Author:
U Binder1, MI Navarro-Mendoza2, FE Nicolas2, V Naschberger1, C Kandelbauer1,
I Bauer3, J Pallua4, C Lass-Flörl1, V Garre2
Author address:
1Institute of Hygiene and Medical Microbiology, Medical University Innsbruck, Innsbruck, Austria
2Fungal Genomics and Molecular Biotechnology, University of Murcia, Murcia, Spain
3Institute of Molecular Biology, Medical University Innsbruck, Innsbruck, Austria
4Institute of Pathology, Medical University Innsbruck, Innsbruck, Austria
Full conference title:
9th Advances Against Aspergillosis
Date: 26 February 2020
Abstract:
Purpose: Invasive infections caused by mucormycetes are increasingly seen in the clinics and are still associated with unacceptable high mortality rates. Still, little is known about the biology of the pathogens, the establishment and progression of the infection, antifungal resistance mechanisms and successful therapy. Therefore, we aimed to generate a tool for (1) alternative methods of drug testing in vitro, (2) non-invasive monitoring of the infection in different model hosts, and (3) visualization of antifungal efficacy.
Methods: Firefly luciferase, both mammalian or codon-optimized without the peroxisomal target sequence was cloned in the pMAT1477 vector under the control of different promoters. Linear plasmid was used to transfect M. circinelloides protoplasts of auxotrophic strains. Positive transformants were checked for gene integration and then light emission measured under various conditions. Selected strains were used to determine antifungal susceptibility, virulence potential and in vivo monitoring of mucormycosis in Galleria mellonella.
Results: Firefly luciferase was successfully expressed in M. circinelloides with a single integration and light emission detected by imaging and luminometer. Codon optimization was critical to enhance light emission, making these strains usable for allow real-time, non-invasive infection monitoring in insect and murine models, and the testing of antifungal efficacy by means other than survival. Phenotype, virulence potential in G. mellonella and antifungal susceptibility are indifferent to the wild-type strains.
Conclusion: The construction and further optimization of bioluminescent Mucor strains allows for the visualization of temporal and spatial progression of infection by a non-invasive method in insect and murine models, and the testing of antifungal efficacy by other means than survival only. This will give valuable new insights in the pathogenesis of Mucorales infections.
Abstract Number: 53
Link to conference website:
Link Conference abstract:
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