A Functional Genomics Approach for Understanding Aspergillus fumigatus Pathogenicity

Abstract

A unifying feature of all pulmonary infections caused by the opportunistic fungus Aspergillus fumigatus is damage to the lung epithelium. However, the mechanistic basis and the regulatory control of such damage is poorly characterized. This study employed a functional genomic approach combined with an in vitro epithelial cell model to better understand the pathobiology of A. fumigatus-mediated lung damage. Previous in vitro observations have identified A. fumigatus mediated lung cell damage occurring at early phase (≤ 16 hours) via epithelial cell detachment or late phase (>24 hours) via epithelial cell lysis. This study sought to identify and characterize the A. fumigatus transcriptional regulators modulating epithelial cell damage during these phases. Hence, the suitability and performance of two epithelial damage assays in high throughput format were assessed and validated. Screening 479 A. fumigatus transcription factor null mutants for their capacity to cause epithelial cell detachment and cell lysis revealed a total of 17 transcription factor mutants with reproducible deficits in causing epithelial damage. Of these, 10 mutants were defective in causing early damage via detachment and 8 mutants were defective in causing late damage via lysis. 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 hyphal extension rates, and filtrates of 7 out of 8 mutants deficient in late phase damage were less cytotoxic. Remarkably, the ΔpacC strain was defective in both phases of epithelial damage and was also defective in all tested phenotypes. Thus, by integrating two genome-wide next generation sequencing approaches, this study aimed to uncover the biological processes and the transcriptional network directly regulated by A. fumigatus PacC at acidic, neutral, and alkaline pH environments. Aligning with the critical role of PacC during host tissue invasion and virulence, PacC reiterated as a master regulator not only directing genes involved in cell wall and secreted products, as well as 26 transcription factors, but also interacting with 14 of the 17 regulators of epithelial damage. This study delivers the first overview of A. fumigatus regulatory genes governing lung epithelial cell damage and identified distinct and temporal yet coordinated host-damaging activities and a hierarchical regulatory network governing epithelial damage.

Date of Award	31 Dec 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Michael Bromley (Supervisor), Elaine Bignell (Supervisor) & Margherita Bertuzzi (Supervisor)