Date of Award

5-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Bioanalysis

Research Advisor

P. David Rogers, Pharm.D., Ph.D.

Committee

Ramin Homayouni, Glen E. Palmer, Brian M. Peters, C. Ryan Yates

Keywords

Azole, Candida albicans, ERG11, ERG3, MRR2, Resistance

Abstract

"Candida albicans is a commensal organism commonly colonizing the human gut and skin. As an opportunistic pathogen, it can cause persistent and serious infections in individuals with compromised immune systems, including the very young and elderly. Moreover, C. albicans can cause a wide spectrum of diseases ranging from superficial mucosal infections to life-threatening invasions of the organs and bloodstream. Candida species are the most common cause of invasive fungal disease, which is associated with high mortality and imposes a heavy toll on the healthcare system. Over the last 30 years, the azole antifungals have been a mainstay of antifungal therapy, being effective in a wide variety of fungal infections and serving as the primary oral treatment option. However, increased use, inappropriate dosing, and prolonged treatments have given rise to azole-resistant Candida albicans and other Candida species. Resistance in C. albicans results from a combination of different mechanisms. Increased expression of the efflux pump encoding genes CDR1, CDR2, and MDR1 as well as increased expression of ERG11, encoding the azole target (14α-lanosterol demethylase, also known as CYP51) are all primary mechanisms of azole resistance that arise in azole-resistant clinical isolates. These changes are known to be mediated through gain-of-function mutations in the genes of a fungal-specific transcription factor family known as the zinc cluster transcription factors. Furthermore, genetic changes in the ergosterol biosynthesis genes, ERG11 and ERG3, encoding a C-5 sterol desaturase, also contribute to clinical azole resistance in C. albicans. The interplay of these mechanisms can result in azole-resistance, treatment failure, and ultimately, poorer outcomes in patients. Therefore, to improve healthcare outcomes, understanding resistance development and the mechanisms that drive them in C. albicans is crucial. Within a collection of predominantly fluconazole-resistant clinical isolates of C. albicans, our lab had previously characterized most known mechanisms of azole resistance present in each isolate. Increased CDR1 expression in isolates lacking TAC1 gain-of-function mutations coupled with recent literature suggesting a role of the Mrr2 zinc cluster transcription factor in azole resistance lead us to sequence and test mutations in the MRR2 gene in across this collection. By placing mutant MRR2 alleles in azole- susceptible backgrounds, we hoped to measure the contribution of MRR2 mutations to azole resistance through changes in CDR1 expression and fluconazole minimum inhibitory concentrations (MICs). Counter to what has been recorded in the literature, we found no evidence that mutations in MRR2 impact either CDR1 expression or azole susceptibility in C. albicans. This is a novel finding correcting a previous mistaken paradigm of a clinically relevant mechanism driving resistance in C. albicans. Next we more closely examined the role of ERG11 mutations found in clinical isolates. Though the contributory effects of ERG11 mutations to azole resistance had been quantified, the specific biochemical impact of these mutations on enzyme function and ligand-binding interaction have only recently come to light. Here we introduced additional CaCYP51 amino acid substitution mutants (D278N and Y132H) in C. albicans" "vi" "and tested our entire collection of CaCYP51 mutant strains to determine their in vitro azole susceptibilities in the context of these findings. In general, we observed differences in the fluconazole and voriconazole MICs between CaCYP51 amino acid substitutions. In contrast, MICs to itraconazole showed a small, fairly consistent increase in MIC across tested CaCYP51 strains and MICs to posaconazole did not increase at all over the wild type except for the G448E substitution, suggesting posaconazole possesses the best in vitro activity against these CaCYP51 mutants. Overall, we also revealed that not all ERG11 mutations confer azole resistance through decreased binding interactions with the target and the azole drug, suggesting that CaCYP51 amino acid substitutions may instead interact with other associated proteins to confer resistance. Furthermore, it was discovered that many ERG11 mutations from clinical isolates result in low catalytic turnover of the enzyme, which is crucial to normal rates of ergosterol production in a healthy cell. Though preliminary results of growth in CaCYP51 mutant strains does not support attenuated fitness in competitive assay, the findings here prove that some clinical ERG11 mutations result in diminished enzyme function." We also sequenced the collection of clinical isolates and discovered an A351V Erg3 amino acid substitution in our azole-resistant isolates and predominantly in those with multiple ERG11 mutations. This suggested a possible connection between CaCYP51 mutants and amino acid substitutions in Erg3. As the proteins encoded for by the ERG11 and ERG3 genes are involved in the same ergosterol biosynthesis pathway, defects in Erg11 enzyme function might be expected to impact accumulation of substrates of Erg3, specifically, precursors of the toxic sterol metabolite 14α-methylergosta- 8,24(28)-dien-3β, 6α-diol. By testing ERG11 mutant strains with and without the ERG3A351V allele in growth competition experiments, we hoped to observe a conferred fitness benefit by the ERG3 mutation. Interestingly, we were unable to generate one of our selected ERG11 mutants with the poorest catalytic turnover in the absence of the A351V amino acid change in Erg3. Future investigation by other lab members is needed to determine if ERG3 mutations can indirectly influence azole susceptibility through permissive mutation." "Lastly, we tested susceptibility of our clinical collection to the new tetrazole antifungals VT-1161 and VT-1598, which have been reported to exhibit potent activity against azole-resistant C. albicans and a host of other fungal species. We additionally investigated determinants of resistance to the two new agents by obtaining susceptibilities to C. albicans strains containing individual known mechanisms of azole resistance. While susceptibility to VT-1161 was reduced when CDR1 and MDR1 were overexpressed, VT-1598 seemed unaffected by any tested resistance mechanism. Importantly, both retained activity against a significant portion of mutant ERG11 strains. VT-1598 MICs were not affected by any single mechanism of resistance. However, screening of our azole-resistant clinical isolates identified five isolates with greatly elevated MICs to all tested agents. While one of these isolates possesses an ERG3 nonsense mutation that likely explains its pan-azole resistant profile, the other four isolates do not uniquely overexpress known resistance genes or possess known gene mutations that might explain their resistance. This finding suggests that there are determinants of azole resistance that are as yet undiscovered in C. albicans."

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

0000-0003-1244-4656

DOI

10.21007/etd.cghs.2019.0486

Additional Files
2019-007-Nishimoto-DOA.pdf (451 kB)
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