Pleiotropic Effects of Deubiquitinating Enzymes on Growth and Pathogenesis of Cryptococcus neoformans
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ABSTRACT:
ORGANISM(S): Cryptococcus neoformans var. grubii H99
SUBMITTER: Michael Price
PROVIDER: E-GEOD-36184 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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.
2012-04-10 | E-GEOD-36182 | biostudies-arrayexpress
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 strain H99, mutant strains 1A10 (ena1M-bM-^HM-^F, CNAG_00531), and 14A4 (pik1M-bM-^HM-^F, CNAG_07744) were incubated in filter-sterilized human CSF or serum from anonymous human donors at 37M-BM-0C with shaking, and harvested at either 1 or 24 hours post-resuspension in each biological fluid for RNA extraction.
2012-04-10 | E-GEOD-36181 | biostudies-arrayexpress
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 ubp5∆ 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 (doa4∆ and ubp13∆) exhibit similar phenotypes to the ubp5∆ 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. New Zealand White rabbits were inoculated with WT strain H99 and harvested by intracisternal spinal tap on days 1, 3, 4, and 7. For the human sample, RNA was extracted from CSF obtained from a de-identified patient with cryptococcal meningitis.
2012-04-10 | E-GEOD-36183 | biostudies-arrayexpress
Project description:Cryptococcus neoformans causes meningoencephalitis and is an increasing human health threat. C. neoformans is neurotropic, and persists in the cerebrospinal fluid (CSF) of the mammalian host during infection. In order to survive in the host, pathogenic fungi must procure nutrients such as carbon and nitrogen. To enhance our understanding of nutrient acquisition during infection by Cryptococcus species, we examined utilization of nitrogen sources available in CSF. We screened for growth and capsule production of 817 global environmental and clinical isolates on various sources of nitrogen. Capsule production was assessed using ammonium and urea in the presence or absence of benomyl to determine the relationship of urea exposure to capsule production. Since urea is metabolized to ammonia and CO2 (a known signal for capsule induction), we examined urea metabolism mutants for their response to urea regarding capsule production. Non-preferred nitrogen sources were found to greatly affect capsule production in pathogenic species of Cryptococcus. Urea induced the greatest magnitude of capsule production. Capsule induction by urea was greater in Cryptococcus gattii strains than in C. neoformans strains. In addition, both environmental and clinical strains grew robustly on uric acid, casamino acids, creatinine, and asparagine as sole nitrogen sources. While substantial growth on nitrate was not apparent at day 3, growth was apparent by day 6 for all serotypes. In this study, transcription profiles of urea pathway mutants (ure1 and amt1/2) and WT Cryptococcus neoformans strains were compared in a dye-swap experiment following 1hr exposure to proline or proline + urea (.25g/L).
2013-09-25 | E-GEOD-46829 | biostudies-arrayexpress
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. In this study, transcription profiles of ubp5 mutant and WT Cryptococcus neoformans strains were compared in a dye-swap experiment following 1hr exposure to either 30C or 37C.
2012-04-10 | E-GEOD-35067 | biostudies-arrayexpress
Project description:The opportunistic pathogen Cryptococcus neoformans causes fungal meningoencephalitis in immunocompromised individuals. In previous studies, we found that the Hap complex in this pathogen represses genes encoding mitochondrial respiratory functions and TCA cycle components under low-iron conditions. The orthologous Hap2/3/4/5 complex in Saccharomyces cerevisiae exerts a regulatory influence on mitochondrial functions and Hap4 is subject to glucose repression via the carbon catabolite repressor Mig1. In this study, we explored the regulatory link between a candidate ortholog of the Mig1 protein and the HapX component of the Hap complex in C. neoformans. This analysis revealed repression of MIG1 by HapX and activation of HAPX by Mig1 in low iron conditions, and Mig1 regulation of mitochondrial functions including respiration, tolerance for reactive oxygen species, and expression of genes for iron-consuming and iron-acquisition functions. Consistent with these regulatory functions, a mig1Î mutant had impaired growth on inhibitors of mitochondrial respiration and ROS inducers. Furthermore, deletion of MIG1 provoked a dysregulation in nutrient sensing via the TOR pathway and impacted the pathway for cell wall remodeling. Importantly, loss of Mig1 increased susceptibility to fluconazole thus further establishing a link between azole antifungal drugs and mitochondrial function. Mig1 and HapX were also required together for survival in macrophages, but Mig1 alone had a minimal impact on virulence in mice. Overall, these studies provide novel insights into a HapX/Mig1 regulatory network and reinforce an association between mitochondrial dysfunction and drug susceptibility that may provide new targets for the development of antifungal drugs. In this study, transcription profiles of WT and mig1D mutant strains of Cryptococcus neoformans were compared in a dye-swap experiment following 6hr exposure to Low Iron Medium (LIM) or LIM + 100mM FeCl3.
2015-12-17 | E-GEOD-76063 | biostudies-arrayexpress
Project description:Investigation of changes in global gene expression patterns as Ignicoccus hospitalis becomes increasingly populated by Nanoarchaeum equitans These microarray data are complemented by shot gun proteomics (see manuscript) A total of 16 chips were used (each experimental sample was done in duplicate, totally 8 samples) to examine total RNA from I. hospiatalis over a time course grown in pure culture and in co-culture with N. equitans.
2014-04-24 | E-GEOD-57033 | biostudies-arrayexpress
Project description:Clostridium thermocellum is a promising CBP candidate organism capable of directly converting lignocellulosic biomass to ethanol. Low yields, productivities and growth inhibition prevent industrial deployment of this organism for commodity fuel production. Symptoms of potential redox imbalance such as incomplete substrate utilization, and fermentation products characteristic of overflow metabolism, have been observed during growth. This perceived redox imbalance may be in part responsible for the mentioned bioproductivity limitations. Toward better understanding the redox metabolism of C. thermocellum, we analyzed gene expression, using microarrays, during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which we observed to change fermentation redox potential. High quality RNA was extracted from C. thermocellum grown on cellobiose in chemostat culture and exposed, separately, to methyl viologen and hydrogen peroxide. Transcriptome profiles were obtained at seven time points during actively growing fermentations, 3 minutes, 15 minutes, 35 minutes, 7 hours, 14 hours, 50 hours, and 60 hours after beginning exposure to each stressor. Exposure treatments were carried out in duplicate and reference/untreated samples were taken before and between treatments, after flushing of stressor chemicals and re-equilibration of growth conditions.
2015-07-29 | E-GEOD-71465 | biostudies-arrayexpress
Project description:The high cellulose digestion capability of the anaerobic thermophilic bacterium, Clostridium thermocellum, can be attributed to its efficient glycoside hydrolase system, consisting of both a free-enzyme system and a cellulosomal system, wherein carbohydrate active enzymes (CAZymes) are organized by primary and secondary scaffoldin proteins into large protein complexes bound to the bacterial cell wall. Using an integrated series of experiments encompassing polysaccharide depolymerization assays, direct biochemical analyses and transcriptomics, we show herein that, in addition to cell-bound cellulosomes and M-bM-^@M-^\freeM-bM-^@M-^] enzymes, C. thermocellum naturally employs a system of cell-free cellulosomes that are not designed to be tethered to the cell and can diffuse away to attack polysaccharide substrates at some distance from the cell. By characterizing the cells and the secretomes of a series of mutants in which genes for either individual scaffoldins or combinations thereof are deleted, we demonstrate that the cellulosome is far more important in cellulose degradation than are the free enzymes, and that the primary scaffoldin CipA is much more important for the action of the cellulosomes than is the collective contribution of the secondary scaffoldins. Extensive transcriptomics analyses confirm the above results and extend the study to the effects of scaffoldin deletions on the organism as a whole. To gain insights into the transcriptome profiles of C. thermocellum strains when various cellulosomal scaffoldin genes were deleted. High quality RNA was extracted from C. thermocellum grown on cellulose. Transcriptome profiles were obtained at one time point during actively growing fermentations, mid exponential phase when 50% of the substrate had been consumed.
2015-02-28 | E-GEOD-63883 | biostudies-arrayexpress
Project description:Disproportionate short stature refers to a heterogeneous group of hereditary disorders, which are classified according to their mode of inheritance, their clinical skeletal and non-skeletal manifestations, and their radiological characteristics. In the present study, we report on a novel autosomal recessive osteocutaneous disorder that we termed short stature-onychodysplasia-facial dysmorphism-hypotrichosis (SOFT) syndrome. we identified a homozygous point mutation (p.L171P) in POC1A (Centriolar Protein Homolog A). The mutation affects a highly conserved amino acid residue and is predicted to interfere with protein function. To gain insight into the pathomechanisms underlying the deleterious effect of the causative mutation, we compared transcription profiles of patient and control fibroblasts.
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