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

Ben-Ghazzi, Nagwa; Moreno-Velásquez, Sergio D.; Seidel, Constanze; Thomson, Darren D; Denning, David W.; Read, Nick D.; Bowyer, Paul; Gago, Sara

doi:10.3390/jof7060454

Cited by 16 publications

(23 citation statements)

References 78 publications

(115 reference statements)

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“…In in vitro infections, epithelial uptake can lead to Af killing or, more rarely, intraphagosomal occupancy [33–35, 71–73]. Internalised conidia are quickly trafficked through the endosomal system to the phagolysosomes, as demostrated by the rapid acquisition of late endosomal/lysosomal markers and cathepsin D [29, 72]. Population-scale analyses further indicate that phagosomal acidification results in killing of 97-98% of the internalised conidia within 12-24 hrs, while ∼2-3% of the intracellular Af remain viable and in a third of the cases can eventually germinate by 36 h, without lysis of the host cells [29].…”

Section: Discussionmentioning

confidence: 99%

Epithelial uptake ofAspergillus fumigatusdrives efficient fungal clearancein vivoand is aberrant in Chronic Obstructive Pulmonary Disease (COPD)

Bertuzzi¹,

Howell

Thomson³

et al. 2022

Preprint

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Hundreds of spores of the common mould Aspergillus fumigatus (Af) are inhaled daily by human beings, representing a constant, often fatal, threat to our respiratory health. The small size of Af spores suggest that interactions with Airway Epithelial Cells (AECs) are frequent and we and others have previously demonstrated that AECs are able to internalise Af spores. We thus hypothesised that Af spore uptake and killing by AECs is important for driving efficient fungal clearance in vivo and that defective spore uptake and killing would represent major risk factors for Aspergillus-related diseases. In order to test this, we utilised single-cell approaches based on Imaging Flow Cytometry (IFC) and live-cell microfluidic imaging to measure spore uptake and outcomes in vitro, in vivo and using primary human AECs. In vitro, viability of immortalised AECs was largely unaffected by Af uptake and AECs were able to significantly curtail the growth of internalised spores. Applying our approach directly to infected mouse lungs we demonstrated, for the first time, that Af spores are internalised and killed by AECs during whole animal infection, whereby only ~3% of internalised spores remained viable after 8 hours of co-incubation with murine AECs. Finally, in vitro analysis of primary human AECs from healthy and at-risk donors revealed significant alterations in the uptake and consequent outcomes in Chronic Obstructive Pulmonary Disease (COPD), whereby gorging COPD-derived AECs were unable to quell intracellular Af as efficiently as healthy primary AECs. We have thus demonstrated that AECs efficiently kill Af spores upon uptake in vivo and that this process is altered in COPD, a well-known risk factor for debilitating fungal lung disease, thereby suggesting that AECs critically contribute to the efficient clearance of inhaled Af spores and that dysregulation of curative AEC responses represents a potent driver of Aspergillus-related diseases.

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Section: Discussionmentioning

confidence: 99%

Epithelial uptake ofAspergillus fumigatusdrives efficient fungal clearancein vivoand is aberrant in Chronic Obstructive Pulmonary Disease (COPD)

Bertuzzi¹,

Howell

Thomson³

et al. 2022

Preprint

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“…Numerous studies have demonstrated that A. fumigatus conidia are readily internalised by AECs in vitro, with both alveolar and bronchial epithelial cells internalising 30–50% of the conidia they encounter [ 17 , 31 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 ]. In vitro A. fumigatus uptake by AECs is a time-dependent process, which increases proportionally with longer co-incubations or a higher multiplicity of infections [ 17 , 126 , 128 ]. AECs exert antifungal activity, with intracellular conidia showing impaired germination relative to extracellular conidia [ 17 , 126 ].…”

Section: Are Airway Respiratory Cells Internalising a Fumig...mentioning

confidence: 99%

Novel Insights into Aspergillus fumigatus Pathogenesis and Host Response from State-of-the-Art Imaging of Host–Pathogen Interactions during Infection

Ortiz

Pennington

Thomson

et al. 2022

JoF

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Aspergillus fumigatus spores initiate more than 3,000,000 chronic and 300,000 invasive diseases annually, worldwide. Depending on the immune status of the host, inhalation of these spores can lead to a broad spectrum of disease, including invasive aspergillosis, which carries a 50% mortality rate overall; however, this mortality rate increases substantially if the infection is caused by azole-resistant strains or diagnosis is delayed or missed. Increasing resistance to existing antifungal treatments is becoming a major concern; for example, resistance to azoles (the first-line available oral drug against Aspergillus species) has risen by 40% since 2006. Despite high morbidity and mortality, the lack of an in-depth understanding of A. fumigatus pathogenesis and host response has hampered the development of novel therapeutic strategies for the clinical management of fungal infections. Recent advances in sample preparation, infection models and imaging techniques applied in vivo have addressed important gaps in fungal research, whilst questioning existing paradigms. This review highlights the successes and further potential of these recent technologies in understanding the host–pathogen interactions that lead to aspergillosis.

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“…Among these 8 TFKOs, only 4 TF-encoding genes have been previously characterised. This includes HapX (AFUB_052420), a component of the CCAAT-binding complex (Hortschansky et al ., 2017) which is involved in iron acquisition and metabolism in Aspergillus (Gsaller et al ., 2014), PacC (AFUB_037210) previously reported to regulate A. fumigatus epithelial invasion (Bertuzzi et al ., 2014), Ace1/SltA (AFUB_041100) which regulates conidial formation including cell wall architecture and secretion of mycotoxins and secondary metabolites (H. Liu et al ., 2021) and AtfD (AFUB_078150) which is involved in conidial stress responses (Silva, Horta and Goldman, 2021).…”

Section: Resultsmentioning

confidence: 99%

“…Consistent with such functionality, the colonial phenotypes of all four mutants exhibit compact morphology (Fig 3 and Fig 6). It is therefore feasible that the secondary metabolite composition of the spores of these mutants differs sufficiently from that of the parental isolate to cause deficits in epithelial detachment, either via aberrancies in one or several of contact-mediated toxicity, epithelial uptake of and phagolysomal fusion, the latter of which has recently been shown to determine efficiency of intracellular fungal killing (Ben-Ghazzi et al, 2021). Remarkably, TFKOs that are deficient in causing epithelial cell lysis universally exhibit culture filtrates that are less cytolytic than the parental progenitor (Fig7).…”

Section: Discussionmentioning

confidence: 99%

Distinct cohorts of Aspergillus fumigatus transcription factors are required for epithelial damage occurring via contact- or soluble effector-mediated mechanisms

Rahman

Rhijn

Papastamoulis

et al. 2022

Preprint

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Damage to the lung epithelium is a unifying feature of disease caused by the saprophytic fungus Aspergillus fumigatus. However, the mechanistic basis and the regulatory control of such damage is poorly characterized. Previous studies have identified A. fumigatus mediated pathogenesis as occurring at early (≤ 16 hours) or late (>16 hours) phases of the fungal interaction with epithelial cells, and respectively involve direct contact with the host cell or the action of soluble factors produced by mature fungal hyphae. Both early and late phases of epithelial damage have been shown to be subject to genetic regulation by the pH-responsive transcription factor PacC. This study sought to determine whether other transcriptional regulators play a role in modulating epithelial damage. In particular, whether the early and late phases of epithelial damage are governed by same or distinct regulators. Furthermore, whether processes such as spore uptake and hyphal adhesion, that have previously been documented to promote epithelial damage, are governed by the same cohorts of epithelial regulators. Using 479 strains from the recently constructed library of A. fumigatus transcription factor null mutants, two high-throughput screens assessing epithelial cell detachment and epithelial cell lysis were conducted. A total of 17 transcription factor mutants were found to exhibit reproducible deficits in epithelial damage causation. Of these, 10 mutants were defective in causing early phase damage via epithelial detachment and 8 mutants were defective in causing late phase damage via epithelial lysis. Remarkably only one transcription factor, PacC, was required for causation of both phases of epithelial damage. The 17 mutants exhibited varied and often unique phenotypic profiles with respect to fitness, epithelial adhesion, cell wall defects, and rates of spore uptake by epithelial cells. Strikingly, 9 out of 10 mutants deficient in causing early phase damage also exhibited reduced rates of hyphal extension, and culture supernatants of 7 out of 8 mutants deficient in late phase damage were significantly less cytotoxic. Our study delivers the first high-level overview of A. fumigatus regulatory genes governing lung epithelial damage, suggesting highly coordinated genetic orchestration of host-damaging activities that govern epithelial damage in both space and time.

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

Cited by 16 publications

References 78 publications

Epithelial uptake ofAspergillus fumigatusdrives efficient fungal clearancein vivoand is aberrant in Chronic Obstructive Pulmonary Disease (COPD)

Epithelial uptake ofAspergillus fumigatusdrives efficient fungal clearancein vivoand is aberrant in Chronic Obstructive Pulmonary Disease (COPD)

Novel Insights into Aspergillus fumigatus Pathogenesis and Host Response from State-of-the-Art Imaging of Host–Pathogen Interactions during Infection

Distinct cohorts of Aspergillus fumigatus transcription factors are required for epithelial damage occurring via contact- or soluble effector-mediated mechanisms

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