Ubiquitin degradation of the Ribonuclease H2 subunit B Rnh2B regulates the virulence of <i>Cryptococcus neoformans</i>

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 <i>C. neoformans</i>, 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 <i>RNH2B</i> 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 <i>RNH2B</i> 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 <i>rnh2B</i>Δ mutants maintained a low yet stable fungal burden, in contrast to a significant reduction in fungal load observed in conjunction with <i>RNH2B</i> overexpression, ultimately leading to complete clearance. Analysis of host immune responses indicated a correlation between diminished virulence in the <i>RNH2B</i>^OE strain and robust host immune activation. Notably, both the <i>RNH2B</i>^OE strain and the <i>cdc4</i>Δ mutant induced analogous adaptive immune responses, facilitating the clearance of <i>C. neoformans</i> infection. Collectively, our findings propose a model wherein Cdc4 mediates the ubiquitination and degradation of Rnh2B, thereby exerting regulatory control over the pathogenicity of <i>C. neoformans</i>.