Date of Award

5-2023

Document Type

Thesis

Department

Microbiology

Committee Chair/Advisor

Dr. Emily Rosowski

Second Advisor

Dr. Savini Thrikawala

Abstract

Invasive fungal infections account for nearly 1.5 million deaths a year and therefore are considered one of the leading causes of death worldwide. The first line of therapy towards these infections are antifungal drugs in the triazole class such as voriconazole, posaconazole, itraconazole, and isavuconazole. However, these antifungal drug therapies fail in a large number of patients and can produce toxic side effects, therefore increasing the risk of mortality worldwide. While prior research has demonstrated the efficacy of these drugs in mitigating fungal growth in vitro, it is unclear why these drugs fail to protect patients infected with fungal pathogens. Cytochrome P450 (CYP-P450) enzymes are involved in the metabolism of triazole antifungal drugs. Particularly, CYP3A4 and CYP1A are responsible for a large majority of triazole drug metabolism in the human body. I hypothesized that CYP3A4 and CYP1A proteins mediate triazole drug efficacy and toxicity in a whole organism. I utilized a larval zebrafish model to study how these genes affect the efficacy and toxicity of triazole drug treatments in vivo. I identified orthologs of the human CYP3A4 and CYP1A genes in zebrafish using the Zebrafish Information Network (ZFIN): cyp3c1 and cyp1a, respectively. I first tested if these genes are expressed in zebrafish larvae by performing RT-PCR of RNA extracted from zebrafish larvae. I find that both cyp3c1 and cyp1a are expressed in the larvae at 2-, 3-, and 5-days post fertilization. To determine the effects of antifungal drug treatment on the expression of these genes, I treated zebrafish larvae with four antifungal drugs at 2 days post fertilization: voriconazole, posaconazole, itraconazole, and isavuconazole. The most notable difference was the significant upregulation of expression in the cyp3c1 gene at one day post treatment with isavuconazole. However, there was a significant upregulation of this gene’s expression at 2 days post treatment with itraconazole. To determine how this could impact the drug toxicity of the triazole antifungal drugs, CRISPR-Cas9 system was utilized to mutate the cyp3c1gene. To do so, I generated two gRNAs to target this gene as well as

forward and reverse primers to check that the gRNAs induce double strand breaks at the targeted regions of the gene. An injection mixture containing both gRNAs for the genes as well as Cas9 protein was injected into the zebrafish embryos at the one cell stage. At two days post injection the larvae were treated with the four drugs and survival of the larvae was tracked. The findings thus far demonstrate that the CYP3A4 homolog in zebrafish, cyp3c1, protects against isavuconazole drug toxicity in vivo. In the future we will infect cyp3c1 mutant larvae with Aspergillus fumigatus and treat infected larvae with triazole drugs to determine the role of cyp3c1 in drug efficacy against a fungal infection.

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