Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans
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ABSTRACT:
ORGANISM(S): Cryptococcus neoformans var. grubii H99
PROVIDER: GSE233612 | GEO | 2023/12/31
REPOSITORIES: GEO
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Project description: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.
2019-04-03 | GSE129227 | GEO
Project description: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. Nevertheless, its role in the human fungal pathogen 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, except for a minor role in diamide and polyene resistance, 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. 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.
2019-10-10 | PXD012983 | Pride
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.
2012-08-23 | E-GEOD-40303 | biostudies-arrayexpress
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.
2012-08-23 | GSE40303 | GEO
Project description:A family of APSES transcription factor is known to be fungal-specific transcriptional regulators and play important roles in governing growth, differentiation, and virulence of diverse fungal pathogens. Yet none of APSES-like transcription factors have been identified and investigated in a basidiomycetous fungal pathogen, Cryptococcus neoformans. In the present study we discovered an APSES-like transcription factor, mbs1 (Mbp1/Swi4-like APSES protein 1), as one of novel flucytosine-responsive genes (total 194 genes) identified through comparative transcriptome analysis of C. neoformans hybrid sensor kinase mutants, tco1 and tco2 mutants, which displayed differential flucytosine-susceptibility. Supporting the microarray data, Northern blot and quantitative RT-PCR analysis confirmed that expression of mbs1 is rapidly induced in response to flucytosine in the wild-type strain, but not in the tco1 and tco2 mutants. Furthermore, C. neoformans with deletion of the mbs1 gene exhibited increased susceptibility to flucytosine. Intriguingly, mbs1 plays pleiotropic roles in diverse cellular process of C. neoformans. mbs1 positively regulates ergosterol biosynthesis and thereby its inhibition confers increased susceptibility and resistance to amphotericin B and azole drugs, respectively. mbs1 is also involved in DNA damage repair counteracting genotoxic stresses. During sexual differentiation mbs1 represses pheromone production, but promotes cell-cell fusion. Furthermore mbs1 is required for production of antioxidant melanin pigment and full virulence of C. neoformans. Finally we also performed DNA microarrray analysis to identify mbs1-regulated genes in C. neoformans. A majority of them were found to be involved in cell cycle regulation and DNA repair. Therefore, this study provides a novel antifungal therapeutic method for treatment of cryptococcosis.
2011-06-23 | GSE30156 | GEO
Project description:Cryptococcus neoformans is an invasive human fungal pathogen, which causes pneumonia and meningoencephalitis in both healthy and immunodeficient individuals, resulting 181,100 deaths each year. Studies have demonstrated that copper detoxification machineries play critical functions in modulating C. neoformans fitness and pathogenicity in the host lung tissue. Pulmonary C. neoformans infection provokes the generation of toxic copper bombardment, which in return activate detoxification process in fungal cells. However, the molecular mechanism on how Cu inhibits C. neoformans in proliferation remains unclear. Here, using Cu detoxification gene knockout strains we demonstrated that exogenous Cu ions inhibit cell growth of the metallothionein mutant, which was significantly rescue when supplementing with the ROS scavenger, N-acetylcysteine. To future characterize the molecular mechanism of C. neoformans response to Cu toxicity, we employ iTRAQ with LC-MS/MS analysis, in the presence of exogenous Cu or NAC. Our data showed that an increased Cu level repressed expression of factors involved in protein translation but activates expression of important players in ubiquitin-degradation process. We proposed that the downregulation of protein synthesis and the upregulation of protein degradation were the main strategy of Cu toxicity. The metallothionein mutant showed a higher ubiquitination level under Cu treatment. In addition, MG132 could partially restore the Cu toxicity effect on metallothionein mutant. These results shed new light on the Cu antifungal mechanisms from proteomic profile.
2021-02-09 | PXD024098 | iProX
Project description:C. neoformans is an opportunistic human pathogen whose polysaccharide capsule anchored to the cell wall is critical for virulence. Biogenesis of both cell wall and capsule relies on the secretory pathway. Protein secretion begins with protein translocation across the endoplasmic reticulum (ER) membrane through a highly conserved channel formed by three proteins, Sec61, Sbh1, and Sss1. Sbh1 is most divergent and contains multiple phosphorylation sites which may allow it to control entry into the secretory pathway in a regulated, species- and protein-specific manner. We show here that in contrast to S. cerevisiae, C. neoformans lacking SBH1 are barely temperature-sensitive, but that absence of SBH1 causes a cell-wall defect in both species. Comparison of the proteomes of wildtype and Δsbh1 C. neoformans revealed a small set of secretory and transmembrane proteins whose expression under infection-like conditions was upregulated in wildtype, but not in the Δsbh1 mutant. These proteins are mostly involved in cell-wall biogenesis. We found that adhesion to macrophages was compromised in the Δsbh1 strain and in mice, the C. neoformans Δsbh1 mutant was virtually avirulent. We conclude that upon contact with the host Sbh1 controls entry of virulencefactors into the secretory pathway of Cryptococcus neoformans.
2023-01-27 | PXD013894 | Pride
Project description:Comparison of wild-type serotype A C. neoformans cells grown at 21% and 3% oxygen for 2 hours in rich medium. Also comparison of wild-type and Sre1-pathway mutant strains grown at 3 % oxygen for 2 hours in rich medium.
2009-09-29 | GSE18282 | GEO
Project description:This SuperSeries is composed of the following subset Series: GSE29627: Chromosome copy number variation in Cryptococcus neoformans influences virulence and occurs in isolates from AIDS patients: CBS7779 black and white strains GSE29671: Chromosome copy number variation in Cryptococcus neoformans influences virulence and occurs in isolates from AIDS patients: clinal and environmental strains Refer to individual Series
2011-05-31 | E-GEOD-29675 | biostudies-arrayexpress
Project description:The human fungal pathogen Cryptococcus neoformans undergoes many phenotypic changes to promote its survival in specific ecological niches and inside the host. To explore the role of chromatin remodeling on the expression of virulence-related traits, we identified and deleted seven genes encoding predicted class I/II histone deacetylases (HDACs) in the C. neoformans genome. Our results identified the HDA1 HDAC gene as a central mediator controlling several cellular processes, including mating and virulence. A global gene expression profile comparing the hda1Δ mutant versus wild-type revealed altered transcription of specific genes associated with the most prominent virulence attributes in this fungal pathogen. This study directly correlates the effects of Class I/II HDAC-mediated chromatin remodeling on the marked phenotypic plasticity and virulence potential of this microorganism. Furthermore, our results provide insights into regulatory mechanisms involved in virulence gene expression that are likely shared with other microbial pathogens.
2018-06-06 | GSE109582 | GEO