Date of Award
August 2021
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics and Biochemistry
Committee Member
Lukasz Kozubowski
Committee Member
Kimberly Paul
Committee Member
James Morris
Committee Member
Hong Luo
Abstract
Cryptococcus neoformans is an environmental basidiomycetous fungus and opportunistic pathogen that primarily infects the immunocompromised patients. The ability to adapt to mammalian body temperature is essential for survival and successful virulence. Cryptococcus neoformans relies on a multiple complex signaling network and virulence factors for the adaptation and survival in the environment of human host. This dissertation presents result of genetic screen assay performed on 4,031 gene knockout strains of C. neoformans. The analysis has resulted in identification of 46 genes with significant contribution to the maximum temperature at which C. neoformans can proliferate. Our research described previously uncharacterized genes that contribute to adaptation to growth at elevated temperature. A mutant that lacked septin Cdc11 was among those with the highest impact on high temperature tolerance. Septins are conserved filament - forming GTPases that play important role in cytokinesis, cell surface organization, and morphogenesis. Previous studies have demonstrated that septins are essential for growth of C. neoformans at the human body temperature. Analysis of changes in gene expression in wild type and septin cdc12Δ strains grown at 24°C and 37°C revealed that loss of septin compromises cellular response to heat and osmotic stress, metal ion transport and autophagy. Additionally, we discovered that deletion of septins in C. neoformans is compensated by changes in expression of over 130 genes. Gene described as CNAG_05305 presented the highest contribution to cell survival after loss of functional septin complex. Cryptococcal transient receptor potential channel, Flc1 is one of that contribute to adaptation to growth at elevated temperature. In budding yeast, Flc1 homologues has been shown to play significant role in in the cell growth regulation, calcium homeostasis, cell wall integrity, response to osmotic shock and flavin transport. Using molecular genetics methods, I discovered that Flc1 is important for regulation of calcineurin pathway, vacuolar fusion, and response to osmotic stress. Microscopic observation revealed that Flc1 plays critical role in capsule production and organization of the cell wall. Finally, in vivo experiments in macrophages, Galleria mellonella and mouse model demonstrated that Flc1 is essential for virulence of C. neoformans. These results demonstrate complexity of system involved in adaptation to human host in Cryptococcus neoformans.
Recommended Citation
Stempinski, Piotr Ryszard, "Genetic Contribution to Growth of Cryptococcus neoformans in the Environment of the Human Host" (2021). All Dissertations. 2889.
https://open.clemson.edu/all_dissertations/2889