Unraveling the Mechanism of Thermotolerance in Cryptococcus neoformans by Set3C Complex
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ABSTRACT: 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: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:Discovering the Roles of the Casein Kinase 2 Complex in the Growth, Differentiation, Stress Responses, and Pathogenicity of Cryptococcus neoformans
Project description:WD40 motif-containing Msi1-like (MSIL) proteins play pleiotropic cellular functions as a negative regulator of the Ras/cAMP-pathways and a component of chromatin assembly factor-I (CAF-I), and yet have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans, which can cause fatal meningoencephalitis in humans. Notably, Msl1 was not a functional ortholog for the yeast Msi1 but played pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners but mainly Ras-independently. Msl1 negatively controlled antioxidant melanin production and sexual differentiation, which can be repressed by inhibiting the cAMP-signaling pathways. In contrast, Msl1 controlled thermotolerance, diverse stress responses, and antifungal drugs resistance in Ras/cAMP-independent manners. Cac2, which is the second CAF-I component, appeared to play both redundant and distinct function with Msl1. Msl1 is required for full virulence of C. neoformans. Transcriptome and proteomic analysis identified a group of Msl1-regulated genes or -interacting proteins, respectively, which mostly include stress-related genes, including HSP12, HSP78, SSA1, SSA4, and STM1. Furthermore, we identified the third putative component of CAF-1, Rlf2, in C. neoformans. In conclusion, this study demonstrated the pleiotropic roles of Msl1 in human fungal pathogen C. neoformans, providing a novel antifungal therapeutic target. There is more than 95% genome homology between JEC21 and H99. Therefore, 6 slides of JEC21 (Cryptococcus neoformans var. neoformans serotype D) 70-mer oligos are used in this analysis. Total RNAs are extracted from 2 strains from H99 (H99 wild-type strain (Cryptococcus neoformans var. grubii serotype A), msl1M-NM-^T). 3 biological replicate experiments are performed for each strain. We use the mix of all total RNAs from this experiment as the control RNA. We use Cy3 as the test sample dye and Cy5 as the control dye.
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.
Project description:Rho-GDP dissociation inhibitors (RDI) are repressors of Rho-type monomeric GTPases that allow for precise control of their target processes, e.g. cytoskeletal arrangement, vesicle trafficking, and polarized growth. In the human pathogenic yeast Cryptococcus neoformans, maintenance of normal cell morphology is vital for pathogenicity. We identified and deleted the gene encoding an RDI homolog in the human fungal pathogen Cryptococcus neoformans and investigated its impact on pathogenicity in animal models of cryptococcosis. Rdi1 deletion resulted in altered vacuole size in tissue culture medium, with corresponding alterations in expression of vesicle trafficking related genes. The rdi1∆ mutant strain showed reduced intracellular survival in macrophages, and severe attenuation of virulence in murine models of cryptococcosis. This reduction in virulence of the rdi1 mutant occurs in the absence of major defects in growth, morphology, or classical virulence-associated phenotypes. Keywords: mutant response, macrophage co-culture
Project description:Cryptococcus neoformans is a type of pathogenic fungi that can cause infections in people with weakened immune systems. This fungal pathogen can survive in the body by growing at the host's body temperature (37\'b0C), forming a carbohydrate capsule, and producing melanin. When Cryptococcus neoformans strains lack certain septin proteins, they cannot grow at 37\'b0C and are not able to cause infections in mice. However, it is not yet clear how septins help the yeast to grow at the host's body temperature. Septins are a group of proteins that are important for cell division and morphogenesis. In the model organism, Saccharomyces cerevisiae septins are essential. S. cerevisiae septins form a higher-order complex at the mother bud-neck to scaffold over 80 proteins, including those involved in cell wall organization, cell polarity, and cell cycle control. In C. neoformans, septins also form a complex at the mother bud neck but the septin interacting proteome in this species remains largely unknown. In this study, the entire septin complex in C. neoformans was uncovered as downregulated during the stationary growth phase and heat stress. In addition, we investigated the septin interactome in C. neoformans, shedding light on the proteins that interact with septins Cdc3 and Cdc10 under ambient temperature and heat stress, respectively. Our findings unveiled a diverse array of interacting proteins, including components of Golgi to plasma membrane transport, cell division, and single-stranded DNA binding. \'a0Overall, this study delineates septins in C. neoformans as part of the cytoskeleton and cytoskeleton-dependent cytokinesis and provides a landscape of septin interactors to investigate and further understand septin biology in fungal systems.
Project description:Ubiquitination is a reversible protein modification involved in various cellular processes in eukaryotic cells. Deubiquitinating enzymes, the proteins responsible for the removal of ubiquitin, act as essential regulators to maintain ubiquitin homeostasis and to exquisitely regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily within the immunocompromised population. In order to understand the possible influence deubiquitinating enzymes have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of 7 putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5M-bM-^HM-^F mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 appears to be the major deubiquitinating enzyme in C. neoformans for maintaining a pool of free ubiquitin for stress responses, supports the evolutionary divergence of Cryptococcus sp. from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. In addition, other putative deubiquitinase mutants (doa4M-bM-^HM-^F and ubp13M-bM-^HM-^F) exhibit similar phenotypes to the ubp5M-bM-^HM-^F mutant, illustrating possible functional overlap among deubiquitinating enzymes in C. neoformans. Certain functioning deubiquitinating enzymes are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host. WT Cryptococcus neoformans strain H99 was incubated in pooled ex vivo human CSF obtained from de-identified patients and harvested after 24hr for RNA extraction. New Zealand White rabbits were inoculated with WT strain H99 and harvested by intracisternal spinal tap on days 1 and 7 for RNA extraction.
Project description:The high osmolarity glycerol response (HOG) pathway plays a pivotal role in the stress response, virulence regulation, and differentiation of fungi, including Cryptococcus neoformans that causes fatal meningoencephalitis. Core signaling components of in the HOG pathway, including the Tco-Ypd1-Ssk1 phosphorelay system and the Ssk2-Pbs2-Hog1 MAPK module, have been elucidated but its downstream transcription factors remain unclear. Here we demonstrated that Atf1 with a basic leucine zipper domain is the transcription factor downstream of Hog1 in C. neoformans. We found that ATF1 expression was differentially regulated by oxidative damaging agents, mainly in a Hog1-dependent, but Mpk1-independent, manner. Interestingly, Atf1 not only promoted oxidative stress response and adaptation, but also played an opposing role to Hog1 in the process. Atf1 primarily localized to the nucleus under both unstressed and oxidative stress conditions in a Hog1-independent manner. Our data demonstrated that Atf1 promoted pheromone production and sexual differentiation under negative control by Hog1. Finally, a DNA microarray-based transcriptome analysis of the atf1M-bM-^HM-^F mutant under unstressed and oxidative stress conditions revealed that Atf1 regulated oxidative stress response genes, including a sulfiredoxin gene (SRX1). Intriguing, the array data further demonstrated that Atf1 modulated basal expression of genes involved in DNA repair and genotoxic stress response. Supporting this, we found that the atf1M-bM-^HM-^F mutant was highly sensitive to genotoxic agents. In conclusion, this study provided a further insight into the Hog1-dependent oxidative and genotoxic stress response and differentiation mechanism of C. neoformans. There are more than 95% of genome homology between JEC21 and H99. Therefore 6 slides of JEC21 (Cryptococcus neoformans var. neoformans serotype D) 70-mer oligo are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted 2 conditions (with or without treatment of Hydroten peroxide) from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), and atf1M-NM-^T). We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy3 as Sample dye and Cy5 as a control dye.