Molecular insights into Candida auris glycosylphosphatidylinositol transamidase

<i>Candida auris</i> poses an escalating global health threat due to its rapid dissemination and extensive multidrug resistance, underscoring the urgent need for new antifungal strategies. The glycosylphosphatidylinositol transamidase (GPI-T) complex, a central component of the GPI biosynthesis pathway, is essential for fungal viability and virulence and therefore represents a compelling antifungal target. Here, we reconstituted the <i>C. auris</i> GPI-T complex in human cells and demonstrated that none of its subunits can functionally substitute for their human counterparts, revealing intrinsic species-specific incompatibility. To elucidate the molecular basis of this divergence, we determined a 3.2 Å cryo-electron microscopy structure of <i>C. auris</i> GPI-T bound to a GPI substrate-the first structure of a pathogenic fungal GPI-T complex.Our study reveals a pentameric architecture of GPI-T stabilized by extensive intersubunit interactions and lipid-mediated contacts between Gab1 and Gaa1, and defines the precise composition of the binding site of GPI substrate. Comparative analysis with the human enzyme uncovers substantial divergence at key interfaces, most notably within the catalytic subunit Gpi8, including a fungal-specific loop related to substrate engagement and a conserved C-terminal anchoring loop essential for catalytic activity.Together, these findings provide a structural framework for understanding GPI-T function, define species-specific human-fungal divergences, establish a rational foundation for designing pathogen-selective antifungal therapies targeting <i>C. auris</i> GPI-T.