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Background: Aspergillus fumigatus is a pathogenic fungus which causes a range of diseases, particularly in the human lung. The pathological mechanism is unknown but may involve a complex mixture of biomolecules which can diffuse from the spore surface. This material is known as A fumigatus diffusate (AfD) and has previously been shown to have a range of immunosuppressive functions. It is hypothesised that AfD may influence the binding of spores to extracellular matrix (ECM) proteins and lung epithelial cells, thereby affecting the ability of the fungus to cause infection., Methods: The binding of spores to ECM proteins and to epithelial cells was carried out using a direct binding assay in microtitre plates and spores were counted by phase contrast microscopy. Rat bronchoalveolar lavage (BAL) fluid was enriched for surfactant protein D (SP-D) using maltose agarose affinity chromatography. The effects of AfD and the SP-D enriched BAL fluid were assessed by pre-incubation with ECM proteins or epithelial cells in the direct binding assay., Results: AfD enhanced the binding of spores to laminin by 137% and to A549 epithelial cells by 250%. SP-D enriched BAL fluid inhibited spore binding to ECM proteins and epithelial cells. Pre-incubation of ECM proteins and epithelial cells with SP-D enriched BAL fluid prevented the enhancement of spore binding by AfD, and pre-incubation of ECM proteins and epithelial cells with AfD prevented the inhibition of spore binding by SP-D enriched BAL fluid. This pretreatment did not prevent the enhancement of spore binding, giving an increase of 95% for collagen I, 80% for fibronectin, 75% for laminin, and 150% for A549 cells., Conclusions: The hypothesis that AfD would affect spore binding to ECM proteins and epithelial cells was confirmed. Rat BAL fluid, with SP-D as the possible bioactive agent, prevented this enhancement. The in vivo significance is unclear but the enhanced binding of spores may increase the chance of fungal infection in the lung which could be prevented by the protective effects of lung surfactant components (possibly SP-D). The results suggest that there may be competition between AfD and a BAL fluid component (possibly SP-D) for the same or similar binding sites on ECM proteins and epithelial cells. Whether this competition occurs in vivo requires further investigation.

Background: The fungus Aspergillus fumigatus, whose spores are present ubiquitously in the air, causes a range of diseases in the human lung. A small molecular weight (< 10 kD) heat stable toxin released from the spores of clinical and environmental isolates of A fumigatus within minutes of deposition in aqueous solution has previously been described. A key effect of the toxin was to inhibit the oxidative burst of macrophages as measured by superoxide anion release. It was hypothesised that the toxin was one of the commonly found A fumigatus hyphal toxins such as gliotoxin. This inhibitor may be an important factor which allows the fungus to colonise the lung., Methods: The spore derived inhibitor was shown to inhibit the respiratory burst of rat alveolar macrophages, as measured by the generation of superoxide anion. Samples of the spore diffusate were subject to reversed phase high performance liquid chromatography (HPLC), thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), or organic extraction followed by TLC or HPLC to identify the presence of gliotoxin, fumagillin, helvolic acid, fumigaclavine-C, and aurasperone-C. Commercially obtained preparations of the toxins gliotoxin, fumagillin and helvolic acid and extracts enriched for fumigaclavine-C and aurasperone-C were used as internal and external standards and in the respiratory burst measurements., Results: Gliotoxin, fumagillin, helvolic acid, fumigaclavine-C, and aurasperone-C were not detected in spore derived diffusate using PHLC or TLC. Using extraction procedures with solvents known to extract gliotoxin from A fumigatus culture supernatants, no gliotoxin was detected in the spore derived diffusate. Commercial gliotoxin, fumagillin, and helvolic acid or extracts enriched for fumigaclavine-C and aurasperone-C did not inhibit the oxidative burst of macrophages., Conclusions: The hypothesis that the spore derived toxin is one of the toxins derived from hyphae such as gliotoxin, helvolic acid, fumagillin, fumigaclavine-C, or aurasperone-C is not proved. The spore toxin may exert its effect through its ability to diffuse rapidly into the lung lining fluid, diminish the macrophage oxidative burst, and play a part in allowing A fumigatus to persist in the lung and manifest its well known pathogenic effects.

Background: Aspergillus fumigatus is a fungus that grows on dead and decaying organic matter in the environment and whose spores are present ubiquitously in the air. The fungus causes a range of diseases in the human lung. A study was undertaken to demonstrate and partially characterise an inhibitor of the macrophage respiratory burst from the surface of A fumigatus spores that could be an important factor in allowing the fungus to colonise the lung., Methods: The spore-derived inhibitor of the respiratory burst of rat alveolar macrophages, as measured by generation of superoxide anion, was demonstrated in Hank's balanced salt solution extracts of four clinical isolates and an environmental isolate of A fumigatus. The time course of the release of the inhibitor into aqueous solution was assessed and the cytotoxic potential of the spore-derived inhibitor towards macrophages was tested using the propidium iodide method. An oxygen electrode was used to confirm the superoxide anion measurements. Molecular weight cutoff filters were used to determine the size of the inhibitor as assessed in the respiratory burst assay and also by its ability to inhibit macrophage spreading on glass. The crude diffusate from the spore surface was fractionated by reversed phase high pressure liquid chromatography (HPLC) and the fractions analysed for inhibitory activity, protein, and carbohydrate content., Results: A small molecular weight (< 10 kD) heat stable toxin was released from the spores of clinical and environmental isolates of A fumigatus within minutes of deposition in aqueous solution. The key effect of the toxin demonstrated here was its ability to inhibit the oxidative burst of macrophages as measured by superoxide anion release. The inhibition was not due to cell death or detectable loss of membrane integrity as measured by permeability to propidium iodide. The toxin was not a scavenger of superoxide anion. Oxygen electrode studies suggested indirectly that the inhibitor acted to inhibit the assembly of the macrophage NADPH-oxidase complex. Fractions of < 10 kD also inhibited the spreading of alveolar macrophages, confirming that the toxin had an additional effect on macrophages that leads to loss of adherence or impairment of cytoskeletal function. In reversed phase HPLC fractions the inhibitory activity eluted with an associated carbohydrate, although the exact chemical nature of the toxin remains to be elucidated., Conclusions: This spore toxin may, through its ability to diffuse rapidly into lung lining fluid, diminish the macrophage respiratory burst and play a part in allowing A fumigatus to persist in the lung and manifest its well known pathogenic effects. Future research will be focused on further molecular characterisation of the toxin and elaboration of the effect of the toxin on intracellular signalling pathways involved in the activation of alveolar macrophages.