Date of Award
Doctor of Philosophy (PhD)
Cryptococcus neoformans is an environmental basidiomycetous fungus with a worldwide distribution and a wide range of habitats. Inhalation of the desiccated yeasts or spores of C. neoformans often leads to opportunistic pulmonary infections in immunocompromised individuals, and in severe cases causes lethal meningitis following hematogenous dissemination. During infection, depending on the tissue and disease state, the invading fungi experience a range of nutrient microenvironments within the host body. As a result, rapid metabolic adaptations geared towards efficient utilization of carbon sources alternative to glucose become one of the prime determinants of survival and growth for the pathogen. Incidentally, cryptococcal infection has a long-standing association with acetate metabolism as underlined by previous cryptococcal transcriptomic studies in infection models that revealed an increased activity of alternative carbon metabolism during infection, and highlighted by the observation that cryptococcomas (infectious granulomas) are enriched with the two-carbon metabolite acetate, a physiologically important alternative carbon source. In this work I investigated the direct transcriptomic impacts of acetate utilization compared to glucose utilization via RNA-sequencing, and noted that cryptococcal transcriptome bears signatures of nutrient starvation and metabolic adaptations in acetate-grown conditions, as well as a remarkable upregulation of virulence-associated genes. Moreover, to investigate the importance of acetate production in C. neoformans we biochemically and kinetically characterized acetate kinase (Ack), an enzyme involved in acetate metabolism, and found that kinetically Ack has a preference in the acetate forming direction compared acetyl phosphate forming direction. This observation is consistent with the previously discovered biochemical role of another cryptococcal enzyme xylulose-5-phosphate/ fructose-6-phosphate phosphoketolase (Xfp2), which produces acetyl phosphate from phosphoketose sugars. Taken together, here I propose that Ack and Xfp2 forms a pathway of acetate production in C. neoformans, and hence are likely to be involved in acetate homeostasis. Possibility of the Xfp-Ack pathway of acetate production in the fungal species C. neoformans prompted us to search for Ack sequences in other eukaryotic genomes. Here I employed a hidden Markov model based strategy to predict Ack sequences in publicly available and curated eukaryotic genomes, and found that an overwhelming majority of the predicted Ack sequences can be observed in dikaryotic fungi. Maximum likelihood based phylogeny of the predicted eukaryotic Ack sequences suggest an evolutionary trajectory involving divergence from a common ancestor and loss in most eukaryotic lineages. Finally, to interpret the observation that acetate utilization appears to be connected with the upregulation of virulence-associated genes in C. neoformans, I have proposed a new theoretical framework to explain the emergence of virulence in fungal pathogens. The hypothesis posits that molecular response to various environmental stresses and virulence phenotypes are modular in nature and hence are often not connected in most fungal species. Occasionally, species may co-opt virulence modules, and hierarchically nest it with stress-response modules, thereby acquiring the ability of opportunistic pathogenicity in mammalian hosts. Overall, this work furthers our current understanding of the impacts of acetate metabolism in the human fungal pathogen C. neoformans.
Ahmed, Oly, "Acetate Metabolism in the Fungal Pathogen Cryptococcus Neoformans" (2023). All Dissertations. 3324.
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