Transcriptome analysis of cryptococcal IPK mutants
Ontology highlight
ABSTRACT:
ORGANISM(S): Cryptococcus neoformans var. grubii H99
SUBMITTER: Julianne Djordjevic
PROVIDER: E-GEOD-78824 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Transcriptome profiling of wild type and cfo1 mutant with fluconazole treatment in Cryptococcus neoformans var. grubii H99 Purpose: The goals of this study are to compare cfo1 mutant transcriptome profiling (RNA-seq) to wild-type with or without fluconazole treatment in Cryptococcus neoformans var. grubii H99. Methods: mRNA profiles of wild-type and cfo1 mutant with or without fluconazole treatment were generated by RNA-Seq, using Illumina GAIIx. The sequence reads that passed quality filters were mapped to reference genome and the normalized RPKM values were calculated by CLC Genomics Workbench. Results: Compared to wild-type, a number of genes were differentially expressed in the cfo1 mutant, especially genes involved in iron homeostasis and transport, ergosterol biosynthesis, mitochondrial function and respiration. Conclusions: Our data suggested reduced expression of the genes in the respiratory chain is the main reason for altered antifungal sensitivity of the cfo1 mutant. The results of our study revealed that iron uptake plays a key role in fluconazole sensitivity of C. neoformans. mRNA profiles of wild-type and cfo1 mutant with fluconazole treatment were generated by RNA-Seq, using Illumina GAIIx.
2013-05-31 | E-GEOD-37875 | biostudies-arrayexpress
Project description:Purpose: The purpose of this study is to define the mechanism of antifungal action of the vanillin derivative, o-vanillin, against Cryptococcus neoformans Methods: mRNA profiles of C. neoformasn cells cultured with or without o-vanillin were generated by RNA-Seq, using Illumina GAIIx. The sequence reads that passed quality filters were mapped to reference genome and the normalized RPKM values were calculated by CLC Genomics Workbench. Results: o-vanillin significantly altered global transcript profiles, induces oxidative stress, and interferes mitochondrial functions in C. neoformans. Conclusions: o-vanillin can be considered as the effective antifungal drug candidate. mRNA profiles of the cells grown in the presence or absence of o-vanillin were generated by RNA-Seq using Illumina GAIIx.
2014-10-22 | E-GEOD-52645 | biostudies-arrayexpress
Project description:BACKGROUND: Lithospermum erythrorhizon, popularly known as Gromwell, is a perennial herb belonging to the family of Boraginaceae and its extract is known to have diverse pharmacological, cosmetic, and nutritional values for human. Nevertheless the biological influence of Gromwell extract on general physiology of eukaryotic cells remains unknown. In this study, we for the first time performed a global transcriptome analysis by using DNA microarray analysis to identify genes affected by the addition of Gromwell extract (GE) by using a eukaryotic microorganism, basidiomycete fungus Cryptococcus neoformans, as a model system. RESULTS: The transcriptome analysis revealed that a total of 1932 genes are differentially regulated in a statistically significant manner and expressions of 715 genes are up- (315 genes) or down-regulated (457 genes) more than twofold upon GE treatment. In response to GE treatment, genes involved in signal transduction genes are immediately regulated and the evolutionarily conserved set of genes involved in the core cellular functions, including DNA replication, RNA transcription/processing, and protein translation/processing, are generally upregulated. In contrast, a number of genes involved in carbohydrate metabolism and transport, inorganic ion transport and metabolism, post-translational modification/protein turnover/chaperone functions, and signal transduction are downregulated by the GE treatment. Among the GE-responsive genes that are also evolutionarily conserved in the human genome, we confirmed expression patterns of YSA1, TPO2, FET5 (CFO1), and PZF1 by Northern blot analysis. Based on the functional characterization of some GE-responsive genes through phenotypic analysis of gene knockout mutants, we found that GE treatment may promote cellular tolerance against a variety of environmental stresses in eukaryotes. CONCLUSIONS: A significant portion of the Cryptococcus genome is affected in expression levels by treatment of GE, implying that the general physiology of eukaryotic cells is significantly affected by the GE treatment. 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 from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A)) with 6.4 mg/ml Gromwell extract in 0, 10, 30, 60 min. 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.
2013-01-10 | E-GEOD-43370 | biostudies-arrayexpress
Project description:In eukaryotes, inositol polyphosphates perform essential metabolic and signaling functions. Using human fungal pathogen Cryptococcus neoformans as a model, we created mutants in three inositol polyphosphates kinases: Arg1, Ipk1 and Kcs1. Each of the mutants produces a unique repertoire of inositol polyphosphates, different from the wild type strain. Comparative phenotypic and transcriptome analyses of wild type and mutant strains indicates that inositol polyphosphate PP-IP5 (IP7) is the key regulator of gene expression, fitness and virulence in C. neoformans.
2016-03-03 | GSE78824 | GEO
Project description:Purpose: To explore the function of VIB1 in regulating cellulase production in Neurospora crassa. Method: mRNA from vib-1 mutants were collected after a culture shift from sucrose to Avicel (crystaline cellulose) or carbon-free media. Expression profiles of vib-1 mutants were compared with published profiles of wild type created under the same conditions. Results: We found that many genes that specifically upregulated in wild type upon exposure to Avicel were expressed at low levels in M-bM-^HM-^Fvib-1 and many other genes involved in metabolism and energy were expressed at high levels compared to wild type. Conclusions: Our study shows that VIB1 is required for suppression of glucose response and carbon catabolite repression to allow proper expression of clr-2 and subsequent cellulase production in response to cellulosic induction. Biological triplicates of liquid culture N. crassa were harvested at 4 hours after a switch from sucrose media to media of interest. Global mRNA abundances were measured by sequencing on the Illumina Genome Analyzer Iix and HiSeq2000 platforms.
2014-07-08 | E-GEOD-52316 | biostudies-arrayexpress
Project description:Cryptococcus neoformans interactions with murine macrophages are critical for disease. In this project we analyzed fungal proteins which were co-purified with murine host proteins after interaction. H99 C. neoformans was opsonized with mAb 18B7 and addedd to murine macrophages. Then murine cells were lysed and cell extracts submitted to proteomics.
2023-03-11 | PXD024951 | Pride
Project description:Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work by us and others have shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely PKC1, BCK1, MKK2 and MPK1 results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors and, for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional response of gene deletions of BCK1, MKK2 and MPK1 compared to wild-type cells. We found over 1000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cAMP/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion stains revealed significantly less cAMP in all of the deletion strains compared to wild-type cells. The deletion strains also produced significantly smaller capsules than wild type KN99 under capsule inducing conditions, although they shed similar levels of capsule as wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to near wild-type levels. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis. Comparison of three gene deletion strains to wild-type, with 3 biological replicates for each sample.
2014-07-01 | E-GEOD-57217 | biostudies-arrayexpress
Project description:The cAMP-pathway plays a central role in regulation of growth, differentiation, and virulence of human pathogenic fungi, including Cryptococcus neoformans. Three major upstream signaling regulators of the adenylyl cyclase (Cac1), Ras, Aca1 (Adenylyl cyclase-associated protein 1) and G-alpha subunit protein (Gpa1), have been identified to control the cAMP-pathway in C. neoformans, but their functional relationship remains elusive. Here we performed genome-wide transcriptome analysis with C. neoformans ras1, gpa1, cac1, aca1, and pka1 pka2 mutants by DNA microarray. The aca1, gpa1, cac1, and pka1 pka2 mutants displayed similar transcriptome patterns to each other whereas the ras1 mutant exhibited distinctive transcriptome patterns compared to WT and the cAMP mutants. Interestingly, a number of environmental stress response genes are differentially modulated in the ras1 and cAMP mutants. In fact, the Ras1-signaling pathway was found to be involved in osmotic and genotoxic stress response, and maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Through this microarray analysis, we have identified a number of cAMP-dependent genes, including GRE2, HSP12, ENA1, TCO2, PKP2, CAT1, in C. neoformans. Notably, a majority of ergosterol biosynthesis genes were found to be upregulated in the cAMP mutants. Interestingly, the gpa1, cac1, and pka1 mutants, but not the aca1 and pka2 mutants were hypersensitive to amphotericin B, but resistant to fluconazole. In conclusion, we demonstrated in this study that the Ras1- and cAMP-signaling pathways are involved in stress response and sterol biosynthesis of C. neoformans. There are more than 95% of genome homology between JEC21 and H99. Therefore 100 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 with 6 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), ras1Î, aca1Î, gpa1Î, cac1Î, pka1Îpka2Î), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye. several sample are dye swaped.
2010-05-22 | E-GEOD-17338 | biostudies-arrayexpress
Project description:Purpose: Evaluate the transcriptional profile of Neurospora crassa cells treated with a group of novel antifungal compounds Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium for 6 hours (26ºC, 140 rpm, constant light) followed by the addition of the indicated compound (or DMSO) and growth for 1 more hour. Cells were harvested using 0.45 μm filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 μg RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturerâs protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analysed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Results: XP13 promoted a particularly active transcriptional response. Functional enrichment analysis of the genes with altered expression revealed novel pathways that are affected by these compounds. Conclusions: These compounds are active in Neurospora crassa and we anticipate that this and future studies will ascertain their potential as antifungal agents. The transcriptional profile of Neurospora crassa wild type cells exposed with a group of novel compounds with antifungal activity was analysed using illumina HiSeq2000 and single reads of 50 bp.
2015-11-25 | E-GEOD-53040 | biostudies-arrayexpress
Project description:Purpose: Compare the transcriptional profile of staurosporine-treated cells in Neurospora crassa wild type and M-NM-^Tczt-1 (M-NM-^TNCU09974) cells Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium for 6 hours (26M-BM-:C, 140 rpm, constant light) followed by the addition of staurosporine (or DMSO) and growth for 1 more hour. Cells were harvested using 0.45 M-NM-<m filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 M-NM-<g RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturerM-bM-^@M-^Ys protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analysed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments/Reads Per Kilobase of transcript per Million mapped reads (FPKM/RPKM). Results: Transcriptional profiling of staurosporine-treated cells wild type by RNA-sequencing showed that genes encoding the machinery for protein synthesis are enriched among the genes repressed by the drug. Functional category enrichment analyses also show that genes encoding components of the mitochondrial respiratory chain are downregulated by staurosporine, whereas genes involved in endoplasmic reticulum activities are upregulated. In contrast, a staurosporine-treated czt-1 deletion strain is unable to repress the genes for the respiratory chain and to induce the genes related with the endoplasmic reticulum, indicating a role for CZT-1 on the regulation of activity of these organelles. Conclusions: This transcriptional profiling study is framed in a comprehensive study on the role of the novel transcription factor CZT-1 (NCU09974) in Neurospora crassa. Transcriptional profile of wild type and M-NM-^Tczt-1 cells staurosporine-treated cells in Neurospora crassa was compared using illumina HiSeq2000 and single reads of 50 bp.
2014-07-01 | E-GEOD-52153 | biostudies-arrayexpress