Author:
CA Zacharias1*, FN Gravelat1, FA Le Mauff1, H Liu2, SG Filler2,3, DC Sheppard1,4
Author address:
1Infectious Diseases and Immunity in Global Health, Research Institute of the McGill University Health Centre, Montreal, Canada
2Division of Infectious Diseases, Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, USA
3David Geffen School of Medicine, University of California Los Angeles, Los Angeles, USA
4Medicine, McGill University, Montreal, Canada
Full conference title:
10th Advances Against Aspergillosis and Mucormycosis
Date: 2 February 2022
Abstract:
Purpose:
Aspergillus fumigatus is a ubiquitous filamentous mold that causes necrotizing pneumonia in immunosuppressed individuals. One of the virulence factors of A. fumigatus is the synthesis of the adhesive, cationic exopolysaccharide GAG, which is mediated by the products of a five gene cluster. One of these genes, ega3 encodes a glycosyl hydrolase that is specific for deacetylated GAG that is anchored to the cell membrane of A. fumigatus. We hypothesized that Ega3 is necessary for GAG synthesis, and to test this hypothesis, we sought to disrupt ega3 in A. fumigatus.
Methods:
CRISPR/Cas9 was used to replace the ega3 allele of Aspergillus fumigatus Af293 with a hygromycin resistance cassette. CRISPR/Cas9 was also used to complement the ega3-null strain at the native locus. Deletion and complementation were confirmed using reverse transcription quantitative PCR (RT-qPCR) and Western blot. Strains were characterized using a crystal violet biofilm assay. The ega3-null strain was transformed with a tetracycline-inducible agd3 construct. Fungal membrane permeability was measured using an ATP release assay. Mammalian cell viability was measured via radioactive chromium release or propidium iodide staining.
Results:
Multiple attempts to disrupt ega3 by conventional methods were unsuccessful. Switching to CRISPR/Cas9 generated a single ega3-null clone. As with other mutations in the GAG cluster, this mutant did not produce deacetylated GAG. Complementation with an ega3 allele failed to restore deacetylated GAG production, suggesting the presence of a secondary mutation in this strain. Analysis of the expression of the rest of the GAG cluster genes revealed that agd3, encoding the GAG deacetylase, was not expressed in the ega3 null mutant. We therefore hypothesized that ega3 is conditionally essential in the presence of deacetylated GAG production. To test this, agd3 was expressed in the ega3 null mutant under the control of a tetracycline-inducible promotor (ega3null-Tet ON-agd3). Under agd3-expressing conditions, GAG production was restored, however fungal growth was inhibited. We hypothesized that GAG may be toxic to the cell membrane of A. fumigatus and that Ega3 is necessary for degrading GAG near the membrane. To test this, ATP was measured in fungal culture supernatants as a proxy for cell leakage. Induction of agd3 expression in the ega3null-Tet ON-agd3 mutant causes increased ATP release, suggesting that deacetylated GAG disrupts the fungal cell membrane.
Since GAG causes membrane permeability in the ega3 null mutant, we hypothesized that it may cause host cell injury. This was tested in a cell damage assay where epithelial cells were loaded with radioactive chromium and exposed to culture supernatants of wild-type A. fumigatus with and without recombinant Ega3. A. f umigatus-induced epithelial cell damage was nearly abolished in the presence of Ega3. Similar findings were observed using propidium iodide staining of bone marrow-derived macrophages exposed to GAG with or without Ega3.
Conclusion:
This data suggests that cationic GAG is cytotoxic to both fungal and host cells, and that Ega3 can serve as an antitoxin to mitigate these effects.
Abstract Number: 48
Conference Year: 2022
Link to conference website: https://aaam2022.org/
URL Conference abstract: