Aspergillus fumigatus LaeA-mediated phagocytosis is associated with a decreased hydrophobin layer

Abstract

Aspergillus fumigatus is the causal agent of the life-threatening disease invasive aspergillosis. A. fumigatus laeA deletants, aberrant in toxin biosynthesis and spore development, are decreased in virulence. Among other characteristics, the decreased virulence is associated with increased spore susceptibility to macrophage phagocytosis. Three characteristics, cell wall microbe-associated molecular patterns (MAMPs), secreted metabolites, and rodlet content, thought to be important in macrophage-Aspergillus spore interactions were examined. Flow cytometry analysis of wild-type and DeltalaeA spores did not reveal any differences in surface-accessible MAMPs, including beta-(1,3)-glucan, alpha-mannose, chitin, and other carbohydrate ligands. Blocking experiments with laminarin and mannan supported the conclusion that differences in cell wall carbohydrates were not responsible for enhanced DeltalaeA spore phagocytosis. Aspergillus spores have been reported to secrete metabolites affecting phagocytosis. Neither spent culture exchange, transwell, nor coincubation internalization experiments supported a role for secreted metabolites in the differential uptake of wild-type and DeltalaeA spores. However, sonication assays implicated a role for surface rodlet protein/hydrophobin (RodAp) in differential spore phagocytosis. A possible role of RodAp in enhanced DeltalaeA spore uptake was further assessed by RodAp extraction and quantification, where wild-type spores were found to contain 60% more RodAp than DeltalaeA spores. After removal of the surface rodlet layer, wild-type spores were phagocytosed at similar rates as DeltalaeA spores. We conclude that increased uptake of DeltalaeA resting spores is not associated with changes in secreted metabolite production of this mutant or surface carbohydrate availability but, rather, due to a decrease in the surface RodAp content of DeltalaeA spores. We theorize that RodAp acts as an antiphagocytic molecule, possibly via physicochemical means and/or by impeding MAMP recognition by macrophage receptors.

FIG. 1.

Surface carbohydrate analysis of A. fumigatus wild-type and ΔlaeA spores. (A) Flow cytometry was used to assess carbohydrate accessibility on the cell wall of resting spores. Shown are the results of six carbohydrate moieties analyzed (19 total). The gray lined and tinted histogram represents relative signal on wild-type spores, whereas the unfilled black histogram represents signal on ΔlaeA spores. At least 10,000 spores were counted per histogram, and the percentage of maximum on the x axis refers to the percentage of total counts for a given fluorescence intensity. Relative fluorescence intensity is shown on the y axis. (B) Soluble laminarin and mannan were used in phagocytosis assays to block peritoneal macrophage receptor recognition of carbohydrate ligands (n = 4 mice). Results are presented as the mean ± SEM of two independent experiments.

FIG. 2.

Macrophage phagocytosis comparison of live and dead spores. Spores were killed by formaldehyde fixation and washed at least five times before use. Peritoneal macrophages (n = 4 mice) were combined with spores for 1 h, washed, and incubated an additional 2 h before counterstaining with calcofluor white. Percent uptake was calculated as follows: (number of macrophages containing 1 or more spores/total number of macrophages counted) × 100. Results are presented as the mean percent uptake ± SEM of two independent experiments. *, P < 0.05.

FIG. 3.

Increased uptake of ΔlaeA spores is not related to a secreted product. (A) Secondary metabolites extracted from wild-type or ΔlaeA GMM had no effect on uptake of wild-type spores. Results are shown for wild-type metabolite (WT met) and ΔlaeA metabolite (ΔlaeA met) extracts added to wild-type spores. Similarly, wild-type and ΔlaeA extracts added to ΔlaeA spores had no effect (data not shown). (B) At a 100-fold excess, ΔlaeA spores did not produce transwell-diffusible components to influence the phagocytosis of either live or dead wild-type spores. (C) When ΔlaeA and wild-type spores were combined in the same well with macrophages, phagocytosis of ΔlaeA spores remained significantly higher than wild type. At least two independent experiments were performed with peritoneal macrophages (n = 4 to 8 mice) for each panel. Results are presented as the mean percent uptake ± SEM.

FIG. 4.

An altered rodlet layer may be responsible for increased uptake of ΔlaeA spores. (A) Swollen spores were prepared by incubating in RPMI 10 for 7 h at 300 rpm. Conidial swelling, which results in loss of the rodlet layer, eliminated the difference in peritoneal macrophage uptake between wild-type and ΔlaeA spores. (B) Resting spores were sonicated to remove the rodlet layer, and uptake of sonicated spores was compared to resting spores. The difference in uptake between wild-type and ΔlaeA spores decreased following sonication. Results of two independent experiments per figure (n = 4 mice per experiment) are shown as the mean percent uptake ± SEM. *, P < 0.05.

FIG. 5.

RodAp content is reduced in ΔlaeA spores compared to wild type. An SDS-PAGE (15%) profile is shown for the rodlets extracted from 1 × 10⁷ wild-type or ΔlaeA spores using hydrofluoric acid, wherein the RodAp level was decreased in the ΔlaeA spores.

FIG. 6.

Removal of the RodAp layer from wild-type spores results in similar phagocytosis rates as with ΔlaeA spores. Resting wild-type and ΔlaeA conidia were treated with hydrofluoric acid to remove the surface rodlet layer, fixed with p-formaldehyde, and examined for phagocytic uptake by peritoneal macrophages (n = 5 mice). Results from two independent experiments are shown as the mean percent uptake ± SEM.