Date of Award
Doctor of Philosophy (PhD)
Kerry S. Smith, Committee Chair
Cryptococcus neoformans is a basidiomycetous fungal pathogen that claims 625,000 lives annually worldwide. Particularly among the immunocompromised, it is the leading cause of fungal meningitis following pulmonary colonization and dissemination to the central nervous system via the blood-brain barrier. The success of C. neoformans as a pathogen is largely attributable to metabolic scavenging during starvation conditions within the host's environment. Lung alveolar macrophages, which are among the first immune effectors to combat an initial pulmonary infection, present a glucose- and amino acid-poor environment that likely necessitates metabolism of nonpreferred carbon sources such as lactate and acetate for establishment of a pulmonary infection.Â While acetate transporters have been characterized in ascomycetous fungi such as Saccharomyces cerevisiae, Candida albicans, Aspergillus nidulans and Yarrowia lipolytica the role of acetate production and transport in C. neoformans is not well understood and its importance in infection has not been established. Putative acetate transporter genes, designated as ADY2 and ATO2, from the GPR1/FUN34/YAAH family have been identified in C. neoformans and are highly expressed during infection in the lung and during growth on acetate as sole carbon source. Studies have identified acetate as one of the metabolites in brain tissue biopsies of infected rats and in culture supernatant. We propose that Ady2 and Ato2 have a role in acetate transport as a metabolic adaptation during starvation and stressful environmental conditions. Growth of the ady2 single mutant, and the ady2ato2 double mutant, but not ato2 single mutant, was severely impaired in 1 mM versus 10 mM acetate plates, suggesting that Ady2 is the essential acetate importer during growth on low acetate. Intensity of growth defects was emphasized by a 137-fold overexpression of ADY2 during growth on acetate in relation to growth on glucose. The role of Ato2 during prolonged starvation was highlighted by diminished acetate uptake in the ato2 single mutant after glucose-grown cells were passaged into minimal media with acetate as sole carbon source. This study also uncovered a role of Ady2 and Ato2 in ammonia export and that they preferentially import acetate versus other carboxylates. Major C. neoformans virulence factors: growth at 37Â°C and capsule synthesis were impaired by loss of Ady2 and Ato2. Moreover, Ady2 and Ato2 were essential for virulence in mice such that ady2ato2 infected mice survived as long as uninfected controls. Additionally, Ady2 and Ato2 were additively required during coculture with human and mouse-derived phagocytes. Therefore, acetate transporters play a significant role in the survival and virulence of C. neoformans.
Kisirkoi, Grace Naanyu, "Acetate Transport Is Essential for Survival and Virulence of Cryptococcus neoformans" (2017). All Dissertations. 1915.