Date of Award
8-2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics and Biochemistry
Committee Chair/Advisor
Dr. Lukasz Kozubowski
Committee Member
Dr. James Morris
Committee Member
Dr. Kimberly Paul
Committee Member
Dr. Kerry Smith
Abstract
Cryptococcus neoformans is a globally distributed fungal pathogen responsible for causing cryptococcal meningitis in immunocompromised individuals. This pathogenic yeast must adapt to changes in temperature upon entering the human host. Septin proteins are conserved filament-forming GTPases that assemble as higher-order complexes at the cell cortex to support cytokinesis and morphogenesis in fungal and animal cells. In C. neoformans, four septin homologs (Cdc3, Cdc10, Cdc11, and Cdc12) assemble at the mother-bud neck, contributing to cytokinesis through poorly understood mechanisms. C. neoformans strains lacking the septins Cdc3 or Cdc12 are viable at 25°C, but fail to proliferate at 37°C, and are avirulent in the Galleria mellonella model of infection. Our observations indicate that the entire septin complex also congregates at the plasma membrane, particularly in response to temperature stress at 37°C. This investigation used co-immunoprecipitation (co-IP) and tandem-mass spectrometry to examine the proteins associated with Cdc3 and Cdc10 in C. neoformans at both 25 and 37°C. A total of 196 and 40 proteins were identified through Cdc3 co-IP at 25 and 37°C, respectively. In contrast, 112 and 46 proteins were associated with Cdc10 at 25 and 37°C, respectively. Additionally, 14 proteins were found to interact with both Cdc3 and Cdc10 at both temperatures, while 9 proteins were identified as exclusively associating with both Cdc3 and Cdc10 at 37°C. The identified proteins specific to heat stress are involved in GDP-GTP exchange, mRNA processing, microtubule binding, and plasma membrane transport. The study also delves into the role septins play in upholding cell wall integrity and plasma membrane stability in C. neoformans. Septin deletion mutants of Cdc3 and Cdc12 in C. neoformans are susceptible to cell wall and plasma membrane-disrupting agents, emphasizing the pivotal function of septins in stress resistance. Moreover, the absence of Cdc3 or Cdc12 resulted in abnormal septum structure morphology and compromised the composition of the cell wall and plasma membrane. These deletion mutants also displayed heightened sensitivity to sphingolipid synthesis inhibitors, indicating the involvement of septins in membrane lipid homeostasis. Additionally, this study explored the interplay between septins and the cell wall integrity-MAPK pathway. The strains lacking Cdc3 or Cdc12 exhibited elevated levels of phosphorylated Mpk1, a homolog of Saccharomyces cerevisiae Mpk1/Slt2 MAP kinase, during optimal temperature growth (similar to the levels observed in wild-type cells during heat stress). Furthermore, C. neoformans cells lacking both Cdc3 and Cdc12 showed a significant decrease in Mpk1 phosphorylation, suggesting a failure to properly activate the MAPK signaling pathway during heat stress. Together, these findings suggest septins play an important role in C. neoformans coordinating stress response, especially to stressors that disrupt the cell wall and plasma membrane.
Recommended Citation
Martinez Barrera, Stephani S., "Elucidating the Roles of Septin Proteins in Thermotolerance and Cell Wall Integrity in Cryptococcus neoformans" (2024). All Dissertations. 3700.
https://open.clemson.edu/all_dissertations/3700
Author ORCID Identifier
0000-0002-9350-3576
Included in
Biochemistry Commons, Cell Biology Commons, Molecular Biology Commons, Molecular Genetics Commons, Pathogenic Microbiology Commons