Unravelling the pathobiological role of the fungal KEOPS complex in Cryptococcus neoformans
Ontology highlight
ABSTRACT:
ORGANISM(S): Cryptococcus neoformans
PROVIDER: GSE208333 | GEO | 2022/10/18
REPOSITORIES: GEO
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Project description:Cryptococcus neoformans is an important pathogen that annually kills 200,000 people worldwide. It survives in the environment as a yeast or spore and can also proliferate within host macrophages after being inhaled into the lungs. In conditions of immunocompromise, cryptococcal cells can escape from the lungs to the brain, where they cause a deadly meningoencephalitis that is both difficult and expensive to treat. Cryptococcal adaptation to the harsh lung environment is a critical first step in its pathogenesis, and consequently a compelling topic of study. This adaptation is mediated by a complex transcriptional program that integrates cellular responses to environmental stimuli. Although several key regulators in this process have been examined, one important protein complex that modulates transcription yet remains understudied in C. neoformans is the Mediator complex. In other organisms, this complex promotes transcription of specific genes by increasing the assembly of the RNA Polymerase II pre-initiation complex. We have focused on the Kinase Module of the Mediator complex, which consists of cyclin C (Ssn801), cyclin-dependent kinase 8 (Cdk8), and two other proteins (Med12 and Med13). This module provides important inhibitory control of Mediator complex assembly and activity. Using a transcriptomics approach, we discovered that Cdk8 and Ssn801 together regulate cryptococcal functions relevant to pathogenesis, including the response to oxidative stress. Deletion of CDK8 resulted in altered mitochondrial morphology and the dysregulation of genes involved in oxidation-reduction processes, such that this strain exhibited increased susceptibility to oxidative stress. This resulted in an inability of mutant cells to proliferate within phagocytes, decreased lung burdens, and attenuated virulence in in vivo studies. These findings increase our understanding of cryptococcal adaptation to the host environment and its regulation of oxidative stress resistance and virulence.
2019-01-18 | GSE125281 | GEO
Project description:Cryptococcus neoformans (Cn) is an opportunistic fungal microorganism that causes life-threatening meningoencephalitis. During the infection, the microbial population is heterogeneously composed of cells with varying generational ages, with older cells accumulating during chronic infections. This is attributed to their enhanced resistance to phagocytic killing and tolerance of antifungals like fluconazole (FLC). Using RNA-seq, we investigated how transcriptomic changes in different aspects of metabolism contribute to age-associated FLC tolerance in Cn.
2024-03-06 | GSE260902 | GEO
Project description:Casein kinase 2 (CK2) is a potential therapeutic target for several human diseases due to its crucial roles in growth, differentiation, and metabolic homeostasis. This study delves into the role of the CK2 complex in Cryptococcus neoformans, a key fungal pathogen causing meningitis. In C. neoformans, the CK2 complex is made up of a main catalytic subunit (Cka1) and two regulatory subunits (Ckb1 and Ckb2). The primary role of Cka1 is as a protein kinase, while Ckb1/2 assist in various cellular functions. Triple mutants without all three subunits exhibited more pronounced defects than mutants lacking only Cka1, hinting at potential independent functions of Ckb1/2. In animal studies, mutants without these subunits showed a sharp decline in virulence. Moreover, CK2 disruption affected many effector proteins and disrupted key signaling pathways crucial for the pathogenicity of C. neoformans. Overall, the findings highlight the importance of the CK2 complex in fungal biology and its potential as a target for new antifungal treatments.
2023-12-21 | GSE245849 | GEO
Project description:Cryptococcal meningoencephalitis caused by C. neoformans infection is the most common cause of death in HIV/AIDS patients. Macrophages are recognized as key players in promoting the growth, proliferation, and dissemination of Cryptococcus. However, macrophage-derived factors that underlie these pathogenic responses remain unclear. Thus, the aim of this study is to investigate the permissive factors of macrophages that support the pathogenicity of Cryptococcus infection. We generated mRNA expression profiles of alveolar macrophage isolated from BALB/c mice intranasally treated with PBS or infected with C. neoformans H99 at 7 and 14 days postinfection and performed a comparative transcriptomic analysis to investigate the transcriptomic changes of macrophages during infection. Alveolar macrophages isolated from C. neoformans-infected mice showed dynamic gene expression patterns which were altered from a protective M1-like cells to a pathogenic M2-like phenotype. Interestingly, Arg1, encoded for enzyme arginase-1, was a predominant up-regulated gene observed in alveolar macrophages after infection at 14 days and the activation of macrophage arginase activity is required for the development of M2 macrophages that supports cryptococcal growth, proliferation, and dissemination.
2022-11-30 | GSE214001 | GEO
Project description:The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and other functions during fungal adaptation to the intracellular environment by SAGE analysis. We focused on the ACL1 gene for ATP-citrate lyase, which converts citrate to acetyl-CoA, because this gene showed elevated transcript levels in macrophages and because of the importance of acetyl-CoA as a central metabolite. Mutants lacking ACL1 showed delayed growth on medium containing glucose, reduced cellular levels of acetyl-CoA, defective production of virulence factors, increased susceptibility to the antifungal drug fluconazole and decreased survival within macrophages. Importantly, acl1 mutants were unabl e to cause disease in a murine inhalation model, a phenotype that was more extreme than other mutants with defects in acetyl-CoA production (e.g., an acetyl-CoA synthetase mutant). Loss of virulence is likely due to perturbation of critical physiological interconnections between virulence factor expression and metabolism in C. neoformans. Phylogenetic analysis and structural modeling of cryptococcal Acl1 identified three indels unique to fungal protein sequences; these differences may provide opportunities for the development of pathogen-specific inhibitors. SAGE analysis of C. neoformans cells isolated from macrophages The murine macrophage-like cell line J774A.1 was maintained at 37°C in 10% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FBS), 1% nonessential amino acids, 100 µg mL-1 penicillin-streptomycin, and 4 mM L-glutamine (Invitrogen). Cryptococcus cells were opsonized with monoclonal antibody 18B7 against capsule (1 µg mL-1), and macrophages were treated with recombinant mouse gamma interferon (IFN-gamma) (50 U mL-1) and lipopolysaccharide (LPS) (0.3 µg mL-1) prior to co-incubation at a multiplicity of infection (MOI) of 1:1. Macrophages were inoculated with H99 cells and washed after 1 h of inoculation to remove unattached, extracellular fungal cells. After 6 h of incubation, sterile, ice-cold distilled H2O was applied to each well to lyse the macrophages, and the fungal cells (4 x 107) were harvested by centrifugation. H99 control cells were prepared by growth under the same condition but without macrophages, and 108 cell s were harvested.
2013-08-15 | GSE41066 | GEO
Project description:Cryptococcus neoformans is a fungal pathogen responsible for an increased mortality among immunocompromised individuals. Long antifungal therapies to treat cryptococcal infections have compounded the occurrence of resistant strains that threaten the efficacy of current treatments. In this senseDue to resistance mechanisms, discovery of compounds that inhibit virulence factors, rather than kill the fungus, have emerged as potential new strategies to combat infection and reduce the rate of resistance due to lower selective pressure. Invertebrates rely solely on an effective innate immune system to prevent infections, provide providing a potential one health approach for discovery of novel antifungal and antibacterial compounds. Here, we demonstrate a differentiated extraction of proteins from three mollusks (freshwater and terrestrial) and evaluate extract their effects against the growth and virulence factor production (thermotolerance, melanin, capsule, and biofilm) in C. neoformans. We show that clarified extracts of Planorbella pilsbryi have a fungicidal effect on cryptococcal cells in a comparable way to Fluconazolefluconazole. Similarly, crude and clarifiedall extracts of Cipangopaludina chinensis not only affects cryptococcal thermotolerance but also impairs biofilm and capsule production. In addition, incubation of C. neoformans with clarified extracts of Cepaea nemoralis extracts reduced capsule production. Using inhibitory activity of extracts against peptidases related with virulence factors and Quantitative quantitative proteomics arose distinct proteome signatures for each extract and proposed proteins driving the observed anti-virulence properties. Overall, this work demonstratesproves the potential of compounds derived from natural sources to inhibit virulence factor production in a clinically important fungal pathogen.
2023-03-06 | PXD038987 | Pride
Project description:Cryptococcus neoformans is an opportunistic basidiomycete pathogen that is a major etiological agent of fungal meningoencephalitis leading to more than 180,000 deaths worldwide annually. For this pathogen, the polysaccharide capsule is a key virulence factor, which interferes with the phagocytosis by host innate immune cells, but its complex signaling networks remain elusive. In this study, we systematically analyzed capsule biosynthesis and signaling networks by using C. neoformans transcription factor (TF) and kinase mutant libraries under diverse capsule-inducing conditions, such as Dulbecco’s Modified Eagle’s (DME), Littman’s medium (LIT) and fetal bovine serum (FBS) medium. We found that deletion of GAT201, YAP1, BZP4, and ADA2 consistently causes capsule production defects in all tested media, indicating that they are capsule-regulating core TFs. Epistatic and expression analysis showed that Yap1 and Ada2 control Gat201 upstream, whereas Bzp4 and Gat201 regulate capsule production independently. We next searched for potential upstream kinases and found that mutants deleted of PKA1, BUD32, POS5, IRE1 or CDC2801 showed reduced capsule production under all three capsule induction conditions, whereas mutants deleted of HOG1 and IRK5 displayed enhanced capsule production. Notably, Pka1 and Irk5 controls induction of GAT201 and BZP4, respectively, under capsule induction condition. Finally, we monitored transcriptome profiles governed by Bzp4, Gat201, and Ada2 under capsule-inducing condition and demonstrated that these TFs regulate redundant and unique sets of downstream target genes. In conclusion, this study provides further insight into the complex regulatory mechanism of capsule production related signaling pathways in C. neoformans.
2022-10-18 | GSE208163 | GEO
Project description:Systemic infection with Cryptococcus neoformans, a dangerous and contagious pathogen found throughout the world, frequently results in lethal cryptococcal pneumonia and meningoencephalitis, and no effective treatments of cryptococcosis are available. Here, we describe Prm1, a novel regulator of virulence in C. neoformans. C. neoformans prm1 cells exhibit extreme sensitivity to various environmental stress conditions. Furthermore, prm1 cells show deficiencies in the biosynthesis of chitin, chitosan, and mannoprotein, which in turn result in impairment of cell wall integrity. Treatment of mice with heat-killed prm1 cells was found to facilitate the host immunological defence against infection with wild-type C. neoformans. Further investigation demonstrated that prm1 cells strongly promote pulmonary production of interferon- and Th1 responses, leading to activation of macrophage M1 differentiation and inhibition of M2 polarization. Therefore, our findings suggest that C. neoformans Prm1 may be a viable target for the development of anti-cryptococcosis medications and, cells lacking Prm1 represent a promising candidate for a vaccine.
2023-10-11 | PXD038840 | Pride
Project description:Melanin is an antioxidant polyphenol pigment, which is required for the pathogenicity of many human and plant fungal pathogens. Nevertheless, its comprehensive regulatory mechanism still remain elusive. In this study, we systematically analyzed melanin-regulating signaling pathways in Cryptococcus neoformans. Here we identified four core melanin-regulating TFs, Bzp4, Usv101, Mbs1, and Hob1, which are required for induction of the laccase gene (LAC1). Bzp4, Usv101 and Mbs1 independently regulate LAC1 induction while Hob1 controls Bzp4 and Usv101 expression. Both Bzp4 and Usv101 are translocated from the cytoplasm into the nucleus upon nutrient starved condition, while Mbs1 is constitutively localized in the nucleus. Notably, the cAMP pathway is not involved in regulation of the four TFs, while the HOG pathway negatively regulates induction of BZP4 and LAC1. As potential kinases upstream of the core TFs, we identified the nine core kinases, whose deletion leads to defective melanin production and LAC1 induction. Deletion of GSK3 or KIC1 abolishes induction of LAC1 and BZP4, and also perturbs nuclear translocation of Bzp4. Notably, Gsk3 also regulates the expression of HOB1, USV101, and MBS1, indicating that it is a critical melanin-regulating kinase. Finally, the RNA-sequencing based transcriptome analysis of the wild-type and bzp4Δ, usv101Δ, hob1Δ, and mbs1Δ strains under nutrient rich and starved conditions reveals that the core melanin-regulating TFs govern redundant and distinct classes of genes, which are involved in a variety of biological processes.
2019-05-30 | GSE131891 | GEO
Project description:Cryptococcus neoformans is an invasive human fungal pathogen, which causes pneumonia and meningoencephalitis in both healthy and immunodeficient individuals, resulting 181,100 deaths each year. Studies have demonstrated that copper detoxification machineries play critical functions in modulating C. neoformans fitness and pathogenicity in the host lung tissue. Pulmonary C. neoformans infection provokes the generation of toxic copper bombardment, which in return activate detoxification process in fungal cells. However, the molecular mechanism on how Cu inhibits C. neoformans in proliferation remains unclear. Here, using Cu detoxification gene knockout strains we demonstrated that exogenous Cu ions inhibit cell growth of the metallothionein mutant, which was significantly rescue when supplementing with the ROS scavenger, N-acetylcysteine. To future characterize the molecular mechanism of C. neoformans response to Cu toxicity, we employ iTRAQ with LC-MS/MS analysis, in the presence of exogenous Cu or NAC. Our data showed that an increased Cu level repressed expression of factors involved in protein translation but activates expression of important players in ubiquitin-degradation process. We proposed that the downregulation of protein synthesis and the upregulation of protein degradation were the main strategy of Cu toxicity. The metallothionein mutant showed a higher ubiquitination level under Cu treatment. In addition, MG132 could partially restore the Cu toxicity effect on metallothionein mutant. These results shed new light on the Cu antifungal mechanisms from proteomic profile.
2021-02-09 | PXD024098 | iProX