Abstract
Fungal infections represent a staggering global health crisis, affecting over 1 billion people annually and resulting in more than 3.75 million deaths. The clinical utility of current antifungal classes including azoles, echinocandins, and polyenes is increasingly compromised by the rapid evolution of antifungal resistance, driven by the inherent genomic plasticity of fungal pathogens and the emergence of new pathogens. As traditional therapies face these limitations, there is an urgent need for novel therapeutic strategies that exploit unique fungal biological pathways. Killer yeasts, which produce and excrete proteinaceous toxins to inhibit the growth of competing fungi, offer a promising alternative. Among these, the K1 killer toxin from Saccharomyces cerevisiae has demonstrated potent activity against pathogens such as Nakaseomyces glabratus (syn. Candida glabrata) and is thought to bind to the cell wall protein Kre1. Understanding the molecular interface between these toxins and their targets is essential for developing next-generation antimycotics.The fungal cell wall is a dynamic, essential organelle that serves as the primary interface between the fungus and its environment, providing structural integrity and mediating host-pathogen interactions. Within this complex architecture, the protein Kre1 (Killer Toxin Resistant 1) occupies a critical regulatory and structural node. Kre1 is a glycosylphosphatidylinositol (GPI)-anchored protein that undergoes extensive post-translational processing through the secretory pathway, including O-glycosylation. Deletion of the KRE1 gene results in a profound reduction of approximately 40% in cell wall beta-1,6-glucan content, leading to severe aberrations in cell wall ultrastructure and increased susceptibility to environmental stressors. However, its deletion also leads to resistance to the K1 toxin..
Crucially, Kre1 serves a dual role: it is both a biosynthetic effector of cell wall assembly and the hypothesized secondary cell wall receptor for the K1 killer toxin. K1 is thought to function as an ionophore, binding to Kre1 to facilitate the formation of lethal ion channels in the plasma membrane, which results in cellular death via ion leakage. Strains lacking the KRE1 gene exhibit complete immunity to K1, yet the influence of genetic variation across different fungal species on this susceptibility remains poorly understood.
This thesis investigates the role of Kre1 orthologs in mediating K1 toxin susceptibility and cell wall integrity. By engineering S. cerevisiae strains to express KRE1 variants from related species, including the human pathogen Nakasomyces glabratus, this research characterizes how evolutionary divergence in protein structure dictates toxin binding and resistance. Through phenotypic assays involving cell wall stressors, calcium chloride (CaCl), and calcofluor white (CFW), the study of cell-wall-depleted spheroplasts, and investigation of key protein domains, this work tests the hypothesis that orthologous Kre1 compositions are the primary determinants of K1 resistance. By elucidating these mechanisms, this research aims to bridge the gap between basic yeast genetics and medical mycology, identifying Kre1 as a high-value target for novel antifungal interventions and further defining the molecular interactions of the fungal cell surface