Date of Award
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Program
Pharmaceutical Sciences
Track
Bioanalysis
Research Advisor
Brian Peters, PhD
Committee
Camaron Hole, PhD; Glen Palmer, PhD; Jarrod Fortwendel, PhD; Michael Kruppa, PhD
Keywords
Candida albicans, cell wall, glycogen, host immune response, metabolism
Abstract
Candida albicans is an opportunistic human fungal pathogen that presents in healthy persons colonizing the oropharyngeal, esophageal, and gastrointestinal mucosa. This polymorphic fungal pathogen remains a leading cause of both invasive and superficial mycoses. C. albicans can cause oropharyngeal candidiasis (OPC) and disseminated candidiasis when normal host immunity is disturbed, while also causing vulvovaginal candidiasis (VVC) in healthy women. Adaptation to different host niches requires rapid and coordinated changes in Candida metabolism and physiology to cope with host immune surveillance and to counteract host-imposed constraints such as glucose limitation. Although largely unexplored in C. albicans, the glycogen metabolism pathway is a vital carbon assimilation and utilization system shown to be metabolically important in the model yeast Saccharomyces cerevisiae. Aside from the soluble glycogen that potentially contributes to Candida metabolism, glycogen has also been reported to be covalently linked to the immunogenic pathogen-associated molecular pattern (PAMP) b-(1,3)-glucan via a b-(1,6)-linked side chain to form a glucan-glycogen macromolecular complex at the cell wall interface of C. albicans. However, the mechanism driving its incorporation, and the potential immunological consequence of cell wall glycogen content are still unclear. This work focuses on C. albicans glycogen, including its metabolism and regulatory mechanisms, closing knowledge gaps on their impact on Candida fitness, virulence and host-Candida interaction. In C. albicans, there are S. cerevisiae orthologs involved in glycogen metabolism but these proteins and pathways have not been functionally validated. We characterized glycogen metabolism pathways in C. albicans and investigated whether these impact C. albicans long-term survival both in vitro and in vivo during murine VVC or virulence during systemic infection. Genetic deletion and complementation of key genes involved in glycogen metabolism in S. cerevisiae confirmed that glycogen synthase (encoded by GSY1) and branching enzyme (encoded by GLC3), glycogen phosphorylase (encoded by GPH1) and debranching enzyme (encoded by GDB1) are essential for glycogen synthesis and catabolism in C. albicans, respectively. Potential compensatory roles for a glucoamylase encoded by SGA1 were also explored. Competitive survival assays revealed that gsy1∆/∆, gph1Δ/Δ and gph1∆/∆ sga1∆/∆ mutants exhibited long-term survival defects in vitro under starvation conditions and in vivo during vaginal colonization concomitant with reduced neutrophil recruitment. Complete inability to catabolize glycogen (gph1∆/∆sga1∆/∆) also rendered C. albicans significantly less virulent during disseminated candidiasis. Using nuclear magnetic resonance (NMR) profiling, we confirmed that dysregulation of glycogen synthesis ablated cell wall glycogen content that is typically covalently linked to the cell wall PAMP b-(1,3)-glucan. Challenge of human immune cells with fixed or live glycogen synthase mutant gsy1Δ/Δ led to exacerbated pro-inflammatory cytokine secretion (e.g., IL-1β) from macrophages or neutrophil swarming as compared to a WT or revertant strain. Antibody-mediated receptor neutralization confirmed that elevated cytokine release observed in human macrophages challenged with gsy1Δ/Δ was partially dependent on hDectin-1 signaling. To further establish translational impact of our findings, a collection of C. albicans clinical isolates was screened for glycogen content and significant intra-species heterogeneity between inflammatory status and level of glycogen content was revealed. Remarkably, overexpression of GSY1 in a subset of reduced glycogen accumulation isolates reversed their hyper-inflammatory phenotype and deletion of GSY1 in a subset of WT-like glycogen accumulation isolates induced a hyper-inflammatory phenotype during human macrophage challenge. Further analysis of cell wall components by fluorescence staining and flow cytometry revealed that levels of total glucan, total mannan and total chitin remained similar, while reduced glycogen content significantly correlated with increased cell wall β-(1,3)-glucan exposure. As the dysregulation of glycogen synthesis greatly affects C. albicans fitness both in vitro and in vivo and alters host innate immune recognition, we wished to further explore glycogen synthesis regulatory mechanisms in C. albicans. We determined that the protein encoded by the uncharacterized gene C1_01140C, and not the currently annotated C. albicans Gac1p, is the major type 1 protein phosphatase regulatory (PPP1R) subunit involved in glycogen synthesis. C1_01140Cp contains a conserved GVNK motif observed across multiple starch/glycogen binding proteins in various species and alanine substitution of each residue in this motif significantly impaired glycogen accumulation in C. albicans. Moreover, fluorescent protein tagging, microscopy, and co-immunoprecipitation (Co-IP) indicated that C1_01140Cp colocalized and associated with the PPP1 catalytic (PPP1C) subunit of the CaGlc7p phosphatase and CaGsy1p glycogen synthase CaGsy1p. We demonstrated that C. albicans retains a PPP1-mediated glycogen synthase regulatory mechanism that is highly orthologous to S. cerevisiae. Given these observations, we propose that C1_01140C be named as GAC1 and that C7_00660W (previously annotated GAC1) be renamed GAC2. Collectively, this work demonstrates the impact of C. albicans glycogen metabolism on fitness, virulence and host-Candida interaction. Glycogen catabolism positively impacts C. albicans long-term fitness in nutrient deficient environments and is important for full virulence. The dysregulation of glycogen synthesis in C. albicans also impacts host-Candida interactions through altering β-(1,3)-glucan exposure. C1_01140Cp was newly characterized and identified as the major PPP1R binding subunit that associates with PPP1C CaGlc7p to initialize glycogen synthesis in C. albicans.
ORCID
0000-0001-7013-3800
DOI
10.21007/etd.cghs.2024.0676
Recommended Citation
Miao, Jian (0000-0001-7013-3800), "The Impact of Candida albicans Glycogen on Fitness, Virulence and Host Response" (2024). Theses and Dissertations (ETD). Paper 696. http://dx.doi.org/10.21007/etd.cghs.2024.0676.
https://dc.uthsc.edu/dissertations/696
Declaration of Authorship
Included in
Bacterial Infections and Mycoses Commons, Medical Pathology Commons, Pharmacy and Pharmaceutical Sciences Commons
