Germination of Aspergillus fumigatus conidia in the lungs of normal and cortisone-treated mice.

Inhaled conidia of Aspergillus fumigatus germinated in the lungs of mice at a low rate but both germinated and ungerminated spores were cleared. Spores germinated at a high rate in the lungs of cortisone-treated mice.     

Elimination of Aspergillus fumigatus conidia from the airways of mice with allergic airway inflammation

Background

Aspergillus fumigatus conidia can exacerbate asthma symptoms. Phagocytosis of conidia is a principal component of the host antifungal defense. We investigated whether allergic airway inflammation (AAI) affects the ability of phagocytic cells in the airways to internalize the resting fungal spores.

Methods

Using BALB/c mice with experimentally induced AAI, we tested the ability of neutrophils, macrophages, and dendritic cells to internalize A. fumigatus conidia at various anatomical locations. We used light microscopy and differential cell and conidium counts to determine the ingestion potential of neutrophils and macrophages present in bronchoalveolar lavage (BAL). To identify phagocyte-conidia interactions in conducting airways, conidia labeled with tetramethylrhodamine-(5-(and-6))-isothiocyanate were administered to the oropharyngeal cavity of mice. Confocal microscopy was used to quantify the ingestion potential of Ly-6G⁺ neutrophils and MHC II⁺ antigen-presenting cells located in the intraepithelial and subepithelial areas of conducting airways.

Results

Allergen challenge induced transient neutrophil recruitment to the airways. Application of A. fumigatus conidia at the acute phase of AAI provoked recurrent neutrophil infiltration, and consequently increased the number and the ingestion potential of the airway neutrophils. In the absence of recurrent allergen or conidia provocation, both the ingestion potential and the number of BAL neutrophils decreased. As a result, conidia were primarily internalized by alveolar macrophages in both AAI and control mice at 24 hours post-inhalation. Transient influx of neutrophils to conducting airways shortly after conidial application was observed in mice with AAI. In addition, the ingestion potential of conducting airway neutrophils in mice with induced asthma exceeded that of control mice. Although the number of neutrophils subsequently decreased, the ingestion capacity remained elevated in AAI mice, even at 24 hours post-conidia application.

Conclusions

Aspiration of allergen to sensitized mice enhanced the ingestion potential of conducting airway neutrophils. Such activation primes neutrophils so that they are sufficient to control dissemination of non-germinating A. fumigatus conidia. At the same time, it can be a reason for the development of sensitivity to fungi and subsequent asthma exacerbation.     

Detection of circulating antigen of Aspergillus fumigatus in sera of mice and rabbits by enzyme-linked immunosorbent assay

Circulating antigen of Aspergillus fumigatus was demonstrated in the sera of experimentally infected, cortisone-treated mice and rabbits by enzyme-linked immunosorbent assay (micro-ELISA), confirming earlier results where fungal antigen had been detected by counter-immunoelectrophoresis (CIE). Peaks of detection of circulating antigen by CIE and micro-ELISA in mice were not simultaneous suggesting that the nature of the predominant antigens may have altered during the course of infection. CIE failed to detect fungal antigen in infected rabbits whereas micro-ELISA monitored antigenemia until death. Both CIE and micro-ELISA demonstrated the rapid clearance of intravenously inoculated Aspergillus-antigen from the rabbit circulation.     

Detection of early phase specific surface appendages during germination of Aspergillus fumigatus conidia

During the past 15 years the saprophytic fungus Aspergillus fumigatus has become the most prevalent airborne fungal pathogen, causing severe and often fatal infections especially in immuno-compromised patients. Germination of inhaled conidia is an early and crucial event in the infection process of A. fumigatus. In this study we have analyzed morphological changes that take place during this differentiation process using scanning electron microscopy. Our data show that (i) the hydrophobic surface layer of resting conidia seems to be shed before the cells start to swell and (ii) that filamentous surface appendages are expressed at a very early phase of the germtube formation. These surface structures were only found on the first few microm of the germtube, but were absent from the surface of mycelial hyphae and resting or swollen conidia. The highly regulated expression of these novel surface organelles suggests that they may play an important role during early germination and represent a potential target for future anti-A. fumigatus therapies.     

Characterisation of the phagocytic uptake of Aspergillus fumigatus conidia by macrophages

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for severe, life-threatening infections in immunocompromised patients. Airborne conidia are the infectious agent and can reach the lower parts of the respiratory system. In the lung, phagocytes represent the first line of defence. Resident macrophages are able to track down, engulf and kill the invading spores. Phagocytosis of the conidia is therefore a prerequisite for their efficient elimination. Using human and murine macrophages we analysed the phagocytic uptake of A. fumigatus conidia. We found that conidial phagocytosis is an actin-depending process that additionally requires myosin motor, phosphoinositide-3-phosphate kinase and tyrosine kinase activity. Both broad range tyrosine kinase inhibitors and inhibitors that specifically block src kinases had a strong impact on the conidial uptake. Immunofluorescence data demonstrate the recruitment of tyrosine-phosphorylated proteins to the vicinity of engulfed conidia. Uptake of the conidia was accompanied by a strong and local reorganisation of the actin cytoskeleton, whereas no prominent reorganisation was apparent for the microtubules. Both confocal immunofluorescence and electron microscopic data revealed the presence of large ruffle-like structures engaged in the uptake of conidia. This suggests that the internalisation of A. fumigatus spores can be mediated by a process resembling macropinocytosis, which is furthermore supported by the detection of intracellular conidia within spacious vacuoles. Taken together, our data provide new insights into the internalisation of A. fumigatus spores by macrophages, a key process in the early immune defence against an Aspergillus infection.     

High-resolution cell surface dynamics of germinating Aspergillus fumigatus conidia

We used real-time atomic force microscopy with a temperature-controlled stage (37°C) to probe the structural and physicochemical dynamics of single Aspergillus fumigatus conidia during germination. Nanoscale topographic images of dormant spores revealed the presence of a layer of rodlets made of hydrophobins, in agreement with earlier electron microscopy observations. Within the 3-h germination period, progressive disruption of the rodlet layer was observed, revealing hydrophilic inner cell wall structures. Using adhesion force mapping with hydrophobic tips, these ultrastructural changes were shown to correlate with major differences in cell surface hydrophobicity. That is, the rodlet surface was uniformly hydrophobic due to the presence of hydrophobins, whereas the cell wall material appearing upon germination was purely hydrophilic. This study illustrates the potential of real-time atomic force microscopy imaging and force spectroscopy for tracking cell-surface dynamics.     

Sterol metabolism during germination of conidia of Aspergillus fumigatus.

The sterol content of germinating conidia of the opportunistic pathogenic fungus Aspergillus fumigatus has been correlated with germination phase and sensitivity to polyene antibiotics. The sterol and sterol ester contents of walls did not change during germination. The sterol ester content of membranes and cell sap remained constant during germination, whereas the sterol content increased during the outgrowth of germ tubes. On the basis of differential extraction studies it was concluded that the loss of resistance to polyenes that occurred in the early stages of swelling of conidia during germination was not due to a movement of sterol or sterol ester out of the wall. Radioactive-labelling experiments demonstrated that, although the amounts of conidial wall sterol and sterol ester did not change during germination, they were metabolically active. Changes in the turnover rate of wall and membrane sterol and sterol ester during germination were investigated and their relationship to a possible mechanism for the change from resistance to sensitivity to polyene antibiotics is discussed.     

The absence of VPAC2 leads to aberrant antibody production in Aspergillus fumigatus sensitized and challenged mice

Vasoactive intestinal peptide (VIP) facilitates a “pro-allergy” phenotype when signaling through its G protein-coupled receptor, VPAC₂. We have shown that VPAC₂ knock-out (KO) mice developed an allergic phenotype marked by eosinophilia and elevated serum IgE. Therefore, we hypothesized that the humoral response to allergen challenge in these mice was T_H2 dominant similar to wild-type (WT) C57BL/6 mice. Antibody responses in WT and KO mice were measured after Aspergillus fumigatus conidia inhalation. In contrast to previous reports, basal levels of serum IgG_2a and IgA were significantly higher in naïve VPAC₂ KO animals. Antibody availability in the serum as well as the bronchoalveolar lavage fluid after fungal challenge was dominated by the pro-inflammatory isotype IgG_2a and the mucosal isotype, IgA. IgA localizing cells dominated in the peribronchovascular areas of allergic KO mice while IgE immune complexes were found in WT allergic lungs. This research shows for the first time that VPAC₂ has a significant effect on antibody regulation, in the context of allergy.     

A comparison of aspergillus fumigatus antigens by counterimmunoelectrophoresis

Counterimmunoelectrophoresis (CIE) has been evaluated for routine diagnostic work, using antigens prepared from 2 different isolates of Aspergillus fumigatus, at 2 different concentrations. Additional antigens prepared in a variety of ways and 2 commercially available antigens (Bencard, London; Institut Pasteur, Paris, France) have been compared with the routine antigens. It has been shown that the use of the routine antigens will detect the majority of positive reactions. At optimal reacting concentrations antibodies were detected in 75% of asthmatic patients. In sera from patients with a presumptive diagnosis of allergic aspergillosis, CIE will detect twice as many positive reactions as a conventional agar gel diffusion test.     

Detection of Aspergillus fumigatus hyphae by solid phase cytometry

A rapid method based on solid phase cytometry (SPC) for the detection of Aspergillus fumigatus hyphae is described. With an enzymatic "viability" staining procedure, fungal hyphae can be detected non-specifically within the hour. By combining this procedure with an immunofluorescence labelling, a distinction between Aspergillus spp. and other clinically important fungi is possible, except for Penicillium spp. due to cross-reactivity. To differentiate both genera, microcolonies are generated by incubation at 45 degrees C prior to viability staining. The latter approach in conjunction with immunofluorescence labelling allows a quasi-specific detection of A. fumigatus hyphae and has shown its applicability to samples of bronchoalveolar lavage liquid (BAL).     

PKSP-dependent reduction of phagolysosome fusion and intracellular kill of Aspergillus fumigatus conidia by human monocyte-derived macrophages

Previously, we described the isolation of an Aspergillus fumigatus mutant producing non-pigmented conidia, as a result of a defective polyketide synthase gene, pksP (polyketide synthase involved in pigment biosynthesis). The virulence of the pksP mutant was attenuated in a murine animal infection model and its conidia showed enhanced susceptibility towards damage by monocytes in vitro. Because macrophage-mediated killing is critical for host resistance to aspergillosis, the interaction of both grey-green wild-type conidia and white pksP mutant conidia with human monocyte-derived macrophages (MDM) was studied with respect to intracellular processing of ingested conidia. After phagocytosis, the percentage of wild-type conidia residing in an acidic environment was approximately fivefold lower than that observed for non-pigmented pksP mutant conidia. The phagolysosome formation, as assessed by co-localization of LAMP-1 and cathepsin D with ingested conidia, was significantly lower for wild-type conidia compared with pksP mutant conidia. Furthermore, the intracellular kill of pksP mutant conidia was significantly higher than of wild-type conidia, which was markedly increased by chloroquine, a known enhancer of phagolysosome fusion. Taken together, these findings suggest that the presence of a functional pksP gene in A. fumigatus conidia is associated with an inhibition of phagolysosome fusion in human MDM. These data show for the first time that a fungus has the capability to inhibit the fusion of the phagosome with the lysosome. This finding might help explain the attenuated virulence of pksP mutant strains in a murine animal model and provides a conceptual frame to understand the virulence of A. fumigatus.     

Methods for reducing particle concentrations of Aspergillus fumigatus conidia and mouldy hay dust

The effectiveness of commercially available domestic air purifiers to reduce airborne Aspergillus fumigatus conidia and mouldy hay dust was investigated. It was found that the rate of particle clearance is a function of the volume of air passing through the purifiers but that the low throughflow of air makes their use of little value in clearing particles from a normal sized room. Vacuum cleaners were more effective than air purifiers because of their higher air throughput, so too were high volume fan systems in conjunction with simple filtration units. Ionizers had no effect but steam condensation was very efficient at clearing airborne particles.     

Rapid germination of Aspergillus fumigatus conidia in mouse kidneys and a kidney extract.

Intravenously injected conidia of Aspergillus fumigatus germinated rapidly in the kidneys of untreated and cortisone-treated specific-pathogen-free (SPF) mice and in the livers of cortisone-treated SPF mice. Extracts of kidneys from untreated and cortisone-treated mice stimulated germination of A. fumigatus conidia in vitro. The possible roles of a germination stimulant and host defences in the kidney localisation of A. fumigatus infection are discussed.     

Aspergillus fumigatus adhesion factors in dormant conidia revealed through comparative phenotypic and transcriptomic analyses

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K‐means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high‐adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin‐like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.     

Mastitis by Aspergillus fumigatus in sheep

Several outbreaks of sheep mastitis by Aspergillus fumigatus in Castilla y Leon (Spain), were studied. Only sheep that were treated intramammarily with antibacterial antibiotics during the dry period suffered this mastitis. Mastitis was acute with a morbidity up to 14 % and mortality near 100 %. The udder was markedly enlarged in size, fibrotic, haemorrhagic and with multiple compact nodules, some with purulent material inside; after 30-50 days postpartum, cheesy abscess of several centimetres in diameter were present. Some sheep had granulomatous nodules in the lung. Microscopy and culture shown the presence of A. fumigatus in milk, udder and lung. The route of infection was by intramammary via as a consequence of unhygienic intramammary treatment in the dry period.     

Detection of Aspergillus fumigatus mycotoxins: immunogen synthesis and immunoassay development

Immunological detection of secreted low molecular weight toxins represents a potentially novel means of diagnosing infection by the fungus Aspergillus fumigatus. Two such metabolites, gliotoxin and helvolic acid, were selected and conjugated to thyroglobulin for antisera generation in rabbits. Gliotoxin was initially activated using N-[p-maleimidophenyl] isocyanate (PMPI) and subsequently conjugated to S-acetyl thioglycolic acid N-hydroxysuccinimide-activated thyroglobulin, whereas helvolic acid was activated with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in the presence of thyroglobulin prior to immunisation. To facilitate subsequent antisera evaluation, both toxins were similarly conjugated to bovine serum albumin (BSA). Matrix-Assisted Laser Desorption Ionisation-Time Of Flight (MALDI-TOF) mass spectrometry and SDS-PAGE analysis confirmed covalent attachment of toxins to BSA in the ratios of 15 and 2.4 mol per mol BSA for gliotoxin and helvolic acid, respectively. Resultant high titer antisera were capable of detecting both BSA-conjugated toxins (inhibitory concentration (IC)(50): 4-5 microg/ml). Free toxins were also detectable by competitive immunoassay, whereby 10 microg/ml free gliotoxin (30 microM) and helvolic acid (17 microM), respectively, inhibited antibody binding to cognate toxin-BSA previously immobilised on microwells. This work confirms that sensitive and specific antisera can be raised against fungal toxins and may have an application in diagnosing fungal infection.