Date of Graduation

8-2022

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Pinto, Ines

Committee Member

Lewis, Jeffrey A.

Second Committee Member

Sakon, Joshua

Keywords

Antifungal; Antimicrobial peptide; Candida glabrata; Cas9; CRISPR

Abstract

Antibiotic resistance is increasing prevalence, particularly in Candida glabrata. This opportunistic pathogen is closely phylogenetically related to Saccharomyces cerevisiae; however, its characterization is limited. C. glabrata is only second to Candida albicans as a fungal pathogen in immunocompromised patients. Commonly resistant to azoles, the most common fungal therapy, it has become costly and challenging to treat. A histatin 5 derived antifungal peptide, KM29, has a high degree of efficacy in Candida species and S. cerevisiae. The objective of this work is to advance our understanding of the mechanism of action of KM29 against C. glabrata. Previous work in the lab used the S. cerevisiae deletion library as a related fungal system to identify mutants that resulted in increased resistance to KM29, with the goal of discovering pathways that could then be tested in C. glabrata. In this work we focused on plasma membrane transporters identified in the S. cerevisiae screen. Validation of the KM29 killing activity showed no significant increased resistance in deletions of four hexose transporters (HXT2, HXT3, HXT5, and HXT10) or two metal transporters (FET4 and ZRT2). Deletion of the membrane sterol regulator LAM1 and its paralog SIP3 showed significant resistance to KM29, as well as the polyamine transporter AGP2. We concentrated on setting up the CRISPR-Cas9 system to delete C. glabrata AGP2. A successful deletion was confirmed by DNA sequencing and positive mutants displayed increased resistance to KM29 in C. glabrata, although no increased resistance to polyamine toxicity was observed. Further analyses are needed to investigate AGP2 function. We conclude that the screen in S. cerevisiae can be useful to identify pathways in C. glabrata to understand the fungicidal mechanism of KM29.

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MS Thesis Supplemental.pdf (98520 kB)
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