Date of Award

8-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics and Biochemistry

Committee Chair/Advisor

Kerry Smith

Committee Member

Cheryl Ingram-Smith

Committee Member

Lukasz Kozubowski

Committee Member

Hong Luo

Abstract

The basidiomycete Cryptococcus neoformans is an invasive opportunistic fungal pathogen and the most frequent cause of fungal meningitis resulting in approximately 112,000 deaths per year worldwide. Recently, the World Health Organization systematically prioritized fungal pathogens in which C. neoformans was among four fungal pathogens deemed critically important. Current methods of treatment are inadequate for most of the infected population; therefore, it is imperative to find novel therapeutic targets. Previous studies have shown that acetate is a major metabolite found in biopsies of C. neoformans infected mouse brain and lung tissues. Two potential pathways for acetate production have been identified. The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) - acetate kinase (Ack) pathway, previously thought to be present only in bacteria, converts phosphoketose sugars to acetate through acetyl-phosphate. The pyruvate decarboxylase (Pdc) – acetaldehyde dehydrogenase (Ald) pathway, found in other fungi, converts pyruvate to acetate through acetaldehyde. We observed the growth of gene deletion mutants of ACK, XFP1, XFP2, and XFP1/XFP2 on various carbon sources and stress conditions at both 30ºC and 37ºC. The XFP-ACK mutants displayed various phenotypes under these conditions. All the mutants produced less melanin and a significantly smaller capsule, two key virulence determinants. During primary infection C. neoformans faces unfavorable conditions such as low glucose availability. As a result, C. neoformans may assimilate other readily available alternative carbon sources such as acetate. After entry into the cell, cytosolic acetate is converted into acetyl-CoA by acetyl-CoA synthetase. The acetyl unit can then be transported into the mitochondria via the carnitine shuttle. In C. neoformans carnitine is generated de novo, however the carnitine biosynthesis pathway has yet to be investigated in C. neoformans. We report the first genetic characterization of a partial carnitine biosynthesis pathway in C. neoformans. Through genetic analysis and metabolic complementation, we have identified the first (trimethyllysine hydroxylase), third (trimethylaminobutyrlaldehyde dehydrogenase), and fourth (γ-butyrobetaine hydroxylase) steps in this pathway. C. neoformans deletion mutants of genes encoding these enzymes display reduced growth on acetate and reduced production of melanin.

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