[Show abstract][Hide abstract] ABSTRACT: 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 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 remained viable. In vivo studies were performed in a leucopenic murine model. Mice were challenged by intranasal instillation with a bioluminescent reporter strain (5 × 10(5) and 2.5 × 10(5) conidia) and therapy efficacy of liposomal amphotericin B, voriconazole and anidulafungin was followed. In monotherapy, highest treatment efficacy was observed with liposomal amphotericin B, whereas efficacy of voriconazole and anidulafungin was strongly dependent on the infectious dose. When therapy efficacy was studied by different drug combinations, all combinations improved treatment success compared to monotherapy. 100% survival was obtained under treatment with a combination of liposomal amphotericin B and anidulafungin, which not only prevented 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.
Antimicrobial Agents and Chemotherapy 04/2013; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Infectious Diseases 04/2012; 205(8):1268-77. · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Microbes and Infection 11/2010; 12(12-13):1035-41. · 2.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Applied and Environmental Microbiology 10/2008; 74(22):7023-35. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Fungal Genetics and Biology 08/2007; 44(7):682-90. · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Infection and Immunity 04/2007; 75(3):1237-44. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Immunology 10/2006; 177(6):3994-4001. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aspergillus fumigatus is a human pathogen, able to cause invasive aspergillosis in immunosuppressed patients. In the immunocompetent situation inhaled conidia are easily cleared by the immune system. Knowledge of the cellular pathways involved in the innate immunity against A. fumigatus is poorly represented. Therefore, we aimed to investigate the immune response against A. fumigatus in murine alveolar macrophages in terms of MAP kinases, NF-kappaB and cytokine signalling. Our investigations revealed that in murine alveolar macrophages, MAP kinases, ERK and p38 are activated under in vitro conditions, following addition of A. fumigatus conidia. In vivo experiments, however, showed that only ERK is directly involved, because activation of p38 was negligible. Immunosuppression with corticosteroids inhibited phosphorylation of ERK and was directly accompanied with a strongly decreased level of TNF-alpha and additional cytokines. In addition, killing of A. fumigatus conidia is reduced using the ERK inhibitor. Therefore, ERK appears to be an essential MAP kinase in the defence against A. fumigatus. Activation of the transcription factor NFkappaB appeared only at late times after infection suggesting an association with the intracellular swelling of conidia.
Medical mycology: official publication of the International Society for Human and Animal Mycology 09/2006; 44(Supplement_1):S213-S217. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Applied and Environmental Microbiology 06/2006; 72(5):3259-67. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
International Immunology 02/2006; 18(1):139-50. · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phagocytosis and mechanisms of killing of Aspergillus fumigatus conidia by murine alveolar macrophages (AM), which are the main phagocytic cells of the innate immunity of the lung, were investigated. Engulfment of conidia by murine AM lasts 2 h. Killing of A. fumigatus conidia by AM begins after 6 h of phagocytosis. Swelling of the conidia inside the AM is a prerequisite for killing of conidia. The contributions of NADPH oxidase and inducible nitric oxide synthase to the conidicidal activity of AM were studied using AM from OF1, wild-type and congenic p47phox(-/-) 129Sv, and wild-type and congenic iNOS(-/-) C57BL/6 mice. AM from p47phox(-/-) mice were unable to kill A. fumigatus conidia. Inhibitors of NADPH oxidase that decreased the production of reactive oxidant intermediates inhibited the killing of A. fumigatus without altering the phagocytosis rate. In contrast to NADPH oxidase, nitric oxide synthase does not play a role in killing of conidia. Corticosteroids did not alter the internalization of conidia by AM but did inhibit the production of reactive oxidant intermediates and the killing of A. fumigatus conidia by AM. Impairment of production of reactive oxidant intermediates by corticosteroids is responsible for the development of invasive aspergillosis in immunosuppressed mice.
Infection and Immunity 07/2003; 71(6):3034-42. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aspergillus fumigatus is the most prevalent airborne fungal pathogen responsible for fatal invasive aspergillosis in immunocompromised patients. Upon arrival in the lung alveolus, conidia of A. fumigatus are phagocytosed by alveolar macrophages, the major phagocytic cells of the lung. Engulfment and intracellular trafficking of A. fumigatus conidia in alveolar macrophages of two different origins, the murine cell line MH-S and human pulmonary alveolar macrophages, were analyzed by electron microscopy and immunofluorescence. Phagocytosis of A. fumigatus conidia required actin polymerization and phosphatidylinositol 3-kinase activity. Fusion of A. fumigatus phagosomes with early and late endosomes was shown by immunolabeling with specific markers for the transferrin receptor, early endosome antigen, and Rab7. Maturation of A. fumigatus phagolysosomes was monitored by using a fixable acidotropic probe, LysoTracker Red DND-99, and an anti-cathepsin D antibody. Bafilomycin A-induced inhibition of lysosomal acidification abolished the conidial killing by the macrophages. These data suggest that the maturation of A. fumigatus phagosomes results from fusion with the compartments of the endocytic pathway and that the killing of conidia depends on phagolysosome acidification. A model for the phagocytosis of A. fumigatus conidia by alveolar macrophages is proposed on the basis of these results.
Infection and Immunity 03/2003; 71(2):891-903. · 4.07 Impact Factor