Endocytic Markers Associated with the Internalization and Processing of Aspergillus fumigatus Conidia by BEAS-2B Cells

Abstract

Conidia from the fungus Aspergillus fumigatus are notorious for their ability to stay airborne. This characteristic is believed to allow conidia to penetrate into the cleanest environments. Several hundred conidia are thought to be inhaled each day by a given individual and then expelled by mucociliary clearance. Given that airway epithelial cells make up a significant portion of the pulmonary-air interface, we set out to determine the percentage of conidia that are actually internalized after initial contact with airway epithelial cells. We determined this through an in vitro assay using an immortalized bronchial airway epithelial cell line known as BEAS-2B. Our results suggest a small fraction of conidia are internalized by BEAS-2B cells, while the majority stay adherent to the surface of cells or are washed away during sample processing. Internalization of conidia was observed at 6 h postchallenge and not prior. Our data also indicate conidia are rendered metabolically inactive within 3 h of challenge, suggesting BEAS-2B cells process a large number of conidia without internalization in this early time frame. We have also identified several host endocytosis markers that localize around internalized conidia as well as contribute to the processing of conidia. Understanding how these host endocytosis markers affect the processing of internal and/or external conidia may provide a novel avenue for therapeutic development.

Bronchial Epithelial Cells on the Front Line to Fight Lung Infection-Causing Aspergillus fumigatus

“…This antifungal activity of BECs is fungistatic and occurs via a mechanism that is phosphoinositide 3-kinase dependent. The same kind of observation was made by Clark et al who found that after infecting BEAS-2B cells with A. fumigatus conidia, a large subset of conidia is rendered metabolically inactive, as measured with the metabolic marker FUN-1, while not being internalized by the cells (71).…”

“…Indeed, Clark et al observed the internalization of 10 to 20% of the conidia in contact with BEAS-2B cells at 6 and 9 h after challenge. In comparison, 70% of conidia in contact with macrophages were internalized after 1 h incubation (71). A 41% internalization rate has also been reported with 16HBE cells after 6 h incubation (70).…”

“…First, Paris et al, showed by microscopic observations that rabbit tracheal epithelial cells were able to internalize A. fumigatus conidia after 6 h of incubation and that conidia were enclosed in membrane-bound vacuoles (69). Then, other studies reported that the conidia of A. fumigatus were taken up in vitro by a human bronchial epithelial cell line cultured in monolayers (70,71). Indeed, Clark et al observed the internalization of 10 to 20% of the conidia in contact with BEAS-2B cells at 6 and 9 h after challenge.…”

“…Adhesion of conidia onto BECs induces actin polymerization (75), a phenomenon dependent on the activity of the human actin reorganization complex 2 and 3, regulated by Wiskott-Aldrich syndrome protein-interacting proteins (76). Using the BEAS-2B cell line, Clark et al identified 7 host markers-caveolin, flotillin-2, RAB5C, RAB8B, RAB7A, 2xFYVE, and FAPP1-that consistently localized around the internalized conidia (71).…”

Aspergillus fumigatus and Aspergillosis in 2019

“…The role of the lung epithelium during Aspergillus infection has been insufficiently analyzed to date, and the published data are often contradictory. These discrepancies come from the use of epithelial cells of different origins (cell lines originating from type II ECs [e.g., A549] or bronchial epithelial adenocarcinoma, which are very different from primary ECs [204,212,213,215,216]) or the use of different mutants and morphotypes (resting conidia, swollen germinated conidia, or germ tubes) and the limited number of in vivo studies.…”