Dataset Information

Phagocytosis of Aspergillus fumigatus by Human Bronchial Epithelial Cells Is Mediated by the Arp2/3 Complex and WIPF2.

ABSTRACT: Aspergillus fumigatus is an opportunistic fungal pathogen capable of causing severe infection in humans. One of the limitations in our understanding of how A. fumigatus causes infection concerns the initial stages of infection, notably the initial interaction between inhaled spores or conidia and the human airway. Using publicly-available datasets, we identified the Arp2/3 complex and the WAS-Interacting Protein Family Member 2 WIPF2 as being potentially responsible for internalization of conidia by airway epithelial cells. Using a cell culture model, we demonstrate that RNAi-mediated knockdown of WIPF2 significantly reduces internalization of conidia into airway epithelial cells. Furthermore, we demonstrate that inhibition of Arp2/3 by a small molecule inhibitor causes similar effects. Using super-resolution fluorescence microscopy, we demonstrate that WIPF2 is transiently localized to the site of bound conidia. Overall, we demonstrate the active role of the Arp2/3 complex and WIPF2 in mediating the internalization of A. fumigatus conidia into human airway epithelial cells.

SUBMITTER: Culibrk L

PROVIDER: S-EPMC6375057 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Phagocytosis of Aspergillus fumigatus by Human Bronchial Epithelial Cells Is Mediated by the Arp2/3 Complex and WIPF2.

Culibrk Luka L Croft Carys A CA Toor Amreen A Yang S Jasemine SJ Singhera Gurpreet K GK Dorscheid Delbert R DR Moore Margo M MM Tebbutt Scott J SJ

Frontiers in cellular and infection microbiology 20190207

Aspergillus fumigatus is an opportunistic fungal pathogen capable of causing severe infection in humans. One of the limitations in our understanding of how A. fumigatus causes infection concerns the initial stages of infection, notably the initial interaction between inhaled spores or conidia and the human airway. Using publicly-available datasets, we identified the Arp2/3 complex and the WAS-Interacting Protein Family Member 2 WIPF2 as being potentially responsible for internaliza ...[more]

PMID: 30792969

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Project description:Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus which reproduces asexually by releasing abundant airborne conidia (spores), which are easily respirable. In allergic and immunocompromised individuals A. fumigatus can cause a wide spectrum of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis. Previous studies have demonstrated that A. fumigatus conidia are internalized by macrophages and lung epithelial cells; however the exact transcriptional responses of airway epithelial cells to conidia are currently unknown. Thus, the aim of this study was to determine the transcriptomic response of the human bronchial epithelial cell line (16HBE14o-) following interaction with A. fumigatus conidia. We used fluorescence-activated cell sorting (FACS) to separate 16HBE14o- cells having bound and/or internalized A. fumigatus conidia expressing green fluorescent protein from cells without spores. Total RNA was then isolated and the transcriptome of 16HBE14o- cells was evaluated using Agilent Whole Human Genome microarrays.Immunofluorescent staining and nystatin protection assays demonstrated that 16HBE14o- cells internalized 30-50% of bound conidia within six hrs of co-incubation. After FAC-sorting of the same cell culture to separate cells associated with conidia from those without conidia, genome-wide analysis revealed a set of 889 genes showing differential expression in cells with conidia. Specifically, these 16HBE14o- cells had increased levels of transcripts from genes associated with repair and inflammatory processes (e.g., matrix metalloproteinases, chemokines, and glutathione S-transferase). In addition, the differentially expressed genes were significantly enriched for Gene Ontology terms including: chromatin assembly, G-protein-coupled receptor binding, chemokine activity, and glutathione metabolic process (up-regulated); cell cycle phase, mitosis, and intracellular organelle (down-regulated).We demonstrate a methodology using FACs for analyzing the transcriptome of infected and uninfected cells from the same cell population that will provide a framework for future characterization of the specific interactions between pathogens such as A. fumigatus with human cells derived from individuals with or without underlying disease susceptibility.

Project description:Lung infection with the fungus Aspergillus fumigatus (Af) is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function. We established a fungal epithelial co-culture model to examine the impact of Af infection on CF bronchial epithelial barrier function using Af strains 10AF and AF293-GFP, and the CFBE41o- cell line homozygous for the F508del mutation with (CF+CFTR) and without (CF) normal CFTR expression. Following exposure of the epithelial surface to Af conidia, formation of germlings (early stages of fungal growth) was detected after 9-12 hours and hyphae (mature fungal growth) after 12-24 hours. During fungal morphogenesis, bronchial epithelial cells showed signs of damage including rounding, and partial detachment after 24 hours. Fluorescently labeled conidia were internalized after 6 hours and more internalized conidia were observed in CF compared to CF+CFTR cells. Infection of the apical surface with 10AF conidia, germlings, or hyphae was performed to determine growth stage-specific effects on tight junction protein zona occludens protein 1 (ZO-1) expression and transepithelial electrical resistance (TER). In response to infection with conidia or germlings, epithelial barrier function degraded time-dependently (based on ZO-1 immunofluorescence and TER) with a delayed onset in CF+CFTR cell monolayers and required viable fungi and apical application. Infection with hyphae caused an earlier onset and faster rate of decline in TER compared to conidia and germlings. Gliotoxin, a major Af virulence factor, caused a rapid decline in TER and induced a transient chloride secretory response in CF+CFTR but not CF cells. Our findings suggest growth and internalization of Af result in deleterious effects on bronchial epithelial barrier function that occurred more rapidly in the absence of CFTR. Bronchial epithelial barrier breakdown was time-dependent and morphotype-specific and mimicked by acute administration of gliotoxin. Our study also suggests a protective role for CFTR by turning on CFTR-dependent chloride transport in response to gliotoxin, a mechanism that will support mucociliary clearance, and could delay the loss of epithelial integrity during fungal development in vivo.

Dataset Information

Phagocytosis of Aspergillus fumigatus by Human Bronchial Epithelial Cells Is Mediated by the Arp2/3 Complex and WIPF2.

Publications

Phagocytosis of <i>Aspergillus fumigatus</i> by Human Bronchial Epithelial Cells Is Mediated by the Arp2/3 Complex and WIPF2.

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