Cellular proteome profiling of Cryptococcus neoformans KN99 and C8
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Exploris 240
ORGANISM(S): Cryptococcus Neoformans
TISSUE(S): Cell Suspension Culture, Fungal Cell
SUBMITTER: 
Arjun Sukumaran
LAB HEAD: Dr. Jennifer Geddes-McAlister
PROVIDER: PXD045163 | Pride | 2023-10-23
REPOSITORIES: Pride
Dataset's files
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| July_24_2023_MSF_TD_1.raw | Raw | |||
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| July_24_2023_MSF_TD_3.raw | Raw | |||
| July_24_2023_MSF_TD_4.raw | Raw | |||
| July_24_2023_MSF_TD_5.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.
2024-10-13 | PXD056761 | Pride
Project description:In this study, we use mass spectrometry (MS)-based proteomics to define the host response under single and dual infection states of macrophages with C. neoformans and K. pneumoniae to define critical responses of each biological system. This high-resolution comparative analysis illustrates how protein abundance deviates during a transition from an acute to chronic infectious state with exposure to additional microbial stimuli. We reveal global changes upon infection followed by pathogen-specific host response signatures. Additionally, we define regulatory changes within C. neoformans as the fungi adapts to the host environment and stabilizes prior to further disruption in the presence of chronic bacterial infection. We validate our findings with host cytokine detection and phenotypic profiling of the fungi throughout the host and bacterial exposures. Overall, our study provides an in-depth analysis of cross-kingdom protein level changes during macrophage infection. This information provides new insight into fungal modulation of the immune response, including a stabilization and adaptation of the host and fungi during a chronic infection, which is disrupted upon chronic co-infection with a bacterial pathogen.
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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.
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Project description:Mass spectrometry-based proteomics supports investigation into diverse biological systems, providing insights into regulation, signaling, modifications, and interactions. Optimized sample preparation is critical to ensure robust and reproducible analyses balanced with time and cost. In this article, we evaluated trypsin digestion parameters for the human fungal pathogen, Cryptococcus neoformans, and determined conditions for optimal peptide identification
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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.
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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.
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