Abstract
The ubiquitin-proteasome system (UPS) regulates protein degradation in eukaryotes by polyubiquitinating substrate proteins, with F-box proteins serving as key components for substrate recognition. Research has shown that the absence of Cdc4, an F-box protein in C. neoformans, decreases virulence, yet the mechanisms by which Cdc4 affects these processes remain unclear. Using an iTRAQ-based proteomic strategy, we recognized Ribonuclease H2 subunit B (Rnh2B) as a target substrate for Cdc4. Protein-protein interaction assays have confirmed the interaction between Cdc4 and Rnh2B, indicating that Cdc4 regulates Rnh2B stability through ubiquitination. Functional analyses revealed that both the knockout and overexpression of RNH2B impair growth at 37°C, with the latter also affecting cellular membrane integrity and growth under osmotic stress conditions. Pathogenicity assays revealed that the deletion of RNH2B results in reduced virulence, whereas its overexpression leads to a complete loss of pathogenicity. Longitudinal tracking of fungal infection progression illustrated that mice lungs infected with rnh2BΔ mutants maintained a low yet stable fungal burden, in contrast to a significant reduction in fungal load observed in conjunction with RNH2B overexpression, ultimately leading to complete clearance. Analysis of host immune responses indicated a correlation between diminished virulence in the RNH2BOE strain and robust host immune activation. Notably, both the RNH2BOE strain and the cdc4Δ mutant induced analogous adaptive immune responses, facilitating the clearance of C. neoformans infection. Collectively, our findings propose a model wherein Cdc4 mediates the ubiquitination and degradation of Rnh2B, thereby exerting regulatory control over the pathogenicity of C. neoformans.