Project description:Thermotolerance is a crucial virulence attribute for Cryptococcus neoformans, which causes fatal fungal meningitis in humans. A protein kinase, Sch9, suppresses the thermotolerance of C. neoformans but its regulatory mechanism remains unknown. Here we elucidated the Sch9-dependent and -independent signaling networks for modulating the thermotolerance of C. neoformans through a genome-wide transcriptome analysis and reverse genetics approaches. We found that more than 1,800 genes were under transcriptional control during temperature upshift. Genes encoding for molecular chaperones and heat shock proteins were mainly upregulated, while those for translation, transcription, and sterol biosynthesis were highly suppressed. In this process Sch9 was found to regulate basal or induced expression levels of some temperature-responsive genes. Interestingly we found that Sch9 was involved in transcriptional regulation of the Ire1 kinase, which is a key sensor for the unfolded protein response pathway, and was found to be involved in ER stress response. Most notably, our data demonstrated that expression of HSF1, encoding a heat shock transcription factor 1, was downregulated during temperature upshift and Sch9 suppresses its downregulation. In spite of such expression patterns, Hsf1 was essential for growth and its overexpression indeed promoted the thermotolerance of C. neoformans, suggesting dual roles of Hsf1 in thermotolerance. This idea was supported by additional transcriptome analysis with HSF1 overexpression strain, which revealed that Hsf1 served as both activator and repressor. Hsf1 promoted genes such as Hsp104 and Hsp70 (Ssa1 and Ssa2), both of which were found to be highly upregulated during temperature upshift and required for thermotolerance, while Hsf1 repressed genes involved in oxidative stress and thermotolerance.
Project description:Thermotolerance is a crucial virulence attribute for Cryptococcus neoformans, which causes fatal fungal meningitis in humans. A protein kinase, Sch9, suppresses the thermotolerance of C. neoformans but its regulatory mechanism remains unknown. Here we elucidated the Sch9-dependent and -independent signaling networks for modulating the thermotolerance of C. neoformans through a genome-wide transcriptome analysis and reverse genetics approaches. We found that more than 1,800 genes were under transcriptional control during temperature upshift. Genes encoding for molecular chaperones and heat shock proteins were mainly upregulated, while those for translation, transcription, and sterol biosynthesis were highly suppressed. In this process Sch9 was found to regulate basal or induced expression levels of some temperature-responsive genes. Interestingly we found that Sch9 was involved in transcriptional regulation of the Ire1 kinase, which is a key sensor for the unfolded protein response pathway, and was found to be involved in ER stress response. Most notably, our data demonstrated that expression of HSF1, encoding a heat shock transcription factor 1, was downregulated during temperature upshift and Sch9 suppresses its downregulation. In spite of such expression patterns, Hsf1 was essential for growth and its overexpression indeed promoted the thermotolerance of C. neoformans, suggesting dual roles of Hsf1 in thermotolerance. This idea was supported by additional transcriptome analysis with HSF1 overexpression strain, which revealed that Hsf1 served as both activator and repressor. Hsf1 promoted genes such as Hsp104 and Hsp70 (Ssa1 and Ssa2), both of which were found to be highly upregulated during temperature upshift and required for thermotolerance, while Hsf1 repressed genes involved in oxidative stress and thermotolerance.
Project description:Thermotolerance, a key factor essential for the virulence of pathogenic fungi including Cryptococcus neoformans, remains largely unexplored in terms of its underlying mechanism. In this study, our findings demonstrate that Set3C, a widely distributed and conserved histone deacetylase complex, is required for thermotolerance in Cryptococcus neoformans. Specifically, the deletion of the core subunit Set302, responsible for the integrity of the complex, results in a significant reduction in the growth ability under high stress and the viability at extreme temperature. Moreover, the absence of Set302 leads to a decrease in the production of capsule and melanin. Transcriptomics analysis revealed that Set302 regulates a large number of genes compared to normal condition, and their expression is responsive to heat stress. Notably, we observed that Set302 positively influences the expression of genes related to ubiquitin-proteasome system (UPS) at high temperature. Using GFP-α-synuclein overexpression model, we observed a pronounced accumulation of misfolded proteins under heat stress, consequently inhibiting the thermotolerance of Cryptococcus neoformans. Furthermore, the loss of Set302 exacerbates this inhibition of thermotolerance. Interestingly, set302∆ strain exhibits a similar phenotype under proteasome stress as it does under high temperature. We also found that set302∆ strain displayed significantly reduced pathogenicity and colonization ability compared to the wild-type strain in the murine infection model. Collectively, our findings indicate that Set302 modulates the degradation of misfolded proteins through the UPS pathway, thereby affecting the thermotolerance and pathogenicity of Cryptococcus neoformans.
Project description:Comparison of transcriptional profiles of WT Cryptococcus neoformans (H99) and strain CM126 (pRPL2b-GAT201) which overexpresses the transcription factor GAT201 using a ribosomal protein promoter Keywords: Genetic modification
Project description:Comparison of transcriptional profiles of WT Cryptococcus neoformans (H99) and strain CM126 (pRPL2b-GAT201) which overexpresses the transcription factor GAT201 using a ribosomal protein promoter Keywords: Genetic modification WT vs. CM126 competitive hybridization. 4 biological replicates including 2 dye flips. Cultures grown at 37 degress Celsius in minimal (YNB) medium. Cultures independently grown and harvested during exponential growth.
Project description:We measured protein translation (by ribosome profiling) and RNA levels (by polyA-enriched RNA-seq) in Cryptococcus neoformans strain H99 and Cryptococcus neoformans strain JEC21. This is the first transcriptome-wide map of translation in this species complex.
