Date of Award

5-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

P. David Rogers, PharmD, PhD

Committee

Elizabeth A. Fitzpatrick, Jarrod R. Fortwendel, Ramin Homayouni, Glen E. Palmer, Brian M. Peters

Abstract

Invasive aspergillosis is a leading cause of morbidity and mortality among immunocompromised populations and is predicted to cause more than 200,000 life- threatening infections each year. Aspergillus fumigatus is the most prevalent pathogen isolated from patients with invasive aspergillosis, accounting for more than 60% of all cases. Currently, the only antifungal agents available with consistent activity against A. fumigatus are the mold-active triazoles and amphotericin B, of which the triazoles commonly represent both front-line and salvage therapeutic options. Unfortunately, the treatment of infections caused by A. fumigatus has recently been further complicated by the global emergence of triazole resistance among both clinical and environmental isolates, and a large proportion of this resistance remains unexplained. In this work, we characterize the contributions of previously identified mechanisms of triazole resistance, including mutations in the sterol-demethylase- encoding gene cyp51A, overexpression of sterol-demethylase genes, and overexpression of the efflux pump-encoding gene abcC, among a large collection of highly triazole- resistant clinical A. fumigatus isolates. Upon revealing that these mechanisms alone cannot substantiate the majority of triazole resistance exhibited by this collection, we then characterize the direct contribution of two additional efflux pump-encoding genes, abcA and atrI. Increased expression of abcA and atrI has previously been associated with triazole resistance in clinical isolates of A. fumigatus, and both of these genes exhibit a high degree of homology with the well characterized Candida albicans triazole efflux pump-encoding gene, CDR1. However, deletion of either abcA or atrI in triazole-resistant clinical isolates which overexpress these genes, did not result in a significant change in triazole susceptibility. Finally, upon demonstrating that the canonical mechanisms of triazole resistance poorly explain the high level of triazole resistance observed in this collection of clinical isolates, we subsequently describe the identification and characterization of a novel genetic determinant of triazole resistance. Mutations in the HMG-CoA reductase encoding gene, hmg1, were identified in a majority of triazole-resistant clinical isolates in our collection. Introduction of three different hmg1 mutations, predicted to encode residue alterations in the conserved sterol sensing domain of Hmg1, resulted in significantly increased resistance to the triazole class of agents. Additionally, correction of an hmg1 mutation in a pan-triazole-resistant clinical isolate of A. fumigatus with a novel Cas9-ribonucleoprotein (RNP) mediated system, was shown to restore clinical susceptibility to all triazole agents. Mutations in hmg1 were also shown to lead to the accumulation of ergosterol precursors, such as eburicol, by sterol profiling, while not altering the expression of sterol-demethylase genes. Taken together, the findings described in this work serve to demonstrate that mutations in hmg1 are a common and significant genetic determinant of triazole resistance in clinical isolates of A. fumigatus."

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0000-0002-9317-0935

DOI

10.21007/etd.cghs.2019.0487

2019-011-Rybak-DOA.pdf (461 kB)
Declaration of Authorship

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