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NMD-mediated control of Tor influences adaptation to nutrient and temperature conditions in Cryptococcus neoformans

ABSTRACT: The yeast Cryptococcus neoformans is an opportunistic human pathogen capable of surviving within various environmental conditions. The repertoire of antifungal agents effective in treating cryptococcal infection is limited, necessitating the identification of alternative treatment strategies. Nonsense-mediated decay (NMD) is an RNA decay mechanism that serves as a post-transcriptional regulator of gene expression. While the absence of NMD in C. neoformans sensitizes cells to the antifungal fluconazole, the mechanism underlying this sensitivity and role of NMD in C. neoformans biology remained unexplored. Using phenotypic analysis and RNA-sequencing analysis, we identify basal dysregulation of thermal- and nutrient-adaptive genes and demonstrate temperature- and/or nutrient-dependent phenotypic suppression of upf1Δ phenotypes, including fluconazole sensitivity and resistance to rapamycin. We determine rapamycin co-treatment also suppresses the upf1Δ fluconazole sensitivity, implicating dysregulation of Tor signaling in phenotypic outcomes when NMD is absent. We then investigate Tor-sensitive signaling in the upf1Δ mutant, finding inhibition of cell wall integrity (CWI) signaling and hyperactivation of the kinase Gcn2, both of which returned to wildtype-like levels by either rapamycin treatment, nutrient limitation, or constitutive thermal stress. These results indicated NMD is required for appropriate regulation of Tor signaling in unstressed conditions and suggested upf1Δ phenotypes are driven in part by Tor hyperactivation. A phenotypic screen of mutants lacking Tor regulators revealed that deletion of the Tor-suppressing IML1 gene recapitulates upf1Δ phenotypes and signaling defects, consistent with Tor hyperactivation. Taken together, our results suggest NMD participates in the regulation of Tor signaling in C. neoformans. Future work will investigate how specific targets of NMD impact Tor signaling and promote fluconazole sensitivity in C. neoformans.

ORGANISM(S): Cryptococcus neoformans

PROVIDER: GSE319882 | GEO | 2026/03/17

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.

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.

Project description:Two nutrient sensing and regulatory pathways, the general amino acid control (GAAC) and the target of rapamycin (TOR), control yeast growth and metabolism in response to changes in nutrient availability. Starvation for amino acids activates the GAAC pathway, involving Gcn2p phosphorylation of eIF2 and preferential translation of GCN4, a transcription activator of genes involved in amino acid metabolism. TOR senses nitrogen availability and regulates gene expression through transcription factors, such as Gln3p. We used microarray analyses to address the integration of the GAAC and TOR pathways in directing the yeast transcriptome in response to amino acid starvation and rapamycin treatment. Of the ~2500 genes whose expression was changed by 2-fold or greater, Gcn4p and Gln3p were required for 542 and 657 genes, respectively. While Gcn4p activates a common core of 57 genes in response to amino acid starvation or rapamycin treatment, the different stress arrangements allow for variations in Gcn4p-directed transcription. With few exceptions, genes requiring Gcn2p eIF2 kinase for induced expression also required Gcn4p, emphasizing the role of Gcn2p as an upstream activator of Gcn4p-directed transcription. There is also significant coordination between the GAAC and TOR pathways, with Gcn4p being required for activation of more genes during rapamycin treatment than Gln3p. Importantly, TOR regulates the GAAC-directed transcription of genes required for assimilation of nitrogen sources, such as Î³-amino-butyric acid. Therefore, yeast has integrated gene expression responses to amino acid abundance and nitrogen source quality through the control of Gcn2p phosphorylation of eIF2 and GCN4 translation. Keywords: gene expression In this study, we carried out microarray analyses in a collection of yeast strains deleted for GCN2, GCN4, and GLN3, individually or in combinations, to explore the importance of the TOR and GAAC pathways in directing the transcriptome in response to amino acid starvation and rapamycin treatment.

Project description:We show that the sensitivity of tsc mutant cells to rapamycin is mediated by TORC1 and can be suppressed by overexpression of the 2-oxoglutarate-Fe(II) dependent oxygenase, Isp7. We show that Isp7 is a novel regulator of amino acids uptake that acts via regulation of gene expression, both upstream and downstream of TOR signaling. suppressed by overexpression of the putative 2-oxoglutarate-Fe(II) dependent oxygenase, Isp7. We show that Isp7 is a novel master regulator of amino acids uptake that acts via regulation of gene expression, both upstream and downstream of TOR signaling. TOR proteins reside in two distinct complexes, TOR complex 1 and 2 (TORC1 and TORC2) that are central for the regulation of cellular growth, proliferation and survival. TOR is also the target for the immunosuppressive and anti-cancer drug rapamycin. In Schizosaccharaomyces pombe, disruption of the TSC complex, mutations in which can lead to the Tuberous Sclerosis syndrome in humans, results in a rapamycin sensitive phenotype under poor nitrogen conditions. We show here that the sensitivity to rapamycin is mediated via inhibition of TORC1 and suppressed by overexpression of isp7+, a member of the family of 2-oxoglutarate-Fe(II) dependent oxygenases. The transcript level of isp7+ is negatively regulated by TORC1 but positively regulated by TORC2. Yet, we find extensive similarity between the transcriptome of cells disrupted for isp7+ and cells mutated in the catalytic subunit of TORC1. Moreover, Isp7 regulates amino acid permease expression similarly to TORC1 and in contrast to TORC2. Overexpression of isp7+ induces TORC1-dependent phosphorylation of ribosomal protein Rps6, while inhibiting TORC2-dependent phosphorylation and activation of the AGC-like kinase Gad8. Taken together, our findings suggest a central role for Isp7 in amino acid homeostasis and the presence of isp7+-dependent regulatory loops that affect both TORC1 and TORC2. 6 Samples (arrays) were performed. We generated pairwise comparison between DISP7 and WT, using Partek Genomics Suite. Genes with pâ¤5%[FDR] and a fold-change difference of â¥2\1.5 or <-2\-1.5 were selected.

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NMD-mediated control of Tor influences adaptation to nutrient and temperature conditions in Cryptococcus neoformans

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