Candida auris is a rising global health threat due to multidrug resistance, emphasizing the urgent need for novel antifungal strategies. The C. auris glycosylphosphatidylinositol transamidase (GPI-T) complex is the key enzyme that mediates the attachment of GPI anchors to target proteins. This essential step in the fungal GPI biosynthetic pathway is critical for fungal growth and virulence, which underscores the GPI-T complex's potential as an underexploited target for novel antifungal therapies. In this work, we determined the cryo-EM structure of C. auris GPI-T bound to a GPI substrate—the first structure of a pathogenic fungal GPI-T complex. Our structural and functional analyses of the C. auris GPI-T complex illuminate its assembly, substrate recognition, and catalytically relevant features, while identifying key divergences from the human GPI-T.
Key findings from the study include: (1) We reconstituted the C. auris 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. (2) We determined a 3.2 Å cryo-EM structure of C. auris GPI-T bound to a GPI substrate and revealed the pentameric organization of C. auris GPI-T complex, involving extensive contacts among Gpi8, Gpi16, Gaa1, Gpi17, and Gab1 and lipid-mediated inter-subunit stabilization between Gab1 and Gaa1. (3) We delineated a conserved GPI-binding pocket formed by Gaa1, Gab1, Gpi8 and Gpi16, and mutational analysis confirmed the functional importance of key residues within this pocket, establishing their essential role in substrate recognition and enzymatic activity. (4) Comparative analysis with the human GPI-T 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 C. auris GPI-T.
DOI：10.1093/procel/pwag023