Author:
TF Furukawa1*, NvR van Rhijn1, JR Rhodes2, RFG Fortune-Grant1, EB Bignell1, MF Fisher2, MB Bromley1
Author address:
1School of Biological Sciences, The University of Manchester, Manchester, UK
2School of Public Health, Imperial College London, London, UK
Full conference title:
10th Advances Against Aspergillosis and Mucormycosis
Date: 2 February 2022
Abstract:
Purpose:
Aspergillus fumigatus is the most important airborne fungal pathogen and allergen of humans causing more than 300,000 life-threatening infections annually. However, our ability to treat such infections remains critically challenged by limited understanding of the molecular processes involved in the pathogenicity of A. fumigatus. In this context, development of novel molecular tools are necessary to fully explore the molecular landscape of A. fumigatus virulence and to facilitate the design of new therapeutic strategies. In this study, we explored the potential of a genetically conserved inactive transposon-encoding locus, aft4, as a defined DNA integration site for gene complementation and transgene expression.
Methods:
A deletion mutant of the aft4 inactive transposable element (Δaft4-hyg) was constructed in A. fumigatus MFIG001 by replacing the open reading frame (ORF) of aft4 with the hygromycin B resistance marker expression cassette. Detailed genetic, biochemical, and pathological characterization of the generated mutant were conducted to asses the ability of the aft4 locus as a universal DNA integration site in A. fumigatus.
Results:
A genomic survey of potential transposable elements in the genome of A. fumigatus identified the aft4 locus as a unique single copy element with a genetically inactivated class II transposon. Bioinformatic analysis revealed that the aft4 locus is highly conserved in the genome of a wide range of clinical and environmental isolates of A. fumigatus suggesting its potential as a universal DNA integration site for various applications. Our genetic and biochemical characterization of the Δaft4-hyg mutants demonstrated that the deletion of the aft4 element as well as the transgene expression from the aft4 locus do not have any significant impact on the growth characteristics of A. fumigatus under infection relevant stress conditions. Furthermore, we observed that the virulence of the Δaft4-hyg mutant remains unchanged compared to the isogenic control in both in vitro and in vivo infection models. Finally, we showed that the aft4 locus can be used as an integration site for expression of a transgenic construct.
Conclusion:
In this study, we identified a new genomic safe-haven site, a non-functional class II transposable element defined here as aft4. Our study provides evidence that the aft4 locus can serve as a novel molecular tool for genetic manipulation of A. fumigatus to aid functional genomics studies of this important human fungal pathogen.
Abstract Number: 65
Conference Year: 2022
Link to conference website: https://aaam2022.org/
URL Conference abstract: