus section Fumigati contains 12 clinically relevant species. Among these Aspergillus species, A. fumigatus is the most frequent agent of invasive aspergillosis, followed by A. lentulus and A. viridinutans. Genealogical concordance and mating experiments were performed to examine the relationship between phylogenetic distance and mating success in these three heterothallic species. Analyses of 19 isolates from section Fumigati revealed the presence of three previously unrecognized species within the broadly circumscribed species A. viridinutans. A single mating type was found in the new species Aspergillus pseudofelis and Aspergillus pseudoviridinutans, but in Aspergillus parafelis, both mating types were present. Reciprocal interspecific pairings of all species in the study showed that the only successful crosses occurred with the MAT1-2 isolates of both A. parafelis and A. pseudofelis. The MAT1-2 isolate of A. parafelis was fertile when paired with the MAT1-1 isolates of A. fumigatus, A. viridinutans, A. felis, A. pseudoviridinutans, and A. wyomingensis but was not fertile with the MAT1-1 isolate of A. lentulus. The MAT1-2 isolates of A. pseudofelis were fertile when paired with the MAT1-1 isolate of A. felis but not with any of the other species. The general infertility in the interspecies crossings suggests that genetically unrelated species are also biologically incompatible, with the MAT1-2 isolates of A. parafelis and A. pseudofelis being the exception. Our findings underscore the importance of genealogical concordance analysis for species circumscription, as well as for accurate species identification, since misidentification of morphologically similar pathogens with differences in innate drug resistance may be of grave consequences for disease management. PMID:25100816

  2. Genetic Relatedness versus Biological Compatibility between Aspergillus fumigatus and Related Species

    PubMed Central

    Sugui, Janyce A.; Peterson, Stephen W.; Figat, Abigail; Hansen, Bryan; Samson, Robert A.; Mellado, Emilia; Cuenca-Estrella, Manuel

    2014-01-01

    Aspergillus section Fumigati contains 12 clinically relevant species. Among these Aspergillus species, A. fumigatus is the most frequent agent of invasive aspergillosis, followed by A. lentulus and A. viridinutans. Genealogical concordance and mating experiments were performed to examine the relationship between phylogenetic distance and mating success in these three heterothallic species. Analyses of 19 isolates from section Fumigati revealed the presence of three previously unrecognized species within the broadly circumscribed species A. viridinutans. A single mating type was found in the new species Aspergillus pseudofelis and Aspergillus pseudoviridinutans, but in Aspergillus parafelis, both mating types were present. Reciprocal interspecific pairings of all species in the study showed that the only successful crosses occurred with the MAT1-2 isolates of both A. parafelis and A. pseudofelis. The MAT1-2 isolate of A. parafelis was fertile when paired with the MAT1-1 isolates of A. fumigatus, A. viridinutans, A. felis, A. pseudoviridinutans, and A. wyomingensis but was not fertile with the MAT1-1 isolate of A. lentulus. The MAT1-2 isolates of A. pseudofelis were fertile when paired with the MAT1-1 isolate of A. felis but not with any of the other species. The general infertility in the interspecies crossings suggests that genetically unrelated species are also biologically incompatible, with the MAT1-2 isolates of A. parafelis and A. pseudofelis being the exception. Our findings underscore the importance of genealogical concordance analysis for species circumscription, as well as for accurate species identification, since misidentification of morphologically similar pathogens with differences in innate drug resistance may be of grave consequences for disease management. PMID:25100816

  3. Aspergillus fumigatus enhances elastase production in Pseudomonas aeruginosa co-cultures.

    PubMed

    Smith, Karen; Rajendran, Ranjith; Kerr, Stephen; Lappin, David F; Mackay, William G; Williams, Craig; Ramage, Gordon

    2015-09-01

    In the cystic fibrosis (CF) lung the presence of bacteria and fungi in the airways promotes an inflammatory response causing progressive lung damage, ultimately leading to high rates of morbidity and mortality. We hypothesized that polymicrobial interactions play an important role in promoting airway pathogenesis. We therefore examined the interplay between the most commonly isolated bacterial CF pathogen, Pseudomonas aeruginosa, and the most prevalent filamentous fungi, Aspergillus fumigatus, to test this. Co-culture experiments showed that in the presence of A. fumigatus the production of P. aeruginosa elastase was enhanced. This was confirmed by the presence of zones of clearance on Elastin-Congo Red (ECR) agar, which was identified as elastase by mass spectrometry. When P. aeruginosa were grown in a co-culture model with mature A. fumigatus biofilms, 60% of isolates produced significantly more elastase in the presence of the filamentous fungi than in its absence (P < .05). The expression of lasB also increased when P. aeruginosa isolates PA01 and PA14 were grown in co-culture with A. fumigatus. Supernatants from co-culture experiments were also significantly toxic to a human lung epithelial cell line (19-38% cell cytotoxicity) in comparison to supernatants from P. aeruginosa only cultures (P < .0001). Here we report that P. aeruginosa cytotoxic elastase is enhanced in the presence of the filamentous fungi A. fumigatus, suggesting that this may have a role to play in the damaging pathology associated with the lung tissue in this disease. This indicates that patients who have a co-colonisation with these two organisms may have a poorer prognosis. PMID:26162475

  4. Contributions of Aspergillus fumigatus ATP-Binding Cassette Transporter Proteins to Drug Resistance and Virulence

    PubMed Central

    Paul, Sanjoy; Diekema, Daniel

    2013-01-01

    In yeast cells such as those of Saccharomyces cerevisiae, expression of ATP-binding cassette (ABC) transporter proteins has been found to be increased and correlates with a concomitant elevation in azole drug resistance. In this study, we investigated the roles of two Aspergillus fumigatus proteins that share high sequence similarity with S. cerevisiae Pdr5, an ABC transporter protein that is commonly overproduced in azole-resistant isolates in this yeast. The two A. fumigatus genes encoding the ABC transporters sharing the highest sequence similarity to S. cerevisiae Pdr5 are called abcA and abcB here. We constructed deletion alleles of these two different ABC transporter-encoding genes in three different strains of A. fumigatus. Loss of abcB invariably elicited increased azole susceptibility, while abcA disruption alleles had variable phenotypes. Specific antibodies were raised to both AbcA and AbcB proteins. These antisera allowed detection of AbcB in wild-type cells, while AbcA could be visualized only when overproduced from the hspA promoter in A. fumigatus. Overproduction of AbcA also yielded increased azole resistance. Green fluorescent protein fusions were used to provide evidence that both AbcA and AbcB are localized to the plasma membrane in A. fumigatus. Promoter fusions to firefly luciferase suggested that expression of both ABC transporter-encoding genes is inducible by azole challenge. Virulence assays implicated AbcB as a possible factor required for normal pathogenesis. This work provides important new insights into the physiological roles of ABC transporters in this major fungal pathogen. PMID:24123268

  5. [Farmer's lung with antibodies against Thermoactinomyces vulgaris and Aspergillus fumigatus. Clinical course and treatment].

    PubMed

    Sánchez Palacios, A; Quintero de Juana, A; Paredes, C; Blanco, M; González, J I

    1985-01-01

    We present a case of Farmer's lung with antibodies to Thermoactinomyces vulgaris and Aspergillus fumigatus. A 56-year-old male patient with an atopic family medical history came to our hospital complaining of cough, dyspnea, fever, asthenia and anorexia. His condition worsened after being exposed to cereal powder, becoming symptomatic after 20 minutes or on occasion after 6 hours. Physical examination showed basal crepitant rales in lung auscultation. The radiograph of the thorax showed a bilateral interstitial reticulo-nodular pattern. An obstructive pattern was found on functional respiratory examination. There was also a slight restriction along with the decrease of the VC and a significant reversal of the M.M.E.F. with anticholinergics. The tests for intradermal cutaneous allergies were positive after 20 minutes and for Aspergillus fumigatus Niger and Terreus (Bencard) after 6 hours. With immunoelectrophoresis and double diffusion, precipitation bands in the presence of Aspergillus fumigatus and Thermoactinomyces, were detected. IgG and IgE were high-1570 mg% and 1000 U/ml respectively. The histological study of the transbronchial biopsy showed dilatation of the alveolar septum caused by a lymphocytic infiltration with fragments of collagenous fibres. In bronchoalveolar lavage there was a predominance of lymphocytes and histiocytes. After exertion, arterial blood gases showed desaturation with hypoxemia. The static lung volumes and the flow and diffusion of carbon monoxide (CO) showed a moderate decrease of vital capacity, with the total lung capacity being below normal. Diffusion was markedly attenuated. The provocation test by indirect bronchial inhalation using cereal powder (Alfalfa) was positive. After six hours dyspnea, cough and leukocytosis appeared with an outbreak of fever and an increase in the erythrocyte sedimentation rate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3909794

  6. Primary vocal cord aspergillosis caused by Aspergillus fumigatus and molecular identification of the isolate.

    PubMed

    Ran, Yuping; Yang, Baiyan; Liu, Suling; Dai, Yaling; Pang, Zongguo; Fan, Jiayu; Bai, Haoru; Liu, Shixi

    2008-08-01

    This is a case of vocal cord aspergillosis in a 36-year-old woman whose chief complaint was progressive hoarseness and vocal fatigue of one month duration. These symptoms followed the use of systemic administration of penicillin, cefotaxime natrium and dexamethasone to treat her rhinitis and asthma. Fiberoptic laryngoscopy revealed whitish plaques involving both vocal cords. Microscopic examination of KOH preparations and histopathology studies of tissue revealed dichotomously branching, hyaline, septate hyphae. Morphological and molecular biological identification of the fungal growth in cultures inoculated with clinical specimens from the patient indicated that the etiologic agent was Aspergillus fumigatus. The patient was cured with oral itraconazole without any side effects. PMID:18636395

  7. Azole Resistance in Aspergillus fumigatus Clinical Isolates from an Italian Culture Collection

    PubMed Central

    Lazzarini, Cristina; Esposto, Maria Carmela; Prigitano, Anna; Cogliati, Massimo; De Lorenzis, Gabriella

    2015-01-01

    The aims of the study were to investigate the prevalence of azole resistance among Aspergillus fumigatus clinical isolates. A total of 533 clinical isolates that had been collected between 1995 and 2006, from 441 patients, were screened. No resistance was detected in isolates collected between 1995 and 1997. Starting in 1998, the resistance rate was 6.9%; a total of 24 patients (6.25%) harbored a resistant isolate. The TR34/L98H substitution was found in 21 of 30 tested isolates. PMID:26552980

  8. Pyripyropenes, novel ACAT inhibitors produced by Aspergillus fumigatus. IV. Structure elucidation of pyripyropenes M to R.

    PubMed

    Tomoda, H; Tabata, N; Yang, D J; Namatame, I; Tanaka, H; Omura, S; Kaneko, T

    1996-03-01

    Six new pyripyropenes, M to R, were isolated from the ethyl acetate extracts of the jar fermentation broth of Aspergillus fumigatus FO-1289-2501. Structural elucidation indicated that all the pyripyropenes have the same pyridino-alpha-pyrone sesquiterpene core as pyripyropenes A to L. Among them pyripyropene M showed the most potent inhibition against acyl-CoA : cholesterol acyltransferase activity with an IC50 value of 3.80 microM in rat liver microsomes, but pyripyropenes N to R showed moderate inhibitory activity (IC50 11.0 approximately 78.0 microM). PMID:8626247

  9. Pyripyropenes, Novel ACAT inhibitors produced by Aspergillus fumigatus. III. Structure elucidation of pyripyropenes E to L.

    PubMed

    Tomoda, H; Tabata, N; Yang, D J; Takayanagi, H; Nishida, H; Omura, S; Kaneko, T

    1995-06-01

    Eight new pyripyropenes, E to L, were isolated from the culture broth of Aspergillus fumigatus FO-1289-2501 selected as a higher producer by NTG mutation. Structural elucidation indicated that all the pyripyropenes have the same pyridino-alpha-pyrone sesquiterpene core as pyripyropenes A to D. Among them, pyripyropene L showed the most potent inhibition against acyl-CoA: cholesterol acyltransferase (ACAT) activity with an IC50 value of 0.27 microM in rat liver microsomes. PMID:7622436

  10. Expression, purification and crystallization of an indole prenyltransferase from Aspergillus fumigatus

    PubMed Central

    Chen, Jing; Morita, Hiroyuki; Kato, Ryohei; Noguchi, Hiroshi; Sugio, Shigetoshi; Abe, Ikuro

    2012-01-01

    CdpNPT from Aspergillus fumigatus is a dimethylallyltryptophan synthase/indole prenyltransferase that catalyzes reverse prenylation at position N1 of tryptophan-containing cyclic dipeptides. Residues 38–440 of CdpNPT were expressed in Escherichia coli and crystallized using the sitting-drop vapour-diffusion and microseeding techniques. The crystals belonged to space group P212121, with unit-cell parameters a = 84.4, b = 157.1, c = 161.8 Å, α = β = γ = 90.0°. PMID:22442243

  11. Salvage therapy with topical antifungal for Aspergillus fumigatus empyema complicating extrapleural pneumonectomy

    PubMed Central

    Purohit, Manoj; Guleri, Achyut; Zacharias, Joseph

    2012-01-01

    We describe an unusual case of Aspergillus fumigatus empyema and bronchopleural fistulae after extrapleural pneumonectomy (EPP) and chemoradiotherapy (CRT), which was treated successfully under salvage conditions with debridement, an innovative topical antifungal application and supplemented systemic antifungal therapy and which went on for a definitive surgical procedure. Combinations of CRT and EPP have been recommended in a select group of patients with malignant mesothelioma. Irrespective of the combination, EPP is associated with mortality in the range of 4–15% and a complication rate as high as 62%. PMID:22617507

  12. Beta 1,4-oligoglucosides inhibit the binding of Aspergillus fumigatus conidia to human monocytes.

    PubMed

    Kan, V L; Bennett, J E

    1991-05-01

    The binding of Aspergillus fumigatus conidia to human monocytes is mediated by a barley beta-glucan-inhibitable receptor. The simplest linkages in this glucan are present in the disaccharides laminaribiose (beta 1,3) and cellobiose (beta 1,4). Although laminaribiose gave strong inhibition of conidial binding to monocytes, cellobiose and oligosaccharides with beta 1,4-linked glucose residues were more potent as specific inhibitors of this binding over similar concentrations. Increasing the number of beta 1,4-linked glucose residues led to greater inhibition of conidial binding by human monocytes. PMID:2019764

  13. Effects of Iron Chelators on the Formation and Development of Aspergillus fumigatus Biofilm.

    PubMed

    Nazik, Hasan; Penner, John C; Ferreira, Jose A; Haagensen, Janus A J; Cohen, Kevin; Spormann, Alfred M; Martinez, Marife; Chen, Vicky; Hsu, Joe L; Clemons, Karl V; Stevens, David A

    2015-10-01

    Iron acquisition is crucial for the growth of Aspergillus fumigatus. A. fumigatus biofilm formation occurs in vitro and in vivo and is associated with physiological changes. In this study, we assessed the effects of Fe chelators on biofilm formation and development. Deferiprone (DFP), deferasirox (DFS), and deferoxamine (DFM) were tested for MIC against a reference isolate via a broth macrodilution method. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to DFP, DFM, DFP plus FeCl3, or FeCl3 alone. A preformed biofilm was exposed to DFP with or without FeCl3. The DFP and DFS MIC50 against planktonic A. fumigatus was 1,250 μM, and XTT gave the same result. DFM showed no planktonic inhibition at concentrations of ≤2,500 μM. By XTT testing, DFM concentrations of <1,250 μM had no effect, whereas DFP at 2,500 μM increased biofilms forming in A. fumigatus or preformed biofilms (P < 0.01). DFP at 156 to 2,500 μM inhibited biofilm formation (P < 0.01 to 0.001) in a dose-responsive manner. Biofilm formation with 625 μM DFP plus any concentration of FeCl3 was lower than that in the controls (P < 0.05 to 0.001). FeCl3 at ≥625 μM reversed the DFP inhibitory effect (P < 0.05 to 0.01), but the reversal was incomplete compared to the controls (P < 0.05 to 0.01). For preformed biofilms, DFP in the range of ≥625 to 1,250 μM was inhibitory compared to the controls (P < 0.01 to 0.001). FeCl3 at ≥625 μM overcame inhibition by 625 μM DFP (P < 0.001). FeCl3 alone at ≥156 μM stimulated biofilm formation (P < 0.05 to 0.001). Preformed A. fumigatus biofilm increased with 2,500 μM FeCl3 only (P < 0.05). In a strain survey, various susceptibilities of biofilms of A. fumigatus clinical isolates to DFP were noted. In conclusion, iron stimulates biofilm formation and preformed biofilms. Chelators can inhibit or enhance biofilms. Chelation

  14. Aspergillus fumigatus-specific antibodies in allergic bronchopulmonary aspergillosis and aspergilloma: evidence for a polyclonal antibody response.

    PubMed Central

    Brummund, W; Resnick, A; Fink, J N; Kurup, V P

    1987-01-01

    Patients with the Aspergillus-induced diseases allergic bronchopulmonary aspergillosis (ABPA), aspergilloma (fungus ball), and Aspergillus skin test-positive asthma were differentiated immunologically by radioimmunoassay based on their total immunoglobulin E (IgE) and Aspergillus fumigatus-specific IgE levels. In this study, a new, highly sensitive biotin-avidin-linked immunosorbent assay was used to evaluate A. fumigatus-specific antibodies of all immunoglobulin classes. Studied populations included 13 patients with ABPA, 12 with aspergilloma, 9 with Aspergillus skin test-positive asthma, and 9 normal individuals without asthma. A. fumigatus-specific antibodies of all classes were elevated in patients with ABPA, variably elevated in those with aspergilloma, and lowest in the other two groups. This assay demonstrated significantly higher specific IgE antibody levels in the ABPA group over those of the other groups, even with 1:1,000 dilutions of the sera. This study demonstrated that ABPA is a disease characterized by a polyclonal antibody response to Aspergillus antigen and not just a response to IgE and IgG antibody classes. The measurement of other antibody classes, particularly IgD and IgA, could enhance the immunodiagnosis of ABPA. The biotin-avidin-linked immunosorbent assay was found to be a highly sensitive assay that can be a clinically useful alternative to radioimmunoassay in the measurement of A. fumigatus-specific antibodies. PMID:3539998

  15. PrtT-Regulated Proteins Secreted by Aspergillus fumigatus Activate MAPK Signaling in Exposed A549 Lung Cells Leading to Necrotic Cell Death

    PubMed Central

    Sharon, Haim; Amar, David; Levdansky, Emma; Mircus, Gabriel; Shadkchan, Yana; Shamir, Ron; Osherov, Nir

    2011-01-01

    Aspergillus fumigatus is the most commonly encountered mold pathogen of humans, predominantly infecting the respiratory system. Colonization and penetration of the lung alveolar epithelium is a key but poorly understood step in the infection process. This study focused on identifying the transcriptional and cell-signaling responses activated in A549 alveolar carcinoma cells incubated in the presence of A. fumigatus wild-type and ΔPrtT protease-deficient germinating conidia and culture filtrates (CF). Microarray analysis of exposed A549 cells identified distinct classes of genes whose expression is altered in the presence of germinating conidia and CF and suggested the involvement of both NFkB and MAPK signaling pathways in mediating the cellular response. Phosphoprotein analysis of A549 cells confirmed that JNK and ERK1/2 are phosphorylated in response to CF from wild-type A. fumigatus and not phosphorylated in response to CF from the ΔPrtT protease-deficient strain. Inhibition of JNK or ERK1/2 kinase activity substantially decreased CF-induced cell damage, including cell peeling, actin-cytoskeleton damage, and reduction in metabolic activity and necrotic death. These results suggest that inhibition of MAPK-mediated host responses to treatment with A. fumigatus CF decreases cellular damage, a finding with possible clinical implications. PMID:21412410

  16. The Crystal Structure of Peroxiredoxin Asp f3 Provides Mechanistic Insight into Oxidative Stress Resistance and Virulence of Aspergillus fumigatus.

    PubMed

    Hillmann, Falk; Bagramyan, Karine; Straßburger, Maria; Heinekamp, Thorsten; Hong, Teresa B; Bzymek, Krzysztof P; Williams, John C; Brakhage, Axel A; Kalkum, Markus

    2016-01-01

    Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3's peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target. PMID:27624005

  17. The Fumagillin Gene Cluster, an Example of Hundreds of Genes under veA Control in Aspergillus fumigatus

    PubMed Central

    Dhingra, Sourabh; Lind, Abigail L.; Lin, Hsiao-Ching; Tang, Yi; Rokas, Antonis; Calvo, Ana M.

    2013-01-01

    Aspergillus fumigatus is the causative agent of invasive aspergillosis, leading to infection-related mortality in immunocompromised patients. We previously showed that the conserved and unique-to-fungi veA gene affects different cell processes such as morphological development, gliotoxin biosynthesis and protease activity, suggesting a global regulatory effect on the genome of this medically relevant fungus. In this study, RNA sequencing analysis revealed that veA controls the expression of hundreds of genes in A. fumigatus, including those comprising more than a dozen known secondary metabolite gene clusters. Chemical analysis confirmed that veA controls the synthesis of other secondary metabolites in this organism in addition to gliotoxin. Among the secondary metabolite gene clusters regulated by veA is the elusive but recently identified gene cluster responsible for the biosynthesis of fumagillin, a meroterpenoid known for its anti-angiogenic activity by binding to human methionine aminopeptidase 2. The fumagillin gene cluster contains a veA-dependent regulatory gene, fumR (Afu8g00420), encoding a putative C6 type transcription factor. Deletion of fumR results in silencing of the gene cluster and elimination of fumagillin biosynthesis. We found expression of fumR to also be dependent on laeA, a gene encoding another component of the fungal velvet complex. The results in this study argue that veA is a global regulator of secondary metabolism in A. fumigatus, and that veA may be a conduit via which chemical development is coupled to morphological development and other cellular processes. PMID:24116213

  18. Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus

    PubMed Central

    Bettgenhaeuser, Jan; Iakobachvili, Nino; Bignell, Elaine M.; Read, Nick D.

    2015-01-01

    Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca2+-mediated signalling. Ca2+ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca2+ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuBKU80, were engineered to express the bioluminescent Ca2+-reporter aequorin. An aequorin-mediated method for routine Ca2+ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca2+]c) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca2+, oxidative stress or exposure to human serum) that the fungus might be exposed to during infection, were analysed in living conidial germlings. The ‘signatures’ of the transient [Ca2+]c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca2+-signalling components in each stress response. Concordant with the involvement of Ca2+-calmodulin complexes in these Ca2+-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca2+-signatures to all the challenges. The Ca2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca2+ in initiating the stress responses. PMID:26402916

  19. Particle size distribution of airborne Aspergillus fumigatus spores emitted from compost using membrane filtration

    NASA Astrophysics Data System (ADS)

    Deacon, L. J.; Pankhurst, L. J.; Drew, G. H.; Hayes, E. T.; Jackson, S.; Longhurst, P. J.; Longhurst, J. W. S.; Liu, J.; Pollard, S. J. T.; Tyrrel, S. F.

    Information on the particle size distribution of bioaerosols emitted from open air composting operations is valuable in evaluating potential health impacts and is a requirement for improved dispersion simulation modelling. The membrane filter method was used to study the particle size distribution of Aspergillus fumigatus spores in air 50 m downwind of a green waste compost screening operation at a commercial facility. The highest concentrations (approximately 8 × 10 4 CFU m -3) of culturable spores were found on filters with pore diameters in the range 1-2 μm which suggests that the majority of spores are emitted as single cells. The findings were compared to published data collected using an Andersen sampler. Results were significantly correlated ( p < 0.01) indicating that the two methods are directly comparable across all particles sizes for Aspergillus spores.

  20. Vascular graft infection caused by Aspergillus species: case report and review of the literature.

    PubMed

    Motte, S; Bellens, B; Rickaert, F; Serruys, E; Thys, J P; Dereume, J P

    1993-03-01

    We report an unusual case of vascular graft infection caused by Aspergillus fumigatus that began with a false aneurysm, major arterial emboli, and septic arthritis. Successful treatment included resection of the infected graft, restoration of circulation by extraanatomic bypass, and administration of amphotericin B and itraconazole, a new antifungal agent. Graft infection in the case reported herein most likely occurred during surgery and took place during an insidious outbreak of postoperative infection. PMID:8445760

  1. Glycosylphosphatidylinositol (GPI) anchor is required in Aspergillus fumigatus for morphogenesis and virulence.

    PubMed

    Li, Hong; Zhou, Hui; Luo, Yuanming; Ouyang, Haomiao; Hu, Hongyan; Jin, Cheng

    2007-05-01

    In yeast, glycosylphosphatidylinositol (GPI) is essential for viability and plays an important role in biosynthesis and organization of cell wall. Initiation of the GPI anchor biosynthesis is catalysed by the GPI-N-acetylglucosaminyltransferase complex (GPI-GnT). The GPI3 (SPT14) gene is thought to encode the catalytic subunit of GPI-GnT complex. In contrast to Saccharomyces cerevisiae, little is known about the GPI biosynthesis in filamentous fungi. In this study, the afpig-a gene was identified as the homologue of the GPI3/pig-A gene in Aspergillus fumigatus, an opportunistic fungal pathogen. By replacement of the afpig-a gene with a pyrG gene, we obtained the null mutants. Although the Deltaafpig-a mutant exhibited a significant increased cell lysis instead of temperature-sensitive or conditional lethal phenotype associated to the GPI3 mutant of yeast, they could survive at temperatures from 30 degrees C to 50 degrees C. The analysis of the mutants showed that a completely blocking of the GPI anchor synthesis in A. fumigatus led to cell wall defect, abnormal hyphal growth, rapid conidial germination and aberrant conidiation. In vivo assays revealed that the mutant exhibited a reduced virulence in immunocompromised mice. The GPI anchor was not essential for viability, but required for the cell wall integrity, morphogenesis and virulence in A. fumigatus. PMID:17501924

  2. Characterization of a 5-azacytidine-induced developmental Aspergillus fumigatus variant

    PubMed Central

    Ben-Ami, Ronen; Varga, John; Lewis, Russell E; May, Gregory S; Nierman, William C

    2010-01-01

    The hypomethylating agent 5-azacytidine (5AC) is widely used in patients at risk of invasive mycoses. We sought to determine whether 5AC affects the developmental competence and virulence of Aspergillus fumigatus. Incubation of A. fumigatus strain 293 with 5AC induced high-frequency conversion to a fluffy-variant (Af293FL). The conidiation defect was bypassed by exposing Af293FL to light during the initial 18 hours of growth on solid media. Transcriptional profiling revealed differential expression of multiple genes involved in G-protein signaling, including a putative G-protein coupled photoreceptor (opsin), suggesting that impaired signaling through a light-responsive pathway upstream of brlA is responsible for this phenotype. Af293FL was fully virulent in fruit fly and murine models of invasive aspergillosis. Moreover, Af293FL overexpressed aspergillopepsin F, had increased elastase activity and was more angioinvasive than the parental wild-type strain. The 5AC-induced A. fumigatus fluffy variant illustrates the potential effects of chemotherapeutic agents on the developmental and pathobiologic characteristics of opportunistic fungi. PMID:21178435

  3. Stage-specific innate immune recognition of Aspergillus fumigatus and modulation by echinocandin drugs

    PubMed Central

    HOHL, TOBIAS M.

    2013-01-01

    The pulmonary innate immune system clears inhaled A spergillus fumigatus conidia (spores) from terminal airways. Failure to control conidial germination in immune compromised hosts can result in hyphal tissue invasion and fatal disease. Insight into the molecular recognition of A. fumigatus by host leukocytes indicates that the innate immune system exploits obligate changes in fungal cell wall composition that occur at the first stage of germination, conidial swelling. Germinating spores activate at least two host signal transduction pathways. Surface exposure of fungal β-(1,3) glucan, a polysaccharide constituent of the fungal cell wall, triggers dectin-1 signaling by host phagocytes. Spore germination leads to the induction of Toll-like receptor (TLR) signaling as well. This stage-specific recognition mechanism focuses host antifungal responses on cells with the potential for tissue invasion and may serve to limit potentially deleterious effects of inflammation in space and time. Fungal β-(1,3) glucan not only activates host innate immune responses but also represents the target of echinocandin drugs. The activity of echinocandin drugs has largely been understood on the basis of pharmacologic growth inhibition of yeast and moulds, resulting in lysis of yeast cells and stunting of dysmorphic hyphae. The recognition that fungal β-1,3 glucan activates dectin-1 signaling suggests that echinocandin drugs may exert immune modulatory effects by altering innate immune responses to drug-treated fungal cells, a view supported by recent data from studies on C. albicans, A. fumigatus, and non-Aspergillus moulds. PMID:18608931

  4. Trypacidin, a Spore-Borne Toxin from Aspergillus fumigatus, Is Cytotoxic to Lung Cells

    PubMed Central

    Gauthier, Thierry; Wang, Xiaodi; Sifuentes Dos Santos, Joice; Fysikopoulos, Athanasios; Tadrist, Souria; Canlet, Cécile; Artigot, Marie Pierre; Loiseau, Nicolas; Oswald, Isabelle P.; Puel, Olivier

    2012-01-01

    Inhalation of Aspergillus fumigatus conidia can cause severe aspergillosis in immunosuppressed people. A. fumigatus produces a large number of secondary metabolites, some of which are airborne by conidia and whose toxicity to the respiratory tract has not been investigated. We found that spores of A. fumigatus contain five main compounds, tryptoquivaline F, fumiquinazoline C, questin, monomethylsulochrin and trypacidin. Fractionation of culture extracts using RP-HPLC and LC-MS showed that samples containing questin, monomethylsulochrin and trypacidin were toxic to the human A549 lung cell line. These compounds were purified and their structure verified using NMR in order to compare their toxicity against A549 cells. Trypacidin was the most toxic, decreasing cell viability and triggering cell lysis, both effects occurring at an IC50 close to 7 µM. Trypacidin toxicity was also observed in the same concentration range on human bronchial epithelial cells. In the first hour of exposure, trypacidin initiates the intracellular formation of nitric oxide (NO) and hydrogen peroxide (H2O2). This oxidative stress triggers necrotic cell death in the following 24 h. The apoptosis pathway, moreover, was not involved in the cell death process as trypacidin did not induce apoptotic bodies or a decrease in mitochondrial membrane potential. This is the first time that the toxicity of trypacidin to lung cells has been reported. PMID:22319557

  5. Genetic Engineering Activates Biosynthesis of Aromatic Fumaric Acid Amides in the Human Pathogen Aspergillus fumigatus

    PubMed Central

    Kalb, Daniel; Heinekamp, Thorsten; Lackner, Gerald; Scharf, Daniel H.; Dahse, Hans-Martin; Brakhage, Axel A.

    2014-01-01

    The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both l-tyrosine and l-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-l-tyrosine and fumaryl-l-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds. PMID:25527545

  6. Chemotypic and genotypic diversity in the ergot alkaloid pathway of Aspergillus fumigatus.

    PubMed

    Robinson, Sarah L; Panaccione, Daniel G

    2012-01-01

    Aspergillus fumigatus is an opportunistic human pathogen that synthesizes a group of mycotoxins via a branch of the ergot alkaloid pathway. This fungus is globally distributed, and genetic data indicate that isolates recombine freely over that range; however, previous work on ergot alkaloids has focused on a limited number of isolates. We hypothesized that A. fumigatus harbors variation in the chemotype of ergot alkaloids and genotype of the ergot alkaloid gene cluster. Analysis of 13 isolates by high performance liquid chromatography revealed four distinct ergot alkaloid profiles or chemotypes. Five isolates completed the A. fumigatus branch of the ergot alkaloid pathway to fumigaclavine C. Six independent isolates accumulated fumigaclavine A, the pathway intermediate immediately before fumigaclavine C. One isolate accumulated only the early pathway intermediates chanoclavine-i and chanocla-vine-i aldehyde, and one isolate lacked ergot alkaloids altogether. A genetic basis for each of the observed chemotypes was obtained either by PCR analysis of the ergot alkaloid gene cluster or through sequencing of easL, the gene encoding the prenyl transferase that reverse prenylates fumigaclavine A to fumigaclavine C. Isolates also exhibited differences in pigmentation and sporulation. The ergot alkaloid chemotypes were widely distributed geographically and among substrate of origin. PMID:22453123

  7. GliZ, a Transcriptional Regulator of Gliotoxin Biosynthesis, Contributes to Aspergillus fumigatus Virulence▿

    PubMed Central

    Bok, Jin Woo; Chung, DaWoon; Balajee, S. Arunmozhi; Marr, Kieren A.; Andes, David; Nielsen, Kristian Fog; Frisvad, Jens C.; Kirby, Katharine A.; Keller, Nancy P.

    2006-01-01

    Gliotoxin is a nonribosomal peptide produced by Aspergillus fumigatus. This compound has been proposed as an A. fumigatus virulence factor due to its cytotoxic, genotoxic, and apoptotic properties. Recent identification of the gliotoxin gene cluster identified several genes (gli genes) likely involved in gliotoxin production, including gliZ, encoding a putative Zn2Cys6 binuclear transcription factor. Replacement of gliZ with a marker gene (ΔgliZ) resulted in no detectable gliotoxin production and loss of gene expression of other gli cluster genes. Placement of multiple copies of gliZ in the genome increased gliotoxin production. Using endpoint survival data, the ΔgliZ and a multiple-copy gliZ strain were not statistically different from the wild type in a murine pulmonary model; however, both the wild-type and the multiple-copy gliZ strain were more virulent than ΔlaeA (a mutant reduced in production of gliotoxin and other toxins). A flow-cytometric analysis of polymorphonuclear leukocytes (PMNs) exposed to supernatants from wild-type, ΔgliZ, complemented ΔgliZ, and ΔlaeA strains supported a role for gliotoxin in apoptotic but not necrotic PMN cell death. This may indicate that several secondary metabolites are involved in A. fumigatus virulence. PMID:17030582

  8. Antibody response to low-molecular-weight antigens of Aspergillus fumigatus in allergic bronchopulmonary aspergillosis.

    PubMed Central

    Kurup, V P; Greenberger, P A; Fink, J N

    1989-01-01

    Sera from patients with allergic bronchopulmonary aspergillosis (ABPA) or aspergilloma and normal sera were analyzed for specific antibodies by Western (immuno-) blotting with Aspergillus fumigatus antigens transferred electrophoretically onto polyvinylidene difluoride membranes. Western blot analysis demonstrated consistent reactivity of low-molecular-weight A. fumigatus antigens against ABPA sera but not against uncomplicated aspergilloma or normal sera. None of these low-molecular-weight components had any lectin-binding activity. Sera from patients with aspergilloma, however, frequently reacted with high-molecular-weight components of A. fumigatus. The majority of these high-molecular-weight antigenic components demonstrated concanavalin A-binding activity. The low-molecular-weight bands were discernible in Western blots with sera from all ABPA patients irrespective of disease activities, such as relapse, flare, or treatment. Antibodies detected by methods such as immunodiffusion or enzyme-linked immunosorbent assays demonstrated total antibody responses to most or all antigenic components, while Western blots demonstrated the reactivities of the individual components with the specific antibodies. Western blot analysis thus provided more information for immunodiagnosis of ABPA than other methods, especially when only crude antigens were available. Images PMID:2666440

  9. Determination of Aspergillus fumigatus allergen 1 in poultry farms using the enzyme immunoassay.

    PubMed

    Prester, Ljerka; Macan, Jelena; Matković, Kristina; Vucemilo, Marija

    2010-06-01

    Poultry farms contain high levels of allergenic fungi, and Aspergillus spp. is the most common genus of moulds. Aspergillus fumigatus antigens are responsible for the development of several respiratory diseases including asthma. The aim of this study was to measure the mass fraction of Asp f 1, a major allergen of Asperillus fumigatus in 37 indoor dust samples collected from four poultry farms in a rural area of the Zagreb County (Croatia) using the enzyme-linked immunosorbent assay. More than 62 % of dust samples had detectable Asp f 1 levels (limit of detection 3.6 ng g(-1)). The overall mean Asp f 1 level was 17.9 ng g(-1) [range (3.8 to 72.4) ng g(-1)]. Satisfactory results were obtained for analytical within-run imprecision (6.7 %), between-run imprecision (10.5 %), and accuracy (91 % to 115 %). Microclimate parameters (air temperature, relative humidity, and velocity) were within the recommended ranges in all poultry farms. This study has shown that Asp f 1 settles on dust at poultry farms and that occupational exposure to this allergen deserves monitoring in livestock buildings. PMID:20587390

  10. Identification of Hypoxia-Inducible Target Genes of Aspergillus fumigatus by Transcriptome Analysis Reveals Cellular Respiration as an Important Contributor to Hypoxic Survival

    PubMed Central

    Kroll, Kristin; Pähtz, Vera; Hillmann, Falk; Vaknin, Yakir; Schmidt-Heck, Wolfgang; Roth, Martin; Jacobsen, Ilse D.; Osherov, Nir; Brakhage, Axel A.

    2014-01-01

    Aspergillus fumigatus is an opportunistic, airborne pathogen that causes invasive aspergillosis in immunocompromised patients. During the infection process, A. fumigatus is challenged by hypoxic microenvironments occurring in inflammatory, necrotic tissue. To gain further insights into the adaptation mechanism, A. fumigatus was cultivated in an oxygen-controlled chemostat under hypoxic and normoxic conditions. Transcriptome analysis revealed a significant increase in transcripts associated with cell wall polysaccharide metabolism, amino acid and metal ion transport, nitrogen metabolism, and glycolysis. A concomitant reduction in transcript levels was observed with cellular trafficking and G-protein-coupled signaling. To learn more about the functional roles of hypoxia-induced transcripts, we deleted A. fumigatus genes putatively involved in reactive nitrogen species detoxification (fhpA), NAD+ regeneration (frdA and osmA), nitrogen metabolism (niaD and niiA), and respiration (rcfB). We show that the nitric oxygen (NO)-detoxifying flavohemoprotein gene fhpA is strongly induced by hypoxia independent of the nitrogen source but is dispensable for hypoxic survival. By deleting the nitrate reductase gene niaD, the nitrite reductase gene niiA, and the two fumarate reductase genes frdA and osmA, we found that alternative electron acceptors, such as nitrate and fumarate, do not have a significant impact on growth of A. fumigatus during hypoxia, but functional mitochondrial respiratory chain complexes are essential under these conditions. Inhibition studies indicated that primarily complexes III and IV play a crucial role in the hypoxic growth of A. fumigatus. PMID:25084861

  11. Changes in Atmospheric CO2 Influence the Allergenicity of Aspergillus fumigatus fungal spore

    NASA Astrophysics Data System (ADS)

    Lang-Yona, N.; Levin, Y.; Dannemoller, K. C.; Yarden, O.; Peccia, J.; Rudich, Y.

    2013-12-01

    Increased allergic susceptibility has been documented without a comprehensive understanding for its causes. Therefore understanding trends and mechanisms of allergy inducing agents is essential. In this study we investigated whether elevated atmospheric CO2 levels can affect the allergenicity of Aspergillus fumigatus, a common allergenic fungal species. Both direct exposure to changing CO2 levels during fungal growth, and indirect exposure through changes in the C:N ratios in the growth media were inspected. We determined the allergenicity of the spores through two types of immunoassays, accompanied with genes expression analysis, and proteins relative quantification. We show that fungi grown under present day CO2 levels (392 ppm) exhibit 8.5 and 3.5 fold higher allergenicity compared to fungi grown at preindustrial (280 ppm) and double (560 ppm) CO2 levels, respectively. A corresponding trend is observed in the expression of genes encoding for known allergenic proteins and in the major allergen Asp f1 concentrations, possibly due to physiological changes such as respiration rates and the nitrogen content of the fungus, influenced by the CO2 concentrations. Increased carbon and nitrogen levels in the growth medium also lead to a significant increase in the allergenicity, for which we propose two different biological mechanisms. We suggest that climatic changes such as increasing atmospheric CO2 levels and changes in the fungal growth medium may impact the ability of allergenic fungi such as Aspergillus fumigatus to induce allergies. The effect of changing CO2 concentrations on the total allergenicity per 10^7 spores of A. fumigatus (A), the major allergen Asp f1 concentration in ng per 10^7 spores (B), and the gene expression by RT-PCR (C). The error bars represent the standard error of the mean.

  12. Emergence of Azole Resistance in Aspergillus fumigatus and Spread of a Single Resistance Mechanism

    PubMed Central

    Snelders, Eveline; van der Lee, Henrich A. L; Kuijpers, Judith; Rijs, Anthonius J. M. M; Varga, János; Samson, Robert A; Mellado, Emilia; Donders, A. Rogier T; Melchers, Willem J. G; Verweij, Paul E

    2008-01-01

    Background Resistance to triazoles was recently reported in Aspergillus fumigatus isolates cultured from patients with invasive aspergillosis. The prevalence of azole resistance in A. fumigatus is unknown. We investigated the prevalence and spread of azole resistance using our culture collection that contained A. fumigatus isolates collected between 1994 and 2007. Methods and Findings We investigated the prevalence of itraconazole (ITZ) resistance in 1,912 clinical A. fumigatus isolates collected from 1,219 patients in our University Medical Centre over a 14-y period. The spread of resistance was investigated by analyzing 147 A. fumigatus isolates from 101 patients, from 28 other medical centres in The Netherlands and 317 isolates from six other countries. The isolates were characterized using phenotypic and molecular methods. The electronic patient files were used to determine the underlying conditions of the patients and the presence of invasive aspergillosis. ITZ-resistant isolates were found in 32 of 1,219 patients. All cases were observed after 1999 with an annual prevalence of 1.7% to 6%. The ITZ-resistant isolates also showed elevated minimum inhibitory concentrations of voriconazole, ravuconazole, and posaconazole. A substitution of leucine 98 for histidine in the cyp51A gene, together with two copies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be the dominant resistance mechanism. Microsatellite analysis indicated that the ITZ-resistant isolates were genetically distinct but clustered. The ITZ-sensitive isolates were not more likely to be responsible for invasive aspergillosis than the ITZ-resistant isolates. ITZ resistance was found in isolates from 13 patients (12.8%) from nine other medical centres in The Netherlands, of which 69% harboured the TR/L98H substitution, and in six isolates originating from four other countries. Conclusions Azole resistance has emerged in A. fumigatus and might be more prevalent than currently

  13. Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA

    PubMed Central

    2010-01-01

    Background Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and ΔAfcrzA mutant strains. Results We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively). Decreased mRNA abundance in the ΔcrzA was seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression in A. fumigatus increases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 μM, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl2 25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. GFP::RcnA was mostly detected along the germling, did not accumulate in the nuclei and its location is not affected by the cellular response to calcium chloride. Conclusion We have performed a transcriptional profiling analysis of the A. fumigatus ΔAfcrzA mutant strain exposed to calcium stress. This provided an excellent opportunity to identify genes and pathways that are under the influence of AfCrzA. AfRcnA, one of these selected genes, encodes a modulator of calcineurin activity. Concomitantly with A

  14. Identification of ferrichrome- and ferrioxamine B-mediated iron uptake by Aspergillus fumigatus.

    PubMed

    Park, Yong-Sung; Kim, Ju-Yeon; Yun, Cheol-Won

    2016-05-01

    Aspergillus fumigatus is an opportunistic fungal pathogen for immunocompromised patients, and genes involved in siderophore metabolism have been identified as virulence factors. Recently, we identified the membrane transporters sit1 and sit2, which are putative virulence factors of A. fumigatus; sit1 and sit2 are homologous to yeast Sit1, and sit1 and sit2 gene expression was up-regulated after iron depletion. When expressed heterologously in Saccharomyces cerevisiae, sit1 and sit2 were localized to the plasma membrane; sit1 efficiently complemented ferrichrome (FC) and ferrioxamine B (FOB) uptake in yeast cells, whereas sit2 complemented only FC uptake. Deletion of sit1 resulted in a decrease in FOB and FC uptake, and deletion of sit2 resulted in a decrease in FC uptake in A. fumigatus It is of interest that a sit1 and sit2 double-deletion mutant resulted in a synergistic decrease in FC uptake activity. Both sit1 and sit2 were localized to the plasma membrane in A. fumigatus The expression levels of the sit1 and sit2 genes were dependent on hapX under low-but not high-iron conditions. Furthermore, mirB, and sidA gene expression was up-regulated and sreA expression down-regulated when sit1 and sit2 were deleted. Although sit1 and sit2 failed to affect mouse survival rate, these genes affected conidial killing activity. Taken together, our results suggest that sit1 and sit2 are siderophore transporters and putative virulence factors localized to the plasma membrane. PMID:26929401

  15. Ergot cluster-encoded catalase is required for synthesis of chanoclavine-I in Aspergillus fumigatus.

    PubMed

    Goetz, Kerry E; Coyle, Christine M; Cheng, Johnathan Z; O'Connor, Sarah E; Panaccione, Daniel G

    2011-06-01

    Genes required for ergot alkaloid biosynthesis are clustered in the genomes of several fungi. Several conserved ergot cluster genes have been hypothesized, and in some cases demonstrated, to encode early steps of the pathway shared among fungi that ultimately make different ergot alkaloid end products. The deduced amino acid sequence of one of these conserved genes (easC) indicates a catalase as the product, but a role for a catalase in the ergot alkaloid pathway has not been established. We disrupted easC of Aspergillus fumigatus by homologous recombination with a truncated copy of that gene. The resulting mutant (ΔeasC) failed to produce the ergot alkaloids typically observed in A. fumigatus, including chanoclavine-I, festuclavine, and fumigaclavines B, A, and C. The ΔeasC mutant instead accumulated N-methyl-4-dimethylallyltryptophan (N-Me-DMAT), an intermediate recently shown to accumulate in Claviceps purpurea strains mutated at ccsA (called easE in A. fumigatus) (Lorenz et al. Appl Environ Microbiol 76:1822-1830, 2010). A ΔeasE disruption mutant of A. fumigatus also failed to accumulate chanoclavine-I and downstream ergot alkaloids and, instead, accumulated N-Me-DMAT. Feeding chanoclavine-I to the ΔeasC mutant restored ergot alkaloid production. Complementation of either ΔeasC or ΔeasE mutants with the respective wild-type allele also restored ergot alkaloid production. The easC gene was expressed in Escherichia coli, and the protein product displayed in vitro catalase activity with H(2)O(2) but did not act, in isolation, on N-Me-DMAT as substrate. The data indicate that the products of both easC (catalase) and easE (FAD-dependent oxidoreductase) are required for conversion of N-Me-DMAT to chanoclavine-I. PMID:21409592

  16. A Proteomic Approach to Investigating Gene Cluster Expression and Secondary Metabolite Functionality in Aspergillus fumigatus

    PubMed Central

    Owens, Rebecca A.; Hammel, Stephen; Sheridan, Kevin J.; Jones, Gary W.; Doyle, Sean

    2014-01-01

    A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism. PMID:25198175

  17. Functional characterization of the Woronin body protein WscA of the pathogenic mold Aspergillus fumigatus.

    PubMed

    Leonhardt, Yannik; Beck, Julia; Ebel, Frank

    2016-05-01

    Woronin bodies are fungal-specific organelles that seal damaged hyphal compartments and thereby contribute to the stress resistance and virulence of filamentous fungi. In this study, we have characterized the Aspergillus fumigatus Woronin body protein WscA. WscA is homologous to Neurospora crassa WSC, a protein that was shown to be important for biogenesis, segregation and positioning of Woronin bodies. WscA and WSC both belong to the Mpv17/PMP22 family of peroxisomal membrane proteins. An A. fumigatus ΔwscA mutant is unable to form Woronin bodies, and HexA, the protein that forms the crystal-like core of Woronin bodies, accumulates in large peroxisomes instead. The ΔwscA mutant showed no defect in segregation of HexA containing organelles, as has been reported for the corresponding N. crassa mutant. In the peroxisomes of the A. fumigatus mutant, HexA assembles into compact, donut-shaped structures. Experiments with GFP fusion proteins revealed that WscA function is highly sensitive to these modifications, in particular to an N-terminal fusion of GFP. In N. crassa, WSC was shown to be essentially required for Woronin body positioning, but the respective domain is not conserved in most other Pezizomycotina, including A. fumigatus. We have recently found evidence that HexA may have a direct role in WB positioning, since a HexA-GFP fusion protein, lacking a functional PTS1 motif, is efficiently recruited to the septal pore. In the current study we show that this targeting of HexA-GFP is independent of WscA. PMID:27016805

  18. A proteomic approach to investigating gene cluster expression and secondary metabolite functionality in Aspergillus fumigatus.

    PubMed

    Owens, Rebecca A; Hammel, Stephen; Sheridan, Kevin J; Jones, Gary W; Doyle, Sean

    2014-01-01

    A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism. PMID:25198175

  19. Transcriptomic and proteomic analyses of the Aspergillus fumigatus hypoxia response using an oxygen-controlled fermenter

    PubMed Central

    2012-01-01

    Background Aspergillus fumigatus is a mold responsible for the majority of cases of aspergillosis in humans. To survive in the human body, A. fumigatus must adapt to microenvironments that are often characterized by low nutrient and oxygen availability. Recent research suggests that the ability of A. fumigatus and other pathogenic fungi to adapt to hypoxia contributes to their virulence. However, molecular mechanisms of A. fumigatus hypoxia adaptation are poorly understood. Thus, to better understand how A. fumigatus adapts to hypoxic microenvironments found in vivo during human fungal pathogenesis, the dynamic changes of the fungal transcriptome and proteome in hypoxia were investigated over a period of 24 hours utilizing an oxygen-controlled fermenter system. Results Significant increases in transcripts associated with iron and sterol metabolism, the cell wall, the GABA shunt, and transcriptional regulators were observed in response to hypoxia. A concomitant reduction in transcripts was observed with ribosome and terpenoid backbone biosynthesis, TCA cycle, amino acid metabolism and RNA degradation. Analysis of changes in transcription factor mRNA abundance shows that hypoxia induces significant positive and negative changes that may be important for regulating the hypoxia response in this pathogenic mold. Growth in hypoxia resulted in changes in the protein levels of several glycolytic enzymes, but these changes were not always reflected by the corresponding transcriptional profiling data. However, a good correlation overall (R2 = 0.2, p < 0.05) existed between the transcriptomic and proteomics datasets for all time points. The lack of correlation between some transcript levels and their subsequent protein levels suggests another regulatory layer of the hypoxia response in A. fumigatus. Conclusions Taken together, our data suggest a robust cellular response that is likely regulated both at the transcriptional and post-transcriptional level in response to hypoxia

  20. Aspergillus fumigatus Asp fI DNA is prevalent in sputum from patients with coal workers' pneumoconiosis.

    PubMed

    Nomoto, Y; Kuwano, K; Hagimoto, N; Kunitake, R; Tsuda, M; Hara, N

    1997-01-01

    Aspergillus fumigatus is an apportunistic nosocomial pathogen in immunosuppressed patients or in the lesion where the local defense mechanism was impaired. Patients with pneumoconiosis are known to be susceptible to chronic necrotizing pulmonary aspergillosis. Therefore, we hypothesized that A. fumigatus might be prevalent in sputum from patients with coal workers' pneumoconiosis, and also that asthmatic symptoms in patients with coal workers' pneumoconiosis may be associated with the presence of A. fumigatus. We tested for A. fumigatus in the sputum from patients with coal workers' pneumoconiosis by nested polymerase chain reaction amplification of the Asp fI gene. Sequences specific for this gene were detectable in 5 of 11 (45.5%) patients with coal workers' pneumoconiosis with asthmatic symptoms (group A), 5 of 10 (50.0%) patients with coal workers' pneumoconiosis without asthmatic symptoms (group B) and only 1 of 9 (11.1%) patients with chronic airflow obstruction without pneumoconiosis (group C). The frequency of the Asp fI gene detection was significantly higher in groups A and B than in group C (p < 0.05). The prevalence of A. fumigatus was not associated with asthmatic symptoms. These results demonstrated that A. fumigatus was prevalent in patients with coal workers' pneumoconiosis. We speculate that colonization with A. fumigatus may be associated with this disease. PMID:9257365

  1. Divergent Targets of Aspergillus fumigatus AcuK and AcuM Transcription Factors during Growth In Vitro versus Invasive Disease

    PubMed Central

    Pongpom, Monsicha; Liu, Hong; Xu, Wenjie; Snarr, Brendan D.; Sheppard, Donald C.; Mitchell, Aaron P.

    2014-01-01

    In Aspergillus nidulans, the AcuK and AcuM transcription factors form a complex that regulates gluconeogenesis. In Aspergillus fumigatus, AcuM governs gluconeogenesis and iron acquisition in vitro and virulence in immunosuppressed mice. However, the function of AcuK was previously unknown. Through in vitro studies, we found that A. fumigatus ΔacuK single and ΔacuK ΔacuM double mutants had impaired gluconeogenesis and iron acquisition, similar to the ΔacuM mutant. Also, the ΔacuK, ΔacuM, and ΔacuK ΔacuM mutants had similar virulence defects in mice. However, the ΔacuK mutant had a milder defect in extracellular siderophore activity and induction of epithelial cell damage in vitro than did the ΔacuM mutant. Moreover, overexpression of acuM in the ΔacuK mutant altered expression of 3 genes and partially restored growth under iron-limited conditions, suggesting that AcuM can govern some genes independently of AcuK. Although the ΔacuK and ΔacuM mutants had very similar transcriptional profiles in vitro, their transcriptional profiles during murine pulmonary infection differed both from their in vitro profiles and from each other. While AcuK and AcuM governed the expression of only a few iron-responsive genes in vivo, they influenced the expression of other virulence-related genes, such as hexA and dvrA. Therefore, in A. fumigatus, while AcuK and AcuM likely function as part of the same complex, they can also function independently of each other. Furthermore, AcuK and AcuM have different target genes in vivo than in vitro, suggesting that in vivo infection stimulates unique transcriptional regulatory pathways in A. fumigatus. PMID:25534941

  2. Suppression of Epithelial Signal Transducer and Activator of Transcription 1 Activation by Extracts of Aspergillus fumigatus

    PubMed Central

    Bhushan, Bharat; Homma, Tetsuya; Norton, James E.; Sha, Quan; Siebert, Jason; Gupta, Dave S.; Schroeder, James W.

    2015-01-01

    Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-β or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ–induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-β and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-β or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-β and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-β. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-β, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6

  3. Suppression of epithelial signal transducer and activator of transcription 1 activation by extracts of Aspergillus fumigatus.

    PubMed

    Bhushan, Bharat; Homma, Tetsuya; Norton, James E; Sha, Quan; Siebert, Jason; Gupta, Dave S; Schroeder, James W; Schleimer, Robert P

    2015-07-01

    Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-β or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ-induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-β and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-β or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-β and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-β. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-β, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6

  4. Vaccinations with recombinant variants of Aspergillus fumigatus allergen Asp f 3 protect mice against invasive aspergillosis.

    PubMed

    Ito, James I; Lyons, Joseph M; Hong, Teresa B; Tamae, Daniel; Liu, Yi-Kuang; Wilczynski, Sharon P; Kalkum, Markus

    2006-09-01

    A vaccine that effectively protects immunocompromised patients against invasive aspergillosis is a novel approach to a universally fatal disease. Here we present a rationale for selection and in vivo testing of potential protein vaccine candidates, based on the modification of an immunodominant fungal allergen for which we demonstrate immunoprotective properties. Pulmonary exposure to viable Aspergillus fumigatus conidia as well as vaccination with crude hyphal extracts protects corticosteroid-immunosuppressed mice against invasive aspergillosis (J. I. Ito and J. M. Lyons, J. Infect. Dis. 186:869-871, 2002). Sera from the latter animals contain antibodies with numerous and diverse antigen specificities, whereas sera from conidium-exposed mice contain antibodies predominantly against allergen Asp f 3 (and some against Asp f 1), as identified by mass spectrometry. Subcutaneous immunization with recombinant Asp f 3 (rAsp f 3) but not with Asp f 1 was protective. The lungs of Asp f 3-vaccinated survivors were free of hyphae and showed only a patchy low-density infiltrate of mononuclear cells. In contrast, the nonimmunized animals died with invasive hyphal elements and a compact peribronchial infiltrate of predominantly polymorphonuclear leukocytes. Three truncated versions of rAsp f 3, spanning amino acid residues 15 to 168 [rAsp f 3(15-168)], 1 to 142, and 15 to 142 and lacking the known bipartite sequence required for IgE binding, were also shown to be protective. Remarkably, vaccination with either rAsp f 3(1-142) or rAsp f 3(15-168) drastically diminished the production of antigen-specific antibodies compared to vaccination with the full-length rAsp f 3(1-168) or the double-truncated rAsp f 3(15-142) version. Our findings point to a possible mechanism in which Asp f 3 vaccination induces a cellular immune response that upon infection results in the activation of lymphocytes that in turn enhances and/or restores the function of corticosteroid-suppressed macrophages

  5. Aspergillus infections in cystic fibrosis.

    PubMed

    King, Jill; Brunel, Shan F; Warris, Adilia

    2016-07-01

    Patients with cystic fibrosis (CF) suffer from chronic lung infection and airway inflammation. Respiratory failure secondary to chronic or recurrent infection remains the commonest cause of death and accounts for over 90% of mortality. Bacteria as Staphylococcus aureus, Pseudomonas aeruginosa and Burkholderia cepacia complex have been regarded the main CF pathogens and their role in progressive lung decline has been studied extensively. Little attention has been paid to the role of Aspergillus spp. and other filamentous fungi in the pathogenesis of non-ABPA (allergic bronchopulmonary aspergillosis) respiratory disease in CF, despite their frequent recovery in respiratory samples. It has become more apparent however, that Aspergillus spp. may play an important role in chronic lung disease in CF. Research delineating the underlying mechanisms of Aspergillus persistence and infection in the CF lung and its link to lung deterioration is lacking. This review summarizes the Aspergillus disease phenotypes observed in CF, discusses the role of CFTR (cystic fibrosis transmembrane conductance regulator)-protein in innate immune responses and new treatment modalities. PMID:27177733

  6. Aspergillus fumigatus densities in relation to forest succession and edge effects: implications for wildlife health in modified environments.

    PubMed

    Perrott, John K; Armstrong, Doug P

    2011-09-01

    The hihi (or stitchbird, Notiomystis cincta) is a New Zealand endemic nectivorous forest bird now restricted to one pristine island. Relocation to establish viable hihi populations on other islands has been the main conservation action since the early 1980s. To date, hihi reintroductions to young growth islands have had poor success despite the absence of mammalian predators. It was thought that past failures were due to food limitation, but research suggests that food limitation alone cannot account for their poor survivorship. Post-mortems of dead hihi has shown that aspergillosis caused by Aspergillus fumigatus is a major mortality factor and there is current concern regarding their susceptibility to this fungal disease. In this paper we develop and assess the hypothesis that A. fumigatus limits hihi population viability on modified islands, and suggest that A. fumigatus is a potential indicator species for habitat disturbance. We report that the prevalence of A. fumigatus spores in the soil is much higher in young growth forests and forest edge habitats. Results suggest that hihi mortality rates between islands are potentially due to differential exposure to A. fumigatus spores. We assess relationships between habitat disturbance, A. fumigatus contamination and hihi mortality rates by testing the following predictions: (1) that densities of A. fumigatus spores will be higher on modified islands, (2) that densities of A. fumigatus spores on islands will be correlated with hihi mortality rates and (3) that densities of A. fumigatus spores will be higher at the forest edge than in the interior. We test each of these predictions using soil samples, air samples and samples of nectar from plant species fed on by hihi. PMID:22076057

  7. Interplay between Gliotoxin Resistance, Secretion, and the Methyl/Methionine Cycle in Aspergillus fumigatus

    PubMed Central

    Owens, Rebecca A.; O'Keeffe, Grainne; Smith, Elizabeth B.; Dolan, Stephen K.; Hammel, Stephen; Sheridan, Kevin J.; Fitzpatrick, David A.; Keane, Thomas M.

    2015-01-01

    Mechanistic studies on gliotoxin biosynthesis and self-protection in Aspergillus fumigatus, both of which require the gliotoxin oxidoreductase GliT, have revealed a rich landscape of highly novel biochemistries, yet key aspects of this complex molecular architecture remain obscure. Here we show that an A. fumigatus ΔgliA strain is completely deficient in gliotoxin secretion but still retains the ability to efflux bisdethiobis(methylthio)gliotoxin (BmGT). This correlates with a significant increase in sensitivity to exogenous gliotoxin because gliotoxin trapped inside the cell leads to (i) activation of the gli cluster, as disabling gli cluster activation, via gliZ deletion, attenuates the sensitivity of an A. fumigatus ΔgliT strain to gliotoxin, thus implicating cluster activation as a factor in gliotoxin sensitivity, and (ii) increased methylation activity due to excess substrate (dithiol gliotoxin) for the gliotoxin bis-thiomethyltransferase GtmA. Intracellular dithiol gliotoxin is oxidized by GliT and subsequently effluxed by GliA. In the absence of GliA, gliotoxin persists in the cell and is converted to BmGT, with levels significantly higher than those in the wild type. Similarly, in the ΔgliT strain, gliotoxin oxidation is impeded, and methylation occurs unchecked, leading to significant S-adenosylmethionine (SAM) depletion and S-adenosylhomocysteine (SAH) overproduction. This in turn significantly contributes to the observed hypersensitivity of gliT-deficient A. fumigatus to gliotoxin. Our observations reveal a key role for GliT in preventing dysregulation of the methyl/methionine cycle to control intracellular SAM and SAH homeostasis during gliotoxin biosynthesis and exposure. Moreover, we reveal attenuated GliT abundance in the A. fumigatus ΔgliK strain, but not the ΔgliG strain, following exposure to gliotoxin, correlating with relative sensitivities. Overall, we illuminate new systems interactions that have evolved in gliotoxin-producing, compared

  8. Inhibition of conidiophore development in Aspergillus fumigatus by an Escherichia coli DH5α strain, a promising antifungal candidate against aspergillosis.

    PubMed

    Balhara, M; Ruhil, S; Kumar, M; Dhankhar, S; Chhillar, A K

    2014-03-01

    The opportunistic human pathogen Aspergillus fumigatus produces a massive number of asexual spores (conidia) as the primary means of dispersal, survival, genome protection and infection of hosts. In this report, we investigated secretory and cytosolic proteins of non-pathogenic bacterial species (mostly belonging to human microbiome) for antifungal potential against A. fumigatus, A. flavus and A. niger. Our preliminary results revealed that cytosolic proteins of E. coli DH5α were most active and the less toxic against various pathogenic isolates of A. fumigatus (the major pathogenic species), depicting a minimum inhibitory concentration (MIC) of 62.50 μg/mL, 62.50 μg/mL and 12.50 μg/disc using microbroth dilution assay (MDA), percentage spore germination inhibition assay (PSGI) and disc diffusion assay (DDA), respectively. E. coli protein was non-toxic against human erythrocytes at doses up to 1000 μg/mL as compared to standard drug, amphotericin B which lysed 100% of erythrocytes at a concentration of 37.50 μg/mL. Time kill analysis proved it to be fungicidal in a concentration and time-dependent manner. Scanning electron microscopic studies (SEM) were carried out to prevail what kind of damage it causes to A. fumigatus. SEM results reported that conidiophore (structures forming conidia) development was halted as a major consequence, reducing the number of conidiophores to insignificant values as well as alteration in their morphological attributes. This feature may contribute to the development of new prevention strategies against Aspergillus infections. Hyphal atrophy was also observed, evidenced by shrinking and flattening of hyphal walls and reduced, abrupt hyphal branching. Such actions may effectively reduce the invasive ability of Aspergillus as well as it can sterilize the fungal burden by obstructing the conidiation pathway of A. fumigatus. Hence, E. coli DH5α, being a commensal species, can lead to the development of antifungal molecule with novel

  9. Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan synthase.

    PubMed Central

    Kurtz, M B; Heath, I B; Marrinan, J; Dreikorn, S; Onishi, J; Douglas, C

    1994-01-01

    The lipopeptide antifungal agents, echinocandins, papulacandins, and pneumocandins, kill Candida albicans by inhibiting glucan synthesis. For this fungus, there is a good correlation of in vitro enzyme inhibition with in vitro assays of MICs. Semisynthetic lipopeptides such as cilofungin, LY303366, L-693,989, and L-733,560 have activity in vivo against Aspergillus infections but appear to be inactive in broth dilution in vitro tests (MICs, > 128 micrograms/ml). To understand how compounds which lack activity in vitro can have good in vivo activity, we monitored the effect of pneumocandins on the morphology of Aspergillus fumigatus and A, flavus strains by light microscopy and electron microscopy and related the changes in growth to inhibition of glucan synthesis. Pneumocandin B0 caused profound changes in hyphal growth; light micrographs showed abnormally swollen germ tubes, highly branched hyphal tips, and many cells with distended balloon shapes. Aspergillus electron micrographs confirmed that lipopeptides produce changes in cell walls; drug-treated germlings showed very stubby growth with thick walls and a conspicuous dark outer layer which was much thicker in the subapical regions. The rest of the hyphal tip ultrastructure was unaffected by the drug, indicating considerable specificity for the primary target. The drug-induced growth alteration produced very compact clumps in broth dilution wells, making it possible to score the morphological effect macroscopically. The morphological changes could be assayed quantitatively by using conventional broth microdilution susceptibility assay conditions. We defined the endpoint as the lowest concentration required to produce the morphological effect and called it the minimum effective concentration to distinguish it from the no-growth endpoints used in MIC determinations. The minimum effective concentration assay was related to inhibition of glucan synthase activity in vitro and may provide a starting point for

  10. Identification and Characterization of an Antifungal Protein, AfAFPR9, Produced by Marine-Derived Aspergillus fumigatus R9.

    PubMed

    Rao, Qi; Guo, Wenbin; Chen, Xinhua

    2015-05-01

    A fungal strain, R9, was isolated from the South Atlantic sediment sample and identified as Aspergillus fumigatus. An antifungal protein, AfAFPR9, was purified from the culture supernatant of Aspergillus fumigatus R9. AfAFPR9 was identified to be restrictocin, which is a member of the ribosome-inactivating proteins (RIPs), by MALDI-TOF-TOF-MS. AfAFPR9 displayed antifungal activity against plant pathogenic Fusarium oxysporum, Alternaria longipes, Colletotrichum gloeosporioides, Paecilomyces variotii, and Trichoderma viride at minimum inhibitory concentrations of 0.6, 0.6, 1.2, 1.2, and 2.4 μg/disc, respectively. Moreover, AfAFPR9 exhibited a certain extent of thermostability, and metal ion and denaturant tolerance. The iodoacetamide assay showed that the disulfide bridge in AfAFPR9 was indispensable for its antifungal action. The cDNA encoding for AfAFPR9 was cloned from A. fumigatus R9 by RTPCR and heterologously expressed in E. coli. The recombinant AfAFPR9 protein exhibited obvious antifungal activity against C. gloeosporioides, T. viride, and A. longipes. These results reveal the antifungal properties of a RIP member (AfAFPR9) from marine-derived Aspergillus fumigatus and indicated its potential application in controlling plant pathogenic fungi. PMID:25394604

  11. Multi-azole resistant Aspergillus fumigatus harboring Cyp51A TR46/Y121F/T289A isolated in Japan.

    PubMed

    Hagiwara, Daisuke; Takahashi, Hiroki; Fujimoto, Masanori; Sugahara, Mai; Misawa, Yoshiki; Gonoi, Tohru; Itoyama, Satoru; Watanabe, Akira; Kamei, Katsuhiko

    2016-08-01

    Multi-azole resistant Aspergillus fumigatus carrying TR46/Y121F/T289A was isolated from a patient in Japan in Dec 2013. This strain grouped into the same clade of the ones which were clinically isolated in France and Germany. A. fumigatus harboring this mutation could be rapidly diffused outside the Eurasian continent. PMID:26898666

  12. Structural Requirements for the Activity of the MirB Ferrisiderophore Transporter of Aspergillus fumigatus

    PubMed Central

    Raymond-Bouchard, Isabelle; Carroll, Cassandra S.; Nesbitt, Jason R.; Henry, Kevin A.; Pinto, Linda J.; Moinzadeh, Mina; Scott, Jamie K.

    2012-01-01

    Siderophores have been identified as virulence factors in the opportunistic fungal pathogen Aspergillus fumigatus. The 14-pass transmembrane protein MirB is postulated to function as a siderophore transporter, responsible for uptake of the hydroxamate siderophore N,N′,N″-triacetylfusarinine C (TAFC). Our aim was to identify amino acids of A. fumigatus MirB that are crucial for uptake of TAFC. Site-directed mutagenesis was used to create MirB mutants. Expression of wild-type and mutant proteins in the Saccharomyces cerevisiae strain PHY14, which lacks endogenous siderophore transporters, was confirmed by Western blotting. TAFC transport assays using 55Fe-labeled TAFC and growth assays with Fe-TAFC as the sole iron source identified alanine 125, tyrosine 577, loop 3, and the second half of loop 7 (Loop7Del2) as crucial for function, since their substitution or deletion abrogated uptake completely. Wild-type MirB transported ferricrocin and coprogen as well as TAFC but not ferrichrysin. MirB was localized by fluorescence microscopy using antisera raised against a MirB extracellular loop peptide. Immunofluorescence microscopy showed that in yeast, wild-type MirB had a punctate distribution under the plasma membrane, as did the A125D and Y577A strains, indicating that the defect in transport of these mutants was unlikely to be due to mislocalization or degradation. MirB immunolocalization in A. fumigatus showed that the transporter was found in vesicles which cycled between the cytoplasm and the plasma membrane and was concentrated at the hyphal tips. The location of MirB was not influenced by the presence of the siderophore TAFC but was sensitive to internal iron stores. PMID:22903978

  13. Genomic Context of Azole Resistance Mutations in Aspergillus fumigatus Determined Using Whole-Genome Sequencing

    PubMed Central

    Abdolrasouli, Alireza; Rhodes, Johanna; Beale, Mathew A.; Hagen, Ferry; Rogers, Thomas R.; Chowdhary, Anuradha; Meis, Jacques F.

    2015-01-01

    ABSTRACT A rapid and global emergence of azole resistance has been observed in the pathogenic fungus Aspergillus fumigatus over the past decade. The dominant resistance mechanism appears to be of environmental origin and involves mutations in the cyp51A gene, which encodes a protein targeted by triazole antifungal drugs. Whole-genome sequencing (WGS) was performed for high-resolution single-nucleotide polymorphism (SNP) analysis of 24 A. fumigatus isolates, including azole-resistant and susceptible clinical and environmental strains obtained from India, the Netherlands, and the United Kingdom, in order to assess the utility of WGS for characterizing the alleles causing resistance. WGS analysis confirmed that TR34/L98H (a mutation comprising a tandem repeat [TR] of 34 bases in the promoter of the cyp51A gene and a leucine-to-histidine change at codon 98) is the sole mechanism of azole resistance among the isolates tested in this panel of isolates. We used population genomic analysis and showed that A. fumigatus was panmictic, with as much genetic diversity found within a country as is found between continents. A striking exception to this was shown in India, where isolates are highly related despite being isolated from both clinical and environmental sources across >1,000 km; this broad occurrence suggests a recent selective sweep of a highly fit genotype that is associated with the TR34/L98H allele. We found that these sequenced isolates are all recombining, showing that azole-resistant alleles are segregating into diverse genetic backgrounds. Our analysis delineates the fundamental population genetic parameters that are needed to enable the use of genome-wide association studies to identify the contribution of SNP diversity to the generation and spread of azole resistance in this medically important fungus. PMID:26037120

  14. Transcriptional Regulation of Chemical Diversity in Aspergillus fumigatus by LaeA

    PubMed Central

    Perrin, Robyn M; Fedorova, Natalie D; Bok, Jin Woo; Cramer, Robert A; Wortman, Jennifer R; Kim, H. Stanley; Nierman, William C; Keller, Nancy P

    2007-01-01

    Secondary metabolites, including toxins and melanins, have been implicated as virulence attributes in invasive aspergillosis. Although not definitively proved, this supposition is supported by the decreased virulence of an Aspergillus fumigatus strain, ΔlaeA, that is crippled in the production of numerous secondary metabolites. However, loss of a single LaeA-regulated toxin, gliotoxin, did not recapitulate the hypovirulent ΔlaeA pathotype, thus implicating other toxins whose production is governed by LaeA. Toward this end, a whole-genome comparison of the transcriptional profile of wild-type, ΔlaeA, and complemented control strains showed that genes in 13 of 22 secondary metabolite gene clusters, including several A. fumigatus–specific mycotoxin clusters, were expressed at significantly lower levels in the ΔlaeA mutant. LaeA influences the expression of at least 9.5% of the genome (943 of 9,626 genes in A. fumigatus) but positively controls expression of 20% to 40% of major classes of secondary metabolite biosynthesis genes such as nonribosomal peptide synthetases (NRPSs), polyketide synthases, and P450 monooxygenases. Tight regulation of NRPS-encoding genes was highlighted by quantitative real-time reverse-transcription PCR analysis. In addition, expression of a putative siderophore biosynthesis NRPS (NRPS2/sidE) was greatly reduced in the ΔlaeA mutant in comparison to controls under inducing iron-deficient conditions. Comparative genomic analysis showed that A. fumigatus secondary metabolite gene clusters constitute evolutionarily diverse regions that may be important for niche adaptation and virulence attributes. Our findings suggest that LaeA is a novel target for comprehensive modification of chemical diversity and pathogenicity. PMID:17432932

  15. The pH-Responsive PacC Transcription Factor of Aspergillus fumigatus Governs Epithelial Entry and Tissue Invasion during Pulmonary Aspergillosis

    PubMed Central

    Alcazar-Fuoli, Laura; Cairns, Timothy C.; Muñoz, Alberto; Walker, Louise A.; Herbst, Susanne; Safari, Maryam; Cheverton, Angela M.; Chen, Dan; Liu, Hong; Saijo, Shinobu; Fedorova, Natalie D.; Armstrong-James, Darius; Munro, Carol A.; Read, Nick D.; Filler, Scott G.; Espeso, Eduardo A.; Nierman, William C.; Haas, Hubertus; Bignell, Elaine M.

    2014-01-01

    Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 β-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to

  16. Metabolites Produced by the Endophytic Fungus Aspergillus fumigatus from the Stem of Erythrophloeum fordii Oliv.

    PubMed

    Shi, Yu-Sheng; Zhang, Yan; Chen, Xiao-Zhong; Zhang, Ning; Liu, Yun-Bao

    2015-01-01

    A new diketopiperazine alkaloid named spirotryprostatin K (1), and five known alkaloids, spiro[5H,10H-dipyrrolo[1,2-a:1',2'-d]pyrazine-2(3H),2'-[2H]-indole]-3',5,10(1'H) trione (2), 6-methoxyspirotryprostatin B (3), pseurotin A (4), N-β-acetyltryptamine (5), and lumichrome (6) were isolated from the endophytic fungus Aspergillus fumigatus. The structure and the absolute configuration of spirotryprostatin K were established by extensive spectroscopic analyses, acid hydrolysis and ECD calculations. Pseurotin A exhibited indirect anti-inflammatory activity by suppressing the lipopolysaccharide-induced proinflammatory factors in BV2 microglial cells, with an IC50 of 5.20 µM. PMID:26111169

  17. Nanoscale biophysical properties of the cell surface galactosaminogalactan from the fungal pathogen Aspergillus fumigatus

    NASA Astrophysics Data System (ADS)

    Beaussart, Audrey; El-Kirat-Chatel, Sofiane; Fontaine, Thierry; Latgé, Jean-Paul; Dufrêne, Yves F.

    2015-09-01

    Many fungal pathogens produce cell surface polysaccharides that play essential roles in host-pathogen interactions. In Aspergillus fumigatus, the newly discovered polysaccharide galactosaminogalactan (GAG) mediates adherence to a variety of substrates through molecular mechanisms that are poorly understood. Here we use atomic force microscopy to unravel the localization and adhesion of GAG on living fungal cells. Using single-molecule imaging with tips bearing anti-GAG antibodies, we found that GAG is massively exposed on wild-type (WT) germ tubes, consistent with the notion that this glycopolymer is secreted by the mycelium of A. fumigatus, while it is lacking on WT resting conidia and on germ tubes from a mutant (Δuge3) deficient in GAG. Imaging germ tubes with tips bearing anti-β-glucan antibodies shows that exposure of β-glucan is strongly increased in the Δuge3 mutant, indicating that this polysaccharide is masked by GAG during hyphal growth. Single-cell force measurements show that expression of GAG on germ tubes promotes specific adhesion to pneumocytes and non-specific adhesion to hydrophobic substrates. These results provide a molecular foundation for the multifunctional adhesion properties of GAG, thus suggesting it could be used as a potential target in anti-adhesion therapy and immunotherapy. Our methodology represents a powerful approach for characterizing the nanoscale organization and adhesion of cell wall polysaccharides during fungal morphogenesis, thereby contributing to increase our understanding of their role in biofilm formation and immune responses.

  18. Improved production of melanin from Aspergillus fumigatus AFGRD105 by optimization of media factors.

    PubMed

    Raman, Nitya Meenakshi; Shah, Pooja Harish; Mohan, Misha; Ramasamy, Suganthi

    2015-12-01

    Melanins are indolic polymers produced by many genera included among plants, animals and microorganisms and targeted mainly for their wide range of applications in cosmetics, agriculture and medicine. An approach to analyse the cumulative effect of parameters for enhanced melanin production was carried out using response surface methodology. In this present study, optimization of media and process parameters for melanin production from Aspergillus fumigatus AFGRD105 (GenBank: JX041523; NFCCI accession number: 3826) was carried out by an initial univariate approach followed by statistical response surface methodology. The univariate approach was used to standardise the parameters that can be used for the 12-run Plackett-Burman design that is used for screening for critical parameters. Further optimization of parameters was analysed using Box-Behnken design. The optimum conditions observed were temperature, moisture and sodium dihydrogen phosphate concentration. The yield of every run of both designs were confirmed to be melanin by laboratory tests of analysis in the presence of acids, base and water. This is the first report confirming an increase in melanin production A. fumigatus AFGRD105 without the addition of costly additives. PMID:26597959

  19. Perturbations in small molecule synthesis uncovers an iron-responsive secondary metabolite network in Aspergillus fumigatus

    PubMed Central

    Wiemann, Philipp; Lechner, Beatrix E.; Baccile, Joshua A.; Velk, Thomas A.; Yin, Wen-Bing; Bok, Jin Woo; Pakala, Suman; Losada, Liliana; Nierman, William C.; Schroeder, Frank C.; Haas, Hubertus; Keller, Nancy P.

    2014-01-01

    Iron plays a critical role in survival and virulence of the opportunistic pathogen Aspergillus fumigatus. Two transcription factors, the GATA-factor SreA and the bZip-factor HapX oppositely monitor iron homeostasis with HapX activating iron acquisition pathways (e.g., siderophores) and shutting down iron consumptive pathways (and SreA) during iron starvation conditions whereas SreA negatively regulates HapX and corresponding pathways during iron sufficiency. Recently the non-ribosomal peptide, hexadehydroastechrome (HAS; a tryptophan-derived iron (III)-complex), has been found important in A. fumigatus virulence. We found that HAS overproduction caused an iron starvation phenotype, from alteration of siderophore pools to regulation of iron homeostasis gene expression including sreA. Moreover, we uncovered an iron dependent secondary metabolism network where both SreA and HapX oppositely regulate multiple other secondary metabolites including HAS. This circuitry links iron-acquisition and consumption pathways with secondary metabolism—thus placing HAS as part of a metabolic feedback circuitry designed to balance iron pools in the fungus and presenting iron availability as one environmental trigger of secondary metabolism. PMID:25386169

  20. Tremorgenic mycotoxins from Aspergillus fumigatus as a possible occupational health problem in sawmills.

    PubMed Central

    Land, C J; Hult, K; Fuchs, R; Hagelberg, S; Lundström, H

    1987-01-01

    Wood-trimmers' disease, generally called extrinsic allergic alveolitis, which affects workers in sawmills, is thought to be caused by fungal diaspores. The importance of Aspergillus fumigatus on the surface of wood dried in kilns is accentuated by its ability to produce tremorgenic mycotoxins. Eight strains of A. fumigatus from five different sawmills were isolated and cultivated on liquid media, and one of the strains was also cultivated on wood blocks. Extracts were prepared, and the tremorgenic reactions were induced by oral administration of extracts to rats. Extracts of the strain grown in liquid medium and on wood blocks induced very strong tremorgenic reactions when administered orally to rats. Four other strains induced mild tremorgenic reactions. High-performance liquid chromatography analysis revealed two tremorgenic mycotoxins, verruculogen and fumitremorgen C, in the five toxic strains. One nontoxic strain produced detectable levels of verruculogen. These results, coupled with the known resemblance of the acutely toxic phase of wood-trimmers' disease to the symptoms produced by these tremorgens, imply that wood-trimmers' disease and similar occupational diseases are, at least in part, mycotoxicoses. PMID:3555338

  1. Tremorgenic mycotoxins from Aspergillus fumigatus as a possible occupational health problem in sawmills.

    PubMed

    Land, C J; Hult, K; Fuchs, R; Hagelberg, S; Lundström, H

    1987-04-01

    Wood-trimmers' disease, generally called extrinsic allergic alveolitis, which affects workers in sawmills, is thought to be caused by fungal diaspores. The importance of Aspergillus fumigatus on the surface of wood dried in kilns is accentuated by its ability to produce tremorgenic mycotoxins. Eight strains of A. fumigatus from five different sawmills were isolated and cultivated on liquid media, and one of the strains was also cultivated on wood blocks. Extracts were prepared, and the tremorgenic reactions were induced by oral administration of extracts to rats. Extracts of the strain grown in liquid medium and on wood blocks induced very strong tremorgenic reactions when administered orally to rats. Four other strains induced mild tremorgenic reactions. High-performance liquid chromatography analysis revealed two tremorgenic mycotoxins, verruculogen and fumitremorgen C, in the five toxic strains. One nontoxic strain produced detectable levels of verruculogen. These results, coupled with the known resemblance of the acutely toxic phase of wood-trimmers' disease to the symptoms produced by these tremorgens, imply that wood-trimmers' disease and similar occupational diseases are, at least in part, mycotoxicoses. PMID:3555338

  2. Chemical and immunological characterization of the extracellular galactomannan of Aspergillus fumigatus.

    PubMed Central

    Latgé, J P; Kobayashi, H; Debeaupuis, J P; Diaquin, M; Sarfati, J; Wieruszeski, J M; Parra, E; Bouchara, J P; Fournet, B

    1994-01-01

    The galactomannan (GM) produced extracellularly by Aspergillus fumigatus has been purified by a double sequential hydrazine-nitrous acid treatment of the ethanol precipitate of the culture filtrate. Nuclear magnetic resonance and gas-liquid chromatography-mass spectrometry analysis have been performed on intact GM, acid-hydrolyzed GM, and oligomers resulting from the acetolysis of the acid-hydrolyzed GM. Results show that A. fumigatus GM is composed of a linear mannan core with an alpha-(1-2)-linked mannotetraose repeating unit attached via alpha-(1-6) linkage. Side chains composed of an average of 4 to 5 beta-(1-5)-galactofuranose units are linked to C-6 and C-3 positions of alpha-(1-2)-linked mannose units of the mannan. The immunoreactivity of GM and HCl-hydrolyzed GM was studied by use of human sera from aspergillosis patients and an antigalactofuran monoclonal antibody. The alpha-(1-2) (1-6)-mannan core is not antigenic. The immunogenic galactofuran is found amongst several exocellular glycoproteins. According to a direct enzyme-linked immunosorbent assay with GM as the detector antigen, only 26% of the serum samples from aspergilloma patients (all positive by immunodiffusion assays) give optical density values superior to a cutoff estimated as the mean +/- 3 standard deviations of values obtained with control sera. Images PMID:7960122

  3. Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus's Role in Virulence.

    PubMed

    Losada, Liliana; Sugui, Janyce A; Eckhaus, Michael A; Chang, Yun C; Mounaud, Stephanie; Figat, Abigail; Joardar, Vinita; Pakala, Suman B; Pakala, Suchitra; Venepally, Pratap; Fedorova, Natalie; Nierman, William C; Kwon-Chung, Kyung J

    2015-04-01

    Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence

  4. Genetic and structural validation of Aspergillus fumigatus UDP-N-acetylglucosamine pyrophosphorylase as an antifungal target

    PubMed Central

    Fang, Wenxia; Du, Ting; Raimi, Olawale G; Hurtado-Guerrero, Ramon; Urbaniak, Michael D; Ibrahim, Adel F M; Ferguson, Michael A J; Jin, Cheng; Aalten, Daan M F

    2013-01-01

    The sugar nucleotide UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite in both prokaryotes and eukaryotes. In fungi, it is the precursor for the synthesis of chitin, an essential component of the fungal cell wall. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is the final enzyme in eukaryotic UDP-GlcNAc biosynthesis, converting UTP and N-acetylglucosamine-1-phosphate (GlcNAc-1P) to UDP-GlcNAc. As such, this enzyme may provide an attractive target against pathogenic fungi. Here, we demonstrate that the fungal pathogen Aspergillus fumigatus possesses an active UAP (AfUAP1) that shows selectivity for GlcNAc-1P as the phosphosugar substrate. A conditional mutant, constructed by replacing the native promoter of the A. fumigatus uap1 gene with the Aspergillus nidulans alcA promoter, revealed that uap1 is essential for cell survival and important for cell wall synthesis and morphogenesis. The crystal structure of AfUAP1 was determined and revealed exploitable differences in the active site compared with the human enzyme. Thus AfUAP1 could represent a novel antifungal target and this work will assist the future discovery of small molecule inhibitors against this enzyme. PMID:23750903

  5. Biosynthesis of extracellular and intracellular gold nanoparticles by Aspergillus fumigatus and A. flavus.

    PubMed

    Gupta, Saurabh; Bector, Shruti

    2013-05-01

    Green chemistry is a boon for the development of safe, stable and ecofriendly nanostructures using biological tools. The present study was carried out to explore the potential of selected fungal strains for biosynthesis of intra- and extracellular gold nanostructures. Out of the seven cultures, two fungal strains (SBS-3 and SBS-7) were selected on the basis of development of dark pink colour in cell free supernatant and fungal beads, respectively indicative of extra- and intracellular gold nanoparticles production. Both biomass associated and cell free gold nanoparticles were characterized using X-ray diffractogram (XRD) analysis and transmission electron microscopy (TEM). XRD analysis confirmed crystalline, face-centered cubic lattice of metallic gold nanoparticles along with average crystallite size. A marginal difference in average crystallite size of extracellular (17.76 nm) and intracellular (26 and 22 nm) Au-nanostructures was observed using Scherrer equation. In TEM, a variety of shapes (triangles, spherical, hexagonal) were observed in both extra- and intracellular nanoparticles. 18S rRNA gene sequence analysis by multiple sequence alignment (BLAST) indicated 99 % homology of SBS-3 to Aspergillus fumigatus with 99 % alignment coverage and 98 % homology of SBS-7 to Aspergillus flavus with 98 % alignment coverage respectively. Native-PAGE and activity staining further confirmed enzyme linked synthesis of gold nanoparticles. PMID:23400423

  6. Secretome diversity and quantitative analysis of cellulolytic Aspergillus fumigatus Z5 in the presence of different carbon sources

    PubMed Central

    2013-01-01

    Background Aspergillus fumigatus Z5 has a strong ability to decompose lignocellulose biomass, and its extracellular protein secretion has been reported in earlier studies employing traditional techniques. However, a comprehensive analysis of its secretion in the presence of different carbon sources is still lacking. The goal of this work was to identify, quantify and compare the secretome of A. fumigatus Z5 in the presence of different carbon sources to understand in more details the mechanisms of lignocellulose decomposition by Aspergillus fumigatus Z5. Results Cellulolytic A. fumigatus Z5 was grown in the presence of glucose (Gl), Avicel (Av) and rice straw (RS), and the activities of several lignocellulosic enzymes were determined with chromatometry method. The maximum activities of endoglucanase, exoglucanase, β-glucosidase, laminarinase, lichenase, xylanase and pectin lyase were 12.52, 0.59, 2.30, 2.37, 1.68, 15.02 and 11.40 U·ml-1, respectively. A total of 152, 125 and 61 different proteins were identified in the presence of RS, Av and Gl, respectively, and the proteins were functionally divided into glycoside hydrolases, lipases, peptidases, peroxidases, esterases, protein translocating transporters and hypothetical proteins. A total of 49 proteins were iTRAQ-quantified in all the treatments, and the quantification results indicated that most of the cellulases, hemicellulases and glycoside hydrolases were highly upregulated when rice straw and Avicel were used as carbon sources (compared with glucose). Conclusions The proteins secreted from A. fumigatus Z5 in the present of different carbon source conditions were identified by LC-MS/MS and quantified by iTRAQ-based quantitative proteomics. The results indicated that A. fumigatus Z5 could produce considerable cellulose-, hemicellulose-, pectin- and lignin-degrading enzymes that are valuable for the lignocellulosic bioenergy industry. PMID:24131596

  7. Azole-resistant Aspergillus fumigatus in Denmark: a laboratory-based study on resistance mechanisms and genotypes.

    PubMed

    Jensen, R H; Hagen, F; Astvad, K M T; Tyron, A; Meis, J F; Arendrup, M C

    2016-06-01

    Azole-resistant Aspergillus fumigatus originating from the environment as well as induced during therapy are continuously emerging in Danish clinical settings. We performed a laboratory-based retrospective study (2010-2014) of azole resistance and genetic relationship of A. fumigatus at the national mycology reference laboratory of Denmark. A total of 1162 clinical and 133 environmental A. fumigatus isolates were identified by morphology, thermotolerance and/or β-tubulin sequencing. Screening for azole resistance was carried out using azole agar, and resistant isolates were susceptibility tested by the EUCAST (European Committee on Antimicrobial Susceptibility Testing) E.Def 9.2 reference method and CYP51A sequenced. Genotyping was performed for outbreak investigation and, when appropriate, short tandem repeat Aspergillus fumigatus microsatellite assay. All 133 environmental A. fumigatus isolates were azole susceptible. However, from 2010 to 2014, there was an increasing prevalence of azole resistance (from 1.4 to 6% isolates (p <0.001) and 1.8 to 4% patients (p <0.05)) among the clinical isolates, with the well-known environmental CYP51A variant TR34/L98H responsible for >50% of the azole resistance mechanisms. Among 184 Danish A. fumigatus isolates, 120 unique genotypes were identified and compared to a collection of 1822 international genotypes. Seven (5.8%) Danish genotypes were shared between isolates within Denmark but with different origin, 19 (15.8%) were shared with foreign genotypes, and two (11.8%) of 17 genotypes of isolates carrying the TR34/L98H resistance mechanisms were identical to two Dutch TR34/L98H isolates. Our findings underlines the demand for correct identification and susceptibility testing of clinical mould isolates. Furthermore, although complex, genotyping supported the hypotheses regarding clonal expansion and the potential of a single origin for the TR34/L98H clone. PMID:27091095

  8. Azole Resistance in Aspergillus fumigatus: Can We Retain the Clinical Use of Mold-Active Antifungal Azoles?

    PubMed Central

    Verweij, Paul E.; Chowdhary, Anuradha; Melchers, Willem J. G.; Meis, Jacques F.

    2016-01-01

    Azole resistance in Aspergillus fumigatus has emerged as a global health problem. Although the number of cases of azole-resistant aspergillosis is still limited, resistance mechanisms continue to emerge, thereby threatening the role of the azole class in the management of diseases caused by Aspergillus. The majority of cases of azole-resistant disease are due to resistant A. fumigatus originating from the environment. Patient management is difficult due to the absence of patient risk factors, delayed diagnosis, and limited treatment options, resulting in poor treatment outcome. International and collaborative efforts are required to understand how resistance develops in the environment to allow effective measures to be implemented aimed at retaining the use of azoles both for food production and human medicine. PMID:26486705

  9. Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum*

    PubMed Central

    Wiedner, Susan D.; Burnum, Kristin E.; Pederson, LeeAnna M.; Anderson, Lindsey N.; Fortuin, Suereta; Chauvigné-Hines, Lacie M.; Shukla, Anil K.; Ansong, Charles; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

    2012-01-01

    Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli. PMID:22865858

  10. [Anti-infectious activity of intravitreal injectable voriconazole microspheres on experimental rabbit fungal endophthalmitis of Aspergillus fumigatus].

    PubMed

    Yang, Li-Na; Xin, Meng; Wu, Xiang-Gen; Jiang, Hao-Ran

    2010-06-01

    The therapeutic effect of sustained intravitreal injectable voriconazole microspheres (VCZ-MS) on an experimental endophthalmitis of Aspergillus fumigatus was investigated. VCZ-MS was prepared successfully and its physico-chemical property was also evaluated. Right eyes of albino rabbits were infected with an intravitreal injection of 1 000 CFU x mL(-1) of susceptible Aspergillus fumigatus. All fungal endophthalmitis models were randomly divided into five groups 48 hours later: Group A is control group with no treatment; in group B, vitrectomy was performed combined with intravitreal 3 times injections of 100 microg x 0.1 mL(-1) voriconazole every other day. In group C, D and E, vitrectomy was performed combined with intravitreal injection of 0.5 mg, 1.0 mg and 1.5 mg VCZ-MS respectively. The treatment effect was assessed by slit lamp and indirect ophthalmoscope funduscopy examination, using clinical grading system of inflammation in the anterior chamber and the vitreous opacity. The optical microscopy revealed that microspheres obtained from the experiment design were opaque, discrete and spherical particles with smooth surfaces. The drug content and encapsulation efficiency of microspheres were 29.94% and 73.5%, respectively. Endophthalmitis occurred in all eyes of group A, and rapidly developed to panophthalmitis. The inflammation grade of group B, C, D or E was lower than that of group A (P < 0.05). The grade of vitreous opacity in group C, D, E is lower than group B (P < 0.05). Two eyes in group C developed to panophthalmitis. But in group D and E, all eyes whose inflammation was controlled had no recurrence with vitreous clear. Histopathological examination showed normal structures in the cured eyes, while most uncured eyes were atrophic and with eyeball destroyed. So, it can be safely concluded that the curative effect of intravitreal VCZ-MS is significantly better than that of routine intraocular injection of voriconazole. The optimal dose is the one

  11. [Native valve Aspergillus fumigatus endocarditis with blood culture positive and negative for galactomannan antigen. Case report and literature review].

    PubMed

    Pemán, Javier; Ortiz, Rebeca; Osseyran, Faisa; Pérez-Bellés, Carmen; Crespo, Marisa; Chirivella, Melitina; Frasquet, Juan; Quesada, Anastasio; Cantón, Emilia; Gobernado, Miguel

    2007-06-01

    Native valve endocarditis caused by Aspergillus spp. is an uncommon disease with a high mortality rate. Generally, Aspergillus is isolated from affected valve in post-mortem or biopsy specimens. However, its isolation from blood cultures is exceedingly rare. We report a case of fungal endocarditis in a native mitral valve with the isolation of Aspergillus fumigatus both in valve vegetation and in blood culture bottles. The patient underwent valve replacement and antifungal treatment with voriconazole and caspofungin, but he died on post-operative day 45 with disseminated aspergillosis confirmed by necropsy. Paradoxically, galactomannan antigen detection in serum was negative. This is the third case of Aspergillus endocarditis with positive blood culture reported in the literature. PMID:17604438

  12. Aspergillus Infections in Transplant Recipients

    PubMed Central

    Singh, Nina; Paterson, David L.

    2005-01-01

    Aspergillus infections are occurring with an increasing frequency in transplant recipients. Notable changes in the epidemiologic characteristics of this infection have occurred; these include a change in risk factors and later onset of infection. Management of invasive aspergillosis continues to be challenging, and the mortality rate, despite the use of newer antifungal agents, remains unacceptably high. Performing molecular studies to discern new targets for antifungal activity, identifying signaling pathways that may be amenable to immunologic interventions, assessing combination regimens of antifungal agents or combining antifungal agents with modulation of the host defense mechanisms, and devising diagnostic assays that can rapidly and reliably diagnose infections represent areas for future investigations that may lead to further improvement in outcomes. PMID:15653818

  13. Utility of IgE (total and Aspergillus fumigatus specific) in monitoring for response and exacerbations in allergic bronchopulmonary aspergillosis.

    PubMed

    Agarwal, Ritesh; Aggarwal, Ashutosh N; Sehgal, Inderpaul S; Dhooria, Sahajal; Behera, Digambar; Chakrabarti, Arunaloke

    2016-01-01

    The role of total and specific IgE in monitoring treatment responses in allergic bronchopulmonary aspergillosis (ABPA) remains poorly studied. Here in, we evaluate the utility of total and Aspergillus fumigatus specific IgE in the follow-up of ABPA. Eighty-one consecutive treatment-naïve patients of ABPA (acute stage) with pulmonary infiltrates and bronchiectasis underwent measurement of total and A. fumigatus specific IgE at baseline, after 8 weeks of glucocorticoid therapy, and during exacerbations. There was clinical and radiological improvement after treatment with median decline of total IgE by 51.9%. The total IgE declined by at least 35%, 25% and 20% in 69 (85.2%), 76 (93.6%) and 78 (96.3%) patients, respectively. On the other hand, the A. fumigatus specific IgE increased in 42 (51.9%) subjects, and the mean increase was 1.4%, after 8 weeks. Among 13 patients with exacerbation, 12 (92.3%) had a rise of total IgE by >50%. The A. fumigatus specific IgE increased in only five (38.5%) subjects during exacerbation. Thus, the total IgE is a useful test in monitoring treatment responses in ABPA while A. fumigatus specific IgE has limited utility. PMID:26575791

  14. Verruculogen associated with Aspergillus fumigatus hyphae and conidia modifies the electrophysiological properties of human nasal epithelial cells

    PubMed Central

    Khoufache, Khaled; Puel, Olivier; Loiseau, Nicolas; Delaforge, Marcel; Rivollet, Danièle; Coste, André; Cordonnier, Catherine; Escudier, Estelle; Botterel, Françoise; Bretagne, Stéphane

    2007-01-01

    Background The role of Aspergillus fumigatus mycotoxins in the colonization of the respiratory tract by conidia has not been studied extensively, even though patients at risk from invasive aspergillosis frequently exhibit respiratory epithelium damage. In a previous study, we found that filtrates of A. fumigatus cultures can specifically alter the electrophysiological properties of human nasal epithelial cells (HNEC) compared to those of non pathogenic moulds. Results We fractionated the organic phase of filtrate from 3-day old A. fumigatus cultures using high-performance liquid chromatography. The different fractions were tested for their ability to modify the electrophysiological properties of HNEC in an in vitro primary culture model. The fraction collected between 20 and 30 min mimicked the effects of the whole filtrate, i.e. decrease of transepithelial resistance and increase of potential differences, and contained secondary metabolites such as helvolic acid, fumagillin, and verruculogen. Only verruculogen (10-8 M) had effects similar to the whole filtrate. We verified that verruculogen was produced by a collection of 67 human, animal, plant and environmental A. fumigatus isolates. Using MS-MS analysis, we found that verruculogen was associated with both mycelium and conidia extracts. Conclusion Verruculogen is a secondary metabolite that modifies the electrophysiological properties of HNEC. The role of these modifications in the colonization and invasion of the respiratory epithelium by A. fumigatus on first contact with the epithelium remains to be determined. PMID:17244350

  15. In vitro Protease Inhibition and Cytotoxicity of Aspergillus fumigatus Biomolecules Secreted under Long-Term Aerated Conditions

    PubMed Central

    Arsic Arsenijevic, Valentina S.; Pekmezovic, Marina G.; Rajkovic, Katarina M.; Vekic, Berislav P.; Barac, Aleksandra M.; Tasic-Otasevic, Suzana; Petkovic, Ljubica Dj.

    2014-01-01

    The fatality rate of invasive aspergillosis (IA) is still very high, especially in prolonged and untreated pulmonary cases. Aspergillus fumigatus is the main causative agent of IA and investigation of its metabolites could provide valuable insight into virulence factor(s) associated with this organism. We evaluated the A. fumigatus culture filtrate (CF) products generated during short- and long-term aerated and non-aerated conditions and tested for (i) inhibition of cysteine or serine proteases and (ii) cytotoxicity. In addition, the mathematical model was determined using response surface methodology (RSM) to estimate the influence of different fermentation conditions on A. fumigatus CF characteristics, predict enzyme inhibition and make possible correlations with in vivo conditions. Biosynthesis of A. fumigatus low molecular weight proteinaceous products (from 6.4 to 15.4 kDa) was observed after 6 days of growth under aerated and alkaline conditions. Also, only these CFs showed significant reduction in cell lines survival (Caco-2 and WISH 35.6% and 54.6%, respectively). Obtained results provide solid starting point for further studies that would include: (i) detailed chemical characterization of A. fumigatus CF, (ii) activity relationships and in vivo correlation with pathogenicity of prolonged pulmonary IA and (iii) possible use of biomolecules as diagnostic or therapeutic markers. PMID:25170296

  16. Two C4-sterol methyl oxidases (Erg25) catalyse ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus

    PubMed Central

    Blosser, Sara J.; Merriman, Brittney; Grahl, Nora; Chung, Dawoon

    2014-01-01

    The human pathogen Aspergillus fumigatus adapts to stress encountered in the mammalian host as part of its ability to cause disease. The transcription factor SrbA plays a significant role in this process by regulating genes involved in hypoxia and low-iron adaptation, antifungal drug responses and virulence. SrbA is a direct transcriptional regulator of genes encoding key enzymes in the ergosterol biosynthesis pathway, including erg25A and erg25B, and ΔsrbA accumulates C4-methyl sterols, suggesting a loss of Erg25 activity [C4-sterol methyl oxidase (SMO)]. Characterization of the two genes encoding SMOs in Aspergillus fumigatus revealed that both serve as functional C4-demethylases, with Erg25A serving in a primary role, as Δerg25A accumulates more C4-methyl sterol intermediates than Δerg25B. Single deletion of these SMOs revealed alterations in canonical ergosterol biosynthesis, indicating that ergosterol may be produced in an alternative fashion in the absence of SMO activity. A Δerg25A strain displayed moderate susceptibility to hypoxia and the endoplasmic reticulum stress-inducing agent DTT, but was not required for virulence in murine or insect models of invasive aspergillosis. Inducing expression of erg25A partially restored the hypoxia growth defect of ΔsrbA. These findings implicated Aspergillus fumigatus SMOs in the maintenance of canonical ergosterol biosynthesis and indicated an overall involvement in the fungal stress response. PMID:25107308

  17. The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

    PubMed Central

    Gallagher, Lorna; Owens, Rebecca A.; Dolan, Stephen K.; O'Keeffe, Grainne; Schrettl, Markus; Kavanagh, Kevin; Jones, Gary W.

    2012-01-01

    The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H2O2 (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P = 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P < 0.001) and H2O2 (P < 0.01), unexpectedly, exogenous gliotoxin relieved H2O2-induced growth inhibition in a dose-dependent manner (0 to 10 μg/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P < 0.05) the growth of the ΔgliK26933 deletion mutant. The A. fumigatus ΔgliK26933 mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus ΔgliK26933 mutant than in those of the wild type (P = 0.0024 [fold difference, 24] and P = 0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus ΔgliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus ΔgliK26933 mutant compared to the wild type (P < 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P = 0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the ΔgliK46645 mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus. PMID:22903976

  18. Heterologous Expression of Lysergic Acid and Novel Ergot Alkaloids in Aspergillus fumigatus

    PubMed Central

    Robinson, Sarah L.

    2014-01-01

    Different lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungus Epichloë festucae var. lolii × Epichloë typhina isolate Lp1 (henceforth referred to as Epichloë sp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate genes easH and cloA were expressed in a mutant strain of the mold Aspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with the Epichloë sp. Lp1 allele of easA, which encodes an enzyme that catalyzed the synthesis of agroclavine from an A. fumigatus intermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressing easA and cloA from Epichloë sp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result of Epichloë sp. Lp1 easA expression in the absence of cloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids. PMID:25107976

  19. The Aspergillus fumigatus Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility

    PubMed Central

    Song, Jinxing; Zhai, Pengfei; Zhang, Yuanwei; Zhang, Caiyun; Sang, Hong; Han, Guanzhu; Keller, Nancy P.

    2016-01-01

    ABSTRACT Ergosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11). In this study, we demonstrate that a cytochrome b5-like heme-binding damage resistance protein (Dap) family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapA strains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme. In vivo data demonstrate that inactivated DapA combined with activated DapB yields an A. fumigatus mutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found in Saccharomyces cerevisiae and Schizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli. PMID:26908577

  20. A murine inhalation model to characterize pulmonary exposure to dry Aspergillus fumigatus conidia.

    PubMed

    Buskirk, Amanda D; Green, Brett J; Lemons, Angela R; Nayak, Ajay P; Goldsmith, W Travis; Kashon, Michael L; Anderson, Stacey E; Hettick, Justin M; Templeton, Steven P; Germolec, Dori R; Beezhold, Donald H

    2014-01-01

    Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Δalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×105 twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8+ IL17+ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Δalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants. PMID:25340353

  1. A Murine Inhalation Model to Characterize Pulmonary Exposure to Dry Aspergillus fumigatus Conidia

    PubMed Central

    Buskirk, Amanda D.; Green, Brett J.; Lemons, Angela R.; Nayak, Ajay P.; Goldsmith, W. Travis; Kashon, Michael L.; Anderson, Stacey E.; Hettick, Justin M.; Templeton, Steven P.; Germolec, Dori R.; Beezhold, Donald H.

    2014-01-01

    Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Δalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×105 twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8+ IL17+ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Δalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants. PMID:25340353

  2. Kinetic and structural evaluation of selected active site mutants of the Aspergillus fumigatus KDNase (sialidase).

    PubMed

    Yeung, Juliana H F; Telford, Judith C; Shidmoossavee, Fahimeh S; Bennet, Andrew J; Taylor, Garry L; Moore, Margo M

    2013-12-23

    Aspergillus fumigatus is an airborne fungal pathogen. We previously cloned and characterized an exo-sialidase from A. fumigatus and showed that it preferred 2-keto-3-deoxynononic acid (KDN) as a substrate to N-acetylneuraminic acid (Neu5Ac). The purpose of this study was to investigate the structure-function relationships of critical catalytic site residues. Site-directed mutagenesis was used to create three mutant recombinant enzymes: the catalytic nucleophile (Y358H), the general acid/base catalyst (D84A), and an enlargement of the binding pocket to attempt to accommodate the N-acetyl group of Neu5Ac (R171L). Crystal structures for all enzymes were determined. The D84A mutation had an effect in decreasing the activity of AfKDNase that was stronger than that of the same mutation in the structurally similar sialidase from the bacterium Micromonospora viridifaciens. These data suggest that the catalytic acid is more important in the reaction of AfKDNase and that catalysis is less dependent on nucleophilic or electrostatic stabilization of the developing positive charge at the transition state for hydrolysis. Removal of the catalytic nucleophile (Y358H) significantly lowered the activity of the enzyme, but this mutant remained a retaining glycosidase as demonstrated by nuclear magnetic resonance spectroscopic analysis. This is a novel finding that has not been shown with other sialidases. Kinetic activity measured at pH 5.2 revealed that R171L had higher activity on a Neu5Ac-based substrate than wild-type KDNase; hence, leucine in place of arginine in the binding pocket improved catalysis toward Neu5Ac substrates. Hence, whether a sialidase is primarily a KDNase or a neuraminidase is due in part to the presence of an amino acid that creates a steric clash with the N-acetyl group. PMID:24295366

  3. In vitro and ex vivo effects of cyclosporin A on phagocytic host defenses against Aspergillus fumigatus.

    PubMed Central

    Roilides, E; Robinson, T; Sein, T; Pizzo, P A; Walsh, T J

    1994-01-01

    Because cyclosporin A (CsA) is extensively used as an immunosuppressive agent, its effects on phagocytic defenses against Aspergillus fumigatus were studied in vitro and ex vivo. After incubation with 10 to 250 ng of CsA per ml at 37 degrees C for 60 min, polymorphonuclear leukocytes (PMNs) exhibited unaltered superoxide anion (O2-) production in response to phorbol myristate acetate and N-formylmethionyl leucyl phenylalanine, whereas > or = 500 ng/ml significantly suppressed it (P < 0.01). Moreover, at < 250 ng of CsA per ml, PMNs exhibited no change in their capacity to damage unopsonized hyphae of A. fumigatus compared with controls, whereas at > or = 250 ng/ml, CsA suppressed the function (P < 0.01). Although neither CsA (250 ng/ml) nor hydrocortisone (10 micrograms/ml) suppressed PMN O2- production in response to phorbol myristate acetate and N-formylmethionyl leucyl phenylalanine, combination of the two agents reduced the function compared with that at the baseline (P < 0.05). Incubation of monocytes with 100 ng of CsA per ml for 1 or 2 days suppressed their antihyphal activity. No essential change in phagocytic activity of monocyte-derived macrophages (MDMs) against A. fumigatus conidia, tested as the percentage of phagocytosing MDMs and average number of MDM-associated conidia, was detected after 2 or 4 days of incubation with 10 to 1,000 ng of CsA per ml. Furthermore, in rabbits treated with CsA (up to 20 mg/kg of body weight per day intravenously for 7 days), neither O2- production and hyphal damage caused by PMNs or monocytes against hyphae nor phagocytosis of conidia by pulmonary alveolar macrophages was significantly suppressed. Thus, these results demonstrated that CsA within therapeutically relevant concentrations does not suppress antifungal activity of phagocytes except that of circulating monocytes. However, it may induce significant immunosuppression of phagocytes' antifungal function at relatively high concentrations in vitro, especially when

  4. Triazole Fungicides Can Induce Cross-Resistance to Medical Triazoles in Aspergillus fumigatus

    PubMed Central

    Karawajczyk, Anna; Schaftenaar, Gijs; Kema, Gert H. J.; van der Lee, Henrich A.; Klaassen, Corné H.; Melchers, Willem J. G.; Verweij, Paul E.

    2012-01-01

    Background Azoles play an important role in the management of Aspergillus diseases. Azole resistance is an emerging global problem in Aspergillus fumigatus, and may develop through patient therapy. In addition, an environmental route of resistance development has been suggested through exposure to 14α-demethylase inhibitors (DMIs). The main resistance mechanism associated with this putative fungicide-driven route is a combination of alterations in the Cyp51A-gene (TR34/L98H). We investigated if TR34/L98H could have developed through exposure to DMIs. Methods and Findings Thirty-one compounds that have been authorized for use as fungicides, herbicides, herbicide safeners and plant growth regulators in the Netherlands between 1970 and 2005, were investigated for cross-resistance to medical triazoles. Furthermore, CYP51-protein homology modeling and molecule alignment studies were performed to identify similarity in molecule structure and docking modes. Five triazole DMIs, propiconazole, bromuconazole, tebuconazole, epoxiconazole and difenoconazole, showed very similar molecule structures to the medical triazoles and adopted similar poses while docking the protein. These DMIs also showed the greatest cross-resistance and, importantly, were authorized for use between 1990 and 1996, directly preceding the recovery of the first clinical TR34/L98H isolate in 1998. Through microsatellite genotyping of TR34/L98H isolates we were able to calculate that the first isolate would have arisen in 1997, confirming the results of the abovementioned experiments. Finally, we performed induction experiments to investigate if TR34/L98H could be induced under laboratory conditions. One isolate evolved from two copies of the tandem repeat to three, indicating that fungicide pressure can indeed result in these genomic changes. Conclusions Our findings support a fungicide-driven route of TR34/L98H development in A. fumigatus. Similar molecule structure characteristics of five triazole DMIs

  5. Identification and Deletion of Tft1, a Predicted Glycosyltransferase Necessary for Cell Wall β-1,3;1,4-Glucan Synthesis in Aspergillus fumigatus

    PubMed Central

    Samar, Danial; Kieler, Joshua B.; Klutts, J. Stacey

    2015-01-01

    Aspergillus fumigatus is an environmental mold that causes severe, often fatal invasive infections in immunocompromised patients. The search for new antifungal drug targets is critical, and the synthesis of the cell wall represents a potential area to find such a target. Embedded within the main β-1,3-glucan core of the A. fumigatus cell wall is a mixed linkage, β-D-(1,3;1,4)-glucan. The role of this molecule or how it is synthesized is unknown, though it comprises 10% of the glucans within the wall. While this is not a well-studied molecule in fungi, it has been studied in plants. Using the sequences of two plant mixed linkage glucan synthases, a single ortholog was identified in A. fumigatus (Tft1). A strain lacking this enzyme (tft1Δ) was generated along with revertant strains containing the native gene under the control of either the native or a strongly expressing promoter. Immunofluorescence staining with an antibody against β-(1,3;1,4)-glucan and biochemical quantification of this polysaccharide in the tft1Δ strain demonstrated complete loss of this molecule. Reintroduction of the gene into the knockout strain yielded reappearance in amounts that correlated with expected expression of the gene. The loss of Tft1 and mixed linkage glucan yielded no in vitro growth phenotype. However, there was a modest increase in virulence for the tft1Δ strain in a wax worm model. While the precise roles for β-(1,3;1,4)-glucan within A. fumigatus cell wall are still uncertain, it is clear that Tft1 plays a pivotal role in the biosynthesis of this cell wall polysaccharide. PMID:25723175

  6. Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) from Aspergillus fumigatus and Molecular Basis for the Development of Antifungal Drugs.

    PubMed

    Hargrove, Tatiana Y; Wawrzak, Zdzislaw; Lamb, David C; Guengerich, F Peter; Lepesheva, Galina I

    2015-09-25

    Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths/year. The cytochrome P450 51 (CYP51) inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational development of more efficient agents. A. fumigatus has two CYP51 genes, CYP51A and CYP51B, which share 59% amino acid sequence identity. CYP51B is expressed constitutively, whereas gene CYP51A is reported to be inducible. We expressed, purified, and characterized A. fumigatus CYP51B, including determination of its substrate preferences, catalytic parameters, inhibition, and x-ray structure in complexes with voriconazole and the experimental inhibitor (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI). The enzyme demethylated its natural substrate eburicol and the plant CYP51 substrate obtusifoliol at steady-state rates of 17 and 16 min(-1), respectively, but did not metabolize lanosterol, and the topical antifungal drug miconazole was the strongest inhibitor that we identified. The x-ray crystal structures displayed high overall similarity of A. fumigatus CYP51B to CYP51 orthologs from other biological kingdoms but revealed phylum-specific differences relevant to enzyme catalysis and inhibition. The complex with voriconazole provides an explanation for the potency of this relatively small molecule, whereas the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections. PMID:26269599

  7. Identification and Characterization of a Novel Aspergillus fumigatus Rhomboid Family Putative Protease, RbdA, Involved in Hypoxia Sensing and Virulence.

    PubMed

    Vaknin, Yakir; Hillmann, Falk; Iannitti, Rossana; Ben Baruch, Netali; Sandovsky-Losica, Hana; Shadkchan, Yona; Romani, Luigina; Brakhage, Axel; Kniemeyer, Olaf; Osherov, Nir

    2016-06-01

    Aspergillus fumigatus is the most common pathogenic mold infecting humans and a significant cause of morbidity and mortality in immunocompromised patients. In invasive pulmonary aspergillosis, A. fumigatus spores are inhaled into the lungs, undergoing germination and invasive hyphal growth. The fungus occludes and disrupts the blood vessels, leading to hypoxia and eventual tissue necrosis. The ability of this mold to adapt to hypoxia is regulated in part by the sterol regulatory element binding protein (SREBP) SrbA and the DscA to DscD Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. Loss of the genes encoding these proteins results in avirulence. To identify novel regulators of hypoxia sensing, we screened the Neurospora crassa gene deletion library under hypoxia and identified a novel rhomboid family protease essential for hypoxic growth. Deletion of the A. fumigatus rhomboid homolog rbdA resulted in an inability to grow under hypoxia, hypersensitivity to CoCl2, nikkomycin Z, fluconazole, and ferrozine, abnormal swollen tip morphology, and transcriptional dysregulation-accurately phenocopying deletion of srbA. In vivo, rbdA deletion resulted in increased sensitivity to phagocytic killing, a reduced inflammatory Th1 and Th17 response, and strongly attenuated virulence. Phenotypic rescue of the ΔrbdA mutant was achieved by expression and nuclear localization of the N terminus of SrbA, including its HLH domain, further indicating that RbdA and SrbA act in the same signaling pathway. In summary, we have identified RbdA, a novel putative rhomboid family protease in A. fumigatus that mediates hypoxia adaptation and fungal virulence and that is likely linked to SrbA cleavage and activation. PMID:27068092

  8. Identification and deletion of Tft1, a predicted glycosyltransferase necessary for cell wall β-1,3;1,4-glucan synthesis in Aspergillus fumigatus.

    PubMed

    Samar, Danial; Kieler, Joshua B; Klutts, J Stacey

    2015-01-01

    Aspergillus fumigatus is an environmental mold that causes severe, often fatal invasive infections in immunocompromised patients. The search for new antifungal drug targets is critical, and the synthesis of the cell wall represents a potential area to find such a target. Embedded within the main β-1,3-glucan core of the A. fumigatus cell wall is a mixed linkage, β-D-(1,3;1,4)-glucan. The role of this molecule or how it is synthesized is unknown, though it comprises 10% of the glucans within the wall. While this is not a well-studied molecule in fungi, it has been studied in plants. Using the sequences of two plant mixed linkage glucan synthases, a single ortholog was identified in A. fumigatus (Tft1). A strain lacking this enzyme (tft1Δ) was generated along with revertant strains containing the native gene under the control of either the native or a strongly expressing promoter. Immunofluorescence staining with an antibody against β-(1,3;1,4)-glucan and biochemical quantification of this polysaccharide in the tft1Δ strain demonstrated complete loss of this molecule. Reintroduction of the gene into the knockout strain yielded reappearance in amounts that correlated with expected expression of the gene. The loss of Tft1 and mixed linkage glucan yielded no in vitro growth phenotype. However, there was a modest increase in virulence for the tft1Δ strain in a wax worm model. While the precise roles for β-(1,3;1,4)-glucan within A. fumigatus cell wall are still uncertain, it is clear that Tft1 plays a pivotal role in the biosynthesis of this cell wall polysaccharide. PMID:25723175

  9. H-ficolin binds Aspergillus fumigatus leading to activation of the lectin complement pathway and modulation of lung epithelial immune responses.

    PubMed

    Bidula, Stefan; Sexton, Darren W; Yates, Matthew; Abdolrasouli, Alireza; Shah, Anand; Wallis, Russell; Reed, Anna; Armstrong-James, Darius; Schelenz, Silke

    2015-10-01

    Aspergillus fumigatus is an opportunistic fungal pathogen that typically infects the lungs of immunocompromised patients leading to a high mortality. H-Ficolin, an innate immune opsonin, is produced by type II alveolar epithelial cells and could participate in lung defences against infections. Here, we used the human type II alveolar epithelial cell line, A549, to determine the involvement of H-ficolin in fungal defence. Additionally, we investigated the presence of H-ficolin in bronchoalveolar lavage fluid from transplant patients during pneumonia. H-Ficolin exhibited demonstrable binding to A. fumigatus conidia via l-fucose, d-mannose and N-acetylglucosamine residues in a calcium- and pH-dependent manner. Moreover, recognition led to lectin complement pathway activation and enhanced fungal association with A549 cells. Following recognition, H-ficolin opsonization manifested an increase in interleukin-8 production from A549 cells, which involved activation of the intracellular signalling pathways mitogen-activated protein kinase MAPK kinase 1/2, p38 MAPK and c-Jun N-terminal kinase. Finally, H-ficolin concentrations were significantly higher in bronchoalveolar lavage fluid of patients with lung infections compared with control subjects (n = 16; P = 0·00726). Receiver operating characteristics curve analysis further highlighted the potential of H-ficolin as a diagnostic marker for lung infection (area under the curve = 0·77; P < 0·0001). Hence, H-ficolin participates in A. fumigatus defence through the activation of the lectin complement pathway, enhanced fungus-host interactions and modulated immune responses. PMID:26133042

  10. Aspergillus fumigatus and mesophilic moulds in air in the surrounding environment downwind of non-hazardous waste landfill sites.

    PubMed

    Schlosser, Olivier; Robert, Samuel; Debeaupuis, Catherine

    2016-05-01

    Non-hazardous waste landfilling has the potential to release biological agents into the air, notably mould spores. Some species, such as Aspergillus fumigatus, may be a cause of concern for at-risk nearby residents. However, air concentration in the surrounding environment of non-hazardous waste landfill sites is poorly documented. An extensive sampling programme was designed to investigate the relationship between culturable mesophilic moulds and A. fumigatus concentrations in air and distance downwind of non-hazardous waste landfill sites. On-site and off-site repeated measurements were performed at four landfill sites during cold and warm seasons. A high-flow air-sampler device was selected so as to allow peak concentration measurement. Linear mixed-effects models were used to explain variability in the concentrations in air over time and across sites, seasons, instantaneous meteorological conditions and discharged waste tonnage. Concentrations of mesophilic moulds and A. fumigatus at off-site upwind sampling locations were compared with concentrations at each of the downwind sampling locations. At the tipping face location, peak concentration reached 480,000CFUm(-3) for mesophilic moulds and 9300CFUm(-3) for A. fumigatus. Compared with upwind background levels, these concentrations were, on average, approximately 20 and 40 times higher respectively. A steep decline in the concentration of both mesophilic moulds and A. fumigatus was observed between the tipping face location and the downwind property boundary (reduction by 77% and 84% respectively), followed by a low decline leading to a 90% and 94% reduction in concentration at 200m from the property boundary and beyond. With the 200m and 500m downwind sampling point values added together, the 97.5th percentile of concentration was 6013CFUm(-3) and 87CFUm(-3) for mesophilic moulds and A. fumigatus, respectively. Other determining factors were the discharged waste tonnage, the season, instantaneous temperature

  11. Krüppel-like Factor 4 modulates interleukin-6 release in human dendritic cells after in vitro stimulation with Aspergillus fumigatus and Candida albicans.

    PubMed

    Czakai, Kristin; Leonhardt, Ines; Dix, Andreas; Bonin, Michael; Linde, Joerg; Einsele, Hermann; Kurzai, Oliver; Loeffler, Jürgen

    2016-01-01

    Invasive fungal infections are associated with high mortality rates and are mostly caused by the opportunistic fungi Aspergillus fumigatus and Candida albicans. Immune responses against these fungi are still not fully understood. Dendritic cells (DCs) are crucial players in initiating innate and adaptive immune responses against fungal infections. The immunomodulatory effects of fungi were compared to the bacterial stimulus LPS to determine key players in the immune response to fungal infections. A genome wide study of the gene regulation of human monocyte-derived dendritic cells (DCs) confronted with A. fumigatus, C. albicans or LPS was performed and Krüppel-like factor 4 (KLF4) was identified as the only transcription factor that was down-regulated in DCs by both fungi but induced by stimulation with LPS. Downstream analysis demonstrated the influence of KLF4 on the interleukine-6 expression in human DCs. Furthermore, KLF4 regulation was shown to be dependent on pattern recognition receptor ligation. Therefore KLF4 was identified as a controlling element in the IL-6 immune response with a unique expression pattern comparing fungal and LPS stimulation. PMID:27346433

  12. Krüppel-like Factor 4 modulates interleukin-6 release in human dendritic cells after in vitro stimulation with Aspergillus fumigatus and Candida albicans

    PubMed Central

    Czakai, Kristin; Leonhardt, Ines; Dix, Andreas; Bonin, Michael; Linde, Joerg; Einsele, Hermann; Kurzai, Oliver; Loeffler, Jürgen

    2016-01-01

    Invasive fungal infections are associated with high mortality rates and are mostly caused by the opportunistic fungi Aspergillus fumigatus and Candida albicans. Immune responses against these fungi are still not fully understood. Dendritic cells (DCs) are crucial players in initiating innate and adaptive immune responses against fungal infections. The immunomodulatory effects of fungi were compared to the bacterial stimulus LPS to determine key players in the immune response to fungal infections. A genome wide study of the gene regulation of human monocyte-derived dendritic cells (DCs) confronted with A. fumigatus, C. albicans or LPS was performed and Krüppel-like factor 4 (KLF4) was identified as the only transcription factor that was down-regulated in DCs by both fungi but induced by stimulation with LPS. Downstream analysis demonstrated the influence of KLF4 on the interleukine-6 expression in human DCs. Furthermore, KLF4 regulation was shown to be dependent on pattern recognition receptor ligation. Therefore KLF4 was identified as a controlling element in the IL-6 immune response with a unique expression pattern comparing fungal and LPS stimulation. PMID:27346433

  13. Concomitant presence of Aspergillus fumigatus and Stenotrophomonas maltophilia in the respiratory tract: a new risk for patients with liver disease?

    PubMed

    Cabaret, Odile; Bonnal, Christine; Canoui-Poitrine, Florence; Emirian, Aurélie; Bizouard, Geoffray; Levesque, Eric; Maitre, Bernard; Fihman, Vincent; Decousser, Jean-Winoc; Botterel, Françoise

    2016-05-01

    Concomitant lung colonization by Aspergillus fumigatus and Stenotrophomonas maltophilia was reported mainly in patients with cystic fibrosis (CF) and immunocompromised patients. The aim of the study was to assess the frequency of co-culture of A. fumigatus and S. maltophilia in respiratory samples of hospitalized patients, and to determine its associated factors. Between 2007 and 2011, all patients who had A. fumigatus in their respiratory samples were retrospectively enrolled in the study. Their clinical and laboratory data, including the presence of S. maltophilia in a respiratory sample, were collected within the same month. Of the 257 enrolled patients (372 respiratory samples), 71 % were immunocompromised and 32 % had chronic respiratory disease. S. maltophilia was isolated within the same month in 20 patients (7.8 %). In the univariate analysis, factors associated with concomitant culture of A. fumigatus and S. maltophilia were liver disease (P = 0.009), orotracheal intubation (P = 0.001), ventilator-associated pneumonia (P = 0.006), central venous catheter (P = 0.003), parenteral nutrition (P = 0.008) and culture of Pseudomonas aeruginosa in respiratory samples (P = 0.002). In the multivariate analysis, the simultaneous presence of P. aeruginosa in the respiratory tract (odds ratio (OR) = 3.19, 95 % confidence interval (CI) 1.11-9.14, P = 0.031), liver disease (OR = 3.92, 95 % CI 1.32-11.62, P = 0.014) and orotracheal intubation (OR = 3.42, 95 % CI 1.17-9.96, P = 0.024) were independently associated with the co-culture of S. maltophilia and A. fumigatus. Factors independently associated with the concomitant culture of A. fumigatus and S. maltophilia were identified. These results support a future prospective study focusing on liver disease and its complications. PMID:26872817

  14. Aspergillus fumigatus chronic colonization and lung function decline in cystic fibrosis may have a two-way relationship.

    PubMed

    Noni, M; Katelari, A; Dimopoulos, G; Doudounakis, S-E; Tzoumaka-Bakoula, C; Spoulou, V

    2015-11-01

    Aspergillus fumigatus is commonly found in cystic fibrosis (CF) airways. Our aim was to assess the relationship between A. fumigatus chronic colonization and lung function in CF patients. A case-control study of CF patients born from 1989 to 2002 was performed. Medical records were reviewed from the time of initial diagnosis until December 2013. Chronic colonization was defined as two or more positive sputum cultures in a given year. Each patient chronically colonized with A. fumigatus was matched with three control patients (never colonized by A. fumigatus) for age, sex, and year of birth (±3 years). A number of parameters were recorded and analyzed prospectively. The primary outcome measure was the difference in forced expiratory volume in 1 s (FEV1) in percent predicted between groups. Linear mixed models were used for longitudinal analyses to evaluate the relationship between A. fumigatus chronic colonization and lung function during a 7-year period and study the lung function 4 years before the time of enrollment (t0). Twenty patients had chronic colonization and were matched with 60 controls. A significant difference in lung function was detected throughout the 7-year period after adjustment for confounders (est = 8.66, p = 0.020). Four years before t0, FEV1 baseline was the only factor associated with the course of lung function (est = 0.64, p < 0.001) and was significantly different between groups (p = 0.001). In conclusion, a decreased FEV1 baseline appears to be a risk factor for chronic colonization by A. fumigatus, which, in turn, may cause a faster deterioration of lung function. PMID:26319147

  15. An Old Yellow Enzyme Gene Controls the Branch Point between Aspergillus fumigatus and Claviceps purpurea Ergot Alkaloid Pathways▿

    PubMed Central

    Coyle, Christine M.; Cheng, Johnathan Z.; O'Connor, Sarah E.; Panaccione, Daniel G.

    2010-01-01

    Ergot fungi in the genus Claviceps and several related fungal groups in the family Clavicipitaceae produce toxic ergot alkaloids. These fungi produce a variety of ergot alkaloids, including clavines as well as lysergic acid derivatives. Ergot alkaloids are also produced by the distantly related, opportunistic human pathogen Aspergillus fumigatus. However, this fungus produces festuclavine and fumigaclavines A, B, and C, which collectively differ from clavines of clavicipitaceous fungi in saturation of the last assembled of four rings in the ergoline ring structure. The two lineages are hypothesized to share early steps of the ergot alkaloid pathway before diverging at some point after the synthesis of the tricyclic intermediate chanoclavine-I. Disruption of easA, a gene predicted to encode a flavin-dependent oxidoreductase of the old yellow enzyme class, in A. fumigatus led to accumulation of chanoclavine-I and chanoclavine-I-aldehyde. Complementation of the A. fumigatus easA mutant with a wild-type allele from the same fungus restored the wild-type profile of ergot alkaloids. These data demonstrate that the product of A. fumigatus easA is required for incorporation of chanoclavine-I-aldehyde into more-complex ergot alkaloids, presumably by reducing the double bond conjugated to the aldehyde group, thus facilitating ring closure. Augmentation of the A. fumigatus easA mutant with a homologue of easA from Claviceps purpurea resulted in accumulation of ergot alkaloids typical of clavicipitaceous fungi (agroclavine, setoclavine, and its diastereoisomer isosetoclavine). These data indicate that functional differences in the easA-encoded old yellow enzymes of A. fumigatus and C. purpurea result in divergence of their respective ergot alkaloid pathways. PMID:20435769

  16. Detection of Aspergillus fumigatus in a rat model of invasive pulmonary aspergillosis by real-time nucleic acid sequence-based amplification.

    PubMed

    Zhao, Yanan; Park, Steven; Warn, Peter; Shrief, Raghdaa; Harrison, Elizabeth; Perlin, David S

    2010-04-01

    Rapid and sensitive detection of Aspergillus from clinical samples may facilitate the early diagnosis of invasive pulmonary aspergillosis (IPA). A real-time nucleic acid sequence-based amplification (NASBA) method was investigated by use of an inhalational rat model of IPA. Immunosuppressed male Sprague-Dawley rats were exposed to Aspergillus fumigatus spores for an hour in an aerosol chamber. Bronchoalveolar lavage (BAL) fluid, lung tissues, and whole blood were collected from five infected rats at 1, 24, 48, 72, and 96 h postinfection and five uninfected rats at the end of the experiment. Total nucleic acid (TNA) was extracted on an easyMAG instrument. A primer-molecular beacon set targeting 28S rRNA was designed to detect Aspergillus spp. The results were compared to those of quantitative PCR (qPCR) (18S rDNA) and quantitative culture. The analytical sensitivity of the real-time NASBA assay was <1 CFU/assay. A linear range of detection was demonstrated over 5 log units of conidia (10 to 10(5) spores). Both NASBA and qPCR showed a progressive increase in lung tissue burdens, while the CFU counts were stable over time. The fungal burdens in BAL fluid were more variable and not indicative of a progressive infection. The results of both real-time assays correlated well for both sample types (r = 0.869 and P < 0.0001 for lung tissue, r = 0.887 and P < 0.0001 for BAL fluid). For all whole-blood specimens, NASBA identified Aspergillus-positive samples in the group from which samples were collected at 72 h postinfection (three of five samples) and the group from which samples were collected at 96 h postinfection (five of five samples), but no positive results were obtained by culture or PCR. Real-time NASBA is highly sensitive and useful for the detection of Aspergillus in an experimental model of IPA. PMID:20129972

  17. Opportunistic infection of Aspergillus and bacteria in captive Cape vultures (Gyps coprotheres)

    PubMed Central

    Chege, Stephen; Howlett, Judith; Al Qassimi, Majid; Toosy, Arshad; Kinne, Joerg; Obanda, Vincent

    2013-01-01

    Objective To describe clinical signs, pathology, diagnosis and treatment of Cape vultures in which Aspergillus fumigatus (A. fumigatus) and mixed species of bacteria were isolated. Methods Six Cape vultures sourced from South Africa for exhibition at Al Ain Zoo developed illness manifesting as anorexia, dyspnea, polyuria and lethargy. Three vultures died manifesting “pneumonia-like syndrome”. These three vultures were necropsied and gross lesions recorded, while organ tissues were collected for histopathology. Internal organs were swabbed for bacteriology and mycology. From live vultures, blood was collected for hematology and biochemistry, oropharyngeal and cloacal swabs were collected for mycology and bacteriology. Results A. fumigatus was isolated from the three dead vultures and two live ones that eventually survived. One of the dead vulture and two live vultures were co-infected with A. fumigatus and mixed species of bacteria that included Clostridium perfringens, Pseudomonas, Staphylococcus, Escherichia, Proteus, Enterococcus and Enterbacter. One of the Cape vulture and a Lappet-faced vulture, however, were free of Aspergillus or bacterial infections. At necropsy, intestinal hemorrhages were observed and the lungs were overtly congested with granulomas present on caudal air sac. Histopathological examinations demonstrated granulomatous lesions that were infiltrated by mononuclear cells and giant cells. Conclusions Aspergillosis is a persistent threat to captive birds and we recommend routine health assessments so that early diagnosis may prompt early treatment. It is likely that prompt prophylaxis by broad spectrum antibiotics and antifungals medication contributed to the survival of some of the vultures. PMID:23646305

  18. Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against soil amoeba predation.

    PubMed

    Hillmann, Falk; Novohradská, Silvia; Mattern, Derek J; Forberger, Tilmann; Heinekamp, Thorsten; Westermann, Martin; Winckler, Thomas; Brakhage, Axel A

    2015-08-01

    Filamentous fungi represent classical examples for environmentally acquired human pathogens whose major virulence mechanisms are likely to have emerged long before the appearance of innate immune systems. In natural habitats, amoeba predation could impose a major selection pressure towards the acquisition of virulence attributes. To test this hypothesis, we exploited the amoeba Dictyostelium discoideum to study its interaction with Aspergillus fumigatus, two abundant soil inhabitants for which we found co-occurrence in various sites. Fungal conidia were efficiently taken up by D. discoideum, but ingestion was higher when conidia were devoid of the green fungal spore pigment dihydroxynaphtalene melanin, in line with earlier results obtained for immune cells. Conidia were able to survive phagocytic processing, and intracellular germination was initiated only after several hours of co-incubation which eventually led to a lethal disruption of the host cell. Besides phagocytic interactions, both amoeba and fungus secreted cross inhibitory factors which suppressed fungal growth or induced amoeba aggregation with subsequent cell lysis, respectively. On the fungal side, we identified gliotoxin as the major fungal factor killing Dictyostelium, supporting the idea that major virulence attributes, such as escape from phagocytosis and the secretion of mycotoxins are beneficial to escape from environmental predators. PMID:25684622

  19. Biochemical and antigenic characterization of a new dipeptidyl-peptidase isolated from Aspergillus fumigatus.

    PubMed

    Beauvais, A; Monod, M; Debeaupuis, J P; Diaquin, M; Kobayashi, H; Latgé, J P

    1997-03-01

    A novel dipeptidyl-peptidase (DPP V) was purified from the culture medium of Aspergillus fumigatus. This is the first report of a secreted dipeptidyl-peptidase. The enzyme had a molecular mass of 88 kDa and contained approximately 9 kDa of N-linked carbohydrate. The expression and secretion of dipeptidyl-peptidase varied with the growth conditions; maximal intra- and extracellular levels were detected when the culture medium contained only proteins or protein hydrolysates in the absence of sugars. The gene of DPP V was cloned and showed significant sequence homology to other eukaryotic dipeptidyl-peptidase genes. Unlike the other dipeptidyl-peptidases, which are all intracellular, DPP V contained a signal peptide. Like the genes of other dipeptidyl-peptidases, that of DPP V displayed the consensus sequences of the catalytic site of the nonclassical serine proteases. The biochemical properties of native and recombinant DPP V obtained in Pichia pastoris were unique and were characterized by a substrate specificity limited to the hydrolysis of X-Ala, His-Ser, and Ser-Tyr dipeptides at a neutral pH optimum. In addition, we showed that DPP V is identical to one of the two major antigens used for the diagnosis of aspergillosis. PMID:9045640

  20. Expression and identification of a laminin-binding protein in Aspergillus fumigatus conidia.

    PubMed Central

    Tronchin, G; Esnault, K; Renier, G; Filmon, R; Chabasse, D; Bouchara, J P

    1997-01-01

    Adhesion of Aspergillus fumigatus, the causative agent of human aspergillosis, to the extracellular matrix protein laminin has been previously demonstrated. This study investigated the expression of laminin receptors during swelling of conidia, a step leading to germination and subsequent colonization of tissues. Scanning electron microscopy showed that the laminin binding sites were distributed over the external rodlet layer of resting conidia. During swelling, the characteristic rodlet layer progressively disintegrated and conidia surrounded by a smooth cell wall layer appeared. Flow cytometry using fluorescein isothiocyanate-conjugated laminin demonstrated that expression of laminin receptors at the surface of conidia was swelling dependent. Resting conidia expressed high levels of laminin receptors on their surface. A gradual decrease of laminin binding was then observed as swelling occurred, reaching a minimum for 4-h-swollen conidia. This correlated with a loss of adherence of swollen conidia to laminin immobilized on microtiter plates. Trypsin pretreatment of conidia reduced laminin binding. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting with laminin identified in a cell wall extract a major 72-kDa cell wall glycoprotein which binds laminin. Thus, one of the initial events in the host colonization may be the recognition of basement membrane laminin by this 72-kDa cell wall surface component. PMID:8975886

  1. In Vitro Antifungal Activity and Mode of Action of 2',4'-Dihydroxychalcone against Aspergillus fumigatus

    PubMed Central

    Seo, Young Ho; Kim, Sung-Su

    2015-01-01

    2',4'-Dihydroxychalcone (2',4'-DHC) was identified from a heat shock protein 90 (Hsp90)-targeting library as a compound with Hsp90 inhibitory and antifungal effects. In the presence of 2',4'-DHC (8 µg/mL), radial growth of Aspergillus fumigatus was inhibited 20% compared to the control, and green pigmentation was completely blocked. The expression of the conidiation-associated genes abaA, brlA, and wetA was significantly decreased (approximately 3- to 5-fold) by treatment with 2',4'-DHC. The expression of calcineurin signaling components, cnaA and crzA, was also significantly reduced. The inhibitory effects of 2',4'-DHC on metabolic activity and mycelial growth were significantly enhanced by combination treatment with itraconazole and caspofungin. Docking studies indicated that 2',4'-DHC bind to the ATPase domain of Hsp90. These results suggest that 2',4'-DHC act as an Hsp90-calcinurin pathway inhibitor. PMID:26190922

  2. Fumigaclavines D-H, new ergot alkaloids from endophytic Aspergillus fumigatus.

    PubMed

    Xu, Jing; Song, Yong Chun; Guo, Ye; Mei, Ya Ning; Tan, Ren Xiang

    2014-08-01

    Ergot alkaloids are toxins which are produced biotechnologically on an industrial scale. The chemical investigation of endophytic Aspergillus fumigatus resulted in the isolation of five new ergot alkaloids named fumigaclavines D-H (2-6), along with three known analogues, fumigaclavine C (1), festuclavine (7), and fumigaclavine A (8). Their structures were unequivocally elucidated by extensive spectroscopic analyses in association with X-ray single-crystal diffraction. Fumigaclavines D-H are interesting clavine-type ergot alkaloids featuring a reverse prenyl moiety at C-2, with 1-4, 6, and 8 bearing additional substituents, e.g., an OH or OAc group at C-9. Compounds 2, 4, and 6-8 showed a broad spectrum of antimicrobial activity against a panel of anaerobic microorganisms, of which compounds 4 and 6 were the most active against Veillonella parvula with an MIC=16 µg/mL compared to that (0.12 µg/mL) of tinidazole, co-assayed as a positive reference. PMID:25127024

  3. Evidence of two pathways for the metabolism of phenol by Aspergillus fumigatus.

    PubMed

    Jones, K H; Trudgill, P W; Hopper, D J

    1995-03-01

    Aspergillus fumigatus (ATCC 28282), a thermotolerant fungus, has been shown to be capable of growth on phenol as the sole carbon and energy source. During growth of the organism on phenol, catechol and hydroquinone accumulated transiently in the medium; cells grown on phenol oxidised these compounds without a lag period. Two different routes operating simultaneously, leading to different ring-fission substrates, are proposed for the metabolism of phenol. In one route, phenol undergoes ortho-hydroxylation to give catechol, which is then cleaved by an intradiol mechanism leading to 3-oxoadipate. In the other route, phenol is hydroxylated in the para-position to produce hydroquinone, which is then converted into 1,2,4-trihydroxybenzene for ring fission by ortho-cleavage to give maleylacetate. Cell-free extracts of phenol-grown mycelia were found to contain enzymic activities for the proposed steps. Two ring-fission dioxygenases, one active towards 1,2,4-trihydroxybenzene, but not catechol, and one active towards both ring-fission substrates, were separated by FPLC. Succinate-grown mycelia did not oxidise any of the intermediates until a clear lag period had elapsed and did not contain any of the enzymic activities for phenol metabolism. PMID:7778974

  4. Molecular characterization of an adaptive response to alkylating agents in the opportunistic pathogen Aspergillus fumigatus

    PubMed Central

    O’Hanlon, Karen A.; Margison, Geoffrey P.; Hatch, Amy; Fitzpatrick, David A.; Owens, Rebecca A.; Doyle, Sean; Jones, Gary W.

    2012-01-01

    An adaptive response to alkylating agents based upon the conformational change of a methylphosphotriester (MPT) DNA repair protein to a transcriptional activator has been demonstrated in a number of bacterial species, but this mechanism appears largely absent from eukaryotes. Here, we demonstrate that the human pathogen Aspergillus fumigatus elicits an adaptive response to sub-lethal doses of the mono-functional alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). We have identified genes that encode MPT and O6-alkylguanine DNA alkyltransferase (AGT) DNA repair proteins; deletions of either of these genes abolish the adaptive response and sensitize the organism to MNNG. In vitro DNA repair assays confirm the ability of MPT and AGT to repair methylphosphotriester and O6-methylguanine lesions respectively. In eukaryotes, the MPT protein is confined to a select group of fungal species, some of which are major mammalian and plant pathogens. The evolutionary origin of the adaptive response is bacterial and rooted within the Firmicutes phylum. Inter-kingdom horizontal gene transfer between Firmicutes and Ascomycete ancestors introduced the adaptive response into the Fungal kingdom. Our data constitute the first detailed characterization of the molecular mechanism of the adaptive response in a lower eukaryote and has applications for development of novel fungal therapeutics targeting this DNA repair system. PMID:22669901

  5. Redundant synthesis of a conidial polyketide by two distinct secondary metabolite clusters in Aspergillus fumigatus

    PubMed Central

    Throckmorton, Kurt; Lim, Fang Yun; Kontoyiannis, Dimitrios P.; Zheng, Weifa; Keller, Nancy P.

    2016-01-01

    Summary Filamentous fungi are renowned for the production of bioactive secondary metabolites. Typically, one distinct metabolite is generated from a specific secondary metabolite cluster. Here, we characterize the newly described trypacidin (tpc) cluster in the opportunistic human pathogen Aspergillus fumigatus. We find that this cluster as well as the previously characterized endocrocin (enc) cluster both contribute to the production of the spore metabolite endocrocin. Whereas trypacidin is eliminated when only tpc cluster genes are deleted, endocrocin production is only eliminated when both the tpc and enc non-reducing polyketide synthase-encoding genes, tpcC and encA, respectively, are deleted. EncC, an anthrone oxidase, converts the product released from EncA to endocrocin as a final product. In contrast, endocrocin synthesis by the tpc cluster likely results from incomplete catalysis by TpcK (a putative decarboxylase), as its deletion results in a nearly 10-fold increase in endocrocin production. We suggest endocrocin is likely a shunt product in all related non-reducing polyketide synthase clusters containing homologues of TpcK and TpcL (a putative anthrone oxidase), e.g. geodin and monodictyphenone. This finding represents an unusual example of two physically discrete secondary metabolite clusters generating the same natural product in one fungal species by distinct routes. PMID:26242966

  6. Tolerance to silver of an Aspergillus fumigatus strain able to grow on cyanide containing wastes.

    PubMed

    Sabatini, L; Battistelli, M; Giorgi, L; Iacobucci, M; Gobbi, L; Andreozzi, E; Pianetti, A; Franchi, R; Bruscolini, F

    2016-04-01

    We studied the strategy of an Aspergillus fumigatus strain able to grow on metal cyanide wastes to cope with silver. The tolerance test revealed that the Minimum Inhibitory Concentration of Ag(I) was 6mM. In 1mM AgNO3 aqueous solution the fungus was able to reduce and sequestrate silver into the cell in the form of nanoparticles as evidenced by the change in color of the biomass and Electron Microscopy observations. Extracellular silver nanoparticle production also occurred in the filtrate solution after previous incubation of the fungus in sterile, double-distilled water for 72h, therefore evidencing that culture conditions may influence nanoparticle formation. The nanoparticles were characterized by UV-vis spectrometry, X-ray diffraction and Energy Dispersion X-ray analysis. Atomic absorption spectrometry revealed that the optimum culture conditions for silver absorption were at pH 8.5.The research is part of a polyphasic study concerning the behavior of the fungal strain in presence of metal cyanides; the results provide better understanding for further research targeted at a rationale use of the microorganism in bioremediation plans, also in view of possible metal recovery. Studies will be performed to verify if the fungus maintains its ability to produce nanoparticles using KAg(CN)2. PMID:26705888

  7. RacA-Mediated ROS Signaling Is Required for Polarized Cell Differentiation in Conidiogenesis of Aspergillus fumigatus

    PubMed Central

    Chi, Myoung-Hwan; Craven, Kelly D.

    2016-01-01

    Conidiophore development of fungi belonging to the genus Aspergillus involves dynamic changes in cellular polarity and morphogenesis. Synchronized differentiation of phialides from the subtending conidiophore vesicle is a good example of the transition from isotropic to multi-directional polarized growth. Here we report a small GTPase, RacA, which is essential for reactive oxygen species (ROS) production in the vesicle as well as differentiation of phialides in Aspergillus fumigatus. We found that wild type A. fumigatus accumulates ROS in these conidiophore vesicles and that null mutants of racA did not, resulting in the termination of conidiophore development in this early vesicle stage. Further, we found that stress conditions resulting in atypical ROS accumulation coincide with partial recovery of phialide emergence but not subsequent apical dominance of the phialides in the racA null mutant, suggesting alternative means of ROS generation for the former process that are lacking in the latter. Elongation of phialides was also suppressed by inhibition of NADPH-oxidase activity. Our findings provide not only insights into role of ROS in fungal cell polarity and morphogenesis but also an improved model for the developmental regulatory pathway of conidiogenesis in A. fumigatus. PMID:26890813

  8. Insights from the Molecular Dynamics Simulation of Cellobiohydrolase Cel6A Molecular Structural Model from Aspergillus fumigatus NITDGPKA3.

    PubMed

    Dodda, Subba Reddy; Sarkar, Nibedita; Aikat, Kaustav; Krishnaraj, Navanietha R; Bhattacharjee, Sanchari; Bagchi, Angshuman; Mukhopadhyay, Sudit S

    2016-01-01

    Global demand for bioethanol is increasing tremendously as it could help to replace the conventional fossil fuel and at the same time supporting the bioremediation of huge volume of cellulosic wastes generated from different sources. Ideal genetic engineering approaches are essential to improve the efficacy of the bioethanol production processes for real time applications. A locally isolated fungal strain Aspergillus fumigatus NITDGPKA3 was used in our laboratory for the hydrolysis of lignocellulose with good cellulolytic activity when compared with other contemporary fungal strains. An attempt is made to sequence the cellobiohydrolases (CBHs) of A. fumigatus NITDGPKA3, model its structure to predict its catalytic activity towards improving the protein by genetic engineering approaches. Herein, the structure of the sequenced Cellobiohydrolases (CBHs) of A. fumigatus NITDGPKA3, modelled by homology modelling and its validation is reported. Further the catalytic activity of the modelled CBH enzyme was assessed by molecular docking analysis. Phylogenetic analysis showed that CBH from A. fumigatus NITDGPKA3 belongs to the Glycohydro 6 (Cel6A) super family. Molecular modeling and molecular dynamics simulation suggest the structural and functional mechanism of the enzyme. The structures of both the cellulose binding (CBD) and catalytic domain (CD) have been compared with most widely studied CBH of Trichoderma reesei. The molecular docking with cellulose suggests that Gln 248, Pro 287, Val236, Asn284, and Ala288 are the main amino acids involved in the hydrolysis of the β, 1-4, glycosidic bonds of cellulose. PMID:27109185

  9. The Impact of Aspergillus fumigatus Viability and Sensitization to Its Allergens on the Murine Allergic Asthma Phenotype

    PubMed Central

    Pandey, Sumali; Hoselton, Scott A.; Schuh, Jane M.

    2013-01-01

    Aspergillus fumigatus is a ubiquitously present respiratory pathogen. The outcome of a pulmonary disease may vary significantly with fungal viability and host immune status. Our objective in this study was (1) to assess the ability of inhaled irradiation-killed or live A. fumigatus spores to induce allergic pulmonary disease and (2) to assess the extent to which inhaled dead or live A. fumigatus spores influence pulmonary symptoms in a previously established allergic state. Our newly developed fungal delivery apparatus allowed us to recapitulate human exposure through repeated inhalation of dry fungal spores in an animal model. We found that live A. fumigatus spore inhalation led to a significantly increased humoral response, pulmonary inflammation, and airway remodeling in naïve mice and is more likely to induce allergic asthma symptoms than the dead spores. In contrast, in allergic mice, inhalation of dead and live conidia recruited neutrophils and induced goblet cell metaplasia. This data suggests that asthma symptoms might be exacerbated by the inhalation of live or dead spores in individuals with established allergy to fungal antigens, although the extent of symptoms was less with dead spores. These results are likely to be important while considering fungal exposure assessment methods and for making informed therapeutic decisions for mold-associated diseases. PMID:24063011

  10. Phagocytosis-dependent activation of a TLR9–BTK–calcineurin–NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus

    PubMed Central

    Herbst, Susanne; Shah, Anand; Mazon Moya, Maria; Marzola, Vanessa; Jensen, Barbara; Reed, Anna; Birrell, Mark A; Saijo, Shinobu; Mostowy, Serge; Shaunak, Sunil; Armstrong-James, Darius

    2015-01-01

    Transplant recipients on calcineurin inhibitors are at high risk of invasive fungal infection. Understanding how calcineurin inhibitors impair fungal immunity is a key priority for defining risk of infection. Here, we show that the calcineurin inhibitor tacrolimus impairs clearance of the major mould pathogen Aspergillus fumigatus from the airway, by inhibiting macrophage inflammatory responses. This leads to defective early neutrophil recruitment and fungal clearance. We confirm these findings in zebrafish, showing an evolutionarily conserved role for calcineurin signalling in neutrophil recruitment during inflammation. We find that calcineurin–NFAT activation is phagocytosis dependent and collaborates with NF-κB for TNF-α production. For yeast zymosan particles, activation of macrophage calcineurin–NFAT occurs via the phagocytic Dectin-1–spleen tyrosine kinase pathway, but for A. fumigatus, activation occurs via a phagosomal TLR9-dependent and Bruton's tyrosine kinase-dependent signalling pathway that is independent of MyD88. We confirm the collaboration between NFAT and NF-κB for TNF-α production in primary alveolar macrophages. These observations identify inhibition of a newly discovered macrophage TLR9–BTK–calcineurin–NFAT signalling pathway as a key immune defect that leads to organ transplant-related invasive aspergillosis. PMID:25637383

  11. Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host protection against Aspergillus infection

    PubMed Central

    Karki, Rajendra; Man, Si Ming; Malireddi, R.K. Subbarao; Gurung, Prajwal; Vogel, Peter; Lamkanfi, Mohamed; Kanneganti, Thirumala-Devi

    2015-01-01

    SUMMARY Invasive pulmonary aspergillosis is a leading cause of infection-associated mortality in immunocompromised individuals. Aspergillus fumigatus infection produces ligands that could activate inflammasomes but the contribution of these host defenses remains unclear. We show that two inflammasome receptors, AIM2 and NLRP3, recognize intracellular A. fumigatus and collectively induce protective immune responses. Mice lacking both AIM2 and NLRP3 fail to confine Aspergillus hyphae to inflammatory foci, leading to widespread hyphal dissemination to lung blood vessels. These mice succumb to infection more rapidly than WT mice or mice lacking a single inflammasome receptor. AIM2 and NLRP3 activation initiates assembly of a single cytoplasmic inflammasome platform, composed of the adaptor protein ASC along with caspase-1 and caspase-8. Combined actions of caspase-1 and caspase-8 lead to processing of proinflammatory cytokines IL-1β and IL-18 that critically control the infection. Thus, AIM2 and NLRP3 form a dual cytoplasmic surveillance system that orchestrates responses against A. fumigatus infection. PMID:25704009

  12. A comparative study of antigens of Aspergillus fumigatus isolates from patients and soil of ornamental plants in the immunodiffusion test.

    PubMed

    Staib, F; Folkens, U; Tompak, B; Abel, T; Thiel, D

    1978-11-01

    The strikingly frequent and constant presence of Aspergillus fimigatus in the soil of potted ornamental plants kept in private houses and hospitals has been the reason for studying the antigens of the strains found from the diagnostic and epidemiological angles. Culture-filtrate antigens of A. fumigatus strains isolated from the soil of 4 different ornamental plants, epiphyllum (Epiphyllum truncatum), orange tree (Citrus sinensis), Alpine rose (Azalea indica) and Christmas flower (Euphorbia pulcherrima), were compared, in the immunodiffusion test, with antigens of A. fumigatus strains from aspergillosis patients prepared in an identical way. When tested against 8 different sera from different aspergillosis patients there was a good coincidence of results. Control sera from patients suffering from diseases other than aspergillosis, no false-positive reactions could be observed. The findings are discussed in respect of diagnosis and epidemiology. PMID:83753

  13. Characterization and cilofungin inhibition of solubilized Aspergillus fumigatus (1,3)-beta-D-glucan synthase.

    PubMed

    Beaulieu, D; Tang, J; Yan, S B; Vessels, J M; Radding, J A; Parr, T R

    1994-05-01

    (1,3)-beta-D-Glucan synthase, a major cell wall synthesis enzyme, is the target of antifungal drugs of the lipopeptide class. Aspergillus fumigatus (1,3)-beta-D-glucan synthase was prepared and its activity was measured by incorporation of [14C]glucose from UDP-[U-14C]glucose into an insoluble polymer in the presence of alpha-amylase. Solubilization of the (1,3)-beta-D-glucan synthase was attempted with several detergents, and the maximum percent solubilization was obtained with a polyoxyethylene ether detergent, W-1. Up to 70% of enzyme activity and 50% of total protein were recovered when 1-mg/ml membrane preparations were extracted with 0.045% W-1 at 4 degrees C overnight. Confirmation of the presence of a (1,3)-beta-D-glucose polymer synthesized by this glucan synthase was done by three methods. The first was enzymatic end product degradation by alpha-amylase (no degradation) and beta-glucanase (85 to 95% degradation). The second was gas chromatography-mass spectroscopy analysis of the partially methylated alditol acetate derivatives prepared from total carbohydrate polymers present in the sample. This method identified the presence of (1,3)- and (1,2)-glucosidic linkages. The third was high-performance anion exchange chromatography of radioactive oligosaccharides. This method allowed differentiation of the newly synthesized, radioactive polymers from the contaminating carbohydrates already present in the preparation. The results showed that the polymer synthesized comprised oligosaccharides consistent with beta-(1,3)-linked sugars. Maximal inhibition of the (1,3)-beta-D-glucan synthase by the lipopeptide antifungal agent cilofungin was 80%. Dose-response experiments with this inhibitor showed that the solubilized enzyme was maximally inhibited at a cilofungin concentration of 1.25 microgram/ml and showed <5% inhibition at 0.02 microgram/ml. The apparent K(m) (K(m app)) for the solubilized glucan synthase was 400 +/- 80 microM, and the apparent K(i) (K(i app

  14. ChIP-seq and In Vivo Transcriptome Analyses of the Aspergillus fumigatus SREBP SrbA Reveals a New Regulator of the Fungal Hypoxia Response and Virulence

    PubMed Central

    Merriman, Brittney; Werner, Ernst R.; Lechner, Beatrix E.; Dhingra, Sourabh; Cheng, Chao; Xu, Wenjie; Blosser, Sara J.; Morohashi, Kengo; Mazurie, Aurélien; Mitchell, Thomas K.; Haas, Hubertus; Mitchell, Aaron P.; Cramer, Robert A.

    2014-01-01

    The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence. PMID:25375670

  15. The Gβ-like protein CpcB is required for hyphal growth, conidiophore morphology and pathogenicity in Aspergillus fumigatus.

    PubMed

    Cai, Zhen-dong; Chai, Yan-fei; Zhang, Cai-yun; Qiao, Wei-ran; Sang, Hong; Lu, Ling

    2015-08-01

    CpcB (cross pathway control B) encodes a yeast Cpc2 and mammalian RACK1 (receptor for activated protein kinase C) ortholog, which is a WD repeat protein with functional homology to the β subunit of heterotrimeric G proteins in Aspergillus fumigatus. Previous study has reported that CpcB governs growth and development in both A. fumigatus and Aspergillus nidulans. However, little is known about the functional identities of CpcB orthologs and their relationships with G protein complexes. In this study, we verified that cytoplasmic AfCpcB acts as a Gβ-like protein ortholog and plays important roles in hyphal growth, conidiophore morphology, cell wall integrity, and virulence in A. fumigatus. Furthermore, double deletion of AfcpcB and AfgpaB (Gα) causes a similar phenotype to AfgpaB mutant with abnormal multiple septa conidiophores but exhibits sparse conidiation with white and fluffy colonies. Thus, the exacerbated conidiation defect suggests that AfcpcB has its own specific function compared to the Gα subunit of AfgpaB or the G-protein complex. In addition, complementation assays using AfcpcB orthologs of A. nidulans and yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans) suggest that all tested fungal AfcpcB orthologs under the A. fumigatus native promoter can largely restore hyphal growth defects in AfcpcB deletion mutant, but only the A. nidulans cpcB ortholog completely rescues the ΔAfcpcB conidiation defect, suggesting that CpcB acts as a Gβ-like protein ortholog in the Aspergilli, but may have unique and important unexplored functions that required for conidiation, which is absent in yeast. PMID:25892048

  16. Immunological Characterization of Asp f 2, a Major Allergen from Aspergillus fumigatus Associated with Allergic Bronchopulmonary Aspergillosis

    PubMed Central

    Banerjee, Banani; Greenberger, Paul A.; Fink, Jordan N.; Kurup, Viswanath P.

    1998-01-01

    The 37-kDa recombinant protein Asp f 2, encoding an allergen of Aspergillus fumigatus, was expressed in a prokaryotic expression system and immunologically evaluated for its functional and structural properties. The open reading frame for a 310-amino-acid-long protein was shown to encode a signal peptide of 31 amino acids. A native 37-kDa culture filtrate protein and a 55-kDa mycelial glycoprotein (gp55) exhibited complete N-terminal sequence homology to Asp f 2. A GenBank search for homologous proteins revealed 60 and 44% sequence homologies to the cytosolic protein ASPND1 from Aspergillus nidulans and fibrinogen binding protein from Candida albicans, respectively. The glycosylation sites and cysteine molecules are conserved in all the three proteins. The extracellular matrix protein laminin showed a dose-dependent interaction with Asp f 2. This protein, expressed as a major cell-associated protein within 24 h of in vitro fungal culture, comprises 20 to 40% of total fungal protein. Furthermore, both native and recombinant Asp f 2 exhibited specific immunoglobulin (IgE) binding with allergic bronchopulmonary aspergillosis (ABPA) and cystic fibrosis-ABPA patients, whereas A. fumigatus-sensitized allergic asthma and normal control subjects failed to show IgE binding with Asp f 2. These results indicate that Asp f 2 is a major allergen of A. fumigatus exhibiting IgE antibody binding with sera from patients with ABPA. The antigen should be explored further for its potential role in the differential diagnosis of A. fumigatus-associated allergic diseases. PMID:9784519

  17. The Fumagillin Biosynthetic Gene Cluster in Aspergillus fumigatus Encodes a Cryptic Terpene Cyclase Involved in the Formation of β-trans-Bergamotene

    PubMed Central

    Lin, Hsiao-Ching; Chooi, Yit-Heng; Dhingra, Sourabh; Xu, Wei; Calvo, Ana M.; Tang, Yi

    2013-01-01

    Fumagillin 1 is a meroterpenoid from Aspergillus fumigatus that is known for its anti-angiogenic activity by binding to human methionine aminopeptidase 2. The genetic and molecular basis for biosynthesis of 1 had been an enigma despite the availability of the A. fumigatus genome sequence. Here, we reported the identification and verification of the fma gene cluster, followed by characterization of the polyketide synthase and acyltransferase involved in biosynthesis of the dioic acid portion of 1. More significantly, we uncovered the elusive β-trans-bergamotene synthase in A. fumigatus as a membrane-bound terpene cyclase. PMID:23488861

  18. Molecular Characterization of a Voriconazole-Resistant, Posaconazole-Susceptible Aspergillus fumigatus Isolate in a Lung Transplant Recipient in the United States

    PubMed Central

    Vazquez, Jose A.

    2015-01-01

    Molecular characterization of cyp51A from the azole-resistant Aspergillus fumigatus isolate 50593 from a lung transplant patient showed Y121F/T289A changes coupled with a 46-bp tandem repeat (TR46) on the promoter, whereas cyp51A from the pretherapy isolate, A. fumigatus 47381, showed no changes. This is the first reported case of A. fumigatus azole resistance due to Y121F/T289A/TR46 in the United States, suggesting that multiple mutational alterations of cyp51A resulting in high-level azole resistance could occur during prolonged antifungal therapy. PMID:26574014

  19. In-depth 2-DE reference map of Aspergillus fumigatus and its proteomic profiling on exposure to itraconazole.

    PubMed

    Gautam, Poonam; Mushahary, Dolly; Hassan, Wazid; Upadhyay, Santosh Kumar; Madan, Taruna; Sirdeshmukh, Ravi; Sundaram, Curam Sreenivasacharlu; Sarma, Puranam Usha

    2016-07-01

    Aspergillus fumigatus (A. fumigatus) is a medically important opportunistic fungus that may lead to invasive aspergillosis in humans with weak immune system. Proteomic profiling of this fungus on exposure to itraconazole (ITC), an azole antifungal drug, may lead to identification of its molecular targets and better understanding on the development of drug resistance against ITC in A. fumigatus. Here, proteome analysis was performed using 2-DE followed by mass spectrometric analysis which resulted in identification of a total of 259 unique proteins. Further, proteome profiling of A. fumigatus was carried out on exposure to ITC, 0.154 μg/ml, the minimum inhibitory concentration (MIC50). Image analysis showed altered levels of 175 proteins (66 upregulated and 109 downregulated) of A. fumigatus treated with ITC as compared to the untreated control. Peptide mass fingerprinting led to the identification of 54 proteins (12 up-regulated and 42 down-regulated). The differentially expressed proteins include proteins related to cell stress, carbohydrate metabolism and amino acid metabolism. We also observed four proteins, including nucleotide phosphate kinase (NDK), that are reported to interact with calcineurin, a protein involved in regulation of cell morphology and fungal virulence. Comparison of differentially expressed proteins on exposure to ITC with artemisinin (ART), an antimalarial drug with antifungal activity(1), revealed a total of 26 proteins to be common among them suggesting that common proteins and pathways are targeted by these two antifungal agents. The proteins targeted by ITC may serve as important leads for development of new antifungal drugs. PMID:26868900

  20. Immunomodulatory effect of mesenchymal stem cells on the immune response of macrophages stimulated by Aspergillus fumigatus conidia.

    PubMed

    Cho, Sung-Yeon; Kwon, Eun-Young; Choi, Su-Mi; Lee, Dong-Gun; Park, Chulmin; Park, Sun Hee; Yoo, Jin-Hong; Choi, Jung-Hyun

    2016-05-01

    Mesenchymal stem cells (MSCs) are known to exert potent immunosuppression and anti-inflammatory effects. There is growing interest in their use for immunotherapy for controlling inflammation as well as acute organ injury. However, there are few reports regarding MSC's immunomodulatory effects in the settings of fungal infection. In this study, we attempted to examine the immunomodulatory effects of MSCs in response toAspergillus fumigatus We measured the cytokine response of murine MSCs on the immune response of murine macrophages (J774A.1 cells) evoked byA. fumigatusconidia. In addition, we evaluated the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the MSC-related cytokine response and fungal growth. As a results, after conidia stimulation, tumor necrosis factor (TNF)-α was down-regulated and interleukin (IL)-10 was up-regulated in MSC-treated J774A.1 cells when compared to J774A.1 cells alone. In addition, fungal growth was reduced in MSC-treated J774A.1 cells when compared to J774A.1 cells, which recovered by GM-CSF. However, the effect of MSCs on the cytokine response was not reversed by GM-CSF. NF-κB translocation decreased in MSC-treated J774A.1 cells compared to J774A.1 cells alone. In conclusion, MSCs demonstrate immunomodulatory properties in both aspects of cytokines and fungal growth. The anti-inflammatory effect of MSCs with regard to cytokine response might be associated with decreased NF-κB translocation, and is not reversed by GM-CSF. PMID:26768375

  1. Pyripyropenes, novel inhibitors of acyl-CoA:cholesterol acyltransferase produced by Aspergillus fumigatus. I. Production, isolation, and biological properties.

    PubMed

    Tomoda, H; Kim, Y K; Nishida, H; Masuma, R; Omura, S

    1994-02-01

    Aspergillus fumigatus FO-1289, a soil isolate, was found to produce a series of novel inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). Four active compounds, named pyripyropenes A, B, C and D, were isolated from the fermentation broth of the producing strain by solvent extraction, silica gel column chromatography, ODS column chromatography and preparative HPLC. Pyripyropenes A, B, C and D show very potent ACAT inhibitory activity in an enzyme assay system using rat liver microsomes with IC50 values of 58, 117, 53 and 268 nM, respectively. PMID:8150709

  2. Rice-Field Drowning-Associated Pneumonia in which Pseudomonas spp., Aspergillus fumigatus, and Cunninghamella sp. Are Isolated.

    PubMed

    Yamawaki, Satoshi; Nakashima, Kei; Suzuki, Fumi; Otsuki, Ayumu; Watanabe, Junko; Takai, Motohisa; Katsurada, Masahiro; Katsurada, Naoko; Ohkuni, Yoshihiro; Misawa, Masafumi; Kaneko, Norihiro; Otsuka, Yoshihito; Aoshima, Masahiro

    2016-01-01

    We herein report the case of an 84-year-old who developed pneumonia after drowning in a rice field. Besides Aspergillus fumigatus, many pathogens previously not reported in drowning-associated pneumonia (such as Pseudomonas fluorescens, Pseudomonas putida, Nocardia niigatensis, and Cunninghamella sp.) were isolated from his sputum. He received sulbactam/ampicillin, trimethoprim/sulfamethoxazole, voriconazole, levofloxacin and liposomal amphotericin B, but died due to respiratory failure. Because the patient had drowned in a contaminated stagnant rice field and had multiple lung cavities, zygomycosis was suspected. This report provides invaluable information for the consideration of zygomycosis after an individual drowning in a rice field, even in an immunocompetent patient. PMID:27041173

  3. What makes Aspergillus fumigatus a successful pathogen? Genes and molecules involved in invasive aspergillosis.

    PubMed

    Abad, Ana; Fernández-Molina, Jimena Victoria; Bikandi, Joseba; Ramírez, Andoni; Margareto, Javier; Sendino, Javier; Hernando, Fernando Luis; Pontón, Jose; Garaizar, Javier; Rementeria, Aitor

    2010-01-01

    Aspergillus fumigatus is an opportunistic pathogen that causes 90% of invasive aspergillosis (IA) due to Aspergillus genus, with a 50-95% mortality rate. It has been postulated that certain virulence factors are characteristic of A. fumigatus, but the "non-classical" virulence factors seem to be highly variable. Overall, published studies have demonstrated that the virulence of this fungus is multifactorial, associated with its structure, its capacity for growth and adaptation to stress conditions, its mechanisms for evading the immune system and its ability to cause damage to the host. In this review we intend to give a general overview of the genes and molecules involved in the development of IA. The thermotolerance section focuses on five genes related with the capacity of the fungus to grow at temperatures above 30°C (thtA, cgrA, afpmt1, kre2/afmnt1, and hsp1/asp f 12). The following sections discuss molecules and genes related to interaction with the host and with the immune responses. These sections include β-glucan, α-glucan, chitin, galactomannan, galactomannoproteins (afmp1/asp f 17 and afmp2), hydrophobins (rodA/hyp1 and rodB), DHN-melanin, their respective synthases (fks1, rho1-4, ags1-3, chsA-G, och1-4, mnn9, van1, anp1, glfA, pksP/alb1, arp1, arp2, abr1, abr2, and ayg1), and modifying enzymes (gel1-7, bgt1, eng1, ecm33, afpigA, afpmt1-2, afpmt4, kre2/afmnt1, afmnt2-3, afcwh41 and pmi); several enzymes related to oxidative stress protection such as catalases (catA, cat1/catB, cat2/katG, catC, and catE), superoxide dismutases (sod1, sod2, sod3/asp f 6, and sod4), fatty acid oxygenases (ppoA-C), glutathione tranferases (gstA-E), and others (afyap1, skn7, and pes1); and efflux transporters (mdr1-4, atrF, abcA-E, and msfA-E). In addition, this review considers toxins and related genes, such as a diffusible toxic substance from conidia, gliotoxin (gliP and gliZ), mitogillin (res/mitF/asp f 1), hemolysin (aspHS), festuclavine and fumigaclavine A

  4. Detection of Aspergillus antigens associated with invasive infection.

    PubMed Central

    Haynes, K A; Latge, J P; Rogers, T R

    1990-01-01

    Serial urine samples were collected from 33 neutropenic patients, 10 of whom developed invasive aspergillosis (IA) while undergoing bone marrow transplantation or remission induction therapy for leukemia. Concentrated urine samples from the infected patients were subjected to polyacrylamide gel electrophoresis, blotted, and then incubated with antiserum raised to a cell wall extract of Aspergillus fumigatus (anti-CW) or an immunoglobulin G monoclonal antibody to A. fumigatus galactomannan (EBA1). When IA patient urine blots were probed with anti-CW, major bands at 11 and 18 kilodaltons (kDa); intermediate bands at 13, 14, and 29 kDa; and minor bands at 38 and 44 kDa were seen. In contrast, EBA1 showed diffuse staining at molecular masses larger than 45 kDa and a single weak band at 21 kDa. Urine samples from the 23 patients with no evidence of IA were unreactive with both anti-CW and EBA1. These antigen bands are likely to represent immunodominant antigens which are excreted during IA and should play a valuable role in the development of rapid diagnostic tests for aspergillosis. Images PMID:2229387

  5. Oxygen and an Extracellular Phase Transition Independently Control Central Regulatory Genes and Conidiogenesis in Aspergillus fumigatus

    PubMed Central

    Chi, Myoung-Hwan; Craven, Kelly D.

    2013-01-01

    Conidiogenesis is the primary process for asexual reproduction in filamentous fungi. As the conidia resulting from the conidiogenesis process are primarily disseminated via air currents and/or water, an outstanding question has been how fungi recognize aerial environments suitable for conidial development. In this study, we documented the somewhat complex development of the conidia-bearing structures, termed conidiophores, from several Aspergillus species in a subsurface (gel-phase) layer of solid media. A subset of the isolates studied was able to develop conidiophores in a gel-phase environment, but exposure to the aeriform environment was required for the terminal developmental transition from phialide cells to conidia. The remaining Aspergilli could not initiate the conidiogenesis process until they were exposed to the aeriform environment. Our observations of conidiophore development in high or low oxygen conditions in both aeriform and gel-phase environments revealed that oxygen and the aeriform state are positive environmental factors for inducing conidiogenesis in most of the aspergilli tested in this study. Transcriptional analysis using A. fumigatus strain AF293 confined to either the aeriform or gel-phase environments revealed that expression of a key regulatory gene for conidiophore development (AfubrlA) is facilitated by oxygen while expression of another regulatory gene controlling conidia formation from phialides (AfuabaA) was repressed regardless of oxygen levels in the gel-embedded environment. Furthermore, by comparing the developmental behavior of conidiation-defective mutants lacking genes controlling various regulatory checkpoints throughout the conidiogenesis pathway, we propose that this aerial response by the fungus requires both oxygen and the phase transition (solid to aeriform), with these environmental signals integrating into the upstream regulatory pathway and central regulatory pathway of conidiogenesis, respectively. Our findings

  6. Thermodynamics and kinetic properties of halostable endoglucanase from Aspergillus fumigatus ABK9.

    PubMed

    Das, Arpan; Jana, Arijit; Paul, Tanmay; Halder, Suman Kumar; Ghosh, Kuntal; Maity, Chiranjit; Mohapatra, Pradeep Kumar Das; Pati, Bikas Ranjan; Mondal, Keshab Chandra

    2014-07-01

    An endoglucanase from Aspergillus fumigatus ABK9 was purified from the culture extract of solid-state fermentation and its some characteristics were evaluated. The molecular weight of the purified enzyme (56.3 kDa) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymogram analysis and confirmed by MALDI-TOF mass spectrometry. The enzyme was active optimally at 50 °C, pH 5.0 and stable over a broad range of pH (4.0-7.0) and NaCl concentration of 0-3.0 M. The pKa1 and pKa2 of the ionizable groups of the active sites were 2.94 and 6.53, respectively. The apparent Km , Vmax , and Kcat values for carboxymethyl cellulose were 6.7 mg ml(-1), 775.4 µmol min(-1) , and 42.84 × 10(4)  s(-1), respectively. Thermostability of the enzyme was evidenced by the high activation energy (91.45 kJ mol(-1)), large enthalpy for activation of denaturation (88.77 kJ mol(-1)), longer half-life (T1/2) (433 min at 50 °C), higher melting temperature (Tm ) (73.5 °C), and Q10 (1.3) values. All the characteristics favors its suitability as halotolerant and thermostable enzyme during bioprocessing of lignocellulosic materials. PMID:23832828

  7. Heptahelical Receptors GprC and GprD of Aspergillus fumigatus Are Essential Regulators of Colony Growth, Hyphal Morphogenesis, and Virulence▿ †

    PubMed Central

    Gehrke, Alexander; Heinekamp, Thorsten; Jacobsen, Ilse D.; Brakhage, Axel A.

    2010-01-01

    The filamentous fungus Aspergillus fumigatus normally grows on compost or hay but is also able to colonize environments such as the human lung. In order to survive, this organism needs to react to a multitude of external stimuli. Although extensive work has been carried out to investigate intracellular signal transduction in A. fumigatus, little is known about the specific stimuli and the corresponding receptors activating these signaling cascades. Here, two putative G-protein-coupled receptors, GprC and GprD, were characterized with respect to their cellular functions. Deletion of the corresponding genes resulted in drastic growth defects as hyphal extension was reduced, germination was retarded, and hyphae showed elevated levels of branching. The growth defect was found to be temperature dependent. The higher the temperature the more pronounced was the growth defect. Furthermore, compared with the wild type, the sensitivity of the mutant strains toward environmental stress caused by reactive oxygen intermediates was increased and the mutants displayed an attenuation of virulence in a murine infection model. Both mutants, especially the ΔgprC strain, exhibited increased tolerance toward cyclosporine, an inhibitor of the calcineurin signal transduction pathway. Transcriptome analyses indicated that in both the gprC and gprD deletion mutants, transcripts of primary metabolism genes were less abundant, whereas transcription of several secondary metabolism gene clusters was upregulated. Taken together, our data suggest the receptors are involved in integrating and processing stress signals via modulation of the calcineurin pathway. PMID:20418440

  8. A Sterol-Regulatory Element Binding Protein Is Required for Cell Polarity, Hypoxia Adaptation, Azole Drug Resistance, and Virulence in Aspergillus fumigatus

    PubMed Central

    Willger, Sven D.; Puttikamonkul, Srisombat; Kim, Kwang-Hyung; Burritt, James B.; Grahl, Nora; Metzler, Laurel J.; Barbuch, Robert; Bard, Martin; Lawrence, Christopher B.; Cramer, Robert A.

    2008-01-01

    At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive. Currently, whether hypoxia adaptation is an important virulence attribute of opportunistic pathogenic molds is unknown. Here we report the characterization of a sterol-regulatory element binding protein, SrbA, in the opportunistic pathogenic mold, Aspergillus fumigatus. Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA). Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function. Annotation of these genes implicated SrbA in maintaining sterol biosynthesis and hyphal morphology. Further examination of the SrbA null mutant consequently revealed that SrbA plays a critical role in ergosterol biosynthesis, resistance to the azole class of antifungal drugs, and in maintenance of cell polarity in A. fumigatus. Significantly, the SrbA null mutant was highly susceptible to fluconazole and voriconazole. Thus, these findings present a new function of SREBP proteins in filamentous fungi, and demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds. PMID:18989462

  9. Serum opsonin ficolin-A enhances host-fungal interactions and modulates cytokine expression from human monocyte-derived macrophages and neutrophils following Aspergillus fumigatus challenge.

    PubMed

    Bidula, Stefan; Sexton, Darren W; Schelenz, Silke

    2016-04-01

    Invasive aspergillosis is a devastating invasive fungal disease associated with a high mortality rate in the immunocompromised, such as leukaemia patients, transplant patients and those with HIV/AIDS. The rodent serum orthologue of human L-ficolin, ficolin-A, can bind to and opsonize Aspergillus fumigatus, the pathogen that causes invasive aspergillosis, and may participate in fungal defence. Using human monocyte-derived macrophages and neutrophils isolated from healthy donors, we investigated conidial association and fungal viability by flow cytometry and microscopy. Additionally, cytokine production was measured via cytometric bead arrays. Ficolin-A opsonization was observed to significantly enhance association of conidia, while also inhibiting hyphal growth and contributing to increased fungal killing following incubation with monocyte-derived macrophages and neutrophils. Additionally, ficolin-A opsonization was capable of manifesting a decrease in IL-8, IL-1β, IL-6, IL-10 and TNF-α production from MDM and IL-1β, IL-6 and TNF-α from neutrophils 24 h post-infection. In conclusion, rodent ficolin-A is functionally comparable to human L-ficolin and is capable of modulating the innate immune response to A. fumigatus, down-regulating cytokine production and could play an important role in airway immunity. PMID:26337048

  10. Chronic Aspergillus fumigatus colonization of the pediatric cystic fibrosis airway is common and may be associated with a more rapid decline in lung function.

    PubMed

    Saunders, Rosalind V; Modha, Deborah E; Claydon, Alison; Gaillard, Erol A

    2016-07-01

    Filamentous fungi are commonly isolated from the respiratory tract of CF patients, but their clinical significance is uncertain and the reported incidence variable. We report on the degree of Aspergillus fumigatus airway colonization in a tertiary pediatric CF cohort, evaluate the sensitivity of routine clinical sampling at detecting A. fumigatus, and compare lung function of A. fumigatus-colonized and non-colonized children.We carried out an 8-year retrospective cohort analysis using local databases, examining 1024 respiratory microbiological specimens from 45 children. Nineteen (42%) had a positive A. fumigatus culture at least once during the 8-year period, with 10 (22%) children persistently colonized. Overall, 29% of 48 bronchoalveolar lavage (BAL) samples tested positive for A. fumigatus, compared with 14% of 976 sputum samples. Of 33 children for whom lung function data were available during the study period, seven were classed as having severe lung disease, of whom four (57%) were persistently colonized with A. fumigatus.We conclude that chronic A. fumigatus colonization of the CF airway is common, and may be associated with worse lung function. In our practice, BAL appears superior at detecting lower airway A. fumigatus compared to sputum samples. PMID:26782645

  11. A Putative Mitochondrial Iron Transporter MrsA in Aspergillus fumigatus Plays Important Roles in Azole-, Oxidative Stress Responses and Virulence

    PubMed Central

    Long, Nanbiao; Xu, Xiaoling; Qian, Hui; Zhang, Shizhu; Lu, Ling

    2016-01-01

    Iron is an essential nutrient and enzyme co-factor required for a wide range of cellular processes, especially for the function of mitochondria. For the opportunistic fungal pathogen Aspergillus fumigatus, the ability to obtain iron is required for growth and virulence during the infection process. However, knowledge of how mitochondria are involved in iron regulation is still limited. Here, we show that a mitochondrial iron transporter, MrsA, a homolog of yeast Mrs4p, is critical for adaptation to iron-limited or iron-excess conditions in A. fumigatus. Deletion of mrsA leads to disruption of iron homeostasis with a decreased sreA expression, resulted in activated reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). Furthermore, deletion of mrsA induces hypersusceptibility to azole and oxidative stresses. An assay for cellular ROS content in ΔmrsA combined with rescue from the mrsA-defective phenotype by the antioxidant reagent L-ascorbic acid indicates that the increased sensitivity of ΔmrsA to the azole itraconazole and to oxidative stress is mainly the result of abnormal ROS accumulation. Moreover, site-directed mutation experiments verified that three conserved histidine residues related to iron transport in MrsA are required for responses to oxidative and azole stresses. Importantly, ΔmrsA causes significant attenuation of virulence in an immunocompromised murine model of aspergillosis. Collectively, our results show that the putative mitochondrial iron transporter MrsA plays important roles in azole- and oxidative-stress responses and virulence by regulating the balance of cellular iron in A. fumigatus. PMID:27433157

  12. Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies

    PubMed Central

    Walker, Louise A.; Lee, Keunsook K.; Munro, Carol A.

    2015-01-01

    Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall β-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2 and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca2+-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsG mutant was hypersensitive to caspofungin, and all other ΔAfchs mutants tested remained capable of increasing their chitin content in response to treatment with CaCl2 and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchs mutants tested, with the exception of the ΔAfchsG mutant, which remained sensitive to caspofungin. In vitro exposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was again AfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. These in vitro data demonstrate that A. fumigatus has the potential to survive echinocandin treatment in vivo by AfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections. PMID:26169407

  13. A Putative Mitochondrial Iron Transporter MrsA in Aspergillus fumigatus Plays Important Roles in Azole-, Oxidative Stress Responses and Virulence.

    PubMed

    Long, Nanbiao; Xu, Xiaoling; Qian, Hui; Zhang, Shizhu; Lu, Ling

    2016-01-01

    Iron is an essential nutrient and enzyme co-factor required for a wide range of cellular processes, especially for the function of mitochondria. For the opportunistic fungal pathogen Aspergillus fumigatus, the ability to obtain iron is required for growth and virulence during the infection process. However, knowledge of how mitochondria are involved in iron regulation is still limited. Here, we show that a mitochondrial iron transporter, MrsA, a homolog of yeast Mrs4p, is critical for adaptation to iron-limited or iron-excess conditions in A. fumigatus. Deletion of mrsA leads to disruption of iron homeostasis with a decreased sreA expression, resulted in activated reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). Furthermore, deletion of mrsA induces hypersusceptibility to azole and oxidative stresses. An assay for cellular ROS content in ΔmrsA combined with rescue from the mrsA-defective phenotype by the antioxidant reagent L-ascorbic acid indicates that the increased sensitivity of ΔmrsA to the azole itraconazole and to oxidative stress is mainly the result of abnormal ROS accumulation. Moreover, site-directed mutation experiments verified that three conserved histidine residues related to iron transport in MrsA are required for responses to oxidative and azole stresses. Importantly, ΔmrsA causes significant attenuation of virulence in an immunocompromised murine model of aspergillosis. Collectively, our results show that the putative mitochondrial iron transporter MrsA plays important roles in azole- and oxidative-stress responses and virulence by regulating the balance of cellular iron in A. fumigatus. PMID:27433157

  14. Human Invariant Natural Killer T cells possess immune-modulating functions during Aspergillus infection.

    PubMed

    Beitzen-Heineke, Antonia; Bouzani, Maria; Schmitt, Anna-Lena; Kurzai, Oliver; Hünniger, Kerstin; Einsele, Hermann; Loeffler, Juergen

    2016-02-01

    Aspergillus fumigatus is the most common cause for invasive fungal infections, a disease associated with high mortality in immune-compromised patients. CD1d-restricted invariant natural killer T (iNKT) cells compose a small subset of T cells known to impact the immune response toward various infectious pathogens. To investigate the role of human iNKT cells during A. fumigatus infection, we studied their activation as determined by CD69 expression and cytokine production in response to distinct fungal morphotypes in the presence of different CD1d(+) antigen presenting cells using flow cytometry and multiplex enzyme-linked immunosorbent assay (ELISA). Among CD1d(+) subpopulations, CD1d(+)CD1c(+) mDCs showed the highest potential to activate iNKT cells on a per cell basis. The presence of A. fumigatus decreased this effect of CD1d(+)CD1c(+) mDCs on iNKT cells and led to reduced secretion of TNF-α, G-CSF and RANTES. Production of other Th1 and Th2 cytokines was not affected by the fungus, suggesting an immune-modulating function for human iNKT cells during A. fumigatus infection. PMID:26483428

  15. Identification and characterization of an anti-oxidative stress-associated mutant of Aspergillus fumigatus transformed by Agrobacterium tumefaciens

    PubMed Central

    FAN, ZHONGQI; YU, HUIMEI; GUO, QI; HE, DAN; XUE, BAIJI; XIE, XIANGLI; YOKOYAMA, KOJI; WANG, LI

    2016-01-01

    Aspergillus fumigatus is one of the most common opportunistic pathogenic fungi, surviving in various environmental conditions. Maintenance of the redox homeostasis of the fungus relies upon the well-organized regulation between reactive oxygen species generated by immune cells or its own organelles, and the activated anti-oxidative stress mechanism. To investigate such a mechanism, the present study obtained a number of randomly-inserted mutants of A. fumigatus, mediated by Agrobacterium tumefaciens. In addition, a high throughput hydrogen peroxide screening system was established to examine ~1,000 mutants. A total of 100 mutants exhibited changes in hydrogen peroxide sensitivity, among which a significant increase in sensitivity was observed in the AFM2658 mutant. Further investigations of the mutant were also performed, in which the sequence of this mutant was characterized using thermal asymmetric interlaced-polymerase chain reaction. This revealed that the insertion site was located on chromosome 2 afu1_92, and the 96 bp sequence was knocked out, which partially comprised a sequence localized between the integral membrane protein coding region and the helix-loop-helix transcription factor coding region. A decrease in the levels of anti-oxidative stress-associated mRNAs were observed, and an increase in reactive oxygen species were detected using fluorescence. The results of the present study demonstrated that this sequence may have a protective role in A. fumigatus in the presence of oxidative stress. PMID:26847000

  16. In vitro release by Aspergillus fumigatus of galactofuranose antigens, 1,3-beta-D-glucan, and DNA, surrogate markers used for diagnosis of invasive aspergillosis.

    PubMed

    Mennink-Kersten, Monique A S H; Ruegebrink, Dorien; Wasei, Nazhat; Melchers, Willem J G; Verweij, Paul E

    2006-05-01

    Aspergillus markers are becoming increasingly important for the early diagnosis of invasive aspergillosis. The kinetics of release of these surrogate markers, however, is largely unknown. We investigated the release of beta-(1-5)-galactofuranosyl (galf) antigens (Platelia Aspergillus), 1,3-beta-D-glucan (BG) (Fungitell), and DNA (PCR) in an in vitro model of Aspergillus fumigatus. The results showed that release is correlated to the growth phase of the fungus, which depends on available nutrients. Whereas galf antigens and BG are released during logarithmic growth, DNA is released only after mycelium breakdown. During early logarithmic growth, galf antigens seem to be released somewhat earlier than BG. Furthermore, galf antigen concentrations of more than 120,000 times the serum cutoff value (0.5 ng/ml) can be measured, while BG concentrations reach a value only 978 times the serum cutoff value (60 pg/ml). During lytical growth, release of galf antigens further increased to a maximum level, which depended on pH. After that, the concentration of galf antigens stayed high (pH 7.4) or decreased to zero within 4 days (pH 5.0). In contrast to galf antigens, BG concentration decreased after 1 day of growth. The decrease of galf components seems to be due to the enzyme beta-galactofuranosidase, which is able to destroy galf epitopes and whose activity fluctuates in the culture filtrates in parallel with galf antigen concentration. Fungal DNA seems to be released only due to autolysis caused by nutrient limitation. In conclusion, several factors clearly influence the release of surrogate markers in vitro. These same factors might also play a role at the infection site of Aspergillus disease in humans. PMID:16672397

  17. Characterization of the Aspergillus nidulans aspnd1 gene demonstrates that the ASPND1 antigen, which it encodes, and several Aspergillus fumigatus immunodominant antigens belong to the same family.

    PubMed Central

    Calera, J A; Ovejero, M C; López-Medrano, R; Segurado, M; Puente, P; Leal, F

    1997-01-01

    For the first time, an immunodominant Aspergillus nidulans antigen (ASPND1) consistently reactive with serum samples from aspergilloma patients has been purified and characterized, and its coding gene (aspnd1) has been cloned and sequenced. ASPND1 is a glycoprotein with four N-glycosidically-bound sugar chains (around 2.1 kDa each) which are not necessary for reactivity with immune human sera. The polypeptide part is synthesized as a 277-amino-acid precursor of 30.6 kDa that after cleavage of a putative signal peptide of 16 amino acids, affords a mature protein of 261 amino acids with a molecular mass of 29 kDa and a pI of 4.24 (as deduced from the sequence). The ASPND1 protein is 53.1% identical to the AspfII allergen from Aspergillus fumigatus and 48% identical to an unpublished Candida albicans antigen. All of the cysteine residues and most of the glycosylation sites are perfectly conserved in the three proteins, suggesting a similar but yet unknown function. Analysis of the primary structure of the ASPND1 coding gene (aspnd1) has allowed the establishment of a clear relationship between several previously reported A. fumigatus and A. nidulans immunodominant antigens. PMID:9119471

  18. Proteomic analysis of temperature dependent extracellular proteins from Aspergillus fumigatus grown under solid-state culture condition.

    PubMed

    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2013-06-01

    Fungal species of the genus Aspergillus are filamentous ubiquitous saprophytes that play a major role in lignocellulosic biomass recycling and also are considered as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. Analysis of extracellular secreted biomass degrading enzymes using complex lignocellulosic biomass as a substrate by solid-state fermentation could be a more practical approach to evaluate application of the enzymes for lignocellulosic biorefinery. This study isolated a fungal strain from compost, identified as Aspergillus fumigatus, and further analyzed it for lignocellulolytic enzymes at different temperatures using label free quantitative proteomics. The profile of secretome composition discovered cellulases, hemicellulases, lignin degrading proteins, peptidases and proteases, and transport and hypothetical proteins; while protein abundances and further their hierarchical clustering analysis revealed temperature dependent expression of these enzymes during solid-state fermentation of sawdust. The enzyme activities and protein abundances as determined by exponentially modified protein abundance index (emPAI) indicated the maximum activities at the range of 40-50 °C, demonstrating the thermophilic nature of the isolate A. fumigatus LF9. Characterization of the thermostability of secretome suggested the potential of the isolated fungal strain in the production of thermophilic biomass degrading enzymes for industrial application. PMID:23647126

  19. Detection of Airway Colonization by Aspergillus fumigatus by Use of Electronic Nose Technology in Patients with Cystic Fibrosis.

    PubMed

    de Heer, K; Kok, M G M; Fens, N; Weersink, E J M; Zwinderman, A H; van der Schee, M P C; Visser, C E; van Oers, M H J; Sterk, P J

    2016-03-01

    Currently, there is no noninvasive test that can reliably diagnose early invasive pulmonary aspergillosis (IA). An electronic nose (eNose) can discriminate various lung diseases through an analysis of exhaled volatile organic compounds. We recently published a proof-of-principle study showing that patients with prolonged chemotherapy-induced neutropenia and IA have a distinct exhaled breath profile (or breathprint) that can be discriminated with an eNose. An eNose is cheap and noninvasive, and it yields results within minutes. We determined whether Aspergillus fumigatus colonization may also be detected with an eNose in cystic fibrosis (CF) patients. Exhaled breath samples of 27 CF patients were analyzed with a Cyranose 320. Culture of sputum samples defined the A. fumigatus colonization status. eNose data were classified using canonical discriminant analysis after principal component reduction. Our primary outcome was cross-validated accuracy, defined as the percentage of correctly classified subjects using the leave-one-out method. The P value was calculated by the generation of 100,000 random alternative classifications. Nine of the 27 subjects were colonized by A. fumigatus. In total, 3 subjects were misclassified, resulting in a cross-validated accuracy of the Cyranose detecting IA of 89% (P = 0.004; sensitivity, 78%; specificity, 94%). Receiver operating characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.89. The results indicate that A. fumigatus colonization leads to a distinctive breathprint in CF patients. The present proof-of-concept data merit external validation and monitoring studies. PMID:26677251

  20. Emerging aspergillosis by azole-resistant Aspergillus fumigatus at an intensive care unit in the Netherlands, 2010 to 2013.

    PubMed

    van Paassen, Judith; Russcher, Anne; In 't Veld-van Wingerden, Astrid Wm; Verweij, Paul E; Kuijper, Eduard J

    2016-07-28

    The prevalence of invasive aspergillosis (IA) at the intensive care unit (ICU) is unknown and difficult to assess since IA also develops in patients lacking specific host factors. In the Netherlands, increasing azole-resistance in Aspergillus fumigatus complicates treatment of patients with IA. The aim of this study was to determine the prevalence of IA by azole-resistant A. fumigatus at the ICU among patients receiving antifungal treatment and to follow their clinical outcome and prognosis. A retrospective cohort study was conducted in a university hospital ICU from January 2010 to December 2013. From all patients who received antifungal treatment for suspected IA, relevant clinical and microbiological data were collected using a standardised questionnaire. Of 9,121 admitted ICU-patients, 136 had received antifungal treatment for suspected IA, of which 38 had a positive A. fumigatus culture. Ten of the 38 patients harboured at least one azole-resistant isolate. Resistance mechanisms consisted of alterations in the cyp51A gene, more specific TR34/L98H and TR46/T289A/Y121F. Microsatellite typing did not show clonal relatedness, though isolates from two patients were genetically related. The overall 90-day mortality of patients with IA by azole-resistant A. fumigatus and patients with suspicion of IA by azole-susceptible isolates in the ICU was 100% (10/10) vs 82% (23/28) respectively. We conclude that the changing pattern of IA in ICU patients requires appropriate criteria for recognition, diagnosis and rapid resistance tests. The increase in azole resistance rates also challenges a reconsideration of empirical antifungal therapy. PMID:27541498

  1. Hsp70 and the Cochaperone StiA (Hop) Orchestrate Hsp90-Mediated Caspofungin Tolerance in Aspergillus fumigatus

    PubMed Central

    Juvvadi, Praveen R.; Soderblom, Erik J.; Moseley, M. Arthur; Steinbach, William J.

    2015-01-01

    Aspergillus fumigatus is the primary etiologic agent of invasive aspergillosis (IA), a major cause of death among immunosuppressed patients. Echinocandins (e.g., caspofungin) are increasingly used as second-line therapy for IA, but their activity is only fungistatic. Heat shock protein 90 (Hsp90) was previously shown to trigger tolerance to caspofungin and the paradoxical effect (i.e., decreased efficacy of caspofungin at higher concentrations). Here, we demonstrate the key role of another molecular chaperone, Hsp70, in governing the stress response to caspofungin via Hsp90 and their cochaperone Hop/Sti1 (StiA in A. fumigatus). Mutation of the StiA-interacting domain of Hsp70 (C-terminal EELD motif) impaired thermal adaptation and caspofungin tolerance with loss of the caspofungin paradoxical effect. Impaired Hsp90 function and increased susceptibility to caspofungin were also observed following pharmacologic inhibition of the C-terminal domain of Hsp70 by pifithrin-μ or after stiA deletion, further supporting the links among Hsp70, StiA, and Hsp90 in governing caspofungin tolerance. StiA was not required for the physical interaction between Hsp70 and Hsp90 but had distinct roles in the regulation of their function in caspofungin and heat stress responses. In conclusion, this study deciphering the physical and functional interactions of the Hsp70-StiA-Hsp90 complex provided new insights into the mechanisms of tolerance to caspofungin in A. fumigatus and revealed a key C-terminal motif of Hsp70, which can be targeted by specific inhibitors, such as pifithrin-μ, to enhance the antifungal activity of caspofungin against A. fumigatus. PMID:26014950

  2. Hsp70 and the Cochaperone StiA (Hop) Orchestrate Hsp90-Mediated Caspofungin Tolerance in Aspergillus fumigatus.

    PubMed

    Lamoth, Frédéric; Juvvadi, Praveen R; Soderblom, Erik J; Moseley, M Arthur; Steinbach, William J

    2015-08-01

    Aspergillus fumigatus is the primary etiologic agent of invasive aspergillosis (IA), a major cause of death among immunosuppressed patients. Echinocandins (e.g., caspofungin) are increasingly used as second-line therapy for IA, but their activity is only fungistatic. Heat shock protein 90 (Hsp90) was previously shown to trigger tolerance to caspofungin and the paradoxical effect (i.e., decreased efficacy of caspofungin at higher concentrations). Here, we demonstrate the key role of another molecular chaperone, Hsp70, in governing the stress response to caspofungin via Hsp90 and their cochaperone Hop/Sti1 (StiA in A. fumigatus). Mutation of the StiA-interacting domain of Hsp70 (C-terminal EELD motif) impaired thermal adaptation and caspofungin tolerance with loss of the caspofungin paradoxical effect. Impaired Hsp90 function and increased susceptibility to caspofungin were also observed following pharmacologic inhibition of the C-terminal domain of Hsp70 by pifithrin-μ or after stiA deletion, further supporting the links among Hsp70, StiA, and Hsp90 in governing caspofungin tolerance. StiA was not required for the physical interaction between Hsp70 and Hsp90 but had distinct roles in the regulation of their function in caspofungin and heat stress responses. In conclusion, this study deciphering the physical and functional interactions of the Hsp70-StiA-Hsp90 complex provided new insights into the mechanisms of tolerance to caspofungin in A. fumigatus and revealed a key C-terminal motif of Hsp70, which can be targeted by specific inhibitors, such as pifithrin-μ, to enhance the antifungal activity of caspofungin against A. fumigatus. PMID:26014950

  3. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    PubMed

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis. PMID:27316653

  4. Cytokine and transcription factor expression by Aspergillus fumigatus-stimulated peripheral blood mononuclear cells in dogs with sino-nasal aspergillosis.

    PubMed

    Vanherberghen, M; Bureau, F; Peters, I R; Day, M J; Lynch, A; Fievez, L; Billen, F; Clercx, C; Peeters, D

    2013-08-15

    The causal agent of sino-nasal aspergillosis is usually Aspergillus fumigatus, which is a saprophytic and ubiquitous fungus that causes a severe rhinosinusitis in apparent healthy dogs. Affected dogs do not have systemic immuno-suppression. It has been shown previously that dogs affected by this disease have local over-expression of interleukin (IL)-10 and Th1 cytokines in nasal mucosal tissue. The aim of the present study was to assess the response of peripheral blood mononuclear cells (PBMC) from affected and unaffected dogs to antigen-specific stimulation with heat-inactivated Aspergillus spp. conidia, by quantifying gene expression for specific Th1, Th2, Th17 and Treg cytokines and their related transcription factors. Quantification of IL-4 and IFN-γ protein in culture supernatant was performed by enzyme-linked immunosorbent assay (ELISA). PBMC from dogs with SNA produced adequate mRNA encoding IFN-γ and IFN-γ protein. The expression of IL-17A mRNA was significantly greater in PBMC of affected compared with unaffected dogs. The amount of IL-10 mRNA in PBMC from affected dogs decreased after antigen-specific challenge. These results suggest that the incapacity of affected dogs to clear these fungal infections is not related to a defect in Th1 immunity or to an overwhelming regulatory reaction, but rather to an uncontrolled pro-inflammatory reaction driven by Th17 cells. PMID:23759303

  5. Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae

    PubMed Central

    2013-01-01

    Background Secondary metabolite production, a hallmark of filamentous fungi, is an expanding area of research for the Aspergilli. These compounds are potent chemicals, ranging from deadly toxins to therapeutic antibiotics to potential anti-cancer drugs. The genome sequences for multiple Aspergilli have been determined, and provide a wealth of predictive information about secondary metabolite production. Sequence analysis and gene overexpression strategies have enabled the discovery of novel secondary metabolites and the genes involved in their biosynthesis. The Aspergillus Genome Database (AspGD) provides a central repository for gene annotation and protein information for Aspergillus species. These annotations include Gene Ontology (GO) terms, phenotype data, gene names and descriptions and they are crucial for interpreting both small- and large-scale data and for aiding in the design of new experiments that further Aspergillus research. Results We have manually curated Biological Process GO annotations for all genes in AspGD with recorded functions in secondary metabolite production, adding new GO terms that specifically describe each secondary metabolite. We then leveraged these new annotations to predict roles in secondary metabolism for genes lacking experimental characterization. As a starting point for manually annotating Aspergillus secondary metabolite gene clusters, we used antiSMASH (antibiotics and Secondary Metabolite Analysis SHell) and SMURF (Secondary Metabolite Unknown Regions Finder) algorithms to identify potential clusters in A. nidulans, A. fumigatus, A. niger and A. oryzae, which we subsequently refined through manual curation. Conclusions This set of 266 manually curated secondary metabolite gene clusters will facilitate the investigation of novel Aspergillus secondary metabolites. PMID:23617571

  6. The Potential Inhibitory Effect of Cuminum Cyminum, Ziziphora Clinopodioides and Nigella Sativa Essential Oils on the Growth of Aspergillus Fumigatus and Aspergillus

    PubMed Central

    Khosravi, A.R.; Minooeianhaghighi, M.H.; Shokri, H.; Emami, S.A.; S.M., Alavi; Asili, J.

    2011-01-01

    The goals of this study were to evaluate the effectiveness of Cuminum cyminum, Ziziphora clinopodioides and Nigella sativa essential oils to inhibit the growth of Aspergillus fumigatus and A. flavus and to evoke ultrastructural changes. The fungi were cultured into RPMI 1640 media in the presence of oils at concentrations of 8, 6, 5, 4, 3, 2, 1.5, 1.25, 1, 0.75 and 0.5 mg/ml in broth microdilution and 2, 1.5, 1 and 0.5 mg/ml in broth macrodilution methods with shaking for 48 h at 28oC. Conidial and mycelial samples exposed to 0.25, 0.5, 1, 1.5 and 2 mg essential oils/ml for 5 days in 2% yeast extract granulated plus 15% Saccharose media were processed for transmission electron microscopy (TEM). Based on broth dilution methods, C. cyminum and to a lesser extent Z. clinopodioides oils exhibited the strongest activity against A. fumigatus and A. flavus with MIC90 ranging from 0.25 to 1.5 mg/ml, while the oil from N. sativa exhibited relatively moderate activity against two above fungi with MIC90 ranging from 1.5 to 2 mg/ml. The main changes observed by TEM were in the cell wall, plasma membrane and membranous organelles; in particular, in the nuclei and mitochondria. These modifications in fungal structure were associated with the interference of the essential oils with the enzymes responsible for cell wall synthesis, which disturbed normal growth. Moreover, the essential oils caused high vacuolation of the cytoplasm, detachment of fibrillar layer of cell wall, plasma membrane disruption and disorganization of the nuclear and mitochondrial structures. Aspergillus fumigatus and A. flavus growth inhibition induced by these oils were found to be well-correlated with subsequent morphological changes of the fungi exposed to different fungistatic concentrations of the oils. Our results show the anti-Aspergillus activities of C. cyminum, Z. clinopodioides and N. sativa essential oils, which strengthens the potential use of these substances as anti-mould in the future. PMID

  7. Case report. Mycotic arteritis due to Aspergillus fumigatus in a diabetic with retrobulbar aspergillosis and mycotic meningitis.

    PubMed

    Nenoff, P; Kellermann, S; Horn, L C; Keiner, S; Bootz, F; Schneider, S; Haustein, U F

    2001-11-01

    A 74-year-old man with diabetes mellitus type II, retinopathy and polyneuropathy suffered from exophthalmus, ptosis and diplopia. Magnetic resonance imaging and computer tomography showed a space-occupying process in the right orbital apex. An extranasal ethmoidectomy accompanied by an orbitotomia revealed the presence of septated hyphae. Aspergillus fumigatus was grown from the tissue. After surgical removal of the fungal masses, therapy with amphotericin B (1 mg kg(-1) body weight) plus itraconazole (Sempera, 200 mg per day) over 6 weeks was initiated. Five months later the patient's condition deteriorated again, with vomiting, nausea and pain behind the right eye plus increasing exophthalmus. Antifungal therapy was started again with amphotericin B and 5-fluorocytosine. Neutropenia did not occur. The patient became somnolent and deteriorated, a meningitis was suggested. Aspergillus antigen (titre 1:2, Pastorex) was detected in liquor. Anti-Aspergillus antibodies were not detectable. Both the right eye and retrobulbar fungal masses were eradicated by means of an exenteratio bulbi et orbitae. However, renal insufficiency and an apallic syndrome developed and the patient died. At autopsy, a mycotic aneurysm of the arteria carotis interna dextra was detected. The mycotic vasculitis of this aneurysm had caused a rupture of the blood vessel followed by a massive subarachnoidal haemorrhage. In addition, severe mycotic sphenoidal sinusitis and aspergillosis of the right orbit were seen, which had led to a bifrontal meningitis. PMID:11766108

  8. ADOPTING SELECTED HYDROGEN BONDING AND IONIC INTERACTIONS FROM ASPERGILLUS FUMIGATUS PHYTASE STRUCTURE IMPROVES THE THERMOSTABILITY OF ASPERGILLUS NIGER PHYA PHYTASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although it has been widely used as a feed supplement to reduce manure phosphorus pollution of swine and poultry, Aspergillus niger PhyA phytase is unable to withstand heat inactivation during feed pelleting. Crystal structure comparisons with its close homolog, the thermostable Aspergillus fumigatu...

  9. Transcriptome analysis of cyclic AMP-dependent protein kinase A–regulated genes reveals the production of the novel natural compound fumipyrrole by Aspergillus fumigatus

    PubMed Central

    Macheleidt, Juliane; Scherlach, Kirstin; Neuwirth, Toni; Schmidt-Heck, Wolfgang; Straßburger, Maria; Spraker, Joseph; Baccile, Joshua A.; Schroeder, Frank C.; Keller, Nancy P.; Hertweck, Christian; Heinekamp, Thorsten; Brakhage, Axel A.

    2015-01-01

    Summary Aspergillus fumigatus is an opportunistic human pathogenic fungus causing life-threatening infections in immunocompromised patients. Adaptation to different habitats and also virulence of the fungus depends on signal perception and transduction by modules such as the cyclic AMP-dependent protein kinase A (PKA) pathway. Here, by transcriptome analysis, 632 differentially regulated genes of this important signaling cascade were identified, including 23 putative transcriptional regulators. The highest upregulated transcription factor gene was located in a previously unknown secondary metabolite gene cluster, which we named fmp, encoding an incomplete nonribosomal peptide synthetase, FmpE. Overexpression of the regulatory gene fmpR using the TetOn system led to the specific expression of the other six genes of the fmp cluster. Metabolic profiling of wild type and fmpR overexpressing strain by HPLC-DAD and HPLCHRESI-MS and structure elucidation by NMR led to identification of 5-benzyl-1H-pyrrole-2-carboxylic acid, which we named fumipyrrole. Fumipyrrole was not described as natural product yet. Chemical synthesis of fumipyrrole confirmed its structure. Interestingly, deletion of fmpR or fmpE led to reduced growth and sporulation of the mutant strains. Although fmp cluster genes were transcribed in infected mouse lungs, deletion of fmpR resulted in wild-type virulence in a murine infection model. PMID:25582336

  10. Production of tremorgenic mycotoxins by isolates of Aspergillus fumigatus from sawmills in Sweden.

    PubMed

    Land, C J; Lundström, H; Werner, S

    1993-11-01

    One hundred and six strains of A. fumigatus were isolated from 21 sawmills in Sweden, and 73 of these strains were examined for production of fumitremorgen B and verruculogen (tremorgenic mycotoxins) on YES-medium using thin layer chromatography (TLC). Twenty-three strains (32%) were tremorgen producers and 50 strains (68%) were non-producers. Tremorgenic mycotoxins were detected in conidia of seven A. fumigatus strains. The amount of toxin varied between 0.6-8.0 microgram/10(8) conidia (mean value 2.3 micrograms/10(8) conidia, equivalent with 0.18%). No production of the mycotoxin gliotoxin was detected in 6 strains of A. fumigatus. No tremorgens were detected during mould growth on wood substrates, in spite of the use of different wood species (Scots pine, Pinus sylvestris: Norway spruce, Picea abies and birch, Betula spp.), dried versus non-dried wood, bark (pine), leached wood, and wood after various sterilization methods. PMID:8008045

  11. First Detection of TR34 L98H and TR46 Y121F T289A Cyp51 Mutations in Aspergillus fumigatus Isolates in the United States.

    PubMed

    Wiederhold, Nathan P; Gil, Veronica Garcia; Gutierrez, Felipe; Lindner, Jonathan R; Albataineh, Mohammad T; McCarthy, Dora I; Sanders, Carmita; Fan, Hongxin; Fothergill, Annette W; Sutton, Deanna A

    2016-01-01

    Azole resistance in Aspergillus fumigatus is an increasing problem. The TR34 L98H and TR46 Y121F T289A mutations that can occur in patients without previous azole exposure have been reported in Europe, Asia, the Middle East, Africa, and Australia. Here, we report the detection of both the TR34 L98H and TR46 Y121F T289A mutations in confirmed A. fumigatus isolates collected in institutions in the United States. These mutations, other mutations known to cause azole resistance, and azole MICs are reported here. PMID:26491179

  12. First Detection of TR34 L98H and TR46 Y121F T289A Cyp51 Mutations in Aspergillus fumigatus Isolates in the United States

    PubMed Central

    Gil, Veronica Garcia; Gutierrez, Felipe; Lindner, Jonathan R.; Albataineh, Mohammad T.; McCarthy, Dora I.; Sanders, Carmita; Fan, Hongxin; Fothergill, Annette W.; Sutton, Deanna A.

    2015-01-01

    Azole resistance in Aspergillus fumigatus is an increasing problem. The TR34 L98H and TR46 Y121F T289A mutations that can occur in patients without previous azole exposure have been reported in Europe, Asia, the Middle East, Africa, and Australia. Here, we report the detection of both the TR34 L98H and TR46 Y121F T289A mutations in confirmed A. fumigatus isolates collected in institutions in the United States. These mutations, other mutations known to cause azole resistance, and azole MICs are reported here. PMID:26491179

  13. Comparisons between cellulase production by Aspergillus fumigatus in agitated vessels and in an air-lift fermentor

    SciTech Connect

    Wase, D.A.J.; McManamey, W.J.; Raymahasay, S.; Vaid, A.K.

    1985-08-01

    Aspergillus fumigatus was cultured in disc-turbine-agitated vessels and in an air-lift fermentor. In the agitated vessels the yield of cellulase was reduced when the agitation rate was increased, although extracellular protein levels rose. The enzyme complex itself was shown to be exceptionally stable under conditions similar to those in the agitated vessels, so probably shear damage to the mycelium had occurred, liberating intracellular contents. These appeared to contain an inhibitor that could be removed by fabricated inorganic protein absorbents, such as kieselguhr and alumina. However, the inhibitor was not likely to be protease, since only relatively low levels could be detected and its identity has not been established. The use of an air-lift fermentor avoided the shear effects due to use of the disc turbine agitator in the conventional fermentors, and yields of enzyme were then found to increase by about 20%, maximum yields being obtained at maximum Kla values.

  14. Enhanced production of fumigaclavine C by ultrasound stimulation in a two-stage culture of Aspergillus fumigatus CY018.

    PubMed

    Yao, Ling-Yun; Zhu, Yi-Xiang; Jiao, Rui-Hua; Lu, Yan-Hua; Tan, Ren-Xiang

    2014-05-01

    Stimulation by physical means including ultrasound is important to cell morphology and the product yield. In this work, the effect of ultrasound on the production of fumigaclavine C (FC), a conidiation-associated alkaloid with strong anti-inflammatory activity, was investigated in a newly developed two-stage culture of Aspergillus fumigatus CY018. The optimum ultrasonication conditions consisted of exposing cultures (at 12h of growth phase) to 10-min repeated irradiation (4 times) with a 24-h interval at the fixed power (500 W). Under this condition, FC production reached 118.09 mg/L, which was 89% higher than the control and much higher than previous reported values. Morphological analysis demonstrated that mycelia morphology from ultrasonication was in the form smaller and looser pellets as compared to that of the control. In addition, conidia that is closely related to FC biosynthesis were significantly increased after ultrasound stimulation, with 3 folds of that from the control. PMID:24632633

  15. Influence of essential oil of Hyssopus officinalis on the chemical composition of the walls of Aspergillus fumigatus (Fresenius).

    PubMed

    Ghfir, B; Fonvieille, J L; Dargent, R

    1997-07-01

    The cell walls of the growing hyphae of Aspergillus fumigatus (Fresenius) cultured in the presence or absence of the essential oil of Hyssopus officinalis were isolated and their chemical composition analysed. The presence of the essential oil led to a reduction in levels of neutral sugars, uronic acid and proteins, whereas amino sugars, lipids and phosphorus levels were increased. HPLC analysis of the neutral sugars showed that they consisted mainly of glucose, mannose and galactose, while the amino sugars consisted of glucosamine and galactosamine. The presence of the essential oil in the culture medium induced marked changes in the content of galactose and galactosamine. Cell walls were fractionated by treatment with alkali and acid. The essential oil induced similar alterations in the various fractions with a more marked effect on the major constituents. The alterations were related to changes in the structure of the cells. PMID:16333566

  16. Anethole induces apoptotic cell death accompanied by reactive oxygen species production and DNA fragmentation in Aspergillus fumigatus and Saccharomyces cerevisiae.

    PubMed

    Fujita, Ken-Ichi; Tatsumi, Miki; Ogita, Akira; Kubo, Isao; Tanaka, Toshio

    2014-02-01

    trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum, and antimicrobial activity that is weaker than that of other antibiotics on the market. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to possess significant synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the antifungal mechanism of anethole has not been completely determined. We found that anethole stimulated cell death of a human opportunistic pathogenic fungus, Aspergillus fumigatus, in addition to S. cerevisiae. The anethole-induced cell death was accompanied by reactive oxygen species production, metacaspase activation, and DNA fragmentation. Several mutants of S. cerevisiae, in which genes related to the apoptosis-initiating execution signals from mitochondria were deleted, were resistant to anethole. These results suggest that anethole-induced cell death could be explained by oxidative stress-dependent apoptosis via typical mitochondrial death cascades in fungi, including A. fumigatus and S. cerevisiae. PMID:24393541

  17. Deletion of the msdS/AfmsdC gene induces abnormal polarity and septation in Aspergillus fumigatus.

    PubMed

    Li, Yanjie; Zhang, Lei; Wang, Depeng; Zhou, Hui; Ouyang, Haomiao; Ming, Jia; Jin, Cheng

    2008-07-01

    alpha-Mannosidases play an important role in the processing of mannose-containing glycans in eukaryotes. A deficiency in alpha-mannosidase is lethal in humans and cattle. In contrast to mammals, Saccharomyces cerevisiae does not require the endoplasmic reticulum alpha-mannosidase gene for growth. However, little is known of the consequence of loss of function of class I alpha-mannosidases in filamentous fungi. In this study, the msdS/AfmsdC gene was identified to encode 1,2-alpha-mannosidase MsdS in Aspergillus fumigatus. Soluble MsdS expressed in Escherichia coli was characterized as a typical class I alpha-mannosidase. The msdS gene was deleted by replacement of the msdS gene with a pyrG gene. Although the mutant showed a defect in N-glycan processing, as well as a reduction of cell wall components and a reduced ability of conidiation, it appeared that the rate of hyphal growth was not affected. Morphology analysis revealed abnormal polarity and septation at the stages of germination, hyphal growth and conidiation. Although the mechanism by which the N-glycan processing affects polarity and septation is unclear, our results show that msdS is involved in polarity and septation in A. fumigatus. PMID:18599824

  18. Association of a Myosin Immunoanalogue with Cell Envelopes of Aspergillus fumigatus Conidia and Its Participation in Swelling and Germination

    PubMed Central

    Esnault, Karine; el Moudni, Brahim; Bouchara, Jean-Philippe; Chabasse, Dominique; Tronchin, Guy

    1999-01-01

    A myosin immunoanalogue was identified in conidia of Aspergillus fumigatus by Western blotting, indirect immunofluorescence assay, and gold immunoelectron microscopy with two different antimyosin antibodies. The distribution pattern of this protein was followed during the early stages of germination. A single 180-kDa polypeptide, detected predominantly in a cell envelope extract, was found to cross-react with monoclonal and polyclonal antibodies raised against vertebrate muscle myosin. Immunoelectron microscopy permitted precise localization of this polypeptide, indicating that myosin analogue was mainly distributed along the plasma membrane of resting and swollen conidia. In germinating conidia, indirect immunofluorescence microscopy revealed myosin analogue at the periphery of germ tubes, whereas actin appeared as dispersed punctate structures in the cytoplasm that were more concentrated at the site of germ tube emergence. A myosin ATPase inhibitor, butanedione monoxime, greatly reduced swelling and blocked germination. In contrast, when conidia were treated with cytochalasin B, an inhibitor of actin polymerization, swelling was not affected and germination was only partially reduced. Butanedione monoxime-treated conidia showed accumulation of cytoplasmic vesicles and did not achieve cell wall reorganization, unlike swollen conidia. Collectively, these results suggest an essential role for this myosin analogue in the deposition of cell wall components during germination of A. fumigatus conidia and therefore in host tissue colonization. PMID:10024566

  19. Production of short-chain fatty acids from the biodegradation of wheat straw lignin by Aspergillus fumigatus.

    PubMed

    Baltierra-Trejo, Eduardo; Sánchez-Yáñez, Juan Manuel; Buenrostro-Delgado, Otoniel; Márquez-Benavides, Liliana

    2015-11-01

    The wheat straw lignin-rich fraction (WSLig-RF) can be used as a raw material for the production of metabolites for industrial use if ligninolytic mitosporic fungi are used for its biodegradation into aromatics and short-chain fatty acids (SCFAs, i.e., SCFA2-6). Although methods for the production of SCFA2-6 have been described previously, quantitative data of SCFA2-6 production have not been reported. The objectives of this study were to investigate the biodegradation of different concentrations of WSLig-RF by Aspergillus fumigatus and to identify whether SCFA2-6 production was dependent on the concentration of aromatics. A. fumigatus generated 2805mgL(-1) acetic acid when mixed with WSLig-RF at a concentration of 20gL(-1). Thus, aromatics are a substrate for the biosynthesis of SCFA2-6, and their production depends on the concentration of WSLig-RF aromatics. PMID:26263005

  20. Endoplasmic reticulum localized PerA is required for cell wall integrity, azole drug resistance, and virulence in Aspergillus fumigatus

    PubMed Central

    Chung, Dawoon; Thammahong, Arsa; Shepardson, Kelly M.; Blosser, Sara J.; Cramer, Robert A.

    2014-01-01

    Summary GPI-anchoring is a universal and critical post-translational protein modification in eukaryotes. In fungi, many cell wall proteins are GPI-anchored, and disruption of GPI-anchored proteins impairs cell wall integrity. After being synthesized and attached to target proteins, GPI anchors undergo modification on lipid moieties. In spite of its importance for GPI-anchored protein functions, our current knowledge of GPI lipid remodeling in pathogenic fungi is limited. In this study, we characterized the role of a putative GPI lipid remodeling protein, designated PerA, in the human pathogenic fungus Aspergillus fumigatus. PerA localizes to the endoplasmic reticulum and loss of PerA leads to striking defects in cell wall integrity. A perA null mutant has decreased conidia production, increased susceptibility to triazole antifungal drugs, and is avirulent in a murine model of invasive pulmonary aspergillosis. Interestingly, loss of PerA increases exposure of β-glucan and chitin content on the hyphal cell surface, but diminished TNF production by bone marrow derived macrophages relative to wild type. Given the structural specificity of fungal GPI-anchors, which is different from humans, understanding GPI lipid remodeling and PerA function in A. fumigatus is a promising research direction to uncover a new fungal specific antifungal drug target. PMID:24779420

  1. Surface Structure Characterization of Aspergillus fumigatus Conidia Mutated in the Melanin Synthesis Pathway and Their Human Cellular Immune Response

    PubMed Central

    Bayry, Jagadeesh; Beaussart, Audrey; Dufrêne, Yves F.; Sharma, Meenu; Bansal, Kushagra; Kniemeyer, Olaf; Aimanianda, Vishukumar; Brakhage, Axel A.; Kaveri, Srini V.; Kwon-Chung, Kyung J.

    2014-01-01

    In Aspergillus fumigatus, the conidial surface contains dihydroxynaphthalene (DHN)-melanin. Six-clustered gene products have been identified that mediate sequential catalysis of DHN-melanin biosynthesis. Melanin thus produced is known to be a virulence factor, protecting the fungus from the host defense mechanisms. In the present study, individual deletion of the genes involved in the initial three steps of melanin biosynthesis resulted in an altered conidial surface with masked surface rodlet layer, leaky cell wall allowing the deposition of proteins on the cell surface and exposing the otherwise-masked cell wall polysaccharides at the surface. Melanin as such was immunologically inert; however, deletion mutant conidia with modified surfaces could activate human dendritic cells and the subsequent cytokine production in contrast to the wild-type conidia. Cell surface defects were rectified in the conidia mutated in downstream melanin biosynthetic pathway, and maximum immune inertness was observed upon synthesis of vermelone onward. These observations suggest that although melanin as such is an immunologically inert material, it confers virulence by facilitating proper formation of the A. fumigatus conidial surface. PMID:24818666

  2. Expression, Purification, and Characterization of Aspergillus fumigatus Sterol 14-α Demethylase (CYP51) Isoenzymes A and B▿

    PubMed Central

    Warrilow, Andrew G. S.; Melo, Nadja; Martel, Claire M.; Parker, Josie E.; Nes, W. David; Kelly, Steven L.; Kelly, Diane E.

    2010-01-01

    Aspergillus fumigatus sterol 14-α demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (Ks, 8.6 μM) and eburicol (Ks, 22.6 μM). Membrane suspensions of AF51A bound to both lanosterol (Ks, 3.1 μM) and eburicol (Ks, 4.1 μM). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the Kd (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 μM, respectively, in comparison with a Kd value of 4 μM for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with Kd values ranging from 1 μM for itraconazole to 11.9 μM for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus. PMID:20660663

  3. Phosphorylation of Calcineurin at a Novel Serine-Proline Rich Region Orchestrates Hyphal Growth and Virulence in Aspergillus fumigatus

    PubMed Central

    Juvvadi, Praveen R.; Gehrke, Christopher; Fortwendel, Jarrod R.; Lamoth, Frédéric; Soderblom, Erik J.; Cook, Erik C.; Hast, Michael A.; Asfaw, Yohannes G.; Moseley, M. Arthur; Creamer, Trevor P.; Steinbach, William J.

    2013-01-01

    The fungus Aspergillus fumigatus is a leading infectious killer in immunocompromised patients. Calcineurin, a calmodulin (CaM)-dependent protein phosphatase comprised of calcineurin A (CnaA) and calcineurin B (CnaB) subunits, localizes at the hyphal tips and septa to direct A. fumigatus invasion and virulence. Here we identified a novel serine-proline rich region (SPRR) located between two conserved CnaA domains, the CnaB-binding helix and the CaM-binding domain, that is evolutionarily conserved and unique to filamentous fungi and also completely absent in human calcineurin. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of A. fumigatus CnaA in vivo at four clustered serine residues (S406, S408, S410 and S413) in the SPRR. Mutation of the SPRR serine residues to block phosphorylation led to significant hyphal growth and virulence defects, indicating the requirement of calcineurin phosphorylation at the SPRR for its activity and function. Complementation analyses of the A. fumigatus ΔcnaA strain with cnaA homologs from the pathogenic basidiomycete Cryptococcus neoformans, the pathogenic zygomycete Mucor circinelloides, the closely related filamentous fungi Neurospora crassa, and the plant pathogen Magnaporthe grisea, revealed filamentous fungal-specific phosphorylation of CnaA in the SPRR and SPRR homology-dependent restoration of hyphal growth. Surprisingly, circular dichroism studies revealed that, despite proximity to the CaM-binding domain of CnaA, phosphorylation of the SPRR does not alter protein folding following CaM binding. Furthermore, mutational analyses in the catalytic domain, CnaB-binding helix, and the CaM-binding domains revealed that while the conserved PxIxIT substrate binding motif in CnaA is indispensable for septal localization, CaM is required for its function at the hyphal septum but not for septal localization. We defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation

  4. Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum

    SciTech Connect

    Wiedner, Susan D.; Burnum, Kristin E.; Pederson, Leeanna M.; Anderson, Lindsey N.; Fortuin, Suereta; Chauvigne-Hines, Lacie M.; Shukla, Anil K.; Ansong, Charles; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

    2012-08-03

    Environmental and metabolic adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised lung. We employed an activity-based protein profiling (ABPP) approach utilizing a new aryl vinyl sulfonate probe and a serine hydrolase probe combined with quantitative LC-MS based accurate mass and time (AMT) tag proteomics for the identification of functional pathway adaptation of A. fumigatus to environmental variability relevant to pulmonary Invasive Aspergillosis. When the fungal pathogen was grown with human serum, metabolism and energy processes were markedly decreased compared to no serum culture. Additionally, functional pathways associated with amino acid and protein biosynthesis were limited as the fungus scavenged from the serum to obtain essential nutrients. Our approach revealed significant metabolic adaptation by A. fumigatus, and provides direct insight into this pathogen’s ability to survive and proliferate.

  5. Recombinant expression and antigenic properties of a 32-kilodalton extracellular alkaline protease, representing a possible virulence factor from Aspergillus fumigatus.

    PubMed Central

    Moser, M; Menz, G; Blaser, K; Crameri, R

    1994-01-01

    A 32-kDa nonglycosylated alkaline protease (EC 3.4.1.14) with elastolytic activity, secreted by the opportunistic pathogen Aspergillus fumigatus ATCC 42202, is suggested to be a virulence factor of this fungus. The enzyme is a serine protease of the subtilisin family, and its cDNA nucleotide sequence has recently been reported. We have cloned the cDNA encoding the mature protease into a high-level Escherichia coli expression plasmid and produced the recombinant protease as a fusion protein with a six-adjacent-histidine affinity tag at the carboxy terminus. Subsequently, the recombinant protease was purified to homogeneity, with affinity chromatography yielding 30 to 40 mg of recombinant protease per liter of E. coli culture. Refolded recombinant protease, in comparison with native protease, demonstrated weak enzymatic activity but similar immunochemical characteristics as analyzed by antigen-specific enzyme-linked immunosorbent assay (ELISA), competition ELISA, and immunoblotting assays. To assess the allergenic potential of the protease, sera from patients with allergic bronchopulmonary aspergillosis and sera from healthy control individuals were analyzed by ELISA and immunoblotting techniques. Sera from patients with allergic bronchopulmonary aspergillosis did not have protease-specific immunoglobulin E (IgE) antibodies and, remarkably, did not show significantly elevated protease-specific IgG antibody levels compared with those in sera from healthy control individuals. This suggests that the alkaline protease from A. fumigatus does not elicit IgE antibodies and has weak immunogenicity, a property which may explain fungus persistence in allergic individuals. Images PMID:8112866

  6. Mitogen activated protein kinases SakA(HOG1) and MpkC collaborate for Aspergillus fumigatus virulence.

    PubMed

    Bruder Nascimento, Ariane Cristina Mendes de Oliveira; Dos Reis, Thaila Fernanda; de Castro, Patrícia Alves; Hori, Juliana I; Bom, Vinícius Leite Pedro; de Assis, Leandro José; Ramalho, Leandra Naira Zambelli; Rocha, Marina Campos; Malavazi, Iran; Brown, Neil Andrew; Valiante, Vito; Brakhage, Axel A; Hagiwara, Daisuke; Goldman, Gustavo H

    2016-06-01

    Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen-activated protein kinases of the high-osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild-type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis. PMID:26878695

  7. Four ardeemin analogs from endophytic Aspergillus fumigatus SPS-02 and their reversal effects on multidrug-resistant tumor cells.

    PubMed

    Zhang, Hua-Wei; Ying, Chen; Tang, Yi-Fei

    2014-01-01

    Four ardeemin derivatives, 5-N-acetylardeemin (1), 5-N-acetyl-15bβ-hydroxyardeemin (2), 5-N-acetyl-15b-didehydroardeemin (3), and 5-N-acetyl-16α-hydroxyardeemin (4), were isolated from the fermentation broth of an endophytic Aspergillus fumigatus SPS-02 associated with Artemisia annua L. The structures of these metabolites were elucidated by a combination of spectroscopic data, including 1D-, 2D-NMR and MS. In vitro chemosensitization assay indicated that these ardeemins had different activities of reversing the multidrug-resistant (MDR) phenotype in three cancer cell lines, leukemia doxorubicin resistant cell K562/DOX, human lung adenocarcinoma cis-platin-resistant cell A549/DDP, and ovarian cancer cisplatin-resistant cell SK-OV-S/DDP. Compound 4 exhibited the strongest MDR reversing effect at 5 μM concentration in K562/DOX and A549/DDP cell lines 5.2±0.18-fold, 8.2±0.23-fold, respectively, while compound 2 had the highest reversal capacity in SK-OV-S/DDP cell line with 10.8±0.28 fold. Preliminary investigation of their structureactivity relationship suggested that a OH group at C(15b) or C(16) in ardeemin plays a key role in reversing the MDR effect. It is the first report on ardeemin analogs from endophytic A. fumigatus with reversal effects on MDR cancer cell lines K562/DOX, A549/DDP and SK-OV-S/DDP. PMID:24443428

  8. Scleral Buckle Infection with Aspergillus Flavus

    PubMed Central

    Bouhaimed, Manal; Al-Dhibi, Hassan; Al-Assiri, Abdullah

    2008-01-01

    Purpose: To present a case of scleral buckle infection with Aspergillus flavus in a tertiary eye center in Saudi Arabia. Methods: A retrospective case report of a 28-year-old Saudi male who presented with a six-month history of conjunctival injection and discharge from the left eye which had undergone uncomplicated conventional retinal detachment surgery, at the King Khaled Eye Specialist Hospital in Riyadh, Saudi Arabia, in the form of cryopexy, subretinal fluid drainage and scleral buckle (grooved segmental sponge and circumferential band with sleeve) for a macula on retinal detachment four years earlier. A diagnosis of infected extruded scleral buckle was made and the buckle was removed. Results: The infected scleral buckle was removed under local anesthesia with administration of sub-conjunctival irrigation of 50 mg solution of Vancomycin, and sub-conjunctival injection of 25mg of Vancomycin. Post operative microbiological studies revealed infection with silver staining of moderate Aspergillus flavus hyphae. Visual acuity of the left eye improved from 20/200 before surgery to 20/60 in the two years follow-up visit. Conclusion: This case report indicates the importance of considering infection with multiple organisms – including fungal ones – in cases of scleral buckle infections in our population. PMID:20379425

  9. eNose technology can detect and classify human pathogenic molds in vitro: a proof-of-concept study of Aspergillus fumigatus and Rhizopus oryzae.

    PubMed

    de Heer, K; Vonk, S I; Kok, M; Kolader, M; Zwinderman, A H; van Oers, M H J; Sterk, P J; Visser, C E

    2016-01-01

    Invasive pulmonary mold disease (IPMD) is often fatal in neutropenic patients. This is because IPMD is difficult to diagnose timely, especially when non-Aspergillus molds are the causative agent, as they are usually not associated with a positive galactomannan assay. In 2013 we showed that exhaled breath analysis might be used to diagnose invasive aspergillosis through profiling of patterns in exhaled volatile organic compounds (VOCs) by electronic nose (eNose) technology. The current study aimed to determine (1) whether molds can be discriminated from other microorganisms (using two mold species: Aspergillus fumigatus and a pathogenic mold not associated with a positive galactomannan assay, i.c. Rhizopus oryzae) and (2) whether both molds can be discriminated from each other. First, we cultured strains of Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, A. fumigatus and R. oryzae in separate airtight bottles. We examined whether an eNose (Cyranose 320) could discriminate the headspaces of bottles with molds from those with bacteria/yeasts. Second, we examined whether an eNose could discriminate A. fumigatus and R. oryzae. Diagnostic algorithms were created using canonical discriminant analysis after principle component analysis. Primary outcome parameter was the validated accuracy. The eNose discriminated A. fumigatus from bacteria/yeasts with a cross-validated accuracy of 92.9% (sensitivity 95.2%, specificity 91.9%). The eNose had an accuracy (validated using split-half analysis) of 100% in discriminating A. fumigatus from R. oryzae. Our study suggests that an eNose can identify and classify molds in vitro. This warrants prospective in vivo studies aimed at detecting and classifying IPMD using exhaled breath. PMID:27447026

  10. LC3-associated phagocytosis: a crucial mechanism for antifungal host defence against Aspergillus fumigatus.

    PubMed

    Sprenkeler, Evelien G G; Gresnigt, Mark S; van de Veerdonk, Frank L

    2016-09-01

    LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway involved in the maturation of single-membrane phagosomes and subsequent killing of ingested pathogens by phagocytes. This pathway is initiated following recognition of pathogens by pattern recognition receptors and leads to the recruitment of LC3 into the phagosomal membrane. This form of phagocytosis is utilized for the antifungal host defence and is required for an efficient fungal killing. Here, we provide an overview of the LAP pathway and review the role of LAP in anti-Aspergillus host defence, as well as mechanisms induced by Aspergillus that modulate LAP to promote its survival in the host. PMID:27185357

  11. Aspergillus fumigatus CY018, an endophytic fungus in Cynodon dactylon as a versatile producer of new and bioactive metabolites.

    PubMed

    Liu, J Y; Song, Y C; Zhang, Z; Wang, L; Guo, Z J; Zou, W X; Tan, R X

    2004-11-01

    Aspergillus fumigatus CY018 was recognized as an endophytic fungus for the first time in the leaf of Cynodon dactylon. By bioassay-guided fractionation, the EtOAc extract of a solid-matrix steady culture of this fungus afforded two new metabolites, named asperfumoid (1) and asperfumin (2), together with six known bioactive compounds including monomethylsulochrin, fumigaclavine C, fumitremorgin C, physcion, helvolic acid and 5alpha,8alpha-epidioxy-ergosta-6,22-diene-3beta-ol as well as other four known compounds ergosta-4,22-diene-3beta-ol, ergosterol, cyclo(Ala-Leu) and cyclo(Ala-Ile). Through detailed spectroscopic analyses including HRESI-MS, homo- and hetero-nuclear correlation NMR experiments (HMQC, COSY, NOESY and HMBC), the structures of asperfumoid and asperfumin were established to be spiro-(3-hydroxyl-2,6-dimethoxyl-2,5-diene-4-cyclohexone-(1,3')-5'-methoxyl-7'-methyl-(1'H, 2'H, 4'H)-quinoline-2',4'-dione) and 5-hydroxyl-2-(6-hydroxyl-2-methoxyl-4-methylbenzoyl)-3,6-dimethoxyl-benzoic methyl ester, respectively. All of the 12 isolates were subjected to in vitro bioactive assays against three human pathogenic fungi Candida albicans, Tricophyton rubrum and Aspergillus niger. As a result, asperfumoid, fumigaclavine C, fumitremorgin C, physcion and helvolic acid were shown to inhibit C. albicans with MICs of 75.0, 31.5, 62.5, 125.0 and 31.5 microg/mL, respectively. PMID:15522437

  12. The absence of VPAC2 leads to aberrant antibody production in Aspergillus fumigatus sensitized and challenged mice

    PubMed Central

    Samarasinghe, Amali E.; Hoselton, Scott A.; Schuh, Jane M.

    2010-01-01

    Vasoactive intestinal peptide (VIP) facilitates a “pro-allergy” phenotype when signaling through its G protein-coupled receptor, VPAC2. We have shown that VPAC2 knock-out (KO) mice developed an allergic phenotype marked by eosinophilia and elevated serum IgE. Therefore, we hypothesized that the humoral response to allergen challenge in these mice was TH2 dominant similar to wild-type (WT) C57BL/6 mice. Antibody responses in WT and KO mice were measured after Aspergillus fumigatus conidia inhalation. In contrast to previous reports, basal levels of serum IgG2a and IgA were significantly higher in naïve VPAC2 KO animals. Antibody availability in the serum as well as the bronchoalveolar lavage fluid after fungal challenge was dominated by the pro-inflammatory isotype IgG2a and the mucosal isotype, IgA. IgA localizing cells dominated in the peribronchovascular areas of allergic KO mice while IgE immune complexes were found in WT allergic lungs. This research shows for the first time that VPAC2 has a significant effect on antibody regulation, in the context of allergy. PMID:20923692

  13. Kexin-Like Endoprotease KexB is Required for N-Glycan Processing, Morphogenesis and Virulence in Aspergillus fumigatus

    PubMed Central

    Wang, Jingyang; Zhou, Hui; Lu, Hua; Du, Ting; Luo, Yuanming; Wilson, Iain B. H.; Jin, Cheng

    2015-01-01

    Kexin-like proteins belong to the subtilisin-like family of the proteinases that cleave secretory proproteins to their active forms. Several fungal kexin-like proteins have been investigated. The mutants lacking of kexin-like protein display strong phenotypes such as cell wall defect, abnormal polarity, and, in case of Candida albicans, diminished virulence. However, only several proteins have been confirmed as the substrates of kexin-like proteases in these fungal species. It still remains unclear how kexin-like proteins contribute to the morphogenesis in these fungal species. In this study, a kexB-null mutant of the human opportunistic fungal pathogen Aspergillus fumigatus was constructed and analyzed. The ΔkexB mutant showed retarded growth, temperature-sensitive cell wall defect, reduced conidia formation, and abnormal polarity. Biochemical analyses revealed that deletion of the kexB gene resulted in impaired N-glycan processing, activation of the MpkA-dependent cell wall integrity signaling pathway, and ER-stress. Results from in vivo assays demonstrated that the mutant exhibited an attenuated virulence in immunecompromised mice. Based on our results, the kexin-like endoprotease KexB was involved in the N-glycan processing, which provides a novel insight to understand how kexin-like protein affects the cell-wall modifying enzymes and therefore morphogenesis in fungi. PMID:25687931

  14. Cloning, overexpression and biocatalytic exploration of a novel Baeyer-Villiger monooxygenase from Aspergillus fumigatus Af293

    PubMed Central

    2013-01-01

    The presence of several putative Baeyer-Villiger Monooxygenases (BVMOs) encoding genes in Aspergillus fumigatus Af293 was demonstrated for the first time. One of the identified BVMO-encoding genes was cloned and successfully overexpressed fused to the cofactor regenerating enzyme phosphite dehydrogenase (PTDH). The enzyme named BVMOAf1 was extensively characterized in terms of its substrate scope and essential kinetic features. It showed high chemo-, regio- and stereoselectivity not only in the oxidation of asymmetric sulfides, (S)-sulfoxides were obtained with 99% ee, but also in the kinetic resolution of bicyclo[3.2.0]hept-2-en-6-one. This kinetic resolution process led to the production of (1S,5R) normal lactone and (1R,5S) abnormal lactone with a regioisomeric ratio of 1:1 and 99% ee each. Besides, different reaction conditions, such as pH, temperature and the presence of organic solvents, have been tested, revealing that BVMOAf1 is a relatively robust biocatalyst. PMID:23767684

  15. Preparative separation and purification of fumigaclavine C from fermented mycelia of Aspergillus fumigatus CY018 by macroporous adsorption resin.

    PubMed

    Yao, Ling-Yun; Zhu, Yi-Xiang; Liu, Chang-Qing; Jiao, Rui-Hua; Lu, Yan-Hua; Tan, Ren-Xiang

    2015-05-01

    In this work, the separation and purification of fumigaclavine C (FC), an ergot alkaloid with strong anti-inflammatory activity from fermented mycelia of Aspergillus fumigatus was systematically evaluated. Among the eight tested resins, the non-polar resin D101 displayed the best adsorption and desorption based on of static adsorption and desorption tests. Adsorption isotherms were constructed on D101 resin and fitted well to the Freundlich model. Dynamic adsorption and desorption tests on a column packed with D101 resin have been investigated for optimization of chromatographic parameters. Under optimized conditions, the contents of FC increased from 7.32% (w/w) in the crude extract to 67.54% in the final product with a recovery yield of 90.35% (w/w) via one run. Furthermore, a lab scale-up separation was carried out, in which the FC content and recovery yield were 65.83% and 90.13%, respectively. These results demonstrated that this adsorption-desorption strategy by using D101 resin was simple and efficient, thus showing potential for large scale purification and preparation of FC in the future. PMID:25817261

  16. Fumigaclavine C from a Marine-Derived Fungus Aspergillus Fumigatus Induces Apoptosis in MCF-7 Breast Cancer Cells

    PubMed Central

    Li, Yong-Xin; Himaya, S.W.A.; Dewapriya, Pradeep; Zhang, Chen; Kim, Se-Kwon

    2013-01-01

    Recently, much attention has been given to discovering natural compounds as potent anti-cancer candidates. In the present study, the anti-cancer effects of fumigaclavine C, isolated from a marine-derived fungus, Aspergillus fumigatus, was evaluated in vitro. In order to investigate the impact of fumigaclavine C on inhibition of proliferation and induction of apoptosis in breast cancer, MCF-7 cells were treated with various concentrations of fumigaclavine C, and fumigaclavine C showed significant cytotoxicity towards MCF-7 cells. Anti-proliferation was analyzed via cell mobility and mitogen-activated protein kinase (MAPK) signaling pathway. In addition, fumigaclavine C showed potent inhibition on the protein and gene level expressions of MMP-2, -9 in MCF-7 cells which were manifested in Western blot and reverse transcription polymerase chain reaction (RT-PCR) results. The apoptosis induction abilities of the fumigaclvine C was studied by analyzing the expression of apoptosis related proteins, cell cycle analysis, DNA fragmentation and molecular docking studies. It was found that fumigaclavine C fragmented the MCF-7 cell DNA and arrested the cell cycle by modulating the apoptotic protein expressions. Moreover, fumigaclavine C significantly down-regulated the NF-kappa-B cell survival pathway. Collectively, data suggest that fumigaclavine C has a potential to be developed as a therapeutic candidate for breast cancer. PMID:24351905

  17. Fumigaclavine C from a marine-derived fungus Aspergillus fumigatus induces apoptosis in MCF-7 breast cancer cells.

    PubMed

    Li, Yong-Xin; Himaya, S W A; Dewapriya, Pradeep; Zhang, Chen; Kim, Se-Kwon

    2013-12-01

    Recently, much attention has been given to discovering natural compounds as potent anti-cancer candidates. In the present study, the anti-cancer effects of fumigaclavine C, isolated from a marine-derived fungus, Aspergillus fumigatus, was evaluated in vitro. In order to investigate the impact of fumigaclavine C on inhibition of proliferation and induction of apoptosis in breast cancer, MCF-7 cells were treated with various concentrations of fumigaclavine C, and fumigaclavine C showed significant cytotoxicity towards MCF-7 cells. Anti-proliferation was analyzed via cell mobility and mitogen-activated protein kinase (MAPK) signaling pathway. In addition, fumigaclavine C showed potent inhibition on the protein and gene level expressions of MMP-2, -9 in MCF-7 cells which were manifested in Western blot and reverse transcription polymerase chain reaction (RT-PCR) results. The apoptosis induction abilities of the fumigaclvine C was studied by analyzing the expression of apoptosis related proteins, cell cycle analysis, DNA fragmentation and molecular docking studies. It was found that fumigaclavine C fragmented the MCF-7 cell DNA and arrested the cell cycle by modulating the apoptotic protein expressions. Moreover, fumigaclavine C significantly down-regulated the NF-kappa-B cell survival pathway. Collectively, data suggest that fumigaclavine C has a potential to be developed as a therapeutic candidate for breast cancer. PMID:24351905

  18. Enhanced production of Fumigaclavine C in liquid culture of Aspergillus fumigatus under a two-stage process.

    PubMed

    Zhu, Yi-Xiang; Yao, Ling-Yun; Jiao, Rui-Hua; Lu, Yan-Hua; Tan, Ren-Xiang

    2014-01-01

    Fumigaclavine C (FC) produced by Aspergillus fumigatus is a conidiation associated ergot alkaloid with strong anti-inflammatory activity. However, its wide application has been severely limited by low FC production from submerged culture. In this work, a novel two-stage culture process by combining shake culture with static culture was proposed to enhance the production of FC. After the process optimization, the FC production reached 62.7 mg/L, which was significantly higher than ever report. For scaling up this new culture process, the gas-liquid interfacial area per unit volume (Agas-liq) was identified as the key factor. The results showed that in a combined stirred-static bioreactor system, a maximum FC production (58.97 mg/L) was obtained at an Agas-liq value of 1.30 cm(2)/mL. These results demonstrated that two-stage culture is an efficient strategy to enhance FC production and the information obtained will be useful to production of this powerful bioactive compound on a large scale. PMID:24291794

  19. Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus.

    PubMed

    Tang, Qing; Tian, Shuguang; Yu, Nong; Zhang, Xi; Jia, Xiaodong; Zhai, Hongyan; Sun, Qun; Han, Li

    2016-04-01

    Aspergillus fumigatusis a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection ofA. fumigatusinfection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection ofA. fumigatus The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatusstrains, including 5 species of theAspergillusgenus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 10(2)copies for the same target. Clinical samples from a total of 69 patients with probable IA (n =14) and possible IA (n= 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection ofA. fumigatusin clinical testing has been developed. PMID:26791368

  20. Biofilm Filtrates of Pseudomonas aeruginosa Strains Isolated from Cystic Fibrosis Patients Inhibit Preformed Aspergillus fumigatus Biofilms via Apoptosis

    PubMed Central

    Shirazi, Fazal; Ferreira, Jose A. G.; Stevens, David A.; Clemons, Karl V.; Kontoyiannis, Dimitrios P.

    2016-01-01

    Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) colonize cystic fibrosis (CF) patient airways. Pa culture filtrates inhibit Af biofilms, and Pa non-CF, mucoid (Muc-CF) and nonmucoid CF (NMuc-CF) isolates form an ascending inhibitory hierarchy. We hypothesized this activity is mediated through apoptosis induction. One Af and three Pa (non-CF, Muc-CF, NMuc-CF) reference isolates were studied. Af biofilm was formed in 96 well plates for 16 h ± Pa biofilm filtrates. After 24 h, apoptosis was characterized by viability dye DiBAc, reactive oxygen species (ROS) generation, mitochondrial membrane depolarization, DNA fragmentation and metacaspase activity. Muc-CF and NMuc-CF filtrates inhibited and damaged Af biofilm (p<0.0001). Intracellular ROS levels were elevated (p<0.001) in NMuc-CF-treated Af biofilms (3.7- fold) compared to treatment with filtrates from Muc-CF- (2.5- fold) or non-CF Pa (1.7- fold). Depolarization of mitochondrial potential was greater upon exposure to NMuc-CF (2.4-fold) compared to Muc-CF (1.8-fold) or non-CF (1.25-fold) (p<0.0001) filtrates. Exposure to filtrates resulted in more DNA fragmentation in Af biofilm, compared to control, mediated by metacaspase activation. In conclusion, filtrates from CF-Pa isolates were more inhibitory against Af biofilms than from non-CF. The apoptotic effect involves mitochondrial membrane damage associated with metacaspase activation. PMID:26930399

  1. Surfactant proteins A and D protect mice against pulmonary hypersensitivity induced by Aspergillus fumigatus antigens and allergens.

    PubMed

    Madan, T; Kishore, U; Singh, M; Strong, P; Clark, H; Hussain, E M; Reid, K B; Sarma, P U

    2001-02-01

    Allergic bronchopulmonary aspergillosis (ABPA) is an allergic disorder caused by an opportunistic fungal pathogen, Aspergillus fumigatus (AFU:). Lung surfactant proteins SP-A and SP-D can interact with the glycosylated antigens and allergens of AFU:, inhibit specific IgE binding to these allergens, and block histamine release from sensitized basophils. We have now examined the therapeutic effect of exogenous administration of human SP-A, SP-D, and a recombinant fragment of SP-D (rSP-D), in a murine model of pulmonary hypersensitivity induced by AFU: antigens and allergens, which resembles human ABPA immunologically. The ABPA mice exhibited high levels of AFU:-specific IgG and IgE, blood eosinophilia, extensive infiltration of lymphocytes and eosinophils in the lung sections, and a Th2 cytokine response. Treatment with SP-A, SP-D, and rSP-D lowered blood eosinophilia, pulmonary infiltration, and specific Ab levels considerably, which persisted up to 4 days in the SP-A-treated ABPA mice, and up to 16 days in the SP-D- or rSP-D-treated ABPA mice. The levels of IL-2, IL-4, and IL-5 were decreased, while the level of IFN-gamma was raised in the splenic supernatants of the treated mice, indicating a marked shift from Th2 to Th1 response. These results clearly implicate pulmonary SP-A and SP-D in the modulation of allergic reactions. PMID:11181646

  2. Clinical and radiological signs of ABPA associated with airways infection with Aspergillus in the absence of specific IgE.

    PubMed

    Sunzini, F; Barbato, C; Canofari, C; Lugari, L; Perricone, R; Bergamini, A

    2016-09-01

    Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity reaction to Aspergillus that mainly affects patients with asthma. For diagnosis, elevated serum IgE level are needed according to Greenberger and Patterson criteria. We report a case of 43 years-old woman who developed ABPA with productive cough, fever and radiological findings of multiple confluent areas of consolidation in both upper lobes. Laboratory tests showed elevated peripheral eosinophil counts (9.3 x 10(3)/ml). In bronchial washing A. galactomannans and A. Fumigatus were isolated, although we found normal levels of serum IgE, and the absence of serum IgG and IgE antibodies to Aspergillus and A. galactomannans. In conclusion, clinical and radiological signs of ABPA can be associated with Aspergillus infection also in the absence of a specific serum antibody reaction. PMID:27608478

  3. Sequential serological responses to Aspergillus fumigatus in patients with cystic fibrosis. Use of antigen 'stretching' to delineate IgG and IgE activity.

    PubMed Central

    Edwards, J H; Alfaham, M; Fifield, R; Philpot, C; Clement, M J; Goodchild, M C

    1990-01-01

    Immunogens from Aspergillus fumigatus were fractionated on the basis of molecular weight. Nine fractions ranging from 900 to 10 kDa were used in ELISA and in a radioallergosorbent test (RAST) with sera from cases of allergic bronchopulmonary aspergillosis (ABPA) and from cystic fibrosis (CF) patients with ABPA or other Aspergillus involvement and compared with control subjects. The profile of IgG reactivity to the nine fractions did not vary substantially for all Aspergillus-involved groups producing peaks at greater than 900 kD and 170 kD whereas the profile for control subjects had a peak at greater than 900 kD only. The IgE profile for CF patients with ABPA did not differ from the profile of the RAST-positive CF patients without ABPA and provided only one peak of activity at 24 kD. Recovery from an episode of ABPA in CF patients was accompanied by a fall in both IgG and IgE antibody levels to all nine fractions, whereas increases in IgG and IgE to all fractions were seen during an episode of ABPA. Although there was an exaggerated IgG increase to antigens in the 43-170 kD range during ABPA, a meaningful increase was also observed to unfractionated A. fumigatus antigen preparations. With IgE in one detailed study the 24-kD fraction provided a better indication of Aspergillus involvement than the unfractionated A. fumigatus antigens. Sequential studies of IgG and IgE levels were not able to predict an episode of ABPA but were useful in conjunction with clinical assessment in following the course of the illness. PMID:2165878

  4. Analysis of the Aspergillus fumigatus Proteome Reveals Metabolic Changes and the Activation of the Pseurotin A Biosynthesis Gene Cluster in Response to Hypoxia

    PubMed Central

    2011-01-01

    The mold Aspergillus fumigatus is the most important airborne fungal pathogen. Adaptation to hypoxia represents an important virulence attribute for A. fumigatus. Therefore, we aimed at obtaining a comprehensive overview about this process on the proteome level. To ensure highly reproducible growth conditions, an oxygen-controlled, glucose-limited chemostat cultivation was established. Two-dimensional gel electrophoresis analysis of mycelial and mitochondrial proteins as well as two-dimensional Blue Native/SDS-gel separation of mitochondrial membrane proteins led to the identification of 117 proteins with an altered abundance under hypoxic in comparison to normoxic conditions. Hypoxia induced an increased activity of glycolysis, the TCA-cycle, respiration, and amino acid metabolism. Consistently, the cellular contents in heme, iron, copper, and zinc increased. Furthermore, hypoxia induced biosynthesis of the secondary metabolite pseurotin A as demonstrated at proteomic, transcriptional, and metabolite levels. The observed and so far not reported stimulation of the biosynthesis of a secondary metabolite by oxygen depletion may also affect the survival of A. fumigatus in hypoxic niches of the human host. Among the proteins so far not implicated in hypoxia adaptation, an NO-detoxifying flavohemoprotein was one of the most highly up-regulated proteins which indicates a link between hypoxia and the generation of nitrosative stress in A. fumigatus. PMID:21388144

  5. Overlapping and Distinct Roles of Aspergillus fumigatus UDP-glucose 4-Epimerases in Galactose Metabolism and the Synthesis of Galactose-containing Cell Wall Polysaccharides*

    PubMed Central

    Lee, Mark J.; Gravelat, Fabrice N.; Cerone, Robert P.; Baptista, Stefanie D.; Campoli, Paolo V.; Choe, Se-In; Kravtsov, Ilia; Vinogradov, Evgeny; Creuzenet, Carole; Liu, Hong; Berghuis, Albert M.; Latgé, Jean-Paul; Filler, Scott G.; Fontaine, Thierry; Sheppard, Donald C.

    2014-01-01

    The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis. PMID:24257745

  6. Primary in vitro culture of porcine tracheal epithelial cells in an air-liquid interface as a model to study airway epithelium and Aspergillus fumigatus interactions.

    PubMed

    Khoufache, Khaled; Cabaret, Odile; Farrugia, Cécile; Rivollet, Danièle; Alliot, Annie; Allaire, Eric; Cordonnier, Catherine; Bretagne, Stéphane; Botterel, Françoise

    2010-12-01

    Since the airway epithelium is the first tissue encountered by airborne fungal spores, specific models are needed to study this interaction. We developed such a model using primary porcine tracheal epithelial cells (PTEC) as a possible alternative to the use of primary human cells. PTEC were obtained from pigs and were cultivated in an air-liquid interface. Fluorescent brightener was employed to quantify the internalization of Aspergillus fumigatus conidia. Potential differences (Vt) and transepithelial resistances (Rt) after challenge with the mycotoxin, verruculogen, were studied. Primers for porcine inflammatory mediator genes IL-8, TNF-alpha, and GM-CSF were designed for a quantitative real-time PCR procedure to study cellular responses to challenges with A. fumigatus conidia. TEM showed the differentiation of ciliated cells and the PTEC ability to internalize conidia. The internalization rate was 21.9 ± 1.4% after 8 h of incubation. Verruculogen (10(-6) M) significantly increased Vt without having an effect on the Rt. Exposure of PTEC to live A. fumigatus conidia for 24 h induced a 10- to 40-fold increase in the mRNA levels of inflammatory mediator genes. PTEC behave similarly to human cells and are therefore a suitable alternative to human cells for studying interaction between airway epithelium and A. fumigatus. PMID:20608777

  7. Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies.

    PubMed Central

    Chow, L P; Liu, S L; Yu, C J; Liao, H K; Tsai, J J; Tang, T K

    2000-01-01

    The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens. On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified. This allergen was also present in A. fumigatus culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A. fumigatus and showed 42-49% identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding sequences were expressed in Escherichia coli as a [His](6)-tagged fusion protein which was purified by Ni(2+)-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein. PMID:10677362

  8. Multiple Brain Abscesses Due to Aspergillus Fumigatus in a Patient With Liver Cirrhosis: A Case Report.

    PubMed

    Tang, Hung-Jen; Liu, Wei-Lun; Chang, Tsung Chain; Li, Ming-Chi; Ko, Wen-Chien; Wu, Chi-Jung; Chuang, Yin-Ching; Lai, Chih-Cheng

    2016-03-01

    Invasive cerebral aspergillosis always developed in immunocompromised host. Early diagnosis may save life in this critical condition; however, it is difficult to reach. Herein, we presented an unusual case of invasive cerebral aspergillosis in a cirrhotic patient. A 47-year-old man presented with progressive deterioration of consciousness for three days. The patient had a history of alcoholic liver cirrhosis, Child-Pugh class C. Magnetic resonance imaging (MRI) of brain showed multi-focal parenchymal lesions, which was consistent with multiple brain abscesses. The diagnosis of invasive cerebral aspergillosis was made by molecular based laboratory methods including Aspergillus galactomannan antigen assay and oligonucleotide array. Despite treatment with the antifungal agent, Amphotericin B, the patient died at the ninth day of hospitalization. Our findings suggest that liver cirrhosis can be one of risk factors of invasive cerebral aspergillosis, and support the diagnosing usefulness of MRI, Aspergillus galactomannan antigen assay, and oligonucleotide array. PMID:26945363

  9. Azole-Resistant Aspergillus fumigatus Isolate with the TR34/L98H Mutation in Both a Fungicide-Sprayed Field and the Lung of a Hematopoietic Stem Cell Transplant Recipient with Invasive Aspergillosis

    PubMed Central

    Rocchi, Steffi; Daguindau, Etienne; Grenouillet, Frédéric; Deconinck, Eric; Bellanger, Anne-Pauline; Garcia-Hermoso, Dea; Bretagne, Stéphane; Reboux, Gabriel

    2014-01-01

    A French farmer developed invasive aspergillosis with azole-resistant Aspergillus fumigatus with the TR34/L98H mutation following a hematopoietic stem cell transplantation. He had worked in fungicide-sprayed fields where a non-genetically related A. fumigatus TR34/L98H isolate was collected. If azole resistance detection increases, voriconazole as first-line therapy might be questioned in agricultural areas. PMID:24554754

  10. Biochemical stability and molecular dynamic characterization of Aspergillus fumigatus cystathionine γ-lyase in response to various reaction effectors.

    PubMed

    El-Sayed, Ashraf S A; Abdel-Azeim, Safwat; Ibrahim, Hend M; Yassin, Marwa A; Abdel-Ghany, Salah E; Esener, Sadik; Ali, Gul Shad

    2015-12-01

    Cystathionine γ-lyase (CGL) is a key enzyme in the methionine-cysteine cycle in all living organisms forming cysteine, α-ketobutyrate and ammonia via homocysteine and cystathionine intermediates. Although, human and plant CGLs have been extensively studied at the molecular and mechanistic levels, there has been little work on the molecular and catalytic properties of fungal CGL. Herein, we studied in detail for the first time the molecular and catalytic stability of Aspergillus fumigatus CGL, since conformational instability, inactivation and structural antigenicity are the main limitations of the PLP-dependent enzymes on various therapeutic uses. We examined these properties in response to buffer compositions, stabilizing and destabilizing agents using Differential Scanning Fluorometery (DSF), steady state and gel-based fluorescence of the intrinsic hydrophobic core, stability of internal aldimine linkage and catalytic properties. The activity of the recombinant A. fumigatus CGL was 13.8U/mg. The melting temperature (Tm) of CGL in potassium phosphate buffer (pH 7.0-8.0) was 73.3°C, with ∼3°C upshifting in MES and sodium phosphate buffers (pH 7.0). The conformational thermal stability was increased in potassium phosphate, sodium phosphate and MES buffers, in contrast to Tris-HCl, HEPES (pH 7.0) and CAPS (pH 9.0-10.0). The thermal stability and activity of CGL was slightly increased in the presence of trehalose and glycerol that might be due to hydration of the enzyme backbone, unlike the denaturing effect of GdmCl and urea. Modification of surface CGL glutamic and aspartic acids had no significant effect on the enzyme conformational and catalytic stability. Molecular modeling and dynamics simulations unveil the high conformational stability of the overall scaffold of CGL with high flexibility at the non-structural regions. CGL structure has eight buried Trp residues, which are reoriented to the enzyme surface and get exposed to the solvent under perturbation

  11. Production and partial characterization of serine and metallo peptidases secreted by Aspergillus fumigatus Fresenius in submerged and solid state fermentation.

    PubMed

    da Silva, Ronivaldo Rodrigues; de Freitas Cabral, Tatiana Pereira; Rodrigues, André; Cabral, Hamilton

    2013-01-01

    Enzyme production varies in different fermentation systems. Enzyme expression in different fermentation systems yields important information for improving our understanding of enzymatic production induction. Comparative studies between solid-state fermentation (SSF) using agro-industrial waste wheat bran and submerged fermentation (SmF) using synthetic media were carried out to determinate the best parameters for peptidase production by the fungus Aspergillus fumigatus Fresen. Variables tested include: the concentration of carbon and protein nitrogen sources, the size of the inoculum, the pH of the media, temperature, and the length of the fermentation process. The best peptidase production during SSF was obtained after 96 hours using wheat bran at 30 °C with an inoculum of 1 × 10(6) spores and yielded 1500 active units (U/mL). The best peptidase production using SmF was obtained after periods of 72 and 96 hours of fermentation in media containing 0.5% and 0.25% of casein, respectively, at a pH of 6.0 and at 30 °C and yielded 40 U/mL. We also found examples of catabolite repression of peptidase production under SmF conditions. Biochemical characterization of the peptidases produced by both fermentative processes showed optimum activity at pH 8.0 and 50 °C, and also showed that their proteolytic activity is modulated by surfactants. The enzymatic inhibition profile using phenylmethylsulfonyl fluoride (PMSF) in SmF and SSF indicated that both fermentative processes produced a serine peptidase. Additionally, the inhibitory effect of the ethylene-diaminetetraacetic acid (EDTA) chelating agent on the peptidase produced by SmF indicated that this fermentative process also produced a metallopeptidase. PMID:24159310

  12. New untargeted metabolic profiling combining mass spectrometry and isotopic labeling: application on Aspergillus fumigatus grown on wheat.

    PubMed

    Cano, Patricia M; Jamin, Emilien L; Tadrist, Souria; Bourdaud'hui, Pascal; Péan, Michel; Debrauwer, Laurent; Oswald, Isabelle P; Delaforge, Marcel; Puel, Olivier

    2013-09-01

    Characterization of fungal secondary metabolomes has become a challenge due to the industrial applications of many of these molecules, and also due to the emergence of fungal threats to public health and natural ecosystems. Given that, the aim of the present study was to develop an untargeted method to analyze fungal secondary metabolomes by combining high-accuracy mass spectrometry and double isotopic labeling of fungal metabolomes. The strain NRRL 35693 of Aspergillus fumigatus , an important fungal pathogen, was grown on three wheat grain substrates: (1) naturally enriched grains (99% (12)C), (2) grains enriched 96.8% with (13)C, (3) grains enriched with 53.4% with (13)C and 96.8% with (15)N. Twenty-one secondary metabolites were unambiguously identified by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) analysis. AntiBase 2012 was used to confirm the identity of these metabolites. Additionally, on the basis of tandem mass spectrometry (MS(n)) experiments, it was possible to identify for the first time the formula and the structure of fumigaclavine D, a new member of the fumigaclavines family. Post biosynthesis degradation of tryptoquivaline F by methanol was also identified during HPLC-HRMS analysis by the detection of a carbon atom of nonfungal origin. The interest of this method lies not only on the unambiguous determination of the exact chemical formulas of fungal secondary metabolites but also on the easy discrimination of nonfungal products. Validation of the method was thus successfully achieved in this study, and it can now be applied to other fungal metabolomes, offering great possibilities for the discovery of new drugs or toxins. PMID:23901908

  13. Metabolites from Aspergillus fumigatus, an endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic activities.

    PubMed

    Li, Xiao-Jun; Zhang, Qiang; Zhang, An-Ling; Gao, Jin-Ming

    2012-04-01

    Thirty-nine fungal metabolites 1-39, including two new alkaloids, 12β-hydroxy-13α-methoxyverruculogen TR-2 (6) and 3-hydroxyfumiquinazoline A (16), were isolated from the fermentation broth of Aspergillus fumigatus LN-4, an endophytic fungus isolated from the stem bark of Melia azedarach. Their structures were elucidated on the basis of detailed spectroscopic analysis (mass spectrometry and one- and two-dimensional NMR experiments) and by comparison of their NMR data with those reported in the literature. These isolated compounds were evaluated for in vitro antifungal activities against some phytopathogenic fungi, toxicity against brine shrimps, and antifeedant activities against armyworm larvae (Mythimna separata Walker). Among them, sixteen compounds showed potent antifungal activities against phytopathogenic fungi (Botrytis cinerea, Alternaria solani, Alternaria alternata, Colletotrichum gloeosporioides, Fusarium solani, Fusarium oxysporum f. sp. niveum, Fusarium oxysporum f. sp. vasinfectum, and Gibberella saubinettii), and four of them, 12β-hydroxy-13α-methoxyverruculogen TR-2 (6), fumitremorgin B (7), verruculogen (8), and helvolic acid (39), exhibited antifungal activities with MIC values of 6.25-50 μg/mL, which were comparable to the two positive controls carbendazim and hymexazol. In addition, of eighteen that exerted moderate lethality toward brine shrimps, compounds 7 and 8 both showed significant toxicities with median lethal concentration (LC(50)) values of 13.6 and 15.8 μg/mL, respectively. Furthermore, among nine metabolites that were found to possess antifeedant activity against armyworm larvae, compounds 7 and 8 gave the best activity with antifeedant indexes (AFI) of 50.0% and 55.0%, respectively. Structure-activity relationships of the metabolites were also discussed. PMID:22409377

  14. Characterization of gprK Encoding a Putative Hybrid G-Protein-Coupled Receptor in Aspergillus fumigatus.

    PubMed

    Jung, Mun-Gu; Kim, Sung Su; Yu, Jae-Hyuk; Shin, Kwang-Soo

    2016-01-01

    The G-protein-coupled receptor (GPCR) family represents the largest and most varied collection of membrane embedded proteins that are sensitized by ligand binding and interact with heterotrimeric G proteins. Despite their presumed critical roles in fungal biology, the functions of the GPCR family members in the opportunistic human pathogen Aspergillus fumigatus are largely unknown, as only two (GprC and GprD) of the 15 predicted GPCRs have been studied. Here, we characterize the gprK gene, which is predicted to encode a hybrid GPCR with both 7-transmembrane and regulator of G-protein signaling (RGS) domains. The deletion of gprK causes severely impaired asexual development coupled with reduced expression of key developmental activators. Moreover, ΔgprK results in hyper-activation of germination even in the absence of carbon source, and elevated expression and activity of the protein kinase A PkaC1. Furthermore, proliferation of the ΔgprK mutant is restricted on the medium when pentose is the sole carbon source, suggesting that GprK may function in external carbon source sensing. Notably, the absence of gprK results in reduced tolerance to oxidative stress and significantly lowered mRNA levels of the stress-response associated genes sakA and atfA. Activities of catalases and SODs are severely decreased in the ΔgprK mutant, indicating that GprK may function in proper activation of general stress response. The ΔgprK mutant is also defective in gliotoxin (GT) production and slightly less virulent toward the greater wax moth, Galleria mellonella. Transcriptomic studies reveal that a majority of transporters are down-regulated by ΔgprK. In summary, GprK is necessary for proper development, GT production, and oxidative stress response, and functions in down-regulating the PKA-germination pathway. PMID:27584150

  15. A potential link between TSLP/TSLPR/STAT5 and TLR2/MyD88/NFκB-p65 in human corneal epithelial cells for Aspergillus fumigatus tolerance.

    PubMed

    Ren, Xiaoxiao; Wang, Leyi; Wu, Xinyi

    2016-03-01

    Our previous studies reported that pattern recognition receptors (PRRs), including the cell surface Toll-like receptors (TLRs) and cytoplasmic NOD-like receptors (NLRs), recognize pathogen-associated molecular patterns (PAMPS) to initiate downstream signal cascades to active immunity responses. Thymic stromal lymphopoietin (TSLP) has recently emerged as a key cytokine in the development of type 2 adaptive immune responses. However, the crosstalk between PRRs and TSLP has not been well elucidated in Aspergillus fumigates keratitis. Our studies demonstrated that HCECs not only respond to TSLP, but also initiate immunological regulation through TSLP/TSLPR/STAT5 signaling pathway. In addition, we revealed that zymosan TLR2 agonist enhanced the expression of TSLP and TSLPR and phosphorylation of STAT5. Furthermore, neutralization of TLR2 with monoclonal Ab prevented the production of TSLP and TSLPR and phosphorylation of STAT5 from increasing which induced by A. fumigatus hyphae. Interestingly, we also found that human recombinant TSLP induced the increase of TLR2 downstream signal molecules, and TSLP knockdown could reduce the increase of TLR2 downstream signaling molecules(MyD88 and NF-κB-p65) induced by A. fumigatus hyphae. These studies indicated that HCECs represent a novel target of TSLP, TSLP/TSLPR/STAT5 signaling plays an important role in response to A. fumigatus infection in HCECs, and TLR2 downstream signaling molecules up regulate TSLP/TSLPR/STAT5 signaling as well as TSLP downstream signaling molecules up regulate TLR2/MyD88/NFκB-p65 signaling in this phenomenon. PMID:26874828

  16. Rapid, high-throughput, multiplex, real-time PCR for identification of mutations in the cyp51A gene of Aspergillus fumigatus that confer resistance to itraconazole.

    PubMed

    Balashov, Sergey V; Gardiner, Rebecca; Park, Steven; Perlin, David S

    2005-01-01

    Aspergillus fumigatus is an important cause of life-threatening invasive fungal disease in patients with compromised immune systems. Resistance to itraconazole in A. fumigatus is closely linked to amino acid substitutions in Cyp51A that replace Gly54. In an effort to develop a new class of molecular diagnostic assay that can rapidly assess drug resistance, a multiplexed assay was established. This assay uses molecular beacons corresponding to the wild-type cyp51A gene and seven mutant alleles encoding either Arg54, Lys54, Val54, Trp54, or Glu54. Molecular beacon structure design and real-time PCR conditions were optimized to increase the assay specificity. The multiplex assay was applied to the analysis of chromosomal DNA samples from a collection of 48 A. fumigatus clinical and laboratory-derived isolates, most with reduced susceptibility to itraconazole. The cyp51A allelic identities for codon 54 were established for all of the strains tested, and mutations altering Gly54 in 23 strains were revealed. These mutations included G(54)W (n = 1), G(54)E (n = 12), G(54)K (n = 3), G(54)R (n = 3), and G(54)V (n = 4). Molecular beacon assay results were confirmed by DNA sequencing. Multiplex real-time PCR with molecular beacons is a powerful technique for allele differentiation and analysis of resistance mutations that is dynamic and suitable for rapid high-throughput assessment of drug resistance. PMID:15634974

  17. Humoral and Cell-mediated Autoimmune Reactions to Human Acidic Ribosomal P2 Protein in Individuals Sensitized to Aspergillus fumigatus P2 Protein

    PubMed Central

    Mayer, Christina; Appenzeller, Ulrich; Seelbach, Heike; Achatz, Gernot; Oberkofler, Hannes; Breitenbach, Michael; Blaser, Kurt; Crameri, Reto

    1999-01-01

    A panel of cDNAs encoding allergenic proteins was isolated from an Aspergillus fumigatus cDNA library displayed on the surface of filamentous phage. Solid phase–immobilized serum immunoglobulin E (IgE) from A. fumigatus–allergic individuals was used to enrich phage displaying IgE-binding molecules. One of the cDNAs encoded a 11.1-kD protein that was identified as acidic ribosomal phosphoprotein type 2 (P2 protein). The allergen, formally termed rAsp f 8, shares >62% sequence identity and >84% sequence homology to corresponding eukaryotic P2 proteins, including human P2 protein. The sequences encoding human and fungal P2 protein were subcloned, expressed in Escherichia coli as His6-tagged fusion proteins, and purified by Ni2+–chelate affinity chromatography. Both recombinant P2 proteins were recognized by IgE antibodies from allergic individuals sensitized to the A. fumigatus P2 protein and elicited strong type 1–specific skin reactions in these individuals. Moreover, human and fungal P2 proteins induced proliferative responses in peripheral blood mononuclear cells of A. fumigatus– allergic subjects sensitized to the fungal P2 protein. These data provide strong evidence for in vitro and in vivo humoral and cell-mediated autoreactivity to human P2 protein in patients suffering from chronic A. fumigatus allergy. PMID:10224291

  18. Trehalose-6-Phosphate Phosphatase is required for cell wall integrity and fungal virulence but not trehalose biosynthesis in the human fungal pathogen Aspergillus fumigatus

    PubMed Central

    Puttikamonkul, Srisombat; Willger, Sven D.; Grahl, Nora; Perfect, John R.; Movahed, Navid; Bothner, Brian; Park, Steven; Paderu, Padmaja; Perlin, David S.; Cramer, Robert A.

    2010-01-01

    Summary The trehalose biosynthesis pathway is critical for virulence in human and plant fungal pathogens. In this study, we tested the hypothesis that trehalose-6-phosphate phosphatase (T6PP) is required for Aspergillus fumigatus virulence. A mutant of the A. fumigatus T6PP, OrlA, displayed severe morphological defects related to asexual reproduction when grown on glucose (1%) minimal media. These defects could be rescued by addition of osmotic stabilizers, reduction in incubation temperature, or increase in glucose levels (>4%). Subsequent examination of the mutant with cell wall perturbing agents revealed a link between cell wall biosynthesis and trehalose-6-phosphate (T6P) levels. As expected, high levels of T6P accumulated in the absence of OrlA resulting in depletion of free inorganic phosphate (Pi) and inhibition of hexokinase activity. Surprisingly, trehalose production persisted in the absence of OrlA. Further analyses revealed that A. fumigatus contains two trehalose phosphorylases that may be responsible for trehalose production in the absence of OrlA. Despite a normal growth rate under in vitro growth conditions, the orlA mutant was virtually avirulent in two distinct murine models of invasive pulmonary aspergillosis. Our results suggest that further study of this pathway will lead to new insights into regulation of fungal cell wall biosynthesis and virulence. PMID:20545865

  19. A novel mycovirus from Aspergillus fumigatus contains four unique dsRNAs as its genome and is infectious as dsRNA

    PubMed Central

    Kanhayuwa, Lakkhana; Kotta-Loizou, Ioly; Özkan, Selin; Gunning, A. Patrick; Coutts, Robert H. A.

    2015-01-01

    We report the discovery and characterization of a double-stranded RNA (dsRNA) mycovirus isolated from the human pathogenic fungus Aspergillus fumigatus, Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1), which reveals several unique features not found previously in positive-strand RNA viruses, including the fact that it represents the first dsRNA (to our knowledge) that is not only infectious as a purified entity but also as a naked dsRNA. The AfuTmV-1 genome consists of four capped dsRNAs, the largest of which encodes an RNA-dependent RNA polymerase (RdRP) containing a unique GDNQ motif normally characteristic of negative-strand RNA viruses. The third largest dsRNA encodes an S-adenosyl methionine–dependent methyltransferase capping enzyme and the smallest dsRNA a P-A-S–rich protein that apparently coats but does not encapsidate the viral genome as visualized by atomic force microscopy. A combination of a capping enzyme with a picorna-like RdRP in the AfuTmV-1 genome is a striking case of chimerism and the first example (to our knowledge) of such a phenomenon. AfuTmV-1 appears to be intermediate between dsRNA and positive-strand ssRNA viruses, as well as between encapsidated and capsidless RNA viruses. PMID:26139522

  20. Loss of CclA, required for histone 3 lysine 4 methylation, decreases growth but increases secondary metabolite production in Aspergillus fumigatus

    PubMed Central

    Lee, Seul; Dagenais, Taylor R.T.; Andes, David R.; Kontoyiannis, Dimitrios P.

    2013-01-01

    Secondary metabolite (SM) production in filamentous fungi is mechanistically associated with chromatin remodeling of specific SM clusters. One locus recently shown to be involved in SM suppression in Aspergillus nidulans was CclA, a member of the histone 3 lysine 4 methylating COMPASS complex. Here we examine loss of CclA and a putative H3K4 demethylase, HdmA, in the human pathogen Aspergillus fumigatus. Although deletion of hdmA showed no phenotype under the conditions tested, the cclA deletant was deficient in tri- and di-methylation of H3K4 and yielded a slowly growing strain that was rich in the production of several SMs, including gliotoxin. Similar to deletion of other chromatin modifying enzymes, ΔcclA was sensitive to 6-azauracil indicating a defect in transcriptional elongation. Despite the poor growth, the ΔcclA mutant had wild-type pathogenicity in a murine model and the Toll-deficient Drosophila model of invasive aspergillosis. These data indicate that tri- and di-methylation of H3K4 is involved in the regulation of several secondary metabolites in A. fumigatus, however does not contribute to pathogenicity under the conditions tested. PMID:23638376

  1. A novel mycovirus from Aspergillus fumigatus contains four unique dsRNAs as its genome and is infectious as dsRNA.

    PubMed

    Kanhayuwa, Lakkhana; Kotta-Loizou, Ioly; Özkan, Selin; Gunning, A Patrick; Coutts, Robert H A

    2015-07-21

    We report the discovery and characterization of a double-stranded RNA (dsRNA) mycovirus isolated from the human pathogenic fungus Aspergillus fumigatus, Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1), which reveals several unique features not found previously in positive-strand RNA viruses, including the fact that it represents the first dsRNA (to our knowledge) that is not only infectious as a purified entity but also as a naked dsRNA. The AfuTmV-1 genome consists of four capped dsRNAs, the largest of which encodes an RNA-dependent RNA polymerase (RdRP) containing a unique GDNQ motif normally characteristic of negative-strand RNA viruses. The third largest dsRNA encodes an S-adenosyl methionine-dependent methyltransferase capping enzyme and the smallest dsRNA a P-A-S-rich protein that apparently coats but does not encapsidate the viral genome as visualized by atomic force microscopy. A combination of a capping enzyme with a picorna-like RdRP in the AfuTmV-1 genome is a striking case of chimerism and the first example (to our knowledge) of such a phenomenon. AfuTmV-1 appears to be intermediate between dsRNA and positive-strand ssRNA viruses, as well as between encapsidated and capsidless RNA viruses. PMID:26139522

  2. An in vitro and in vivo study on the synergistic effect and mechanism of itraconazole or voriconazole alone and in combination with tetrandrine against Aspergillus fumigatus.

    PubMed

    Li, Shui-Xiu; Song, Yan-Jun; Zhang, Ling-Ling; Shi, Jian-Ping; Ma, Zheng-Lai; Guo, Hui; Dong, Hui-Yu; Li, Yi-Ming; Zhang, Hong

    2015-09-01

    In this study, we investigated the in vitro antifungal effects of itraconazole/voriconazole (ITR/VRC) alone and in combination with tetrandrine (TET) against 23 clinical isolates of A. fumigatus using a chequerboard microdilution method. The dynamic antifungal effects of TET with ITR/VRC against A. fumigatus were assessed in vivo using time-kill curves following systemic infection of mice with A. fumigatus. After treatment, efflux pump activity was determined by the efflux of rhodamine 6G (R6G). When ITR was combined with TET, ITR MICs were reduced from 0.125-32 to 0.0625-2 μg ml(-1), and TET MICs were reduced from 256-512 to 8-64 μg ml(-1). When VRC was combined with TET, VRC MICs were reduced from 0.125-2 to 0.03125-0.5 μg ml(-1), and TET MICs were reduced from 256-512 to 8-256 μg ml(-1). Time-kill curves revealed that A. fumigatus viability was reduced after treatment with ITR/VRC combined with TET versus ITR/VRC alone. ITR/VRC combined with TET significantly prolonged mouse survival and reduced kidney and brain tissue burdens versus ITR/VRC alone (P < 0.05). Moreover, TET inhibited R6G efflux of A. fumigatus. Thus, in vitro and in vivo, TET acted synergistically with ITR/VRC against A. fumigatus, and the synergistic mechanism was related to inhibition of the drug efflux pump. PMID:26296880

  3. A guide to the recent literature on aspergillosis as caused by Aspergillus fumigatus, a fungus frequently found in self-heating organic matter.

    PubMed

    Marsh, P B; Millner, P D; Kla, J M

    1979-11-30

    Spores of Aspergillus fumigatus have been found to be abundantly present in the outdoor air at a site where large scale experimental composting of sewage sludge is in progress at Beltsville, Maryland. The health significance of this finding, for that site and for others in the future, is still only incompletely understood. Further studies are in progress to characterize absolute concentrations of the spores of the fungus in air at the site, spore dispersal by air from composting operations, and background environmental spore levels in air. The present paper contains a list of references to papers on health effects of A. fumigatus, many published in the past ten years, along with a review of the same designed to assist the reader in finding information on particular aspects of the subject in the literature. It is intended primarily as an aid to individuals interested in sludge composting and wishing to attain an insight into the A. fumigatus-composting situation, but it may also interest others concerned with other substrates which become moldy at 40--50 C. A. fumigatus has been found in great numbers in naturally and artificially heated environments such as decaying leaves, compost heaps, solar heated sloughs, cooling canals for nuclear power generators, silos, grain storage bins, boiler rooms, detritus around steam turbines and sauna baths. The evident practical merits of sludge composting have been described elsewhere; the information presented here has its main significance in respect to requirements for choice of locations for composting sites and to process and design criteria. PMID:396477

  4. Crude cellulase from oil palm empty fruit bunch by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 for fermentable sugars production.

    PubMed

    Ibrahim, M F; Razak, M N A; Phang, L Y; Hassan, M A; Abd-Aziz, S

    2013-07-01

    Cellulase is an enzyme that converts the polymer structure of polysaccharides into fermentable sugars. The high market demand for this enzyme together with the variety of applications in the industry has brought the research on cellulase into focus. In this study, crude cellulase was produced from oil palm empty fruit bunch (OPEFB) pretreated with 2% NaOH with autoclave, which was composed of 59.7% cellulose, 21.6% hemicellulose, and 12.3% lignin using Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2. Approximately 0.8 U/ml of FPase, 24.7 U/ml of CMCase and 5.0 U/ml of β-glucosidase were produced by T. asperellum UPM1 at a temperature of 35 °C and at an initial pH of 7.0. A 1.7 U/ml of FPase, 24.2 U/ml of CMCase, and 1.1 U/ml of β-glucosidase were produced by A. fumigatus UPM2 at a temperature of 45 °C and at initial pH of 6.0. The crude cellulase was best produced at 1% of substrate concentration for both T. asperellum UPM1 and A. fumigatus UPM2. The hydrolysis percentage of pretreated OPEFB using 5% of crude cellulase concentration from T. asperellum UPM1 and A. fumigatus UPM2 were 3.33% and 19.11%, with the reducing sugars concentration of 1.47 and 8.63 g/l, respectively. PMID:23666614

  5. First detection of Aspergillus fumigatus azole-resistant strain due to Cyp51A TR46/Y121F/T289A in an azole-naive patient in Spain.

    PubMed

    Pelaez, T; Monteiro, M C; Garcia-Rubio, R; Bouza, E; Gomez-Lopez, A; Mellado, E

    2015-07-01

    We report the first isolation of a voriconazole-resistant Aspergillus fumigatus strain harbouring the azole resistance mechanism TR46/Y121F/T289A, recovered from an azole-naive patient in Spain with chronic obstructive pulmonary disease. This new finding in Spain suggests the spread of this resistance mechanism and reinforces the need for antifungal susceptibility surveillance. PMID:26082842

  6. First detection of TR46/Y121F/T289A and TR34/L98H alterations in Aspergillus fumigatus isolates from azole-naive patients in Denmark despite negative findings in the environment.

    PubMed

    Astvad, K M T; Jensen, R H; Hassan, T M; Mathiasen, E G; Thomsen, G M; Pedersen, U G; Christensen, M; Hilberg, O; Arendrup, M C

    2014-09-01

    Azole-resistant Aspergillus fumigatus harboring the TR34/L98H or TR46/Y121F/T289A alterations is increasingly found in Europe and Asia. Here, we present the first clinical cases of TR46/Y121/T289A and three cases of TR34/L98H outside the cystic fibrosis (CF) population in Denmark and the results of environmental surveys. Four patients (2012 to 2014) with 11 A. fumigatus and 4 Rhizomucor pusillus isolates and 239 soil samples (spring 2010 and autumn 2013, respectively) with a total of 113 A. fumigatus isolates were examined. Aspergillus isolates were screened for azole resistance using azole-containing agar. Confirmatory susceptibility testing was done using the EUCAST microbroth dilution EDEF 9.1 reference method. For relevant A. fumigatus isolates, CYP51A sequencing and microsatellite genotyping were performed. Three patients harbored TR34/L98H isolates. Two were azole naive at the time of acquisition and two were coinfected with wild-type A. fumigatus or R. pusillus isolates, complicating and delaying diagnosis. The TR46/Y121F/T289A strain was isolated in 2014 from a lung transplant patient. Genotyping indicated that susceptible and resistant Aspergillus isolates were unrelated and that no transmission between patients occurred. Azole resistance was not detected in any of the 113 soil isolates. TR34/L98H and TR46/Y121F/T289A alterations appear to be emerging in the clinical setting in Denmark and now involve azole-naive patients. Two recent soil-sampling surveys in Denmark were unable to indicate any increased prevalence of azole-resistant A. fumigatus in the environment. These findings further support the demand for real-time susceptibility testing of all clinically relevant isolates and for studies investigating the seasonal variation and ecological niches for azole-resistant environmental A. fumigatus. PMID:24936595

  7. Characterization of homocysteine γ-lyase from submerged and solid cultures of Aspergillus fumigatus ASH (JX006238).

    PubMed

    El-Sayed, Ashraf S; Khalaf, Salwa A; Aziz, Hani A

    2013-04-01

    Among 25 isolates, Aspergillus fumigatus ASH (JX006238) was identified as a potent producer of homocysteine gamma- lyase. The nutritional requirements to maximize the enzyme yield were optimized under submerged (SF) and solid-state fermentation (SSF) conditions, resulting in a 5.2- and 2.3-fold increase, respectively, after the last purification step. The enzyme exhibited a single homogenous band of 50 kDa on SDS-PAGE, along with an optimum pH of 7.8 and pH stability range of 6.5 to 7.8. It also showed a pI of 5.0, as detected by pH precipitation with no glycosyl residues. The highest enzyme activity was obtained at 37-40 degrees C, with a Tm value of 70.1 degrees C. The enzyme showed clear catalytic and thermal stability below 40 degrees C, with T1/2 values of 18.1, 9.9, 5.9, 3.3, and 1.9 h at 30 degrees C, 35 degrees C, 40 degrees C, 50 degrees C, and 60 degrees C, respectively. Additionally, the enzyme Kr values were 0.002, 0.054, 0.097, 0.184, and 0.341 S-1 at 30 degrees C, 35 degrees C, 40 degrees C, 50 degrees C, and 60 degrees C, respectively. The enzyme displayed a strong affinity to homocysteine, followed by methionine and cysteine when compared with non-S amino acids, confirming its potency against homocysteinuriarelated diseases, and as an anti-cardiovascular agent and a specific biosensor for homocysteinuria. The enzyme showed its maximum affinity for homocysteine (Km 2.46 mM, Kcat 1.39 × 10(-3) s(-1)), methionine (Km 4.1 mM, Kcat 0.97 × 10(-3) s(-1)), and cysteine (Km 4.9 m M, Kcat 0.77 × 10(-3) s(-1)). The enzyme was also strongly inhibited by hydroxylamine and DDT, confirming its pyridoxal 5'-phosphate (PLP) identity, yet not inhibited by EDTA. In vivo, using Swiss Albino mice, the enzyme showed no detectable negative effects on platelet aggregation, the RBC number, aspartate aminotransferase, alanine aminotransferase, or creatinine titer when compared with negative controls. PMID:23568204

  8. Role of Aspergillus fumigatus in Triggering Protease-Activated Receptor-2 in Airway Epithelial Cells and Skewing the Cells toward a T-helper 2 Bias.

    PubMed

    Homma, Tetsuya; Kato, Atsushi; Bhushan, Bharat; Norton, James E; Suh, Lydia A; Carter, Roderick G; Gupta, Dave S; Schleimer, Robert P

    2016-01-01

    Aspergillus fumigatus (AF) infection and sensitization are common and promote Th2 disease in individuals with asthma. Innate immune responses of bronchial epithelial cells are now known to play a key role in determination of T cell responses upon encounter with inhaled pathogens. We have recently shown that extracts of AF suppress JAK-STAT signaling in epithelial cells and thus may promote Th2 bias. To elucidate the impact of AF on human bronchial epithelial cells, we tested the hypothesis that AF can modulate the response of airway epithelial cells to favor a Th2 response and explored the molecular mechanism of the effect. Primary normal human bronchial epithelial (NHBE) cells were treated with AF extract or fractionated AF extract before stimulation with poly I:C or infection with human rhinovirus serotype 16 (HRV16). Expression of CXCL10 mRNA (real-time RT-PCR) and protein (ELISA) were measured as markers of IFN-mediated epithelial Th1-biased responses. Western blot was performed to evaluate expression of IFN regulatory factor-3 (IRF-3), NF-κB, and tyrosine-protein phosphatase nonreceptor type 11 (PTPN11), which are other markers of Th1 skewing. Knockdown experiments for protease-activated receptor-2 (PAR-2) and PTPN11 were performed to analyze the role of PAR-2 in the mechanism of suppression by AF. AF and a high-molecular-weight fraction of AF extract (HMW-AF; > 50 kD) profoundly suppressed poly I:C- and HRV16-induced expression of both CXCL10 mRNA and protein from NHBE cells via a mechanism that relied upon PAR-2 activation. Both AF extract and a specific PAR-2 activator (AC-55541) suppressed the poly I:C activation of phospho-IRF-3 without affecting activation of NF-κB. Furthermore, HMW-AF extract enhanced the expression of PTPN11, a phosphatase known to inhibit IFN signaling, and concurrently suppressed poly I:C-induced expression of both CXCL10 mRNA and protein from NHBE cells. These results show that exposure of bronchial epithelial cells to AF extract

  9. Diffusible component from the spore surface of the fungus Aspergillus fumigatus which inhibits the macrophage oxidative burst is distinct from gliotoxin and other hyphal toxins

    PubMed Central

    Mitchell, C. G.; Slight, J.; Donaldson, K.

    1997-01-01

    BACKGROUND: The fungus Aspergillus fumigatus, whose spores are present ubiquitously in the air, causes a range of diseases in the human lung. A small molecular weight (< 10 kD) heat stable toxin released from the spores of clinical and environmental isolates of A fumigatus within minutes of deposition in aqueous solution has previously been described. A key effect of the toxin was to inhibit the oxidative burst of macrophages as measured by superoxide anion release. It was hypothesised that the toxin was one of the commonly found A fumigatus hyphal toxins such as gliotoxin. This inhibitor may be an important factor which allows the fungus to colonise the lung. METHODS: The spore derived inhibitor was shown to inhibit the respiratory burst of rat alveolar macrophages, as measured by the generation of superoxide anion. Samples of the spore diffusate were subject to reversed phase high performance liquid chromatography (HPLC), thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), or organic extraction followed by TLC or HPLC to identify the presence of gliotoxin, fumagillin, helvolic acid, fumigaclavine-C, and aurasperone-C. Commercially obtained preparations of the toxins gliotoxin, fumagillin and helvolic acid and extracts enriched for fumigaclavine-C and aurasperone-C were used as internal and external standards and in the respiratory burst measurements. RESULTS: Gliotoxin, fumagillin, helvolic acid, fumigaclavine-C, and aurasperone- C were not detected in spore derived diffusate using PHLC or TLC. Using extraction procedures with solvents known to extract gliotoxin from A fumigatus culture supernatants, no gliotoxin was detected in the spore derived diffusate. Commercial gliotoxin, fumagillin, and helvolic acid or extracts enriched for fumigaclavine-C and aurasperone-C did not inhibit the oxidative burst of macrophages. CONCLUSIONS: The hypothesis that the spore derived toxin is one of the toxins derived from hyphae such as gliotoxin

  10. gfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and A. fumigatus

    PubMed Central

    Futagami, Taiki; Kizjakina, Karina; Sobrado, Pablo; Ekino, Keisuke; Takegawa, Kaoru; Goto, Masatoshi; Nomura, Yoshiyuki; Oka, Takuji

    2013-01-01

    The cell walls of filamentous fungi in the genus Aspergillus have galactofuranose-containing polysaccharides and glycoconjugates, including O-glycans, N-glycans, fungal-type galactomannan, and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple galactofuranose monomers onto other wall components in Aspergillus nidulans. Using reverse-genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in galactofuranose (Galf) antigen biosynthesis. Disruption of gfsA reduced binding of β-Galf-specific antibody EB-A2 to O-glycosylated WscA protein and galactomannoproteins. The results of an in-vitro galactofuranose antigen synthase assay revealed that GfsA has β1,5- or β1,6- galactofuranosyltransferase activity for O-glycans in glycoproteins, uses UDP-D-galactofuranose as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. ΔgfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature, and limited formation of conidia. Several gfsA orthologs were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal β-galactofuranosyltransferase, which was shown to be involved in galactofuranose antigen biosynthesis of O-glycans in the Golgi. PMID:24118544

  11. In vitro antifungal, anti-elastase and anti-keratinase activity of essential oils of Cinnamomum-, Syzygium- and Cymbopogon-species against Aspergillus fumigatus and Trichophyton rubrum.

    PubMed

    Khan, Mohd Sajjad Ahmad; Ahmad, Iqbal

    2011-12-15

    This study was aimed to evaluate effects of certain essential oils namely Cinnamomum verum, Syzygium aromaticum, Cymbopogon citratus, Cymbopogon martini and their major components cinnamaldehyde, eugenol, citral and geraniol respectively, on growth, hyphal ultrastructure and virulence factors of Aspergillus fumigatus and Trichophyton rubrum. The antifungal activity of essential oils and their major constituents was in the order of cinnamaldehyde>eugenol>geraniol=C. verum>citral>S. aromaticum>C. citratus>C. martini, both in liquid and solid media against T. rubrum and A. fumigatus. Based on promising antifungal activity of eugenol and cinnamaldehyde, these oils were further tested for their inhibitory activity against ungerminated and germinated conidia in test fungi. Cinnamaldehyde was found to be more active than eugenol. To assess the possible mode of action of cinnamaldehyde, electron microscopic studies were conducted. The observations revealed multiple sites of action of cinnamaldehyde mainly on cell membranes and endomembranous structures of the fungal cell. Further, test oils were also tested for their anti-virulence activity. More than 70% reduction in elastase activity was recorded in A. fumigatus by the oils of C. verum, C. martini, eugenol, cinnamaldehyde and geraniol. Similar reduction in keratinase activity in A. niger was recorded for the oils of C. martini and geraniol. Maximum reduction (96.56%) in elastase activity was produced by cinnamaldehyde whereas; geraniol caused maximum inhibition (97.31%) of keratinase activity. Our findings highlight anti-elastase and anti-keratinase activity of above mentioned essential oils as a novel property to be exploited in controlling invasive and superficial mycoses. PMID:21893402

  12. The virulence of the opportunistic fungal pathogen Aspergillus fumigatus requires cooperation between the endoplasmic reticulum-associated degradation pathway (ERAD) and the unfolded protein response (UPR)

    PubMed Central

    Richie, Daryl L; Feng, Xizhi; Hartl, Lukas; Aimanianda, Vishukumar; Krishnan, Karthik; Powers-Fletcher, Margaret V; Watson, Douglas S; Galande, Amit K; White, Stephanie M; Willett, Taryn; Latgé, Jean-Paul; Rhodes, Judith C

    2011-01-01

    The filamentous fungal pathogen Aspergillus fumigatus secretes hydrolytic enzymes to acquire nutrients from host tissues. The production of these enzymes exerts stress on the endoplasmic reticulum (ER), which is alleviated by two stress responses: the unfolded protein response (UPR), which adjusts the protein folding capacity of the ER, and ER-associated degradation (ERAD), which disposes of proteins that fail to fold correctly. In this study, we examined the contribution of these integrated pathways to the growth and virulence of A. fumigatus, focusing on the ERAD protein DerA and the master regulator of the UPR, HAcA. A ΔderA mutant grew normally and showed no increase in sensitivity to ER stress. However, expression of the UPR target gene bipA was constitutively elevated in this strain, suggesting that the UPR was compensating for the absence of DerA function. To test this, the UPR was disrupted by deleting the hacA gene. The combined loss of derA and hacA caused a more severe reduction in hyphal growth, antifungal drug resistance and protease secretion than the loss of either gene alone, suggesting that DerA and HacA cooperate to support these functions. Moreover, the ΔderA/ΔhacA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasted the wild-type virulence of ΔderA and the reduced virulence of the ΔhacA mutant. Taken together, these data demonstrate that DerA cooperates with the UPR to support the expression of virulence-related attributes of A. fumigatus. PMID:21217201

  13. Filamentous fungal-specific septin AspE is phosphorylated in vivo and interacts with actin, tubulin and other septins in the human pathogen Aspergillus fumigatus

    SciTech Connect

    Juvvadi, Praveen Rao; Belina, Detti; Soderblom, Erik J.; Moseley, M. Arthur; Steinbach, William J.

    2013-02-15

    Highlights: ► In vivo interactions of the novel septin AspE were identified by GFP-Trap® affinity purification. ► Septins AspA, AspB, AspC and AspD interacted with AspE in vivo. ► Actin and tubulin interacted with AspE in vivo. ► AspE is phosphorylated at six serine residues in vivo. -- Abstract: We previously analyzed the differential localization patterns of five septins (AspA–E), including a filamentous fungal-specific septin, AspE, in the human pathogen Aspergillus fumigatus. Here we utilized the A. fumigatus strain expressing an AspE–EGFP fusion protein and show that this novel septin with a tubular localization pattern in hyphae is phosphorylated in vivo and interacts with the other septins, AspA, AspB, AspC and AspD. The other major proteins interacting with AspE included the cytoskeletal proteins, actin and tubulin, which may be involved in the organization and transport of the septins. This is the first report analyzing the phosphorylation of AspE and localizing the sites of phosphorylation, and opens opportunities for further analysis on the role of post-translational modifications in the assembly and organization of A. fumigatus septins. This study also describes the previously unknown interaction of AspE with the actin-microtubule network. Furthermore, the novel GFP-Trap® affinity purification method used here complements widely-used GFP localization studies in fungal systems.

  14. Application of ZnO Nanoparticles for Improving the Thermal and pH Stability of Crude Cellulase Obtained from Aspergillus fumigatus AA001

    PubMed Central

    Srivastava, Neha; Srivastava, Manish; Mishra, P. K.; Ramteke, Pramod W.

    2016-01-01

    Cellulases are the enzymes which are responsible for the hydrolysis of cellulosic biomass. In this study thermal and pH stability of crude cellulase has been investigated in the presence of zinc oxide (ZnO) nanoparticles. We synthesized ZnO nanoparticle by sol-gel method and characterized through various techniques including, X-ray Diffraction, ultraviolet-visible spectroscope, field emission scanning electron microscope and high resolution scanning electron microscope. The crude thermostable cellulase has been obtained from the Aspergillus fumigatus AA001 and treated with ZnO nanoparticle which shows thermal stability at 65°C up to 10 h whereas it showed pH stability in the alkaline pH range and retained its 53% of relative activity at pH 10.5. These findings may be promising in the area of biofuels production. PMID:27148203

  15. The three-dimensional structure of the cellobiohydrolase Cel7A from Aspergillus fumigatus at 1.5 Å resolution.

    PubMed

    Moroz, Olga V; Maranta, Michelle; Shaghasi, Tarana; Harris, Paul V; Wilson, Keith S; Davies, Gideon J

    2015-01-01

    The enzymatic degradation of plant cell-wall cellulose is central to many industrial processes, including second-generation biofuel production. Key players in this deconstruction are the fungal cellobiohydrolases (CBHs), notably those from family GH7 of the carbohydrate-active enzymes (CAZY) database, which are generally known as CBHI enzymes. Here, three-dimensional structures are reported of the Aspergillus fumigatus CBHI Cel7A solved in uncomplexed and disaccharide-bound forms at resolutions of 1.8 and 1.5 Å, respectively. The product complex with a disaccharide in the +1 and +2 subsites adds to the growing three-dimensional insight into this family of industrially relevant biocatalysts. PMID:25615982

  16. FtmOx1, a non-heme Fe(II) and alpha-ketoglutarate-dependent dioxygenase, catalyses the endoperoxide formation of verruculogen in Aspergillus fumigatus.

    PubMed

    Steffan, Nicola; Grundmann, Alexander; Afiyatullov, Shamil; Ruan, Hanli; Li, Shu-Ming

    2009-10-01

    Verruculogen is a tremorgenic mycotoxin and contains an endoperoxide bond. In this study, we describe the cloning, overexpression and purification of a non-heme Fe(ii) and alpha-ketoglutarate-dependent dioxygenase FtmOx1 from Aspergillus fumigatus, which catalyses the conversion of fumitremorgin B to verruculogen by inserting an endoperoxide bond between two prenyl moieties. Incubation with (18)O(2)-enriched atmosphere demonstrated that both oxygen atoms of the endoperoxide bond are derived from one molecule of O(2). FtmOx1 is the first endoperoxide-forming non-heme Fe(ii) and alpha-ketoglutarate-dependent dioxygenase reported so far. A mechanism of FtmOx1-catalysed verruculogen formation is postulated and discussed. PMID:19763315

  17. Novel mammalian cell cycle inhibitors, tryprostatins A, B and other diketopiperazines produced by Aspergillus fumigatus. I. Taxonomy, fermentation, isolation and biological properties.

    PubMed

    Cui, C B; Kakeya, H; Okada, G; Onose, R; Osada, H

    1996-06-01

    Two novel diketopiperazines named tryprostatins A (1) and B (2) and a new natural product belonging to the diketopiperazine series, designated as demethoxyfumitremorgin C (3), together with four known diketopiperazines, fumitremorgin C (4), 12,13-dihydroxyfumitremorgin C (5), fumitremorgin B (6) and verruculogen (7), were isolated from the fermentation broth of Aspergillus fumigatus BM939 by the combined use of solvent extraction, silica gel column chromatography, preparative TLC and repeated-preparative HPLC. The diketopiperazines showed an inhibitory activity on the cell cycle progression of mouse tsFT210 cells in the M phase with the MIC values of 16.4 microM (1), 4.4 microM (2), 0.45 microM (3), 4.1 microM (4), 60.8 microM (5), 26.1 microM (6) and 12.2 microM (7), respectively. PMID:8698634

  18. Novel mammalian cell cycle inhibitors, tryprostatins A, B and other diketopiperazines produced by Aspergillus fumigatus. II. Physico-chemical properties and structures.

    PubMed

    Cui, C B; Kakeya, H; Osada, H

    1996-06-01

    Two novel diketopiperazines named tryprostatins A and B and a new natural product belonging to the diketopiperazine series, designated as demethoxyfumitremorgin C, together with four known diketopiperazines, fumitremorgin C, 12,13-dihydroxyfumitremorgin C, fumitremorgin B and verruculogen, are new M phase inhibitors of the mammalian cell cycle, which were isolated from the secondary metabolites of Aspergillus fumigatus. The structures of tryprostatins A, B and demethoxyfumitremorgin C were determined mainly by the use of spectroscopic methods especially by detailed analyses of their 1H and 13C NMR spectra with the aid of 2D NMR techniques including pulse field gradient heteronuclear multiple-bond correlation (PFG-HMBC) spectroscopy. Their absolute configurations were determined on the basis of the optical rotational values and CD spectra. PMID:8698635

  19. Deletion of the α-(1,3)-Glucan Synthase Genes Induces a Restructuring of the Conidial Cell Wall Responsible for the Avirulence of Aspergillus fumigatus

    PubMed Central

    Beauvais, Anne; Bozza, Silvia; Kniemeyer, Olaf; Formosa, Céline; Balloy, Viviane; Henry, Christine; Roberson, Robert W.; Dague, Etienne; Chignard, Michel; Brakhage, Axel A.; Romani, Luigina; Latgé, Jean-Paul

    2013-01-01

    α-(1,3)-Glucan is a major component of the cell wall of Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes (AGS1, AGS2 and AGS3) controlling the biosynthesis of α-(1,3)-glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α-(1,3)-glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant. PMID:24244155

  20. A comparison between intratracheal and inhalation delivery of Aspergillus fumigatus conidia in the development of fungal allergic asthma in C57BL/6 mice

    PubMed Central

    Samarasinghe, Amali E.; Hoselton, Scott A.; Schuh, Jane M.

    2010-01-01

    Summary Allergic asthma is a debilitating disease of the airways characterized by airway hyperresponsiveness, eosinophilic inflammation, goblet cell metaplasia with associated mucus hypersecretion, and airway wall remodelling events, particularly subepithelial fibrosis and smooth muscle cell hyperplasia. Animal models that accurately mimic these hallmarks of allergic airways disease are critical for studying mechanisms associated with the cellular and structural changes that lead to disease pathogenesis. Aspergillus fumigatus, is a common aeroallergen of human asthmatics. The intratracheal (IT) delivery of A. fumigatus conidia into the airways of sensitized mice has been described as a model of allergic disease. Here, we compared the IT model with a newly developed inhalation (IH) challenge model. The IH model allowed multiple fungal exposures, which resulted in an exacerbation to the allergic asthma phenotype. Increased recruitment of eosinophils and lymphocytes, the hallmark leukocytes of asthma, were noted with the IH model as compared to the IT model in which macrophages and neutrophils were more prominent. Immunoglobulin E (IgE) production was significantly greater after IH challenge, while that of IgG2a was higher after IT challenge. Airway wall remodelling was pronounced in IH-treated mice, particularly after multiple allergen challenges. Although the IT model may be appropriate for the examination of the played by innate cells in the acute response to fungus, it fails to consistently reproduce the chronic remodelling hallmarks of allergic asthma. The ability of the IH challenge to mimic these characteristics recommends it as a model suited to study these important events. PMID:21215951

  1. Modelling the Survival of Escherichia coli O157:H7 on Raw Portioned Tomatoes, Inoculated with Aspergillus fumigatus and Emericella nidulans

    PubMed Central

    Cardillo, Daniela; Bevilacqua, Antonio; Cibelli, Francesca; Altieri, Clelia; Sinigaglia, Milena

    2009-01-01

    The metabiotic interactions occurring among two fungi (Aspergillus fumigatus and Emericella nidulans) and Escherichia coli O157:H7 on raw portioned tomatoes were studied. Tomatoes, preinoculated with the moulds and inoculated with the pathogen, were packaged in air and stored at 4, 8 and 12∘C for 9 days; pathogen cell number and pH were monitored throughout the storage and the data were modeled using three different equations (Geeraerd, Weibull, and modified Weibull), to assess the shoulder length, the 1-log reduction time, and the death time. Both A. fumigatus and E. nidulans increased the survival of E. coli O157:H7 through the prolongation of the shoulder length; in contrast, the death time was significantly increased. The results of this paper suggested that the metabiotic interactions aspergilli/E. coli O 157:H7 could be of public concern, as the consumption of tomatoes (or other fruits and vegetables) contaminated both by the moulds and the pathogen is a possible scenario. PMID:20037729

  2. Modelling the survival of Escherichia coli O157:H7 on raw portioned tomatoes, inoculated with Aspergillus fumigatus and Emericella nidulans.

    PubMed

    Cardillo, Daniela; Bevilacqua, Antonio; Cibelli, Francesca; Altieri, Clelia; Sinigaglia, Milena

    2009-01-01

    The metabiotic interactions occurring among two fungi (Aspergillus fumigatus and Emericella nidulans) and Escherichia coli O157:H7 on raw portioned tomatoes were studied. Tomatoes, preinoculated with the moulds and inoculated with the pathogen, were packaged in air and stored at 4, 8 and 12( composite function)C for 9 days; pathogen cell number and pH were monitored throughout the storage and the data were modeled using three different equations (Geeraerd, Weibull, and modified Weibull), to assess the shoulder length, the 1-log reduction time, and the death time. Both A. fumigatus and E. nidulans increased the survival of E. coli O157:H7 through the prolongation of the shoulder length; in contrast, the death time was significantly increased. The results of this paper suggested that the metabiotic interactions aspergilli/E. coli O 157:H7 could be of public concern, as the consumption of tomatoes (or other fruits and vegetables) contaminated both by the moulds and the pathogen is a possible scenario. PMID:20037729

  3. Aspergillus fumigatus MADS-Box Transcription Factor rlmA Is Required for Regulation of the Cell Wall Integrity and Virulence

    PubMed Central

    Rocha, Marina Campos; Fabri, João Henrique Tadini Marilhano; Franco de Godoy, Krissia; Alves de Castro, Patrícia; Hori, Juliana Issa; Ferreira da Cunha, Anderson; Arentshorst, Mark; Ram, Arthur F. J.; van den Hondel, Cees A. M. J. J.; Goldman, Gustavo Henrique; Malavazi, Iran

    2016-01-01

    The Cell Wall Integrity (CWI) pathway is the primary signaling cascade that controls the de novo synthesis of the fungal cell wall, and in Saccharomyces cerevisiae this event is highly dependent on the RLM1 transcription factor. Here, we investigated the function of RlmA in the fungal pathogen Aspergillus fumigatus. We show that the ΔrlmA strain exhibits an altered cell wall organization in addition to defects related to vegetative growth and tolerance to cell wall-perturbing agents. A genetic analysis indicated that rlmA is positioned downstream of the pkcA and mpkA genes in the CWI pathway. As a consequence, rlmA loss-of-function leads to the altered expression of genes encoding cell wall-related proteins. RlmA positively regulates the phosphorylation of MpkA and is induced at both protein and transcriptional levels during cell wall stress. The rlmA was also involved in tolerance to oxidative damage and transcriptional regulation of genes related to oxidative stress adaptation. Moreover, the ΔrlmA strain had attenuated virulence in a neutropenic murine model of invasive pulmonary aspergillosis. Our results suggest that RlmA functions as a transcription factor in the A. fumigatus CWI pathway, acting downstream of PkcA-MpkA signaling and contributing to the virulence of this fungus. PMID:27473315

  4. GH16 and GH81 family β-(1,3)-glucanases in Aspergillus fumigatus are essential for conidial cell wall morphogenesis.

    PubMed

    Mouyna, Isabelle; Aimanianda, Vishukumar; Hartl, Lukas; Prevost, Marie-Christine; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Legendre, Rachel; Coppee, Jean-Yves; Latgé, Jean-Paul

    2016-09-01

    The fungal cell wall is a rigid structure because of fibrillar and branched β-(1,3)-glucan linked to chitin. Softening of the cell wall is an essential phenomenon during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosylhydrolases. During the search for glycosylhydrolases acting on β-(1,3)-glucan, we identified seven genes in the Aspergillus fumigatus genome coding for potential endo-β-(1,3)-glucanase. ENG1 (previously characterized and named ENGL1, Mouyna et al., ), belongs to the Glycoside-Hydrolase 81 (GH81) family, while ENG2 to ENG7, to GH16 family. ENG1 and four GH16 genes (ENG2-5) were expressed in the resting conidia as well as during germination, suggesting an essential role during A. fumigatus morphogenesis. Here, we report the effect of sequential deletion of AfENG2-5 (GH16) followed by AfENG1 (GH81) deletion in the Δeng2,3,4,5 mutant. The Δeng1,2,3,4,5 mutant showed conidial defects, with linear chains of conidia unable to separate while the germination rate was not affected. These results show, for the first time in a filamentous fungus, that endo β-(1,3)-glucanases are essential for proper conidial cell wall assembly and thus segregation of conidia during conidiation. PMID:27306610

  5. The Aspergillus fumigatus StuA Protein Governs the Up-Regulation of a Discrete Transcriptional Program during the Acquisition of Developmental CompetenceD⃞

    PubMed Central

    Sheppard, Donald C.; Doedt, Thomas; Chiang, Lisa Y.; Kim, H. Stanley; Chen, Dan; Nierman, William C.; Filler, Scott G.

    2005-01-01

    Members of the Asm1p, Phd1p, Sok2p, Efg1p, and StuAp (APSES) family of fungal proteins regulate morphogenesis and virulence in ascomycetes. We cloned the Aspergillus fumigatus APSES gene encoding StuAp and demonstrated that stuA transcription is markedly up-regulated after the acquisition of developmental competence. A. fumigatus ΔstuA mutants were impaired in their ability to undergo asexual reproduction. Conidiophore morphology was markedly abnormal, and only small numbers of dysmorphic conidia were produced, which exhibited precocious germination. Whole genome transcriptional analysis during the onset of developmental competence was performed and identified a subset of developmentally regulated genes that were stuA dependent, including a cluster of putative secondary metabolite biosynthesis genes, genes encoding proteins implicated in the regulation of morphogenesis, and genes encoding allergens and other antigenic proteins. Additionally, hyphae of the ΔstuA mutant displayed reduced expression of the catalase gene CAT1 and were hypersusceptible to hydrogen peroxide. PMID:16207816

  6. Coordination between BrlA regulation and secretion of the oxidoreductase FmqD directs selective accumulation of fumiquinazoline C to conidial tissues in Aspergillus fumigatus

    PubMed Central

    Lim, Fang Yun; Ames, Brian; Walsh, Christopher; Keller, Nancy

    2014-01-01

    SUMMARY Aerial spores, crucial for propagation and dispersal of the Kingdom Fungi, are commonly the initial inoculum of pathogenic fungi. Natural products (secondary metabolites) have been correlated with fungal spore development and enhanced virulence in the human pathogen Aspergillus fumigatus but mechanisms for metabolite deposition in the spore are unknown. Metabolomic profiling of A. fumigatus deletion mutants of fumiquinazoline (Fq) cluster genes reveal that the first two products of the Fq cluster, FqF and FqA, are produced to comparable levels in all fungal tissues but the final enzymatically-derived product, FqC, predominantly accumulates in the fungal spore. Loss of the sporulation-specific transcription factor, BrlA, yields a strain unable to produce FqA or FqC. Fluorescence microscopy showed FmqD, the oxidoreductase required to generate FqC, was secreted via the Golgi apparatus to the cell wall in an actin-dependent manner. In contrast, all other members of the Fq pathway including the putative transporter, FmqE – which had no effect on Fq biosynthesis – were internal to the hyphae. The coordination of BrlA-mediated tissue specificity with FmqD secretion to the cell wall presents a previously undescribed mechanism to direct localization of specific secondary metabolites to spores of the differentiating fungus. PMID:24612080

  7. A comparison between intratracheal and inhalation delivery of Aspergillus fumigatus conidia in the development of fungal allergic asthma in C57BL/6 mice.

    PubMed

    Samarasinghe, Amali E; Hoselton, Scott A; Schuh, Jane M

    2011-01-01

    Allergic asthma is a debilitating disease of the airways characterized by airway hyperresponsiveness, eosinophilic inflammation, goblet cell metaplasia with associated mucus hypersecretion, and airway wall remodelling events, particularly subepithelial fibrosis and smooth muscle cell hyperplasia. Animal models that accurately mimic these hallmarks of allergic airways disease are critical for studying mechanisms associated with the cellular and structural changes that lead to disease pathogenesis. Aspergillus fumigatus, is a common aeroallergen of human asthmatics. The intratracheal (IT) delivery of A. fumigatus conidia into the airways of sensitized mice has been described as a model of allergic disease. Here, we compared the IT model with a newly developed inhalation (IH) challenge model. The IH model allowed multiple fungal exposures, which resulted in an exacerbation to the allergic asthma phenotype. Increased recruitment of eosinophils and lymphocytes, the hallmark leukocytes of asthma, was noted with the IH model as compared to the IT model in which macrophages and neutrophils were more prominent. Immunoglobulin E (IgE) production was significantly greater after IH challenge, while that of IgG(2a) was higher after IT challenge. Airway wall remodelling was pronounced in IH-treated mice, particularly after multiple allergen challenges. Although the IT model may be appropriate for the examination of the played by innate cells in the acute response to fungus, it fails to consistently reproduce the chronic remodelling hallmarks of allergic asthma. The ability of the IH challenge to mimic these characteristics recommends it as a model suited to study these important events. PMID:21215951

  8. Synthesis of a pentasaccharide and neoglycoconjugates related to fungal α-(1→3)-glucan and their use in the generation of antibodies to trace Aspergillus fumigatus cell wall.

    PubMed

    Komarova, Bozhena S; Orekhova, Maria V; Tsvetkov, Yury E; Beau, Remi; Aimanianda, Vishukumar; Latgé, Jean-Paul; Nifantiev, Nikolay E

    2015-01-12

    3-Aminopropyl α-(1→3)-pentaglucoside, a fragment of α-(1→3)-glucan of the cell wall of Aspergillus fumigatus, has been synthesized in a blockwise approach. The application of mono- and disaccharide N-phenyltrifluoroacetimidates bearing a stereodirecting 6-O-benzoyl group was essential for stereoselective α-glucosylations. In the products, p-methoxyphenyl and levulinoyl groups served as orthogonal protecting groups for the anomeric position and 3-OH group, respectively. Their removal from shared blocks led to donors and acceptors that were used for the synthesis of pentasaccharides. Coupling of free α-(1→3)-pentaglucoside with biotin and bovine serum albumin (BSA) gave glycoconjugate tools for mycological studies. Immunization of mice with the BSA conjugate induced the generation of antibodies that recognize α-(1→3)-glucan on A. fumigatus cell wall and distinguish its morphotypes. This discovery represents a first step to the development of a diagnostic test system and a vaccine to detect and fight this life-threatening pathogen. PMID:25376936

  9. Crystal Structures and Small-angle X-ray Scattering Analysis of UDP-galactopyranose Mutase from the Pathogenic Fungus Aspergillus fumigatus

    SciTech Connect

    Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Karr, Dale B.; Nix, Jay C.; Sobrado, Pablo; Tanner, John J.

    2015-10-15

    UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM has several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 {angstrom} to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.

  10. Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

    SciTech Connect

    Shin, Kwang-Soo; Kim, Young Hwan; Yu, Jae-Hyuk

    2015-07-31

    The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus.

  11. Clonal Expansion and Emergence of Environmental Multiple-Triazole-Resistant Aspergillus fumigatus Strains Carrying the TR34/L98H Mutations in the cyp51A Gene in India

    PubMed Central

    Chowdhary, Anuradha; Kathuria, Shallu; Xu, Jianping; Sharma, Cheshta; Sundar, Gandhi; Singh, Pradeep Kumar; Gaur, Shailendra N.; Hagen, Ferry; Klaassen, Corné H.; Meis, Jacques F.

    2012-01-01

    Azole resistance is an emerging problem in Aspergillus which impacts the management of aspergillosis. Here in we report the emergence and clonal spread of resistance to triazoles in environmental Aspergillus fumigatus isolates in India. A total of 44 (7%) A. fumigatus isolates from 24 environmental samples were found to be triazole resistant. The isolation rate of resistant A. fumigatus was highest (33%) from soil of tea gardens followed by soil from flower pots of the hospital garden (20%), soil beneath cotton trees (20%), rice paddy fields (12.3%), air samples of hospital wards (7.6%) and from soil admixed with bird droppings (3.8%). These strains showed cross-resistance to voriconazole, posaconazole, itraconazole and to six triazole fungicides used extensively in agriculture. Our analyses identified that all triazole-resistant strains from India shared the same TR34/L98H mutation in the cyp51 gene. In contrast to the genetic uniformity of azole-resistant strains the azole-susceptible isolates from patients and environments in India were genetically very diverse. All nine loci were highly polymorphic in populations of azole-susceptible isolates from both clinical and environmental samples. Furthermore, all Indian environmental and clinical azole resistant isolates shared the same multilocus microsatellite genotype not found in any other analyzed samples, either from within India or from the Netherlands, France, Germany or China. Our population genetic analyses suggest that the Indian azole-resistant A. fumigatus genotype was likely an extremely adaptive recombinant progeny derived from a cross between an azole-resistant strain migrated from outside of India and a native azole-susceptible strain from within India, followed by mutation and then rapid dispersal through many parts of India. Our results are consistent with the hypothesis that exposure of A. fumigatus to azole fungicides in the environment causes cross-resistance to medical triazoles. The study

  12. High prevalence of clinical and environmental triazole-resistant Aspergillus fumigatus in Iran: is it a challenging issue?

    PubMed

    Nabili, Mojtaba; Shokohi, Tahereh; Moazeni, Maryam; Khodavaisy, Sadegh; Aliyali, Masoud; Badiee, Parisa; Zarrinfar, Hossein; Hagen, Ferry; Badali, Hamid

    2016-06-01

    Triazole antifungal agents are the mainstay of aspergillosis treatment. As highlighted in numerous studies, the global increase in the prevalence of triazole resistance could hamper the management of aspergillosis. In the present three-year study, 513 samples (213 clinical and 300 environmental samples) from 10 provinces of Iran were processed and screened in terms of azole resistance (4 and 1 mg l-1 of itraconazole and voriconazole, respectively), using selective plates. Overall, 150 A. fumigatus isolates (71 clinical and 79 environmental isolates) were detected. The isolates were confirmed by partial sequencing of the β-tubulin gene. Afterwards, in vitro antifungal susceptibility tests against triazole agents were performed, based on the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document. The CYP51A gene was sequenced in order to detect mutations. The MIC of itraconazole against 10 (6.6 %) strains, including clinical (n=3, 4.2 %) and environmental (n=7, 8.8 %) strains, was higher than the breakpoint and epidemiological cut-off value. Based on the findings, the prevalence of azole-resistant A. fumigatus in Iran has increased remarkablyfrom 3.3 % to 6.6 % in comparison with earlier epidemiological research. Among resistant isolates, TR34/L98H mutations in the CYP51A gene were the most prevalent (n=8, 80 %), whereas other point mutations (F46Y, G54W, Y121F, G138C, M172V, F219C, M220I, D255E, T289F, G432C and G448S mutations) were not detected. Although the number of patients affected by azole-resistant A. fumigatus isolates was limited, strict supervision of clinical azole-resistant A. fumigatus isolates and persistent environmental screening of azole resistance are vital to the development of approaches for the management of azole resistance in human pathogenic fungi. PMID:27008655

  13. Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus.

    PubMed

    Shin, Kwang-Soo; Kim, Young Hwan; Yu, Jae-Hyuk

    2015-07-31

    The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. PMID:26032501

  14. Dephosphorylation of the Core Septin, AspB, in a Protein Phosphatase 2A-Dependent Manner Impacts Its Localization and Function in the Fungal Pathogen Aspergillus fumigatus.

    PubMed

    Vargas-Muñiz, José M; Renshaw, Hilary; Richards, Amber D; Waitt, Greg; Soderblom, Erik J; Moseley, Martin A; Asfaw, Yohannes; Juvvadi, Praveen R; Steinbach, William J

    2016-01-01

    Septins are a conserved family of GTPases that form hetero-oligomeric complexes and perform diverse functions in higher eukaryotes, excluding plants. Our previous studies in the human fungal pathogen Aspergillus fumigatus revealed that the core septin, AspB, a CDC3 ortholog, is required for septation, conidiation, and conidial cell wall organization. Although AspB is important for these cellular functions, nothing is known about the role of kinases or phosphatases in the posttranslational regulation and localization of septins in A. fumigatus. In this study, we assessed the function of the Gin4 and Cla4 kinases and the PP2A regulatory subunit ParA, in the regulation of AspB using genetic and phosphoproteomic approaches. Gene deletion analyses revealed that Cla4 and ParA are indispensable for hyphal extension, and Gin4, Cla4, and ParA are each required for conidiation and normal septation. While deletion of gin4 resulted in larger interseptal distances and hypervirulence, a phenotype mimicking aspB deletion, deletion of cla4 and parA caused hyperseptation without impacting virulence, indicating divergent roles in regulating septation. Phosphoproteomic analyses revealed that AspB is phosphorylated at five residues in the GTPase domain (S134, S137, S247, T297, and T301) and two residues at its C-terminus (S416 and S461) in the wild-type, Δgin4 and Δcla4 strains. However, concomitant with the differential localization pattern of AspB and hyperseptation in the ΔparA strain, AspB remained phosphorylated at two additional residues, T68 in the N-terminal polybasic region and S447 in the coiled-coil domain. Generation of nonphosphorylatable and phosphomimetic strains surrounding each differentially phosphorylated residue revealed that only AspB (mt) -T68E showed increased interseptal distances, suggesting that dephosphorylation of T68 is important for proper septation. This study highlights the importance of septin phosphorylation/dephosphorylation in the regulation of A

  15. Dephosphorylation of the Core Septin, AspB, in a Protein Phosphatase 2A-Dependent Manner Impacts Its Localization and Function in the Fungal Pathogen Aspergillus fumigatus

    PubMed Central

    Vargas-Muñiz, José M.; Renshaw, Hilary; Richards, Amber D.; Waitt, Greg; Soderblom, Erik J.; Moseley, Martin. A.; Asfaw, Yohannes; Juvvadi, Praveen R.; Steinbach, William J.

    2016-01-01

    Septins are a conserved family of GTPases that form hetero–oligomeric complexes and perform diverse functions in higher eukaryotes, excluding plants. Our previous studies in the human fungal pathogen Aspergillus fumigatus revealed that the core septin, AspB, a CDC3 ortholog, is required for septation, conidiation, and conidial cell wall organization. Although AspB is important for these cellular functions, nothing is known about the role of kinases or phosphatases in the posttranslational regulation and localization of septins in A. fumigatus. In this study, we assessed the function of the Gin4 and Cla4 kinases and the PP2A regulatory subunit ParA, in the regulation of AspB using genetic and phosphoproteomic approaches. Gene deletion analyses revealed that Cla4 and ParA are indispensable for hyphal extension, and Gin4, Cla4, and ParA are each required for conidiation and normal septation. While deletion of gin4 resulted in larger interseptal distances and hypervirulence, a phenotype mimicking aspB deletion, deletion of cla4 and parA caused hyperseptation without impacting virulence, indicating divergent roles in regulating septation. Phosphoproteomic analyses revealed that AspB is phosphorylated at five residues in the GTPase domain (S134, S137, S247, T297, and T301) and two residues at its C-terminus (S416 and S461) in the wild-type, Δgin4 and Δcla4 strains. However, concomitant with the differential localization pattern of AspB and hyperseptation in the ΔparA strain, AspB remained phosphorylated at two additional residues, T68 in the N-terminal polybasic region and S447 in the coiled-coil domain. Generation of nonphosphorylatable and phosphomimetic strains surrounding each differentially phosphorylated residue revealed that only AspBmt-T68E showed increased interseptal distances, suggesting that dephosphorylation of T68 is important for proper septation. This study highlights the importance of septin phosphorylation/dephosphorylation in the regulation of A

  16. A comparative study of the timecourse of the expression of the thermo‑inducible HSP70 gene in clinical and environmental isolates of Aspergillus fumigatus.

    PubMed

    Sharafi, Golnaz; Khosravi, Ali Reza; Vahedi, Ghasem; Yahyaraeyat, Ramak; Abbasi, Teimur

    2016-05-01

    The internal environment within animals or humans provides different conditions to invading saprophytic fungal pathogens, requiring the differential regulation of genes in comparison to environmental conditions. Understanding the mechanisms by which pathogens regulate genes within the host may be key in determining pathogen behavior within the host and may additionally facilitate further investigation into novel therapeutic agents. The heat shock protein (HSP)70 gene and its associated proteins have been frequently reported to be among the most highly expressed and dominant proteins present within various locations at physiological temperatures. The present study examined relative gene expression levels of the HSP70 gene in Aspergillus fumigatus isolates from both clinical and environmental origins, at a range of temperature points (20, 30, 37 and 42˚C) over five days, using reverse transcription‑quantitative polymerase chain reaction, comparing with a standard A. fumigatus strain incubated at 25˚C. The results indicated a differential gene expression pattern for the environmental and clinical isolates. During the five days, the HSP70 expression levels in the clinical samples were higher than in the environmental samples. However, the difference in the expression levels between the two groups at 42˚C was reduced. The mean HSP70 expression level over the five incubation days demonstrated a gradual and continual increasing trend by temperature elevation in both groups at 30, 37 and 42˚C, however, at 20˚C both groups demonstrated reduced expression. The temperature shift from 20 to 42˚C resulted in HSP70 induction and up to a 10‑ and 8.6‑fold change in HSP70 expression levels on the fifth day of incubation in the clinical and environmental groups, respectively. In conclusion, incubation at 37 and 42˚C resulted in the highest expression levels in both experimental groups, with these temperature points important for the induction of HSP70

  17. Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source, Phenotype and Growth Conditions of the Bacterium

    PubMed Central

    Ferreira, Jose A. G.; Penner, John C.; Moss, Richard B.; Haagensen, Janus A. J.; Clemons, Karl V.; Spormann, Alfred M.; Nazik, Hasan; Cohen, Kevin; Banaei, Niaz; Carolino, Elisabete; Stevens, David A.

    2015-01-01

    Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment. PMID:26252384

  18. Ergot alkaloid biosynthesis in Aspergillus fumigatus: conversion of chanoclavine-I to chanoclavine-I aldehyde catalyzed by a short-chain alcohol dehydrogenase FgaDH.

    PubMed

    Wallwey, Christiane; Matuschek, Marco; Li, Shu-Ming

    2010-02-01

    Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaDH has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaDH comprises 261 amino acids with a molecular mass of about 27.8 kDa and contains the conserved motifs of classical short-chain dehydrogenases/reductases (SDRs), but shares no worth mentioning sequence similarity with SDRs and other known proteins. The coding region of fgaDH consisting of two exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE60 and overexpressed in E. coli. The soluble tetrameric His(6)-FgaDH was purified to apparent homogeneity and characterized biochemically. It has been shown that FgaDH catalyzes the oxidation of chanoclavine-I in the presence of NAD(+) resulting in the formation of chanoclavine-I aldehyde, which was unequivocally identified by NMR and MS analyzes. Therefore, FgaDH functions as a chanoclavine-I dehydrogenase and represents a new group of short-chain dehydrogenases. K (M) values for chanoclavine-I and NAD(+) were determined at 0.27 and 1.1 mM, respectively. The turnover number was 0.38 s(-1). PMID:20039019

  19. Reverse prenyltransferase in the biosynthesis of fumigaclavine C in Aspergillus fumigatus: gene expression, purification, and characterization of fumigaclavine C synthase FGAPT1.

    PubMed

    Unsöld, Inge A; Li, Shu-Ming

    2006-01-01

    A putative prenyltransferase gene-fgaPT1-has been identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus AF293. The gene was cloned and overexpressed in Escherichia coli, and the His6-fusion FgaPT1 was purified to near homogeneity and characterized biochemically. The enzyme was found to convert fumigaclavine A into fumigaclavine C by attaching a dimethylallyl moiety to C-2 of the indole nucleus in a "reverse" manner, that is, by connection of C-3 of the dimethylallyl moiety to an aromatic nucleus. FgaPT1 is a soluble, dimeric protein with a subunit size of 50 kDa. K m(app) values for fumigaclavine A and dimethylallyl diphosphate were determined to be 6 and 13 microM, respectively, while the turnover number was 0.8 s(-1). Metal ions such as Mg2+ and Ca2+ are not essential for the enzymatic activity. FgaPT1 showed relatively strict substrate specificity towards fumigaclavine A, with only dimethylallyl diphosphate being accepted as a donor under our conditions. FgaPT1 is the first reverse prenyltransferase from fungi to have been purified and characterized in homogenous form after heterologous overproduction. Surprisingly, it shows very low sequence similarity to the recently identified prenyltransferase LtxC from cyanobacteria, which also catalyzes the reverse prenylation of an indole nucleus. PMID:16397874

  20. Banana peel: a potential substrate for laccase production by Aspergillus fumigatus VkJ2.4.5 in solid-state fermentation.

    PubMed

    Vivekanand, V; Dwivedi, Pallavi; Pareek, Nidhi; Singh, Rajesh P

    2011-09-01

    In solid-state fermentation, among various solid supports evaluated, banana peel was found to be an ideal support and resulted into higher levels of laccase (6281.4 ± 63.60 U l(-1)) along with notable levels of manganese peroxidase production (1339.0 ± 131.23 U l(-1)) by Aspergillus fumigatus VkJ2.4.5. Maximum levels of laccase was achieved under derived conditions consisting of 80% of moisture level, 6 days of incubation period, 6% inoculum level, and an aeration level of 2.5 l min(-1). A column-tray bioreactor was designed to scale up and economize the enzyme production in three successive cycles of fermentation using the same fungal biomass. Thermal and pH stability profiles revealed that enzyme was stable up to 50°C and at varying pH range from 5-9 for up to 2 h. The apparent molecular weight of laccase was found to be 34 ± 1 kDa. MALDI-TOF/TOF analysis of the protein showed significant homology with maximum identity of 67% to other laccases reported in database. PMID:21487735

  1. Aspergillus infection monitored by multimodal imaging in a rat model.

    PubMed

    Pluhacek, Tomas; Petrik, Milos; Luptakova, Dominika; Benada, Oldrich; Palyzova, Andrea; Lemr, Karel; Havlicek, Vladimir

    2016-06-01

    Although myriads of experimental approaches have been published in the field of fungal infection diagnostics, interestingly, in 21st century there is no satisfactory early noninvasive tool for Aspergillus diagnostics with good sensitivity and specificity. In this work, we for the first time described the fungal burden in rat lungs by multimodal imaging approach. The Aspergillus infection was monitored by positron emission tomography and light microscopy employing modified Grocott's methenamine silver staining and eosin counterstaining. Laser ablation inductively coupled plasma mass spectrometry imaging has revealed a dramatic iron increase in fungi-affected areas, which can be presumably attributed to microbial siderophores. Quantitative elemental data were inferred from matrix-matched standards prepared from rat lungs. The iron, silver, and gold MS images collected with variable laser foci revealed that particularly silver or gold can be used as excellent elements useful for sensitively tracking the Aspergillus infection. The limit of detection was determined for both (107) Ag and (197) Au as 0.03 μg/g (5 μm laser focus). The selective incorporation of (107) Ag and (197) Au into fungal cell bodies and low background noise from both elements were confirmed by energy dispersive X-ray scattering utilizing the submicron lateral resolving power of scanning electron microscopy. The low limits of detection and quantitation of both gold and silver make ICP-MS imaging monitoring a viable alternative to standard optical evaluation used in current clinical settings. PMID:27060291

  2. Immunohistologic identification of Aspergillus spp. and other hyaline fungi by using polyclonal fluorescent antibodies.

    PubMed Central

    Kaufman, L; Standard, P G; Jalbert, M; Kraft, D E

    1997-01-01

    Isolation and identification of pathogenic Aspergillus and Fusarium spp. from clinical materials provide the most accurate means for establishing a diagnosis of infections by these molds. Such efforts, however, are not always successful. Histologic diagnosis also has its limitations. In vivo the hyphae of Aspergillus and Fusarium spp. are very similar and their in situ manifestations are not pathognomonic. To improve the histologic diagnosis of infections by Aspergillus and Fusarium species, we developed polyclonal fluorescent-antibody reagents to Aspergillus fumigatus and Fusarium solani and evaluated their diagnostic utilities. Our studies revealed that A. fumigatus and F. solani share epitopes not only with one another but also with other Aspergillus and Fusarium spp. as well as with Paecilomyces lilacinus and Pseudallescheria boydii. Adsorption of the A. fumigatus conjugate with cells of Fusarium proliferatum and F. solani and F. solani antiserum with cells of Aspergillus flavus resulted in reagents that distinguished Aspergillus spp. from Fusarium spp. but that still cross-stained P. lilacinus and P. boydii. Adjunctive use of a specific P. boydii conjugate enabled the identification of Aspergillus spp., Fusarium spp., P. lilacinus, and P. boydii in formalin-fixed tissue sections from 19 humans with culture-proven cases of mycotic infection. PMID:9276388

  3. Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33

    PubMed Central

    2012-01-01

    Background Recently, the increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and β-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful β-glucosides. Although the β-glucosidases of different sources have been investigated, the amount of β-glucosidases are insufficient for effective conversion of cellulose. The goal of this work was to search for new resources of β-glucosidases, which was thermostable and with high catalytic efficiency. Results In this study, a thermostable native β-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus Aspergillus fumigatus Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, bgl3, was cloned based on the peptide sequences obtained from the LC-MS/MS results. bgl3 contains an open reading frame (ORF) of 2622 bp and encodes a protein with a predicted molecular weight of 91.47 kDa; amino acid sequence analysis of the deduced protein indicated that nBgl3 is a member of the glycoside hydrolase family 3. A recombinant β-glucosidase (rBgl3) was obtained by the functional expression of bgl3 in Pichia pastoris X33. Several biochemical properties of purified nBgl3 and rBgl3 were determined - both enzymes showed optimal activity at pH 6.0 and 60°C, and they were stable for a pH range of 4-7 and a temperature range of 50 to 70°C. Of the substrates tested, nBgl3 and rBgl3 displayed the highest activity toward 4-Nitrophenyl-β-D-glucopyranoside (pNPG), with specific activities of 103.5 ± 7.1 and 101.7 ± 5.2 U mg-1, respectively. However, these enzymes were inactive toward carboxymethyl cellulose, lactose and xylan. Conclusions An native β-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of A. fumigatus Z5

  4. Aspergillus fumigatus survival in alkaline and extreme zinc-limiting environments relies on the induction of a zinc homeostasis system encoded by the zrfC and aspf2 genes.

    PubMed

    Amich, Jorge; Vicentefranqueira, Rocío; Leal, Fernando; Calera, José Antonio

    2010-03-01

    Aspergillus fumigatus has three zinc transporter-encoding genes whose expression is regulated by both pH and the environmental concentration of zinc. We have previously reported that the zrfA and zrfB genes of A. fumigatus are transcribed at higher levels and are required for fungal growth under acidic zinc-limiting conditions whereas they are dispensable for growth in neutral or alkaline zinc-limiting media. Here we report that the transporter of the zinc uptake system that functions in A. fumigatus growing in neutral or alkaline environments is encoded by zrfC. The transcription of zrfC occurs divergently with respect to the adjacent aspf2 gene, which encodes an immunodominant antigen secreted by A. fumigatus. The two genes-zrfC and aspf2-are required to different extents for fungal growth in alkaline and extreme zinc-limiting media. Indeed, these environmental conditions induce the simultaneous transcription of both genes mediated by the transcriptional regulators ZafA and PacC. ZafA upregulates the expression of zrfC and aspf2 under zinc-limiting conditions regardless of the ambient pH, whereas PacC represses the expression of these genes under acidic growth conditions. Interestingly, the mode of action of PacC for zrfC-aspf2 transcription contrasts with the more widely accepted model for PacC function, according to which under alkaline growth conditions PacC would activate the transcription of alkaline-expressed genes but would repress the transcription of acid-expressed genes. In sum, this report provides a good framework for investigating several important aspects of the biology of species of Aspergillus, including the repression of alkaline genes by PacC at acidic pH and the interrelationship that must exist between tissue pH, metal availability in the host tissue, and fungal virulence. PMID:20038606

  5. Concomitant Aspergillus Species Infection and Squamous Cell Carcinoma Diagnosed on Pap Smear.

    PubMed

    Gupta, Prajwala; Goyal, Snigdha; Kaushal, Manju

    2016-01-01

    Concomitant infection with Aspergillus species and cervical squamous cell carcinoma in the female genital tract is a rare occurrence and attributed to the opportunistic nature of infection in the immunocompromised state due to the underlying malignancy. The contamination of smears with Aspergillus species should be excluded. The diagnosis of Aspergillus species infection along with squamous cell carcinoma was established on cervicovaginal pap smears in a 62-year-old female presented to gynecological clinic with complaints of stress urinary incontinence. Speculum examination revealed first-degree cervical descent. Smears showed features of squamous cell carcinoma along with fungal spores and fruiting body with hyphae of Aspergillus species. The presence of fruiting bodies and hyphae of Aspergillus species with coexisting squamous cell carcinoma is rare in routine pap smears. True infection needs to be distinguished from contamination by Aspergillus species. Early diagnosis can be established on routine cervicovaginal Pap smear examination. PMID:24272933

  6. A Novel C2H2 Transcription Factor that Regulates gliA Expression Interdependently with GliZ in Aspergillus fumigatus

    PubMed Central

    Schoberle, Taylor J.; Nguyen-Coleman, C. Kim; Herold, Jennifer; Yang, Ally; Weirauch, Matt; Hughes, Timothy R.; McMurray, John S.; May, Gregory S.

    2014-01-01

    Secondary metabolites are produced by numerous organisms and can either be beneficial, benign, or harmful to humans. Genes involved in the synthesis and transport of these secondary metabolites are frequently found in gene clusters, which are often coordinately regulated, being almost exclusively dependent on transcription factors that are located within the clusters themselves. Gliotoxin, which is produced by a variety of Aspergillus species, Trichoderma species, and Penicillium species, exhibits immunosuppressive properties and has therefore been the subject of research for many laboratories. There have been a few proteins shown to regulate the gliotoxin cluster, most notably GliZ, a Zn2Cys6 binuclear finger transcription factor that lies within the cluster, and LaeA, a putative methyltransferase that globally regulates secondary metabolism clusters within numerous fungal species. Using a high-copy inducer screen in A. fumigatus, our lab has identified a novel C2H2 transcription factor, which plays an important role in regulating the gliotoxin biosynthetic cluster. This transcription factor, named GipA, induces gliotoxin production when present in extra copies. Furthermore, loss of gipA reduces gliotoxin production significantly. Through protein binding microarray and mutagenesis, we have identified a DNA binding site recognized by GipA that is in extremely close proximity to a potential GliZ DNA binding site in the 5′ untranslated region of gliA, which encodes an efflux pump within the gliotoxin cluster. Not surprisingly, GliZ and GipA appear to work in an interdependent fashion to positively control gliA expression. PMID:24784729

  7. Effect of nickel-cobaltite nanoparticles on production and thermostability of cellulases from newly isolated thermotolerant Aspergillus fumigatus NS (class: Eurotiomycetes).

    PubMed

    Srivastava, Neha; Rawat, Rekha; Sharma, Reetika; Oberoi, Harinder Singh; Srivastava, Manish; Singh, Jay

    2014-10-01

    In the present study, effect of nickel-cobaltite (NiCo2O4) nanoparticles (NPs) was investigated on production and thermostability of the cellulase enzyme system using newly isolated thermotolerant Aspergillus fumigatus NS belonging to the class Euratiomycetes. The NiCo2O4 NPs were synthesized via hydrothermal method assisted by post-annealing treatment and characterized through X-ray diffraction (XRD) and transmission electron microscopy (TEM) techniques. In the absence of NPs in the growth medium, filter paper cellulase (FP) activity of 18 IU/gds was achieved after 96 h, whereas 40 % higher FP activity in 72 h was observed with the addition of 1 mM concentration of NPs in the growth medium. Maximum production of endoglucanase (211 IU/gds), β-glucosidase (301 IU/gds), and xylanase (803 IU/gds) was achieved after 72 h without NPs (control), while in the presence of 1 mM concentration of NPs, endoglucanase, β-glucosidase, and xylanase activity increased by about 49, 53, and 19.8 %, respectively, after 48 h of incubation, against control, indicating a substantial increase in cellulase productivity with the addition of NiCo2O4 NPs in the growth medium. Crude enzyme was thermally stable for 7 h at 80 °C in presence of NPs, as against 4 h at the same temperature for control samples. Significant increase in the activity and improved thermal stability of cellulases in the presence of the NiCo2O4 NPs holds potential for use of NiCo2O4 NPs during enzyme production as well as hydrolysis. From the standpoint of biofuel production, these results hold enormous significance. PMID:24801407

  8. Analytical Comparison of In Vitro-Spiked Human Serum and Plasma for PCR-Based Detection of Aspergillus fumigatus DNA: a Study by the European Aspergillus PCR Initiative

    PubMed Central

    Mengoli, Carlo; Springer, Jan; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Klingspor, Lena; Lagrou, Katrien; Melchers, Willem J. G.; Morton, C. Oliver; Barnes, Rosemary A.; Donnelly, J. Peter; White, P. Lewis

    2015-01-01

    The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardized technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered interchangeable for use in fungal diagnostics, differences in galactomannan enzyme immunoassay (GM-EIA) performance have been reported and are attributed to clot formation. Therefore, it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable and also to compare analytical performance with that of serum PCR. Molecular methods testing serum and plasma were compared through multicenter distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared, and a regression analysis was performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, preextraction-to-postextraction volume ratio, PCR volume, duplicate testing, and the use of an internal control for PCR were positively associated with performance. When whole-blood samples were spiked and then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (P = 0.028). Technically, there are no major differences in the molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease, Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma might improve performance while maintaining the methodological simplicity of serum testing. PMID:26085614

  9. Lung surfactant proteins A and D can inhibit specific IgE binding to the allergens of Aspergillus fumigatus and block allergen-induced histamine release from human basophils

    PubMed Central

    MADAN, T; KISHORE, U; SHAH, A; EGGLETON, P; STRONG, P; WANG, J Y; AGGRAWAL, S S; SARMA, P U; REID, K B M

    1997-01-01

    Aspergillus fumigatus is an opportunistic fungal pathogen which, in the immunocompetent host, causes allergic disorders such as allergic rhinitis, allergic sinusitis, hypersensitivity pneumonitis, and allergic bronchopulmonary Aspergillosis (ABPA). In the present study, the interaction of 3-week culture filtrate (3wcf) allergens and various purified glycosylated and non-glycosylated allergens of A. fumigatus with lung surfactant proteins, SP-A and SP-D, was investigated. Purified SP-A and SP-D, isolated from human bronchoalveolar lavage fluid, bound to the 3wcf allergens and purified allergens, gp55 and gp45, in a carbohydrate-specific and calcium-dependent manner. Both SP-A and SP-D did not bind to deglycosylated allergens, suggesting that the ability of SP-A and SP-D to bind certain allergens is mediated through their carbohydrate recognition domains, interacting with the carbohydrate residues on the allergen. Both SP-A and SP-D could inhibit the ability of allergen-specific IgE from Aspergillosis patients to bind these allergens, suggesting that SP-A and SP-D may be involved in the modulation of allergic sensitization and/or development of allergic reactions. The view that SP-A and SP-D play a protective role against airborne allergens is further supported by the demonstration of their ability to inhibit A. fumigatus allergen-induced histamine release from allergic patients' basophils. PMID:9367408

  10. Lung surfactant proteins A and D can inhibit specific IgE binding to the allergens of Aspergillus fumigatus and block allergen-induced histamine release from human basophils.

    PubMed

    Madan, T; Kishore, U; Shah, A; Eggleton, P; Strong, P; Wang, J Y; Aggrawal, S S; Sarma, P U; Reid, K B

    1997-11-01

    Aspergillus fumigatus is an opportunistic fungal pathogen which, in the immunocompetent host, causes allergic disorders such as allergic rhinitis, allergic sinusitis, hypersensitivity pneumonitis, and allergic bronchopulmonary Aspergillosis (ABPA). In the present study, the interaction of 3-week culture filtrate (3wcf) allergens and various purified glycosylated and non-glycosylated allergens of A. fumigatus with lung surfactant proteins, SP-A and SP-D, was investigated. Purified SP-A and SP-D, isolated from human bronchoalveolar lavage fluid, bound to the 3wcf allergens and purified allergens, gp55 and gp45, in a carbohydrate-specific and calcium-dependent manner. Both SP-A and SP-D did not bind to deglycosylated allergens, suggesting that the ability of SP-A and SP-D to bind certain allergens is mediated through their carbohydrate recognition domains, interacting with the carbohydrate residues on the allergen. Both SP-A and SP-D could inhibit the ability of allergen-specific IgE from Aspergillosis patients to bind these allergens, suggesting that SP-A and SP-D may be involved in the modulation of allergic sensitization and/or development of allergic reactions. The view that SP-A and SP-D play a protective role against airborne allergens is further supported by the demonstration of their ability to inhibit A. fumigatus allergen-induced histamine release from allergic patients' basophils. PMID:9367408

  11. Biogenic synthesis, optimisation and antibacterial efficacy of extracellular silver nanoparticles using novel fungal isolate Aspergillus fumigatus MA.

    PubMed

    Sarsar, Vikas; Selwal, Manjit K; Selwal, Krishan K

    2016-08-01

    To eliminate the elaborate processes employed in other non-biological-based protocols and low cost production of silver nanoparticles (AgNPs), this study reports biogenic synthesis of AgNPs using silver salt precursor with aqueous extract of Aspergillus fumigates MA. Influence of silver precursor concentrations, concentration ratio of fungal extract and silver nitrate, contact time, reaction temperature and pH are evaluated to find their effects on AgNPs synthesis. Ultraviolet-visible spectra gave surface plasmon resonance at 420 nm for AgNPs. Fourier transform infrared spectroscopy and X-ray diffraction techniques further confirmed the synthesis and crystalline nature of AgNPs, respectively. Transmission electron microscopy observed spherical shapes of synthesised AgNPs within the range of 3-20 nm. The AgNPs showed potent antimicrobial efficacy against various bacterial strains. Thus, the results of the current study indicate that optimisation process plays a pivotal role in the AgNPs synthesis and biogenic synthesised AgNPs might be used against bacterial pathogens; however, it necessitates clinical studies to find out their potential as antibacterial agents. PMID:27463792

  12. Differential patterns of activity displayed by two exo-beta-1,3-glucanases associated with the Aspergillus fumigatus cell wall.

    PubMed Central

    Fontaine, T; Hartland, R P; Diaquin, M; Simenel, C; Latgé, J P

    1997-01-01

    Two exo-beta-1,3-glucanases (herein designated exoG-I and exoG-II) were isolated from the cell wall autolysate of the filamentous fungus Aspergillus fumigatus and purified by ion-exchange, hydrophobic-interaction, and gel filtration chromatographies. Molecular masses estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 82 kDa for the monomeric exoG-I and 230 kDa for the dimeric exoG-II. exoG-I and exoG-II were glycosylated, and N glycans accounted, respectively, for 2 and 44 kDa. Their pH optimum is 5.0. Their optimum temperatures are 55 degrees C for exoG-I and 65 degrees C for exoG-II. By a sensitive colorimetric method and high-performance anion-exchange chromatography for product analysis, two patterns of exo-beta-1,3-glucanase activities were found. The 230-kDa exoG-II enzyme acts on p-nitrophenyl-beta-D-glucoside, beta-1,6-glucan, and beta-1,3-glucan. This activity, which retains the anomeric configuration of glucose released, presented a multichain pattern of attack of the glucan chains and a decrease in the maximum initial velocity (Vm) with the increasing size of the substrate. In contrast, the 82-kDa exoG-I, which inverts the anomeric configuration of the glucose released, hydrolyzed exclusively the beta-1,3-glucan chain with a minimal substrate size of 4 glucose residues. This enzyme presented a repetitive-attack pattern, characterized by an increase in Vm with an increase in substrate size and by a degradation of the glucan chain until it reached laminaritetraose, the limit substrate size. The 82-kDa exoG-I and 230-kDa exoG-II enzymes correspond to a beta-1,3-glucan-glucohydrolase (EC 3.2.1.58) and to a beta-D-glucoside-glucohydrolase (EC 3.2.1.21), respectively. The occurrence and functions of these two classes of exo-beta-1,3-glucanases in other fungal species are discussed. PMID:9150209

  13. Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR34/L98H- and TR46/Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014

    PubMed Central

    Mohammadi, Faezeh; Hashemi, Seyed Jamal; Zoll, Jan; Melchers, Willem J. G.; Rafati, Haleh; Dehghan, Parvin; Rezaie, Sasan; Tolooe, Ali; Tamadon, Yalda; van der Lee, Henrich A.; Verweij, Paul E.

    2015-01-01

    We employed an endpoint genotyping method to update the prevalence rate of positivity for the TR34/L98H mutation (a 34-bp tandem repeat mutation in the promoter region of the cyp51A gene in combination with a substitution at codon L98) and the TR46/Y121F/T289A mutation (a 46-bp tandem repeat mutation in the promoter region of the cyp51A gene in combination with substitutions at codons Y121 and T289) among clinical Aspergillus fumigatus isolates obtained from different regions of Iran over a recent 5-year period (2010 to 2014). The antifungal activities of itraconazole, voriconazole, and posaconazole against 172 clinical A. fumigatus isolates were investigated using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. For the isolates with an azole resistance phenotype, the cyp51A gene and its promoter were amplified and sequenced. In addition, using a LightCycler 480 real-time PCR system, a novel endpoint genotyping analysis method targeting single-nucleotide polymorphisms was evaluated to detect the L98H and Y121F mutations in the cyp51A gene of all isolates. Of the 172 A. fumigatus isolates tested, the MIC values of itraconazole (≥16 mg/liter) and voriconazole (>4 mg/liter) were high for 6 (3.5%). Quantitative analysis of single-nucleotide polymorphisms showed the TR34/L98H mutation in the cyp51A genes of six isolates. No isolates harboring the TR46/Y121F/T289A mutation were detected. DNA sequencing of the cyp51A gene confirmed the results of the novel endpoint genotyping method. By microsatellite typing, all of the azole-resistant isolates had genotypes different from those previously recovered from Iran and from the Dutch TR34/L98H controls. In conclusion, there was not a significant increase in the prevalence of azole-resistant A. fumigatus isolates harboring the TR34/L98H resistance mechanism among isolates recovered over a recent 5-year period (2010 to 2014) in Iran. A quantitative assay detecting a single

  14. Rapid Differentiation of Aspergillus Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay

    PubMed Central

    de Aguirre, Liliana; Hurst, Steven F.; Choi, Jong Soo; Shin, Jong Hee; Hinrikson, Hans Peter; Morrison, Christine J.

    2004-01-01

    We developed a PCR-based assay to differentiate medically important species of Aspergillus from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor, differentiated 41 isolates (3 to 9 each of the respective species; P < 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of Acremonium, Exophiala, Candida, Fusarium, Mucor, Paecilomyces, Penicillium, Rhizopus, Scedosporium, Sporothrix, or other aspergilli tested. A single DNA probe to detect all seven of the most medically important Aspergillus species (A. flavus, A. fumigatus, A. nidulans, A. niger, A. terreus, A. ustus, and A. versicolor) was also designed. Identification of Aspergillus species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important Aspergillus species and for their differentiation from other opportunistic fungi. PMID:15297489

  15. 2-hydroxyisocaproic acid is fungicidal for Candida and Aspergillus species.

    PubMed

    Sakko, M; Moore, C; Novak-Frazer, L; Rautemaa, V; Sorsa, T; Hietala, P; Järvinen, A; Bowyer, P; Tjäderhane, L; Rautemaa, R

    2014-04-01

    The amino acid derivative 2-hydroxyisocaproic acid (HICA) is a nutritional additive used to increase muscle mass. Low levels can be detected in human plasma as a result of leucine metabolism. It has broad antibacterial activity but its efficacy against pathogenic fungi is not known. The aim was to test the efficacy of HICA against Candida and Aspergillus species. Efficacy of HICA against 19 clinical and reference isolates representing five Candida and three Aspergillus species with variable azole antifungal sensitivity profiles was tested using a microdilution method. The concentrations were 18, 36 and 72 mg ml(-1) . Growth was determined spectrophotometrically for Candida isolates and by visual inspection for Aspergillus isolates, viability was tested by culture and impact on morphology by microscopy. HICA of 72 mg ml(-1) was fungicidal against all Candida and Aspergillus fumigatus and Aspergillus terreus isolates. Lower concentrations were fungistatic. Aspergillus flavus was not inhibited by HICA. HICA inhibited hyphal formation in susceptible Candida albicans and A. fumigatus isolates and affected cell wall integrity. In conclusion, HICA has broad antifungal activity against Candida and Aspergillus at concentrations relevant for topical therapy. As a fungicidal agent with broad-spectrum bactericidal activity, it may be useful in the topical treatment of multispecies superficial infections. PMID:24125484

  16. Effect of physical and chemical properties of oil palm empty fruit bunch, decanter cake and sago pith residue on cellulases production by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2.

    PubMed

    Zanirun, Zuraidah; Bahrin, Ezyana Kamal; Lai-Yee, Phang; Hassan, Mohd Ali; Abd-Aziz, Suraini

    2014-01-01

    The effect of cultivation condition of two locally isolated ascomycetes strains namely Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 were compared in submerged and solid state fermentation. Physical evaluation on water absorption index, solubility index and chemical properties of lignin, hemicellulose and cellulose content as well as the cellulose structure on crystallinity and amorphous region of treated oil palm empty fruit bunch (OPEFB) (resulted in partial removal of lignin), sago pith residues (SPR) and oil palm decanter cake towards cellulases production were determined. Submerged fermentation shows significant cellulases production for both strains in all types of substrates. Crystallinity of cellulose and its chemical composition mainly holocellulose components was foun