Systems-level phosphoproteomics reveals conserved and subunit-specific STRIPAK signaling networks in <i>Cryptococcus neoformans</i>

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a conserved PP2A-associated signaling hub that integrates kinase-phosphatase networks, yet its roles in human fungal pathogens remain poorly defined. Here, we dissected STRIPAK functions in the opportunistic pathogen <i>Cryptococcus neoformans</i> by combining genetic, genomic, virulence, and systems-level phosphoproteomic analyses across mutants lacking individual STRIPAK subunits. Loss of the core STRIPAK components via <i>PPH22, FAR8, FAR9</i> , or <i>FAR11</i> mutations caused severe defects in growth, stress adaptation, cell-cycle progression, and morphogenesis, accompanied by widespread aneuploidy and genome instability. In murine infection models, <i>far11Δ</i> strains were avirulent, whereas <i>far9Δ</i> mutants caused delayed but ultimately fatal disease and underwent host-associated genome remodeling, with all recovered isolates acquiring chromosome 11 tetraploidy despite no consistent in vitro fitness advantage. In striking contrast, deletion of <i>MOB3</i> produced a hypervirulent phenotype. <i>mob3Δ</i> cells exhibited enhanced transmigration across an <i>in vitro</i> blood-brain barrier model, increased survival in macrophages, and generated abundant small-cell morphotypes <i>in vitro</i> and <i>in vivo</i> , features associated with increased dissemination. Global phosphoproteomic profiling revealed extensive and overlapping phosphorylation changes among core STRIPAK mutants, affecting pathways involved in signaling, cytoskeletal and cell-cycle control, chromatin and transcriptional regulation, RNA metabolism, and stress responses. By contrast, <i>mob3Δ</i> mutants displayed a smaller, largely distinct phosphoproteomic signature. Network and functional enrichment analyses highlighted STRIPAK-dependent regulation of MAPK/GTPase signaling, autophagy, nuclear transport, RNA processing, DNA replication, and ribosome biogenesis. Together, these findings establish STRIPAK as a central coordinator of genome stability, morphological plasticity, and virulence in <i>C. neoformans</i> , and demonstrate that individual STRIPAK subunits drive shared yet divergent signaling outputs that shape host-pathogen interactions.