The Cryptococcus neoformans STRIPAK complex controls genome stability, sexual development, and virulence

Abstract

The eukaryotic serine/threonine protein phosphatase PP2A is a heterotrimeric enzyme composed of a scaffold A subunit, a regulatory B subunit, and a catalytic C subunit. Of the four known B subunits, the B subunit (known as striatin) interacts with the multi-protein striatin-interacting phosphatase and kinase (STRIPAK) complex. Orthologs of STRIPAK components were identified in Cryptococcus neoformans, namely PP2AA/Tpd3, PP2AC/Pph22, PP2AB/Far8, STRIP/Far11, SLMAP/Far9, and Mob3. Structural modeling, protein domain analysis, and detected protein-protein interactions suggest C. neoformans STRIPAK is assembled similarly to the human and fungal orthologs. Here, STRIPAK components Pph22, Far8, and Mob3 were functionally characterized. Whole-genome sequencing revealed that mutations in STRIPAK complex subunits lead to increased segmental and chromosomal aneuploidy, suggesting STRIPAK functions in maintaining genome stability. We demonstrate that PPH22 is a haploinsufficient gene: heterozygous PPH22/pph22{Delta} mutant diploid strains exhibit defects in hyphal growth and sporulation and have a significant fitness disadvantage when grown in competition against a wild-type diploid. Deletion mutants pph22{Delta}, far8{Delta}, and mob3{Delta} exhibit defects in mating and sexual differentiation, including impaired hyphae, basidia, and basidiospore production. Loss of either PPH22 or FAR8 in a haploid background leads to growth defects at 30C, severely reduced growth at elevated temperature, abnormal cell morphology, and impaired virulence. Additionally, pph22{Delta} strains frequently accumulate suppressor mutations that result in overexpression of another putative PP2A catalytic subunit, PPG1. The pph22{Delta} and far8{Delta} mutants are also unable to grow in the presence of the calcineurin inhibitors cyclosporine A or FK506, and thus these mutations are synthetically lethal with loss of calcineurin activity. Conversely, mob3{Delta} mutants display increased thermotolerance, capsule production, and melanization, and are hypervirulent in a murine infection model. Taken together, these findings reveal that the C. neoformans STRIPAK complex plays an important role in genome stability, vegetative growth, sexual development, and virulence in this prominent human fungal pathogen. Author summaryThis study focused on a highly conserved protein signaling complex known as STRIPAK, which is important for various developmental processes in fungi and humans. By investigating the functions of this complex in Cryptococcus neoformans, it was discovered to play crucial roles in maintaining genome stability, sexual development, and pathogenesis. In particular, mutations in the genes encoding two subunits of the STRIPAK complex were found to lead to significant defects, including abnormal growth and cell morphology, compromised stress response, and impaired virulence. Interestingly, mutation of a third STRIPAK complex subunit resulted in hypervirulence, characterized by increased thermotolerance, enhanced production of melanin pigment and polysaccharide capsule, and reduced survival in infected animals. Our findings reveal that the STRIPAK complex is an important regulator of growth and virulence of C. neoformans, highlighting its potential as a target for therapies aimed at combating fungal infections. This work furthers understanding of how the STRIPAK complex functions in Cryptococcus, and also in other organisms including humans, where related protein phosphatase complexes govern key cellular processes.