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. Aspergillus fumigatus is a ubiquitous mold that produces small airborne conidia capable of traversing deep into the respiratory system. Recognition, processing, and clearance of A. fumigatus conidia by bronchial airway epithelial cells are thought to be relevant to host defense and immune signaling. Using z-stack confocal microscopy, we observed that only 10 to 20% of adherent conidia from the AF293 clinical isolate are internalized by BEAS-2B cells 6 h postchallenge and not prior. Similar percentages of internalization were observed for the CEA10 clinical isolate. A large subset of both AF293 and CEA10 conidia are rendered metabolically inactive without internalization at 3 h postchallenge by BEAS-2B cells. A significantly larger percentage of CEA10 conidia are metabolically active at 9 and 12 h postchallenge in comparison to the AF293 isolate, demonstrating heterogeneity among clinical isolates. We identified 7 host markers (caveolin, flotillin-2, RAB5C, RAB8B, RAB7A, 2xFYVE, and FAPP1) that consistently localized around internalized conidia 9 h postchallenge. Transient gene silencing of RAB5C , PIK3C3 , and flotillin-2 resulted in a larger population of metabolically active conidia. Our findings emphasize the abundance of both host phosphatidylinositol 3-phosphate (PI3P) and PI4P around internalized conidia, as well as the importance of class III PI3P kinase for conidial processing. Therapeutic development focused on RAB5C-, PIK3C3-, and flotillin-2-mediated pathways may provide novel opportunities to modulate conidial processing and internalization. Determination of how contacted, external conidia are processed by airway epithelial cells may also provide a novel avenue to generate host-targeted therapeutics. IMPORTANCE 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.