Systems-level phosphoproteomics reveals conserved and subunit-specific STRIPAK signaling networks in Cryptococcus neoformans

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 Cryptococcus neoformans by combining genetic, genomic, virulence, and systems-level phosphoproteomic analyses across mutants lacking individual STRIPAK subunits. Loss of the core STRIPAK components via PPH22, FAR8, FAR9, or FAR11 mutations caused severe defects in growth, stress adaptation, cell-cycle progression, and morphogenesis, accompanied by widespread aneuploidy and genome instability. In murine infection models, far11{Delta} strains were avirulent, whereas far9{Delta} 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 MOB3 produced a hypervirulent phenotype. mob3{Delta} cells exhibited enhanced transmigration across an in vitro blood-brain barrier model, increased survival in macrophages, and generated abundant small-cell morphotypes in vitro and in vivo, 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, mob3{Delta} 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 C. neoformans, and demonstrate that individual STRIPAK subunits drive shared yet divergent signaling outputs that shape host-pathogen interactions. ImportanceFungal pathogens must rapidly adapt their growth, morphology, and stress responses to survive within the host, processes that depend on precise regulation of cellular signaling pathways. The conserved striatin-interacting phosphatase and kinase (STRIPAK) complex controls key developmental programs in eukaryotes, but its roles in fungal pathogenesis are not fully defined. We previously showed that STRIPAK is important for genome stability, development, and virulence in the opportunistic human pathogen Cryptococcus neoformans. Here, we demonstrate that disruption of additional STRIPAK subunits causes severe growth and stress-response defects, and attenuation of virulence, but can also drive genome plasticity and morphological adaptation during infection. In contrast, loss of the Mob3 subunit promotes hypervirulence by enhancing dissemination and persistence within the host. Phosphoproteomic profiling reveals that individual STRIPAK components exert shared yet distinct control over phosphorylation networks that shape host-pathogen interactions, establishing STRIPAK as a central signaling hub and a potential target for antifungal intervention.