Temporal and thermal optimization of trypsin digestion for the cryptococcal proteome
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Exploris 240
ORGANISM(S): Cryptococcus Neoformans Var. Grubii Serotype A (strain H99 / Atcc 208821 / Cbs 10515 / Fgsc 9487) (filobasidiella Neoformans Var. Grubii)
SUBMITTER: 
Jennifer Geddes-McAlister
LAB HEAD: Jennifer Geddes-McAlister
PROVIDER: PXD056755 | Pride | 2024-10-12
REPOSITORIES: Pride
Dataset's files
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| July_5_2024_JM_1.raw | Raw | |||
| July_5_2024_JM_10.raw | Raw | |||
| July_5_2024_JM_11.raw | Raw | |||
| July_5_2024_JM_12.raw | Raw | |||
| July_5_2024_JM_13.raw | Raw |
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Project description:Cryptococcus neoformans is a human fungal pathogen found ubiquitously within the environment and associated with infection of primarily immunocompromised individuals. Without the activation of an effective immune response, the pathogen can survive, proliferate, and disseminate throughout the host through the action of diverse virulence factors. These virulence factors include a polysaccharide capsule to protect the fungus from phagocytosis by macrophages, melanin production to neutralize reactive oxygen species, thermotolerance to survive at human physiological temperatures, and extracellular enzymes for host tissue degradation and invasion. We previously used mass spectrometry-based proteomics to explore the production of fungal virulence factors during infection using in vitro (macrophages) and in vivo (murine) models of disease. Based on our studies, we investigated the proteome response of C. neoformans upon disruption of CipC, a virulence-associated fungal protein.
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2023-10-23 | PXD045163 | Pride
Project description:The high osmolarity glycerol response (HOG) pathway plays a pivotal role in the stress response, virulence regulation, and differentiation of fungi, including Cryptococcus neoformans that causes fatal meningoencephalitis. Core signaling components of in the HOG pathway, including the Tco-Ypd1-Ssk1 phosphorelay system and the Ssk2-Pbs2-Hog1 MAPK module, have been elucidated but its downstream transcription factors remain unclear. Here we demonstrated that Atf1 with a basic leucine zipper domain is the transcription factor downstream of Hog1 in C. neoformans. We found that ATF1 expression was differentially regulated by oxidative damaging agents, mainly in a Hog1-dependent, but Mpk1-independent, manner. Interestingly, Atf1 not only promoted oxidative stress response and adaptation, but also played an opposing role to Hog1 in the process. Atf1 primarily localized to the nucleus under both unstressed and oxidative stress conditions in a Hog1-independent manner. Our data demonstrated that Atf1 promoted pheromone production and sexual differentiation under negative control by Hog1. Finally, a DNA microarray-based transcriptome analysis of the atf1M-bM-^HM-^F mutant under unstressed and oxidative stress conditions revealed that Atf1 regulated oxidative stress response genes, including a sulfiredoxin gene (SRX1). Intriguing, the array data further demonstrated that Atf1 modulated basal expression of genes involved in DNA repair and genotoxic stress response. Supporting this, we found that the atf1M-bM-^HM-^F mutant was highly sensitive to genotoxic agents. In conclusion, this study provided a further insight into the Hog1-dependent oxidative and genotoxic stress response and differentiation mechanism of C. neoformans. 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 2 conditions (with or without treatment of Hydroten peroxide) from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), and atf1M-NM-^T). 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.
2015-01-13 | E-GEOD-64871 | biostudies-arrayexpress
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.
2011-06-23 | E-GEOD-30154 | biostudies-arrayexpress
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. total RNAs are extracted 2 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), mbs1Δ), 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.
2011-06-23 | E-GEOD-30156 | biostudies-arrayexpress
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: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: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:Fungal diseases are critical burdens on the global healthcare system, infecting over 25% of the world’s population. For the opportunistic fungal pathogen, Cryptococcus neoformans, infection leads to cryptococcosis; a disease enabled by elaboration of sophisticated virulence factors, including polysaccharide capsule, melanin, thermotolerance, and extracellular enzymes in the presence of the host. Conversely, the host protects itself from fungal invasion by regulating and sequestering transition metals (e.g., iron, zinc, copper) important for microbial growth and survival. Building upon knowledge of zinc transport regulation at the transcriptional level, here, we explore the intricate relationship between zinc availability and fungal virulence via mass spectrometry-based quantitative proteomics. We observe distinct protein-level responses to zinc-limited and -excess conditions through a shift of the proteome profile towards stress response and metal acquisition, including the upregulation of the zinc transporter, ZIP1. In addition, we reveal a novel connection among zinc availability, protein folding, capsule production, and virulence through the detection of a Wos2-like ortholog in the secretome under replete conditions. Overall, we provide new biological insight into cellular remodeling at the protein level of C. neoformans under regulated zinc conditions and uncover a novel connection between zinc availability and fungal virulence. These findings support the development of new antifungal strategies focused on the enhancement of nutritional immunity within the host.
2022-02-16 | PXD023204 | Pride
Project description:The RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RdRP (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. To further gain insights into the global regulatory circuit governed by the RNAi pathway, comparative transcriptome analysis using the serotype A and D RNAi mutants was performed. Notably, an increase in transcript abundance of active transposons, such as T2 and T3, was observed in the rdp1Δ mutant. Therefore, this study can improve our understanding of the role of the RNAi genes in human fungal pathogens including C. neoformans. RNAi_A: 16 slides are used in this analysis, 3 or 2 biological replicate experiments are performed, total RNAs are extracted from 3 strains (H99 Wild type strain, ago1Δ , rdp1Δ), 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. 2nd experiment sample are dye swaped.
2010-05-01 | E-GEOD-21176 | biostudies-arrayexpress