Publications by authors named "Oumaïma Ibrahim-Granet"

30 Publications

  • Page 1 of 1

Galactosaminogalactan activates the inflammasome to provide host protection.

Nature 2020 12 2;588(7839):688-692. Epub 2020 Dec 2.

Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA.

Inflammasomes are important sentinels of innate immune defence that are activated in response to diverse stimuli, including pathogen-associated molecular patterns (PAMPs). Activation of the inflammasome provides host defence against aspergillosis, which is a major health concern for patients who are immunocompromised. However, the Aspergillus fumigatus PAMPs that are responsible for inflammasome activation are not known. Here we show that the polysaccharide galactosaminogalactan (GAG) of A. fumigatus is a PAMP that activates the NLRP3 inflammasome. The binding of GAG to ribosomal proteins inhibited cellular translation machinery, and thus activated the NLRP3 inflammasome. The galactosamine moiety bound to ribosomal proteins and blocked cellular translation, which triggered activation of the NLRP3 inflammasome. In mice, a GAG-deficient Aspergillus mutant (Δgt4c) did not elicit protective activation of the inflammasome, and this strain exhibited enhanced virulence. Moreover, administration of GAG protected mice from colitis induced by dextran sulfate sodium in an inflammasome-dependent manner. Thus, ribosomes connect the sensing of this fungal PAMP to the activation of an innate immune response.
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http://dx.doi.org/10.1038/s41586-020-2996-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086055PMC
December 2020

conidial metalloprotease Mep1p cleaves host complement proteins.

J Biol Chem 2018 10 23;293(40):15538-15555. Epub 2018 Aug 23.

From the Complement Biology Laboratory and

Innate immunity in animals including humans encompasses the complement system, which is considered an important host defense mechanism against , one of the most ubiquitous opportunistic human fungal pathogens. Previously, it has been shown that the alkaline protease Alp1p secreted from mycelia degrades the complement components C3, C4, and C5. However, it remains unclear how the fungal spores ( conidia) defend themselves against the activities of the complement system immediately after inhalation into the lung. Here, we show that conidia contain a metalloprotease Mep1p, which is released upon conidial contact with collagen and inactivates all three complement pathways. In particular, Mep1p efficiently inactivated the major complement components C3, C4, and C5 and their activation products (C3a, C4a, and C5a) as well as the pattern-recognition molecules MBL and ficolin-1, either by directly cleaving them or by cleaving them to a form that is further broken down by other proteases of the complement system. Moreover, incubation of Mep1p with human serum significantly inhibited the complement hemolytic activity and conidial opsonization by C3b and their subsequent phagocytosis by macrophages. Together, these results indicate that Mep1p associated with and released from conidia likely facilitates early immune evasion by disarming the complement defense in the human host.
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http://dx.doi.org/10.1074/jbc.RA117.001476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177592PMC
October 2018

Genetic deficiency of NOD2 confers resistance to invasive aspergillosis.

Nat Commun 2018 07 6;9(1):2636. Epub 2018 Jul 6.

Department of Experimental Internal Medicine and Radboud Center for Infectious Diseases (RCI), Radboud University Medical Center, Geert Grooteplein zuid 8, 6525GA, Nijmegen, The Netherlands.

Invasive aspergillosis (IA) is a severe infection that can occur in severely immunocompromised patients. Efficient immune recognition of Aspergillus is crucial to protect against infection, and previous studies suggested a role for NOD2 in this process. However, thorough investigation of the impact of NOD2 on susceptibility to aspergillosis is lacking. Common genetic variations in NOD2 has been associated with Crohn's disease and here we investigated the influence of these  genetic variations on the anti-Aspergillus host response. A NOD2 polymorphism reduced the risk of IA after hematopoietic stem-cell transplantation. Mechanistically, absence of NOD2 in monocytes and macrophages increases phagocytosis leading to enhanced fungal killing, conversely, NOD2 activation reduces the antifungal potential of these cells. Crucially, Nod2 deficiency results in resistance to Aspergillus infection in an in vivo model of pulmonary aspergillosis. Collectively, our data demonstrate that genetic deficiency of NOD2 plays a protective role during Aspergillus infection.
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http://dx.doi.org/10.1038/s41467-018-04912-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6035256PMC
July 2018

A Novel Polyaminocarboxylate Compound To Treat Murine Pulmonary Aspergillosis by Interfering with Zinc Metabolism.

Antimicrob Agents Chemother 2018 06 25;62(6). Epub 2018 May 25.

Institut Pasteur, Cytokines & Inflammation Unit, Paris, France

can cause pulmonary aspergillosis in immunocompromised patients and is associated with a high mortality rate due to a lack of reliable treatment options. This opportunistic pathogen requires zinc in order to grow and cause disease. Novel compounds that interfere with fungal zinc metabolism may therefore be of therapeutic interest. We screened chemical libraries containing 59,223 small molecules using a resazurin assay that compared their effects on an wild-type strain grown under zinc-limiting conditions and on a zinc transporter knockout strain grown under zinc-replete conditions to identify compounds affecting zinc metabolism. After a first screen, 116 molecules were selected whose inhibitory effects on fungal growth were further tested by using luminescence assays and hyphal length measurements to confirm their activity, as well as by toxicity assays on HeLa cells and mice. Six compounds were selected following a rescreening, of which two were pyrazolones, two were porphyrins, and two were polyaminocarboxylates. All three groups showed good activity, but only one of the polyaminocarboxylates was able to significantly improve the survival of immunosuppressed mice suffering from pulmonary aspergillosis. This two-tier screening approach led us to the identification of a novel small molecule with fungicidal effects and low murine toxicity that may lead to the development of new treatment options for fungal infections by administration of this compound either as a monotherapy or as part of a combination therapy.
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http://dx.doi.org/10.1128/AAC.02510-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971598PMC
June 2018

Fungal melanin stimulates surfactant protein D-mediated opsonization of and host immune response to spores.

J Biol Chem 2018 03 5;293(13):4901-4912. Epub 2018 Feb 5.

Unite des Aspergillus, Paris 75015, France. Electronic address:

Surfactant protein D (SP-D), a C-type lectin and pattern-recognition soluble factor, plays an important role in immune surveillance to detect and eliminate human pulmonary pathogens. SP-D has been shown to protect against infections with the most ubiquitous airborne fungal pathogen, , but the fungal surface component(s) interacting with SP-D is unknown. Here, we show that SP-D binds to melanin pigment on the surface of dormant spores (conidia). SP-D also exhibited an affinity to two cell-wall polysaccharides of , galactomannan (GM) and galactosaminogalactan (GAG). The immunolabeling pattern of SP-D was punctate on the conidial surface and was uniform on germinating conidia, in accordance with the localization of melanin, GM, and GAG. We also found that the collagen-like domain of SP-D is involved in its interaction with melanin, whereas its carbohydrate-recognition domain recognized GM and GAG. Unlike un-opsonized conidia, SP-D-opsonized conidia were phagocytosed more efficiently and stimulated the secretion of proinflammatory cytokines by human monocyte-derived macrophages. Furthermore, mice challenged intranasally with wildtype conidia or melanin ghosts ( hollow melanin spheres) displayed significantly reduced proinflammatory cytokines in the lung compared with wildtype mice. In summary, SP-D binds to melanin present on the dormant conidial surface, facilitates conidial phagocytosis, and stimulates the host immune response.
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http://dx.doi.org/10.1074/jbc.M117.815852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880149PMC
March 2018

The Absence of NOD1 Enhances Killing of Through Modulation of Dectin-1 Expression.

Front Immunol 2017 13;8:1777. Epub 2017 Dec 13.

Laboratory for Experimental Internal Medicine, Department of Internal Medicine, Radboud University Medical Center, Nijmegen, Netherlands.

One of the major life-threatening infections for which severely immunocompromised patients are at risk is invasive aspergillosis (IA). Despite the current treatment options, the increasing antifungal resistance and poor outcome highlight the need for novel therapeutic strategies to improve outcome of patients with IA. In the current study, we investigated whether and how the intracellular pattern recognition receptor NOD1 is involved in host defense against . When exploring the role of NOD1 in an experimental mouse model, we found that mice were protected against IA and demonstrated reduced fungal outgrowth in the lungs. We found that macrophages derived from bone marrow of mice were more efficiently inducing reactive oxygen species and cytokines in response to . Most strikingly, these cells were highly potent in killing compared with wild-type cells. In line, human macrophages in which NOD1 was silenced demonstrated augmented killing and NOD1 stimulation decreased fungal killing. The differentially altered killing capacity of NOD1 silencing versus NOD1 activation was associated with alterations in dectin-1 expression, with activation of NOD1 reducing dectin-1 expression. Furthermore, we were able to demonstrate that mice have elevated dectin-1 expression in the lung and bone marrow, and silencing of gene expression in human macrophages increases dectin-1 expression. The enhanced dectin-1 expression may be the mechanism of enhanced fungal killing of cells and human cells in which NOD1 was silenced, since blockade of dectin-1 reversed the augmented killing in these cells. Collectively, our data demonstrate that NOD1 receptor plays an inhibitory role in the host defense against . This provides a rationale to develop novel immunotherapeutic strategies for treatment of aspergillosis that target the NOD1 receptor, to enhance the efficiency of host immune cells to clear the infection by increasing fungal killing and cytokine responses.
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http://dx.doi.org/10.3389/fimmu.2017.01777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733348PMC
December 2017

MybA, a transcription factor involved in conidiation and conidial viability of the human pathogen Aspergillus fumigatus.

Mol Microbiol 2017 Sep 21;105(6):880-900. Epub 2017 Jul 21.

Unité des Aspergillus, Institut Pasteur, Paris 75015, France.

Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA-sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane-associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus.
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http://dx.doi.org/10.1111/mmi.13744DOI Listing
September 2017

Treatment of Cyclosporin A retains host defense against invasive pulmonary aspergillosis in a non-immunosuppressive murine model by preserving the myeloid cell population.

Virulence 2017 11 6;8(8):1744-1752. Epub 2017 Jul 6.

b Unité Cytokines & Inflammation , Institut Pasteur , Paris , France.

Cyclosporin A (CsA) is widely used as an immunosuppressive agent for organ transplant recipients. CsA inhibits calcineurin, which is highly conserved in mammals and fungi, and thus affects both types of organism. In mammals, the immunosuppressive effect of CsA is via hampering T cell activation. In fungi, the growth inhibitory effect of CsA is via interference with hyphal growth. The aim of this study was to determine whether CsA renders mice susceptible to invasive pulmonary aspergillosis (IPA) and whether it can protect immunosuppressed mice from infection. We therefore examined both the antifungal and the immunosuppressive activity of CsA in immunosuppressed and in immunocompetent mice infected with Aspergillus fumigatus to model IPA. We found that daily injections of CsA could not produce an antifungal effect sufficient to rescue immunosuppressed mice from lethal IPA. However, a 100% survival rate was obtained in non-immunosuppressed mice receiving daily CsA, indicating that CsA did not render the mice vulnerable to IPA. The lymphocyte subset was significantly suppressed by CsA, while the myeloid subset was not. Therefore, we speculate that CsA does not impair the host defense against IPA since the myeloid cells are preserved.
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http://dx.doi.org/10.1080/21505594.2017.1339007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5810467PMC
November 2017

Administration of Zinc Chelators Improves Survival of Mice Infected with Aspergillus fumigatus both in Monotherapy and in Combination with Caspofungin.

Antimicrob Agents Chemother 2016 10 23;60(10):5631-9. Epub 2016 Sep 23.

Institut Pasteur, Cytokines & Inflammation, Département Infection & Epidemiology, Paris, France

Aspergillus fumigatus can infect immunocompromised patients, leading to high mortality rates due to the lack of reliable treatment options. This pathogen requires uptake of zinc from host tissues in order to successfully grow and cause virulence. Reducing the availability of that micronutrient could help treat A. fumigatus infections. In this study, we examined the in vitro effects of seven chelators using a bioluminescent strain of A. fumigatus 1,10-Phenanthroline and N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) proved to be the chelators most effective at inhibiting fungal growth. Intraperitoneal administration of either phenanthroline or TPEN resulted in a significant improvement in survival and decrease of weight loss and fungal burden for immunosuppressed mice intranasally infected with A. fumigatus In vitro both chelators had an indifferent effect when employed in combination with caspofungin. The use of TPEN in combination with caspofungin also significantly increased survival compared to that when using these drugs individually. Our results suggest that zinc chelation may be a valid strategy for dealing with A. fumigatus infections and that both phenanthroline and TPEN could potentially be used either independently or in combination with caspofungin, indicating that their use in combination with other antifungal treatments might also be applicable.
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http://dx.doi.org/10.1128/AAC.00324-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038287PMC
October 2016

Reducing hypoxia and inflammation during invasive pulmonary aspergillosis by targeting the Interleukin-1 receptor.

Sci Rep 2016 05 24;6:26490. Epub 2016 May 24.

Unité de recherche Cytokines &Inflammation, Institut Pasteur, Paris.

Hypoxia as a result of pulmonary tissue damage due to unresolved inflammation during invasive pulmonary aspergillosis (IPA) is associated with a poor outcome. Aspergillus fumigatus can exploit the hypoxic microenvironment in the lung, but the inflammatory response required for fungal clearance can become severely disregulated as a result of hypoxia. Since severe inflammation can be detrimental to the host, we investigated whether targeting the interleukin IL-1 pathway could reduce inflammation and tissue hypoxia, improving the outcome of IPA. The interplay between hypoxia and inflammation was investigated by in vivo imaging of hypoxia and measurement of cytokines in the lungs in a model of corticosteroid immunocompromised and in Cxcr2 deficient mice. Severe hypoxia was observed following Aspergillus infection in both models and correlated with development of pulmonary inflammation and expression of hypoxia specific transcripts. Treatment with IL-1 receptor antagonist reduced hypoxia and slightly, but significantly reduced mortality in immunosuppressed mice, but was unable to reduce hypoxia in Cxcr2(-/-) mice. Our data provides evidence that the inflammatory response during invasive pulmonary aspergillosis, and in particular the IL-1 axis, drives the development of hypoxia. Targeting the inflammatory IL-1 response could be used as a potential immunomodulatory therapy to improve the outcome of aspergillosis.
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http://dx.doi.org/10.1038/srep26490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877709PMC
May 2016

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

PLoS One 2016 14;11(4):e0153829. Epub 2016 Apr 14.

Institut Pasteur, Unité Cytokines & Inflammation, Paris, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0153829PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4831689PMC
September 2016

Targeting zinc homeostasis to combat Aspergillus fumigatus infections.

Front Microbiol 2015 27;6:160. Epub 2015 Feb 27.

Instituto de Biología Funcional y Genómica, Centro Mixto del Consejo Superior de Investigaciones Científicas y Universidad de Salamanca , Salamanca, Spain ; Departamento de Microbiología y Genética, Universidad de Salamanca , Salamanca, Spain.

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.
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http://dx.doi.org/10.3389/fmicb.2015.00160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343018PMC
March 2015

Assessment of efficacy of antifungals against Aspergillus fumigatus: value of real-time bioluminescence imaging.

Antimicrob Agents Chemother 2013 Jul 15;57(7):3046-59. Epub 2013 Apr 15.

Institut Pasteur Unité de Recherche Cytokines & Inflammation, Paris, France.

Aspergillus fumigatus causes life-threatening infections, especially in immunocompromised patients. Common drugs for therapy of aspergillosis are polyenes, azoles, and echinocandins. However, despite in vitro efficacy of these antifungals, treatment failure is frequently observed. In this study, we established bioluminescence imaging to monitor drug efficacy under in vitro and in vivo conditions. In vitro assays confirmed the effectiveness of liposomal amphotericin B, voriconazole, and anidulafungin. Liposomal amphotericin B and voriconazole were fungicidal, whereas anidulafungin allowed initial germination of conidia that stopped elongation but allowed the conidia to remain viable. In vivo studies were performed with a leukopenic murine model. Mice were challenged by intranasal instillation with a bioluminescent reporter strain (5 × 10(5) and 2.5 × 10(5) conidia), and therapy efficacies of liposomal amphotericin B, voriconazole, and anidulafungin were monitored. For monotherapy, the highest treatment efficacy was observed with liposomal amphotericin B, whereas the efficacies of voriconazole and anidulafungin were strongly dependent on the infectious dose. When therapy efficacy was studied with different drug combinations, all combinations improved the rate of treatment success compared to that with monotherapy. One hundred percent survival was obtained for treatment with a combination of liposomal amphotericin B and anidulafungin, which prevented not only pulmonary infections but also infections of the sinus. In conclusion, combination therapy increases treatment success, at least in the murine infection model. In addition, our novel approach based on real-time imaging enables in vivo monitoring of drug efficacy in different organs during therapy of invasive aspergillosis.
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http://dx.doi.org/10.1128/AAC.01660-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697358PMC
July 2013

Murine infection models for Aspergillus terreus pulmonary aspergillosis reveal long-term persistence of conidia and liver degeneration.

J Infect Dis 2012 Apr;205(8):1268-77

Department of Microbial Pathogenicity Mechanisms and Junior Research Group Microbial Biochemistry and Physiology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany.

Aspergillus terreus is emerging as a causative agent of life-threatening invasive aspergillosis. Prognosis for affected patients is often worse than for A. fumigatus infections. To study A. terreus-mediated disease, we developed 3 infection models. In embryonated hen's eggs and leucopenic mice, the outcome of invasive aspergillosis was similar to that described for A. fumigatus. However, 10(2)- and 10(3)-fold higher conidia concentrations were required for 100% lethality. In corticosteroid-treated mice, only 50% mortality was observed, although bioluminescence imaging revealed transient disease in all infected animals. In surviving animals, we observed persistence of ungerminated but viable conidia. Cytokine levels in these mice were comparable to uninfected controls. In contrast to A. fumigatus infections, all mice infected with A. terreus developed fatty liver degeneration, suggesting the production of toxic secondary metabolites. Thus, at least in mice, persistence and subclinical liver damage are unique features of A. terreus infections.
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http://dx.doi.org/10.1093/infdis/jis193DOI Listing
April 2012

Duality of liver and kidney lesions after systemic infection of immunosuppressed and immunocompetent mice with Aspergillus fumigatus.

Virulence 2012 Jan-Feb;3(1):43-50. Epub 2012 Jan 1.

Institut Pasteur, Unité Histopathologie Humaine et Modèles Animaux, Département Infection et Epidémiologie, Paris, France.

Invasive aspergillosis is a life-threatening disease mainly caused by Aspergillus fumigatus. Patients at risk are generally immunocompromised and lungs are assumed to provide the primary site for infection and invasive disease manifestation. Contrarily, visceral organ involvement appears to result from a subsequent hematogenous spread. To compare the kinetics of dissemination within deep organs in immunosuppressed vs. immunocompetent mice, we used a bioluminescent A. fumigatus strain in an intravenous infection model. By applying an immunosuppressive regimen with corticosteroids, dissemination to the liver and kidneys was observed already 24 h after inoculation accompanied by a marked inflammatory response within the liver. In contrast, in the immunocompetent condition, fungal growth and inflammation were mainly restricted to the kidneys and only small amounts of fungal biomass and a weak inflammatory response were detected in the liver. Additionally, disease progressed much slower compared with the immunosuppressed condition. This is the first study underlying the duality between liver and renal tropism of A. fumigatus in relation to the immune status of the host.
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http://dx.doi.org/10.4161/viru.3.1.18654DOI Listing
September 2012

The crucial role of the Aspergillus fumigatus siderophore system in interaction with alveolar macrophages.

Microbes Infect 2010 Nov 24;12(12-13):1035-41. Epub 2010 Jul 24.

Division of Molecular Biology/Biocenter, Innsbruck Medical University, Fritz-Pregl-Str. 3, 6020 Innsbruck, Austria.

Iron plays a central role in manifestation of infections for a variety of pathogens. To ensure an adequate supply with iron, Aspergillus fumigatus employs extra- and intracellular siderophores (low-molecular mass iron chelators), which are of importance for fungal growth in particular during iron starvation. Here we show that the lack of extracellular siderophores, and especially, the lack of the entire siderophore system cause in immunosuppressed mice in vivo (i) a reduced extracellular growth rate, (ii) a reduced intracellular growth rate in alveolar macrophages, and (iii) an increased susceptibility to conidial growth inhibition by alveolar macrophages. These data underline the crucial role of the fungal siderophore system not only for extracellular growth but also in the interaction with the host immune cells. Moreover, the hyphal growth rate within alveolar macrophages compared to extracellular lavage fluid was significantly decreased indicating that, besides elimination of fungal conidia, inhibition of pathogenic growth is a function of macrophages.
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http://dx.doi.org/10.1016/j.micinf.2010.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2977081PMC
November 2010

In vivo bioluminescence imaging and histopathopathologic analysis reveal distinct roles for resident and recruited immune effector cells in defense against invasive aspergillosis.

BMC Microbiol 2010 Apr 8;10:105. Epub 2010 Apr 8.

Unité de Recherche Cytokines & Inflammation, Institut Pasteur Paris France.

Background: Invasive aspergillosis (IA) is a major cause of infectious morbidity and mortality in immune compromised patients. Studies on the pathogenesis of IA have been limited by the difficulty to monitor disease progression in real-time. For real-time monitoring of the infection, we recently engineered a bioluminescent A. fumigatus strain.

Results: In this study, we demonstrate that bioluminescence imaging can track the progression of IA at different anatomic locations in a murine model of disease that recapitulates the natural route of infection. To define the temporal and functional requirements of distinct innate immune cellular subsets in host defense against respiratory A. fumigatus infection, we examined the development and progression of IA using bioluminescence imaging and histopathologic analysis in mice with four different types of pharmacologic or numeric defects in innate immune function that target resident and recruited phagocyte subsets. While bioluminescence imaging can track the progression and location of invasive disease in vivo, signals can be attenuated by severe inflammation and associated tissue hypoxia. However, especially under non-inflammatory conditions, such as cyclophosphamide treatment, an increasing bioluminescence signal reflects the increasing biomass of alive fungal cells.

Conclusions: Imaging studies allowed an in vivo correlation between the onset, peak, and kinetics of hyphal tissue invasion from the lung under conditions of functional or numeric inactivation of phagocytes and sheds light on the germination speed of conidia under the different immunosuppression regimens. Conditions of high inflammation -either mediated by neutrophil influx under corticosteroid treatment or by monocytes recruited during antibody-mediated depletion of neutrophils- were associated with rapid conidial germination and caused an early rise in bioluminescence post-infection. In contrast, 80% alveolar macrophage depletion failed to trigger a bioluminescent signal, consistent with the notion that neutrophil recruitment is essential for early host defense, while alveolar macrophage depletion can be functionally compensated.
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http://dx.doi.org/10.1186/1471-2180-10-105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2859869PMC
April 2010

Bioluminescent Aspergillus fumigatus, a new tool for drug efficiency testing and in vivo monitoring of invasive aspergillosis.

Appl Environ Microbiol 2008 Nov 26;74(22):7023-35. Epub 2008 Sep 26.

Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Junior Research Group Microbial Biochemistry and Physiology, Beutenbergstr. 11a, 07745 Jena, Germany.

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.
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http://dx.doi.org/10.1128/AEM.01288-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2583481PMC
November 2008

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

BMC Microbiol 2008 Jun 18;8:97. Epub 2008 Jun 18.

Université Paris 12, Créteil, France.

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.
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http://dx.doi.org/10.1186/1471-2180-8-97DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2440385PMC
June 2008

Methylcitrate synthase from Aspergillus fumigatus is essential for manifestation of invasive aspergillosis.

Cell Microbiol 2008 Jan;10(1):134-48

Aspergillus Unit, Institute Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France.

Invasive aspergillosis is a life-threatening disease mainly caused by the fungus Aspergillus fumigatus. In immunocompromised individuals conidia are not efficiently inactivated, which can end in invasive fungal growth. However, the metabolic requirements of the fungus are hardly known. Earlier investigations revealed an accumulation of toxic propionyl-CoA in a methylcitrate synthase mutant, when grown on propionyl-CoA-generating carbon sources. During invasive growth propionyl-CoA could derive from proteins, which are released from infected host tissues. We therefore assumed that a methylcitrate synthase mutant might display an attenuated virulence. Here we show that the addition of propionate to cell culture medium enhanced the ability of alveolar macrophages to kill methylcitrate synthase mutant but not wild-type conidia. When tested in a murine infection model, the methylcitrate synthase mutant displayed attenuated virulence and, furthermore, was cleared from tissues when mice survived the first phase of acute infection. The amplification of cDNA from infected mouse lungs confirmed the transcription of the methylcitrate synthase gene during invasion, which leads to the suggestion that amino acids indeed serve as growth-supporting nutrients during invasive growth of A. fumigatus. Thus, blocking of methylcitrate synthase activity abrogates fungal growth and provides a suitable target for new antifungals.
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http://dx.doi.org/10.1111/j.1462-5822.2007.01025.xDOI Listing
January 2008

The regulation of zinc homeostasis by the ZafA transcriptional activator is essential for Aspergillus fumigatus virulence.

Mol Microbiol 2007 Jun;64(5):1182-97

Instituto de Microbiología-Bioquímica, Centro mixto CSIC/USAL, Departamento de Microbiología y Genética (Universidad de Salamanca). Laboratory 218. Plaza Doctores de la Reina s/n. 37007 Salamanca, Spain.

We have previously shown that Aspergillus fumigatus is able to grow in zinc-limiting media and that this ability is regulated at transcriptional level by both the availability of zinc and pH. When A. fumigatus grows as a pathogen, it must necessarily obtain zinc from the zinc-limiting environment provided by host tissue. Accordingly, the regulation of zinc homeostasis by some zinc-responsive transcriptional regulator in A. fumigatus must be essential for fungal growth within tissues of an immunocompromised host and, in turn, for pathogenicity. Here we provide evidence of the role of the zafA gene in regulating zinc homeostasis and its relevance in the virulence of A. fumigatus. Thus, we observed that (i) zafA can functionally replace the ZAP1 gene from Saccharomyces cerevisiae that encodes the zinc-responsive transcriptional activator Zap1 protein; (ii) the expression of zafA itself is induced in zinc-limiting media and repressed by zinc; (iii) deletion of zafA impairs the germination and growth capacity of A. fumigatus in zinc-limiting media; and (iv) the deletion of zafA abrogates A. fumigatus virulence in a murine model of invasive aspergillosis. In light of these observations, we concluded that ZafA is a zinc-responsive transcriptional activator that represents an essential attribute for A. fumigatus pathogenicity. Consequently, ZafA may constitute a new target for the development of chemotherapeutic agents against Aspergillus, because no zafA orthologues have been found in mammals.
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http://dx.doi.org/10.1111/j.1365-2958.2007.05726.xDOI Listing
June 2007

Clinical, mycological and pathological findings in turkeys experimentally infected by Aspergillus fumigatus.

Avian Pathol 2007 Jun;36(3):213-9

UMR INRA, AFSSA, ENVA, UPVM, 956 BIPAR Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, France.

Experimental aspergillosis was induced in 1-day-old turkeys by intra-air-sac inoculation of a spore suspension of a 3-day-old Aspergillus fumigatus culture (CBS 144.89) containing 10(7) spores. Ten additional poults were used as controls. Infected and non-infected animals were closely observed at least twice a day for the appearance of clinical signs and were sequentially sacrificed at days 1, 2, 3, 5 and 7 post-inoculation. In the infected group, most lung tissues and air sac swabs were culture positive from day 1 to day 5. At 1 day post-inoculation, air sac membranes were multifocally and moderately to severely thickened by an oedema and covered by an exudate. A small number of germinating conidia were present in the superficial exudate, already giving rise to small radiating hyphae. Lung lesions were mild, dominated by a diffuse congestion and a mild heterophilic infiltration. From 2 to 3 days post-inoculation, air sac membranes were more severely affected and several granulomas were observed. Both granulomas and exudates were rich in germinated conidia and hyphae. Pulmonary lesions consisted in a diffuse pneumonia. Five days post-inoculation, air sac membrane lesions progressed to a severe, multifocal, heterophilic and granulomatous inflammation. Seven days post-inoculation, a reduction of the severity of the diffuse pneumonia was detected. Concomitantly, the fungal elements were mainly observed as fragmented tubules in the cytoplasm of multinucleate giant cells. The present study demonstrated that healthy turkey poults might be able to withstand exposure to 10(7) A. fumigatus spores.
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http://dx.doi.org/10.1080/03079450701332337DOI Listing
June 2007

Characterization of the SKN7 ortholog of Aspergillus fumigatus.

Fungal Genet Biol 2007 Jul 30;44(7):682-90. Epub 2007 Jan 30.

Unité des Aspergillus, Institut Pasteur, 25 rue du Docteur Roux, 75724 Cedex 15, France.

Reactive oxidant intermediates play a major role in the killing of Aspergillus fumigatus by phagocytes. In yeasts, SKN7 is a transcription factor contributing to the oxidative stress response. We investigated here the role of afSkn7p in the adaptation of A. fumigatus against oxidative stress. To analyze functionally the afSKN7 in A. fumigatus, we modified a quick PCR fusion methodology for targeted deletion in A. fumigatus. The afskn7Delta mutant was morphologically similar to the wild-type strain, but showed a growth inhibition phenotype associated with hydrogen peroxide and tert-butyl hydroperoxide. However, no significant virulence differences were observed between wild type, mutant and reconstituted strains in a murine model of pulmonary aspergillosis. This result indicated that an increased sensitivity of A. fumigatus to peroxides in vitro is not correlated with a modification of fungal virulence.
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http://dx.doi.org/10.1016/j.fgb.2007.01.009DOI Listing
July 2007

Aspergillus fumigatus does not require fatty acid metabolism via isocitrate lyase for development of invasive aspergillosis.

Infect Immun 2007 Mar 18;75(3):1237-44. Epub 2006 Dec 18.

Leibniz Institute for Natural Products Research and Infection Biology, Beutenbergstr. 11a, 07745 Jena, Germany.

Aspergillus fumigatus is the most prevalent airborne filamentous fungus causing invasive aspergillosis in immunocompromised individuals. Only a limited number of determinants directly associated with virulence are known, and the metabolic requirements of the fungus to grow inside a host have not yet been investigated. Previous studies on pathogenic microorganisms, i.e., the bacterium Mycobacterium tuberculosis and the yeast Candida albicans, have revealed an essential role for isocitrate lyase in pathogenicity. In this study, we generated an isocitrate lyase deletion strain to test whether this strain shows attenuation in virulence. Results have revealed that isocitrate lyase from A. fumigatus is not required for the development of invasive aspergillosis. In a murine model of invasive aspergillosis, the wild-type strain, an isocitrate lyase deletion strain, and a complemented mutant strain were similarly effective in killing mice. Moreover, thin sections demonstrated invasive growth of all strains. Additionally, thin sections of lung tissue from patients with invasive aspergillosis stained with anti-isocitrate lyase antibodies remained negative. From these results, we cannot exclude the use of lipids or fatty acids as a carbon source for A. fumigatus during invasive growth. Nevertheless, test results do imply that the glyoxylate cycle from A. fumigatus is not required for the anaplerotic synthesis of oxaloacetate under infectious conditions. Therefore, an antifungal drug inhibiting fungal isocitrate lyases, postulated to act against Candida infections, is assumed to be ineffective against A. fumigatus.
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http://dx.doi.org/10.1128/IAI.01416-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1828595PMC
March 2007

Aspergillus fumigatus induces innate immune responses in alveolar macrophages through the MAPK pathway independently of TLR2 and TLR4.

J Immunol 2006 Sep;177(6):3994-4001

Unité des Aspergillus, Institut National de la Santé et de la Recherche Médicale E336, Institut Pasteur, Paris, France.

Aspergillus fumigatus causes invasive aspergillosis in immunosuppressed patients. In the immunocompetent host, inhaled conidia are cleared by alveolar macrophages. The signaling pathways of the alveolar macrophage involved in the clearance of A. fumigatus are poorly understood. Therefore, we investigated the role of TLRs in the immune response against A. fumigatus and their contribution to the signaling events triggered in murine alveolar macrophages upon infection with A. fumigatus conidia. Specifically, we examined the MAPKs and NF-kappaB activation and cytokine signaling. Our investigations revealed that immunocompetent TLR2, TLR4, and MyD88 knockout mice were not more susceptible to invasive aspergillosis as compared with wild-type mice and that the in vitro phosphorylation of the MAPKs ERK and p38 was not affected in TLR2, TLR4, or MyD88 knockout mice following stimulation with conidia. In vivo experiments suggest that ERK was an essential MAPK in the defense against A. fumigatus, whereas the activation of NF-kappaB appeared to play only a secondary role. In conclusion, our findings demonstrate that TLR2/4 recognition and MyD88 signaling are dispensable for the clearance of A. fumigatus under immunocompetent situations. Furthermore, our data stress the important role of ERK activation in innate immunity to A. fumigatus.
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http://dx.doi.org/10.4049/jimmunol.177.6.3994DOI Listing
September 2006

Glycosylphosphatidylinositol-anchored Ecm33p influences conidial cell wall biosynthesis in Aspergillus fumigatus.

Appl Environ Microbiol 2006 May;72(5):3259-67

Aspergillus Unit, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.

ECM33 encodes a glycosylphosphatidylinositol-anchored protein whose orthologs in yeast are essential for sporulation. Aspergillus fumigatus Ecm33p is unique and has an apparent mass of 55 kDa. Disruption of A. fumigatus ECM33 results in a mutant with several morphogenetic aberrations, including the following: (i) a defect in conidial separation, (ii) an increase in the diameter of the conidia of the mutant associated with an increase in the concentration of the cell wall chitin, (iii) conidia that were sensitive to the absence of aeration during long-term storage, and (iv) conidia that were more resistant to killing by phagocytes, whereas the mycelium was more easily killed by neutrophils.
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http://dx.doi.org/10.1128/AEM.72.5.3259-3267.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1472355PMC
May 2006

Aspergillus fumigatus conidia inhibit tumour necrosis factor- or staurosporine-induced apoptosis in epithelial cells.

Int Immunol 2006 Jan 15;18(1):139-50. Epub 2005 Dec 15.

INRA, AFSSA, ENVA, UPVM, UMR 956; 22 rue Curie, Maisons Alfort Cedex F-94700, France.

A major innate immune response to inhaled conidia of the opportunistic pathogen Aspergillus fumigatus (Af) is the synthesis of pro-inflammatory cytokines, which include tumour necrosis factor (TNF)-alpha, a known inducer of apoptosis. Modulation of host cell apoptosis has been reported to be one of the mechanisms whereby pathogens overcome host cell defences. Our study was designed to investigate whether or not Af conidia could modulate apoptosis induced by TNF-alpha or staurosporine (STS). Exposure of epithelial cells treated by these inducers and exposed to Af conidia decreased the number of apoptotic cells detected by Annexin V staining, analysis of nuclear morphology, terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end-labelling reaction and immunoblotting. Inhibition of apoptosis by Af conidia was seen in cells of the A549 pneumocyte II line, human tracheal epithelial 16HBE and primary human respiratory cells. Inhibition of apoptosis by Af conidia was also observed when apoptosis was induced by co-cultivating A549 cells with activated human alveolar macrophages. Unlike Af conidia, conidia of Cladosporium cladosporioides as well as latex beads or killed Af conidia have no inhibitory effect on TNF-alpha or STS-induced apoptosis. For TNF-induced apoptosis, the observed anti-apoptotic effect of Af conidia was found to be associated with a significant reduction of caspase-3.
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http://dx.doi.org/10.1093/intimm/dxh356DOI Listing
January 2006

Phosphatidylinositol-dependent phospholipases C Plc2 and Plc3 of Candida albicans are dispensable for morphogenesis and host-pathogen interaction.

Res Microbiol 2005 Aug;156(7):822-9

Unité Postulante Biologie et Pathogénicité Fongiques, INRA USC 2019, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France.

Phospholipases play an important role as virulence factors in human pathogens. Candida albicans, the major fungal pathogen of humans, encodes phospholipases of type A, B, C and D. Type B Plb2 and type D Pld1 phospholipases have been shown to contribute to virulence in this organism. We analyzed, in C. albicans, PLC2 and PLC3, two highly conserved genes coding for phosphatidylinositol-dependent phospholipases C with homology to the known virulence factor PlcA in the human pathogen Listeria monocytogenes. We show that expression of PLC2 and PLC3 is upregulated under different filament-inducing conditions and in the constitutive filamentous mutant tup1Delta. In order to analyze PLC2 and PLC3 function in C. albicans, we constructed strains that carry PLC2 or PLC3 under a constitutive promoter and strains that lack all four PLC2/3 alleles. These strains were not affected in their ability to produce filaments under non-inducing conditions, nor was filamentation modified under inducing conditions, suggesting that PLC2/3 are not critical determinants of the yeast-to-hypha switch. In a cell culture model for macrophage interaction, phagocytosis of C. albicans and subsequent killing were not influenced by PLC2/3. These results demonstrate that C. albicans PLC2 and PLC3 are dispensable for virulence; moreover, they underline the sharp contrast with the function of plcA in L. monocytogenes.
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http://dx.doi.org/10.1016/j.resmic.2005.04.007DOI Listing
August 2005

The Aspergillus fumigatus mepB gene encodes an 82 kDa intracellular metalloproteinase structurally related to mammalian thimet oligopeptidases.

Microbiology (Reading) 1997 Jul;143 ( Pt 7):2247-2253

Laboratoire des Aspergillus, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.

Aspergillus fumigatus produces an 82 kDa intracellular metalloproteinase that hydrolyses the Pz-peptide, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg, a typical substrate of members of the thimet oligopeptidase family which is ubiquitously distributed across animal species. The A. fumigatus mepB gene encoding this 82 kDa metalloproteinase was cloned and sequenced. Analysis of the deduced amino acid sequence of mepB showed that the MepB protein is a cytosolic zinc metalloproteinase of the thimet oligopeptidase family (M3) and as such is probably involved in the intracellular degradation of small peptides. An A. fumigatus mutant that lacks the MepB Pz-peptidolytic activity was constructed by gene disruption at the mepB locus. Analysis of this mutant did not reveal any detectable phenotype.
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http://dx.doi.org/10.1099/00221287-143-7-2247DOI Listing
July 1997
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