Date of Award
Doctor of Philosophy (PhD)
Kerry S Smith
Cheryl J Ingram-Smith
The basidiomycete Cryptococcus neoformans is is an invasive opportunistic pathogen of the central nervous system and the most frequent cause of fungal meningitis. C. neoformans enters the host by inhalation and protects itself from immune assault in the lungs by producing hydrolytic enzymes, immunosuppressants, and other virulence factors. C. neoformans also adapts to the environment inside the host, including producing metabolites that may confer survival advantages. One of these, acetate, can be kept in reserve as a carbon source or can be used to weaken the immune response by lowering local pH or as a key part of immunomodulatory molecules. Thus, cryptococcosis could be treated by targeting acetate production. The Smith laboratory has identified two potential pathways for acetate production. 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) and acetaldehyde dehydrogenase (Ald) pathway, found in other fungi, converts pyruvate to acetate through acetaldehyde. The genes encoding enzymes from these pathways have been shown to be upregulated during infection, suggesting that acetate production may be a necessary and required part of cryptococcal infection. In Saccharomyces cerevisiae, Pdc works with one or more aldehyde dehydrogenases to produce acetate. Eight of the nine C. neoformans aldehyde dehydrogenases and the sole Pdc all contributed to the cellular acetate pool, and loss of some of these enzymes reduced cell survival during growth on various carbon sources, under oxidative or nitrosative stress, under pseudo-hypoxia conditions, and when the cell wall integrity was disrupted. In addition, deletion mutants of of some of these enzymes affected capsule formation and melanization, two primary determinants of Cryptococcus, and led to decreased virulence in macrophages and Galleria mellonella, an invertebrate model of infection.
Metabolic adaptability is an important attribute for fungal pathogens to successfully infect and cause disease. Carbon metabolism is critical for virulence in C. neoformans, but little is known about which carbon sources are utilized during infection. Lung alveolar macrophages, the a first line of host defense against C. neoformans infection, provide a glucose- and amino acid-poor environment, and nonpreferred carbon sources such as lactate and acetate are likely important early in establishment of a pulmonary infection. A global screening was undertaken to identify C. neoformans proteins necessary in acetate utilization, as possible drug targets. From two libraries, together comprising 3936 gene knockouts, 41 mutants failed to grow on media with either glucose or acetate as the carbon source, or on both media. Of the known proteins lacking in these mutants, most function in gluconeogenesis, arginine biosynthesis, or mitochondrial transmembrane transport. Overall, this work elucidated the roles of C. neoformans acetate production and utilization pathways in virulence.
Ammar, Mufida Ahmed Nagi, "Role of Cryptococcus neoformans Pyruvate Decarboxylase and Aldehyde Dehydrogenase Enzymes in Acetate Production and Virulence" (2019). All Dissertations. 2488.