Oumaïma Ibrahim-Granet

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

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.

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

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.

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

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.

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

BackgroundInvasive 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.ResultsIn 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.ConclusionsImaging 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.

Glycosylphosphatidylinositol-Anchored Ecm33p Influences Conidial Cell Wall Biosynthesis in Aspergillus fumigatus

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.

Polymorphism in mitochondrial DNA of several Trichophyton rubrum isolates from clinical specimens

Mitochondrial DNA from six isolates of Trichophyton rubrum (a fungal pathogen of humans) was isolated, purified and digested by restriction endonucleases HindIII, HaeIII, AluI and EcoR1. The isolates could be classified into two groups according to their characteristic fragment patterns. Electron microscopy indicated that T. rubrum mitochondrial DNA are circular molecules of different lengths, 26.85 microns and 21.60 microns respectively, according to the group.

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

BackgroundInvasive 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.ResultsWe 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.ConclusionHNEC 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.

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

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.

Assessment of Efficacy of Antifungals against Aspergillus fumigatus: Value of Real-Time Bioluminescence Imaging

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 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 × 105 and 2.5 × 105 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.

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

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.