GSE129227
The TOR pathway plays pleiotropic roles in growth and stress responses of the fungal pathogen Cryptococcus neoformans
The target of rapamycin (TOR) pathway is an evolutionarily conserved signal transduction system that is activated by varying nutrient and environmental signals and governs a plethora of eukaryotic biological processes, but its role in Cryptococcus neoformans remains elusive. In this study, we investigated the TOR pathway by functionally characterizing two Tor-like kinases, Tor1 and Tlk1, in C. neoformans. We successfully deleted TLK1, but not TOR1. TLK1 deletion did not result in any evident in vitro phenotypes, suggesting that Tlk1 is mainly dispensable for the growth of C. neoformans. We further demonstrated that Tor1, but not Tlk1, is essential and the target of rapamycin by constructing and analyzing conditionally regulated strains and sporulation analysis of heterozygous mutants in the diploid strain background. To analyze the functions of Tor1 in more detail, we constructed constitutive TOR1 overexpression strains. Tor1 negatively regulated thermotolerance and the DNA damage response, which are two important virulence factors of C. neoformans. We also found that TOR1 overexpression reduced Mpk1 phosphorylation, which is required for cell wall integrity and thermoresistance, and Rad53 phosphorylation, which governs the DNA damage response pathway. Tor1 is localized to the cytoplasm but enriched in the vacuole membrane. Phosphoproteomics and transcriptomics revealed that Tor1 regulates a variety of biological processes, including metabolic processes, cytoskeleton organization, ribosome biogenesis, autophagy, and stress response. TOR inhibition by rapamycin caused actin depolarization in a Tor1-dependent manner. Finally, screening rapamycin-sensitive and -resistant kinase and transcription factor mutants revealed that the TOR pathway may crosstalk with a number of signaling pathways, including the Hog1 pathway. In conclusion, our study demonstrates that a single Tor1 kinase plays a variety of roles in the fungal meningitis pathogen C. neoformans. Overall design: To investigate downstream genes regulated by TOR1, total RNA was isolated from the wild-type (H99), TOR1 overexpression (TOR1oe) mutant, and rapamycin treated wild-type strain. Each strains were incubated in YPD liquid medium overnight at 30°C, subcultured in fresh YPD liquid medium, and then further incubated at 30°C until OD600 was approximately 0.6–0.8. The WT cells were divided in half. One-half was used as a WT control while the other half was treated with rapamycin (3 ng/ml) and further incubated for 3 h at 30°C. The WT, TOR1oe, and rapamycin-treated WT cells were harvested and lyophilized. Three biologically independent cultures were prepared for each strain.