Unknown,Transcriptomics,Genomics,Proteomics

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A defect in iron uptake enhances the susceptibility of Cryptococcus neoformans to azole antifungal drugs.

ABSTRACT: 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.

ORGANISM(S): Cryptococcus neoformans var. grubii H99

SUBMITTER: Jeongmi Kim

PROVIDER: E-GEOD-37875 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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.

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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, Msa1 (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 MSA1 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 MSA1 gene exhibited increased susceptibility to flucytosine. Intriguingly, Msa1 plays pleiotropic roles in diverse cellular process of C. neoformans. Msa1 positively regulates ergosterol biosynthesis and thereby its inhibition confers increased susceptibility and resistance to amphotericin B and azole drugs, respectively. Msa1 is also involved in DNA damage repair counteracting genotoxic stresses. During sexual differentiation Msa1 represses pheromone production, but promotes cell-cell fusion. Furthermore Msa1 is required for production of antioxidant melanin pigment and full virulence of C. neoformans. Finally we also performed DNA microarrray analysis to identify Msa1-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. There are more than 95% of genome homology between JEC21 and H99. Therefore 24 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 under 2 conditions (with or without treatment of Flucytosine) with 4 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), tco1Δ , tco2Δ , skn7Δ), 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.

Project description:The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including Cryptococcus neoformans. These cellular responses are evoked by diverse signaling cascades, including the stress-activated HOG pathway. Despite previous analysis of central components of the HOG pathway, its downstream signaling network is poorly characterized in C. neoformans. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in C. neoformans. In this study we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses, but also elucidated novel characteristics of the HOG pathway in C. neoformans. First, inhibition of the HOG pathway increases expression of ergosterol biosynthesis genes and cellular ergosterol content, conferring a striking synergistic antifungal activity with amphotericin B and providing an excellent opportunity to develop a novel therapeutic method for treatment of cryptococcosis. Second, a number of cadmium-sensitive genes are differentially regulated by the HOG pathway, and their mutation causes resistance to cadmium. Finally, we have discovered novel stress-defense and HOG-dependent genes, which encodes a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system. There is more than 95% genome homology between JEC21 (Cryptococcus neoformans var. neoformans serotype D) and H99 (Cryptococcus neoformans var. grubii serotype A). Therefore, 100 slides of JEC21 70-mer oligo arrays are used in this analysis. 3 biological replicate experiments are performed. Total RNAs are extracted under 3 conditions (1M NaCl, 20ug/ul Fludioxonil, 2.5mM Hydrogen peroxide) at 3 time points (0time, 30min, 60min) with 4 strains from H99 (wild type, hog1Î , ssk1Î , skn7Î). We use a mix of all the total RNAs from this experiment as the control RNA. We use Cy5 as the sample dye and Cy3 as the control dye. Several samples are dye swapped.

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A defect in iron uptake enhances the susceptibility of Cryptococcus neoformans to azole antifungal drugs.

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