Human lungs must balance elimination of a myriad of toxic or pathogenic particles inhaled daily with maintenance of tissue homeostasis to avoid lung disease and ultimately respiratory failure. Aspergillus fumigatus spores are major components of the airborne particulate matter and initiate >3,000,000 chronic and >300,000 invasive1,2, potentially fatal, diseases annually worldwide. Thus, there is an urgent need for the development of novel strategies for the clinical management of fungal infection.
Using state-of-the-art single-cell technologies3, Dr Bertuzzi and her research group have demonstrated that airway epithelial cells (AECs) efficiently kill A. fumigatus spores upon uptake and that this process is altered when well-known risk factors for debilitating fungal lung disease are present4. The current working hypothesis of their research is that the respiratory mucosa critically contribute to healthy clearance of inhaled A. fumigatus spores and that dysregulation of protective epithelial responses represents a potent driver of aspergillus-related diseases5,6.
In line with the current research avenues explored by the Bertuzzi’s group and their collaborators (specifically Prof. Paul Bowyer and Prof Lydia Tabernero), the project proposed aims to determine:
1) how healthy AECs recognise and kill A. fumigatus.
To define the epithelial components driving effective fungal clearance by AECs, targeted and global analyses of A. fumigatus uptake and intracellular killing by the model alveolar epithelial cells, A549, will be performed in parallel. The targeted approach will be based on identified A. fumigatus cell wall components mediating uptake, a selection of which will be tagged for co-immunoprecipitation and affinity purification-mass spectrometry. Stable A549 KOs for the putative identified host receptors will be generated via CRISPR/Cas9 genome editing7 and KO A549 cells will be assessed for their ability to internalise and kill A. fumigatus in vitro. A global approach will also be undertaken and two CRISPR/Cas9 KO sub-libraries will be used to KO >1,500 human genes encoding membrane proteins and endocytic proteins via lentivirus transduction8. After pooled CRISPR screens, next generation sequencing and bioinformatic analysis we will define a set of statistically robust drivers of Af uptake to validate by targeted CRISPR-Cas9 genome editing.
2) if these mechanisms are universally exploited for clearance of other respiratory pathogens
1 Brown GD et al. (2012) PMID: 23253612 2 Bongomin F et al. (2017) PMID: 29371573 3 Bertuzzi M and Howell GJ. (2020), PMID: 33405032 4 Bertuzzi M et al. (2022) doi: https://doi.org/10.1101/2022.02.01.478664 5 Bertuzzi M et al. (2019) PMID: 30657899 6 Bertuzzi M* et al. (2018) PMID: 29371501 7 Gago S et al. (2018) PMID: 30237437 8 Palazón-Riquelme P et al. (2018) PMID: 30206189
To identify parallels or divergences in the mechanism and role of epithelial responses against other respiratory pathogens also internalised by the respiratory epithelium9,10, stable A549 KOs generated via CRISPR/Cas9 genome editing above will be assessed for their ability to internalise and kill other respiratory pathogens of bacterial (Mycobacterium tuberculosis) or fungal (Cryptococcus neoformans) origin.
This work will reveal the molecular basis of epithelial activities driving efficient spore containment, providing crucial knowledge and tools for mechanistic studies on how these activities may be universally exploited or subverted by other respiratory pathogens. Understanding how the lung balances mucosal tissue homeostasis and pathogen clearance upon exposure to inhaled pathogens is of major clinical importance and will inform the identification of immune-modulators to facilitate treatment as well as the limitation of damage caused by this and other respiratory pathogens, leading causes of lung diseases.
Candidates are expected to hold (or be about to obtain) a minimum upper second class honours degree (or equivalent) in a related area/subject. Candidates with previous laboratory experience, particularly in cell culture and molecular biology, are particularly encouraged to apply.
How To Apply
For information on how to apply for this project, please visit the Faculty of Biology, Medicine and Health Doctoral Academy website (https://www.bmh.manchester.ac.uk/study/research/apply/). Informal enquiries may be made directly to the primary supervisor. On the online application form select PhD Genetics
For international students, we also offer a unique 4 year PhD programme that gives you the opportunity to undertake an accredited Teaching Certificate whilst carrying out an independent research project across a range of biological, medical and health sciences.
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