Aspergillus fumigatus is a frequent coloniser of the cystic fibrosis (CF) lung and is responsible for the development of several diseases including allergic bronchopulmonary aspergillosis (ABPA) and aspergillosis bronchitis (AB). Our understanding of the factors that drive A. fumigatus pathogenicity in CF is limited by the in vitro models which are currently available. In this study, a new isogenic in vitro model with CF and healthy control airway epithelial cells has been established by using CRISPR/Cas9 mutagenesis to knockout CFTR in A549 cells. These cells have been characterised, revealing that loss of CFTR induces CF-relevant phenotypes including defective ion transport, mucus production and cytokine production in response to infection, as well as decreased ability to internalise A. fumigatus conidia. The relevancy of this isogenic model as a tool for the high-throughput screening of A. fumigatus deletion mutants, to identify fungal drivers of disease in the CF lung has also been demonstrated. Using a library of 110 genetically barcoded protein kinase null mutants and 35 secreted protease null mutants, competitive fitness profiling has been carried out to assess the relative fitness of each mutant under different growth conditions. Comparison of the null mutants identified three protein kinase genes: AFUB_038060, AFUB_012420 and AFUB_020650 which appear to play a role in fungal stress responses via the induction of signalling pathways. Moreover, one secreted protease gene: AFUB_031630, was shown to influence the rate of conidial internalisation. Ultimately, for their first time, an isogenic in vitro model of CF in an alveolar epithelial cell background has been developed, which can be used in conjunction with competitive fitness analysis to facilitate large scale screening of mutant libraries to identify genes which confer fitness in the CF lung.
Date of Award | 31 Dec 2024 |
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Original language | English |
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Awarding Institution | - The University of Manchester
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Supervisor | Michael Bromley (Supervisor), Paul Bowyer (Supervisor) & Sara Gago (Supervisor) |
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- Host-pathogen interaction
- Aspergillus fumigatus
- Cystic fibrosis
- Competitive fitness
Development of an in vitro model to dissect the mechanisms of fungal persistence in the cystic fibrosis lung
Earle, K. R. (Author). 31 Dec 2024
Student thesis: Phd