Date of Award

5-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

Jarrod R. Fortwendel, PhD

Committee

Kevin K. Fuller, PhD; Glen E. Palmer, PhD; Brian M. Peters, PhD; P. David Rogers, Pharm.D., PhD

Keywords

aneuploidy;antifungal susceptibility;Aspergillus fumigatus;ergosterol biosynthesis;spindle assembly checkpoint;triazole resistance

Abstract

The opportunistic pathogen Aspergillus fumigatus represents the chief causative agent of human and animal invasive filamentous fungal infections. Triazoles, the primary therapeutic options to combat invasive aspergillosis (IA), target the fungal biosynthesis of ergosterol, a vital component of the fungal cell membrane. Unfortunately, resistance to this class of medical therapeutic has arisen globally and now threatens the future usefulness of these compounds for antifungal treatment. Infection with A. fumigatus that has acquired triazole resistance increases an already high associated mortality rate and reduces the limited arsenal of therapeutic options to combat IA. Moreover, how this specific fungal pathogen obtains resistance remains poorly understood. In this study, we show that loss of the previously uncharacterized A. fumigatus Spindle Assembly Checkpoint components SldA or SldB results in loss of susceptibility to multiple mold active medical triazoles. Furthermore, these mutants possess reduced susceptibility to antifungal compounds targeting ergosterol biosynthesis at points upstream of the triazole target Cyp51A. Loss of either component also results in signs of aneuploidy within uninucleate conidia in flow cytometry analyses for DNA content. Finally, exposure to voriconazole through laboratory experimental adaptation resulted in decreased triazole susceptibility, increased detection of sub populations of aneuploid spores in all samples of mutant as well as wild type lineages, and selection for a specific duplication in chromosome two in most lineages. Subsequent passages of adapted strains in drug-free media revealed adaptation to be unstable in most cases, with loss of adaptations typically occurring with simultaneous resolution of conidial aneuploidy, suggesting a correlation between aneuploidy and antifungal resistance in this pathogen. Together, these findings illustrate a previously unknown connection in A. fumigatus between defective function of the conserved surveillance system designed to prevent aberrant sorting of genomic material during nuclear division, and loss of susceptibility to antifungal compounds that target ergosterol biosynthesis. This knowledge helps to inform our understanding of how A. fumigatus survives and adapts to medical triazole antifungals.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0000-0002-4969-4192

DOI

10.21007/etd.cghs.2024.0660

Additional Files
2024-006-Nywening-DOA.pdf (1534 kB)
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