Phagolysosomal Survival Enables Non-lytic Hyphal Escape and Ramification Through Lung Epithelium During Aspergillus fumigatus Infection

Abstract

Aspergillus fumigatus is the most important mould pathogen in immunosuppressed patients. Suboptimal clearance of inhaled spores results in the colonisation of the lung airways by invasive hyphae. The first point of contact between A. fumigatus and the host is the lung epithelium. In vitro and ex vivo studies have characterised critical aspects of the interaction of invasive hyphae on the surface of epithelial cells. However, the cellular interplay between internalised A. fumigatus and the lung epithelium remains largely unexplored. Here, we use high-resolution live-cell confocal microscopy, 3D rendered imaging and transmission electron microscopy to define the development of A. fumigatus after lung epithelium internalisation in vitro . Germination, morphology and growth of A. fumigatus were significantly impaired upon internalisation by alveolar (A549) and bronchial (16HBE) lung epithelial cells compared to those growing on the host surface. Internalised spores and germlings were surrounded by the host phagolysosome membrane. Sixty per cent of the phagosomes containing germlings were not acidified at 24 h post infection allowing hyphal development. During escape, the phagolysosomal membrane was not ruptured but likely fused to host plasma membrane allowing hyphal exit from the intact host cell in an non-lytic Manner. Subsequently, escaping hyphae elongated between or through adjacent epithelial lung cells without penetration of the host cytoplasm. Hyphal tips penetrating new epithelial cells were surrounded by the recipient cell plasma membrane. Altogether, our results suggest cells of lung epithelium survive fungal penetration because the phagolysosomal and plasma membranes are never breached and that conversely, fungal spores survive due to phagosome maturation failure. Consequently, fungal hyphae can grow through the epithelial cell layer without directly damaging the host. These processes likely prevent the activation of downstream immune responses alongside limiting the access of professional phagocytes to the invading fungal hypha. Further research is needed to investigate if these events also occur during penetration of fungi in endothelial cells, fibroblasts and other cell types.

Aspergillus fumigatusand aspergillosis: From basics to clinics

“…Indeed, an increasing body of evidence has revealed a critical role of the airway’s epithelium in fungal clearance ( Amich et al. 2020 , Seidel et al. 2020 ) and production of cytokines and antimicrobial peptides ( Bellanger et al.…”

Characterisation of Aspergillus fumigatus Endocytic Trafficking within Airway Epithelial Cells Using High-Resolution Automated Quantitative Confocal Microscopy

“…For all experiments, 10 5 epithelial cells were seeded in 24-glass bottom plates (Greiner Bio-One SensoPlate™, Kremsmünster, Austria) and incubated until confluence was 80-90%, unless otherwise described. Live cell imaging was performed using supplemented phenol red-free DMEM/F12 Medium (Thermo Fisher Scientific, Waltham, MA, USA) [35].…”

“…Digested spores were scored by the assessment of loss of fungal cytoplasmic RFP fluorescence; resident spores were scored as those preserving the same size and fluorescence as the original spore inoculum, while swollen spores were those showing increased volume compared to the original inoculum with comparable fluorescence. Germinated spores were counted when a germ tube of >0.5 spore diameters was visible [35].…”

“…Airway epithelial cells have been shown to efficiently take up fungal spores in a timely manner [26][27][28][29][30][31][32]. In order to prevent fungal spore germination and escape, phagosomes containing internalized spores are acidified and fused with lysosomes [33][34][35]. Although potentially efficient in fungal clearance, previous work by us and others found that during early stages of infection, less than 3% of A. fumigatus spores can survive within the phagolysosome and escape by hyphal elongation without causing any significant host damage in a non-lytic egress mechanism [19,35].…”

“…In order to prevent fungal spore germination and escape, phagosomes containing internalized spores are acidified and fused with lysosomes [33][34][35]. Although potentially efficient in fungal clearance, previous work by us and others found that during early stages of infection, less than 3% of A. fumigatus spores can survive within the phagolysosome and escape by hyphal elongation without causing any significant host damage in a non-lytic egress mechanism [19,35]. However, several respiratory pathogens have developed anti-phagocytic strategies which allow them to survive within the airways and cause disease [36,37].…”