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Cryptococcus neoformans proteins during fungal-macrophage interaction

ABSTRACT: 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.

INSTRUMENT(S): orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Macrophage

SUBMITTER: Carolina Coelho

LAB HEAD: Arturo Casadevall

PROVIDER: PXD024951 | Pride | 2023-03-11

REPOSITORIES: Pride

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Publications

Cryptococcus neoformans releases proteins during intracellular residence that affect the outcome of the fungal-macrophage interaction.

Jung Eric H EH Park Yoon-Dong YD Dragotakes Quigly Q Ramirez Lia S LS Smith Daniel Q DQ Reis Flavia C G FCG Dziedzic Amanda A Rodrigues Marcio L ML Baker Rosanna P RP Williamson Peter R PR Jedlicka Anne A Casadevall Arturo A Coelho Carolina C

microLife 20220921

Cryptococcus neoformans is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with C. neoformans. To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified wa ...[more]

PMID: 36247839

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Proteomics reveals that the antifungal activity of fenbendazole against Cryptococcus neoformans requires protein kinases.

Project description:Cryptococcus neoformans is responsible for over 100,000 deaths annually, and the treatment of this fungal disease is expensive and not consistently effective. Unveiling new therapeutic avenues is crucial. Previous studies have suggested that the anthelmintic drug fenbendazole is an affordable and non-toxic candidate to combat cryptococcosis. However, its mechanism of anticryptococcal activity has been only superficially investigated. In this study, we examined the global cellular response of C. neoformans to fenbendazole using a proteomic approach. Fenbendazole treatment mostly impacted the abundance of proteins related to metabolic pathways, RNA processing, and intracellular traffic. Protein kinases, in particular, were significantly affected by fenbendazole treatment. Experimental validation of the proteomics data using a collection of C. neoformans mutants led to the identification of critical roles of five protein kinases in fenbendazole's antifungal activity. In fact, mutants lacking the expression of genes encoding Chk1, Tco2, Tco3, Bub1, and Sch9 kinases demonstrated greater resistance to fenbendazole compared to wild-type cells. In combination with the standard antifungal drug amphotericin B, fenbendazole reduced the cryptococcal burden in mice. These findings not only contribute to the elucidation of fenbendazole's mode of action but also support its use in combination therapy with amphotericin B. In conclusion, our data suggest that fenbendazole holds promise for further development as an anticryptococcal agent.

2024-05-21 | PXD047041 | Pride

Proteome profiling of virulence-associated CipC from Cryptococcus neoformans

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

The ER protein translocation channel subunit Sbh1 controls virulence of Cryptococcus neoformans

Project description:C. neoformans is an opportunistic human pathogen whose polysaccharide capsule anchored to the cell wall is critical for virulence. Biogenesis of both cell wall and capsule relies on the secretory pathway. Protein secretion begins with protein translocation across the endoplasmic reticulum (ER) membrane through a highly conserved channel formed by three proteins, Sec61, Sbh1, and Sss1. Sbh1 is most divergent and contains multiple phosphorylation sites which may allow it to control entry into the secretory pathway in a regulated, species- and protein-specific manner. We show here that in contrast to S. cerevisiae, C. neoformans lacking SBH1 are barely temperature-sensitive, but that absence of SBH1 causes a cell-wall defect in both species. Comparison of the proteomes of wildtype and Δsbh1 C. neoformans revealed a small set of secretory and transmembrane proteins whose expression under infection-like conditions was upregulated in wildtype, but not in the Δsbh1 mutant. These proteins are mostly involved in cell-wall biogenesis. We found that adhesion to macrophages was compromised in the Δsbh1 strain and in mice, the C. neoformans Δsbh1 mutant was virtually avirulent. We conclude that upon contact with the host Sbh1 controls entry of virulencefactors into the secretory pathway of Cryptococcus neoformans.

2023-01-27 | PXD013894 | Pride

Resolving the temporal and spatial splenic proteome during fungal infection for putative dual perspective biomarker discovery

Project description:Fungal pathogens are emerging threats to global health and the rise of incidence is associated with climate change and increased geographical distribution; factors also influencing host susceptibility to infection. Accurate detection and diagnosis of fungal infections is paramount to align options for rapid and effective therapeutic treatments. For improved diagnostics, the discovery and development of biomarkers presents a promising avenue; however, this approach requires a priori knowledge of markers of infection. To uncover putative novel biomarkers of disease, profiling of the host immune response and pathogen virulence factor production is indispensable. In this study, we use mass spectrometry-based proteomics to resolve the temporal and spatial proteomes of Cryptococcus neoformans infection of the spleen following a murine model of infection. Dual perspective proteome profiling defines global remodeling of the host over a time course of infection, confirming activation of immune associated proteins connected to fungal infections. Conversely, pathogen proteomes detect well-characterized C. neoformans virulence determinants, along with novel mapped patterns of pathogenesis during progression of disease. Together, our systematic approach confirms immune protection against fungal pathogens and explores the discovery of putative biomarkers from complementary biological systems for monitoring the presence and progression of cryptococcal disease.

2023-05-23 | PXD040868 | Pride

Transcription profiling of mouse triplicate samples of RAW 264.7 murine macrophages either untreated, stimulated with 100 ng/ml LPS for 18 hours, or constituitively over-expressing CstF-64

Project description:Triplicate samples of RAW 264.7 murine macrophages either untreated, stimulated with 100 ng/ml LPS for 18 hours, or constituitively over-expressing CstF-64 were analyzed by microarray using Affymetrix murine gene chip 430A.

2007-11-06 | E-GEOD-2002 | biostudies-arrayexpress

Usv101 is a key regulator of cryptococcal virulence and determinant of host response

Project description:Cryptococcus neoformans is a fungal pathogen responsible for hundreds of thousands of deaths per year. Its critical virulence factor is a polysacharride capsule which grows large upon entry into a mammalian host. We previously identified USV101 as a transcription factor whose deletion results in enlarged capsules. Here, we characterize strains lacking or overexpressing USV101 in terms of their virulence-related phenotypes, the altered course of infection by them and immune response to them in a mouse model, the relationship of Usv101 to other transcription factors involved in capsule regulation, and the changes in Usv101 activity during capsule induction. To study the function of Usv101, a key C. neoformans regulator of virulence, and its interactions with downstream capsule-regulating TFs GAT201 and SP1, expression profiles were generated by RNA-seq of a usv101 deletion mutant, a USV101 overexpression mutant, strains expressing GAT201 under various promoters, various double mutants, and wildtype cells grown in a capsule non-inducing condition, a capsule inducing condition for 90 minutes, or a capsule inducing condition for 24 hours.

2016-03-31 | E-GEOD-69532 | biostudies-arrayexpress

The Host Response to a Clinical MDR Mycobacterial Strain Cultured in a Detergent-Free Environment: A Global Transcriptomics Approach

Project description:We infect bone marrow derived macrophages (BMDMs) with Mycobacterium cultured with and without Tween 80 and asses the host response to these bacteria through RNAseq Uninfected BMDMs served as controls, BMDMs infected with detergent-free M.tb (designated RNT), BMDMs infected with Tween cultured m.tb (designated RT). 3 groups with 3 biological replicates, each analysed in triplicate.

2015-08-13 | E-GEOD-72003 | biostudies-arrayexpress

Global transcriptome analysis of fungal genes affected by a novel antifungal agent KR-72, a 13-(4-isopropylbenzyl)berberine derivative a 13-(4-isopropylbenzyl)berberine derivative

Project description:A 13-(4-isopropylbenzyl)berberine derivative (named KR-72) was synthesized and examined for antifungal activities against various human pathogenic fungi. The synthesized compound exhibited remarkably enhanced antifungal activity than berberine and berberrubine. Regardless of the potent antifungal activity of KR-72, its mode of action and the physiological impacts of the drug on fungal metabolism remain elusive. In this study, we performed the DNA microarray-based transcriptome analysis to identify KR-72 responsive genes and employed reverse genetics approaches to characterize their functions in Cryptococcus neoformans, which causes fatal meningoencephalitis in humans. First, KR-72 treatment altered in remodeling of transcriptome profiles in C. neoformans. Genes involved in translation and transcription were mostly upregulated, while those involved in cytoskeleton, intracellular trafficking, lipid and carbohydrate metabolism and energy production were downregulated. Supporting this, KR-72 has a strong synergistic effect with a calcineurin inhibitor FK506, while it has an antagonistic effect with polyene drug. Finally, KR-72 treatment promoted expression of ECM16, NOP14, HSP10, and MGE1, which we proved to be essential for the growth of C. neoformans. Among them, KR-72 mediated induction of MGE1 also appeared to hamper the viability of C. neoformans, potentially through impaired cell cycle or DNA repair system. This study will proposed mode of action for KR-72. The six slides of Cryptococcus_neoformans 3X20K are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted under 2 conditions (with or without treatment of KR-72 with H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A). We use the KR-72 non-treated RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye.

2014-02-26 | E-GEOD-55337 | biostudies-arrayexpress

Real time quantitative PCR analysis of murine macrophages

Project description:Murine macrophages were isolated from the lungs of mice given a pulmonary challenge with C. neoformans strain H99. Mice were either given a protective (H99γ) or a mock (HKCn) immunization prior to C. neoformans H99 challenge, and macrophages were isolated from the lungs of mice 24 hours, 3 days, or 7 days post-challenge using anti-CD11b microbeads according to the Miltenyi cell sorting system. We used SA Biosciences Toll-like Receptor PCR assay panel to quantitate gene expression of signal transduction factors in total RNA isolated from macrophages derived from immunized mice compared to non-immunized.

2012-08-09 | GSE39990 | GEO

Microarray experiment of murine macrophages co-cultured with neural stem cells in the presences of LPS

Project description:This experiment aimed at characterising the modulatory role of murine induced Neural Stem Cells (iNSCs) and Neural Stem Cells (NSCs) on macrophages (MPs) exposed to LPS in vitro. NaÃ¯ve MPs were polarized into an M1-like phenotype with LPS, and then co-cultured with 1:1 ratios of iNSCs in a trans-well system that avoids cell-to-cell contacts. NaÃ¯ve MPs, LPS-stimulated MPs and LPS-stimulated MPs co-cultured with NSCs were used as controls.

2018-01-01 | E-MTAB-5579 | biostudies-arrayexpress