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Ribosomal Protein L40e Fused With a Ubiquitin Moiety Is Essential for the Vegetative Growth, Morphological Homeostasis, Cell Cycle Progression, and Pathogenicity of Cryptococcus neoformans.

ABSTRACT: Ubiquitin is a highly conserved protein required for various fundamental cellular processes in eukaryotes. Herein, we first report the contribution of the ubiquitin fusion protein Ubi1 (a ubiquitin monomer fused with the ribosome protein L40e, Rpl40e) in the growth and pathogenicity of Cryptococcus neoformans. UBI1 deletion resulted in severe growth restriction of C. neoformans, whose growth rate was positively correlated with UBI1 expression level. The growth defect of the ubi1? strain could be closely associated with its morphological abnormalities, such as its reduced ribosome particles. In addition, the ubi1? mutant also displayed increased cell ploidy, cell cycle arrest, and decreased intracellular survival inside macrophages. All these phenotypes were reversed by the reconstitution of the full-length UBI1 gene or RPL40a domain. Mouse survival and fungal burden assays further revealed a severely attenuated pathogenicity for the ubi1? mutant, which is probably associated with its reduced stress tolerance and the induction of T-helper 1-type immune response. Taken together, Ubi1 is required for maintaining the vegetative growth, morphological homeostasis, cell cycle progression, and pathogenicity in vivo of C. neoformans. The pleiotropic roles of Ubi1 are dependent on the presence of Rpl40e and associated with its regulation of cryptococcal ribosome biogenesis.

SUBMITTER: Zhao J

PROVIDER: S-EPMC7674629 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Ribosomal Protein L40e Fused With a Ubiquitin Moiety Is Essential for the Vegetative Growth, Morphological Homeostasis, Cell Cycle Progression, and Pathogenicity of Cryptococcus neoformans.

Zhao Jingyu J Yang Yali Y Fan Yibin Y Yi Jiu J Zhang Chao C Gu Zhongkai Z Pan Weihua W Gu Julin J Liao Wanqing W Fang Wei W

Frontiers in microbiology 20201105

Ubiquitin is a highly conserved protein required for various fundamental cellular processes in eukaryotes. Herein, we first report the contribution of the ubiquitin fusion protein Ubi1 (a ubiquitin monomer fused with the ribosome protein L40e, Rpl40e) in the growth and pathogenicity of Cryptococcus neoformans. UBI1 deletion resulted in severe growth restriction of C. neoformans, whose growth rate was positively correlated with UBI1 expression level. The growth defect ...[more]

PMID: 33224112

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Project description:Cryptococcus neoformans, an opportunistic yeast pathogen, relies on a complex network of stress response pathways that allow for proliferation in the host. In Saccharomyces cerevisiae, stress responses are regulated by integral membrane proteins containing a transient receptor potential (TRP) domain, including the flavin carrier protein 1 (Flc1), which regulates calcium homeostasis and flavin transport. Here, we report that deletion of C. neoformans FLC1 results in cytosolic calcium elevation and increased nuclear content of calcineurin-dependent transcription factor Crz1, which is associated with an aberrant cell wall chitin overaccumulation observed in the flc1Δ mutant. Absence of Flc1 or inhibition of calcineurin with cyclosporine A prevents vacuolar fusion under conditions of combined osmotic and temperature stress, which is reversed in the flc1Δ mutant by the inhibition of TORC1 kinase with rapamycin. Flc1-deficient yeasts exhibit compromised vacuolar fusion under starvation conditions, including conditions that stimulate formation of carbohydrate capsule. Consequently, the flc1Δ mutant fails to proliferate under low nutrient conditions and displays a defect in capsule formation. Consistent with the previously uncharacterized role of Flc1 in vacuolar biogenesis, we find that Flc1 localizes to the vacuole. The flc1Δ mutant presents a survival defect in J774A.1 macrophage cell-line and profound virulence attenuation in both the Galleria mellonella and mouse pulmonary infection models, demonstrating that Flc1 is essential for pathogenicity. Thus, cryptococcal Flc1 functions in calcium homeostasis and links calcineurin and TOR signaling with vacuolar biogenesis to promote survival under conditions associated with vacuolar fusion required for this pathogen's fitness and virulence. IMPORTANCE Cryptococcosis is a highly lethal infection with limited drug choices, most of which are highly toxic or complicated by emerging antifungal resistance. There is a great need for new drug targets that are unique to the fungus. Here, we identify such a potential target, the Flc1 protein, which we show is crucial for C. neoformans stress response and virulence. Importantly, homologues of Flc1 exist in other fungal pathogens, such as Candida albicans and Aspergillus fumigatus, and are poorly conserved in humans, which could translate into wider spectrum therapy associated with minimal toxicity. Thus, Flc1 could be an "Achille's heel" of C. neoformans to be leveraged therapeutically in cryptococcosis and possibly other fungal infections.

Project description:Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills almost 200,000 people worldwide each year. It is acquired when mammalian hosts inhale the infectious propagules; these are deposited in the lung and, in the context of immunocompromise, may disseminate to the brain and cause lethal meningoencephalitis. Once inside the host, C. neoformans undergoes a variety of adaptive processes, including secretion of virulence factors, expansion of a polysaccharide capsule that impedes phagocytosis, and the production of giant (Titan) cells. The transcription factor Pdr802 is one regulator of these responses to the host environment. Expression of the corresponding gene is highly induced under host-like conditions in vitro and is critical for C. neoformans dissemination and virulence in a mouse model of infection. Direct targets of Pdr802 include the quorum sensing proteins Pqp1, Opt1, and Liv3; the transcription factors Stb4, Zfc3, and Bzp4, which regulate cryptococcal brain infectivity and capsule thickness; the calcineurin targets Had1 and Crz1, important for cell wall remodeling and C. neoformans virulence; and additional genes related to resistance to host temperature and oxidative stress, and to urease activity. Notably, cryptococci engineered to lack Pdr802 showed a dramatic increase in Titan cells, which are not phagocytosed and have diminished ability to directly cross biological barriers. This explains the limited dissemination of pdr802 mutant cells to the central nervous system and the consequently reduced virulence of this strain. The role of Pdr802 as a negative regulator of Titan cell formation is thus critical for cryptococcal pathogenicity.IMPORTANCE The pathogenic yeast Cryptococcus neoformans presents a worldwide threat to human health, especially in the context of immunocompromise, and current antifungal therapy is hindered by cost, limited availability, and inadequate efficacy. After the infectious particle is inhaled, C. neoformans initiates a complex transcriptional program that integrates cellular responses and enables adaptation to the host lung environment. Here, we describe the role of the transcription factor Pdr802 in the response to host conditions and its impact on C. neoformans virulence. We identified direct targets of Pdr802 and also discovered that it regulates cellular features that influence movement of this pathogen from the lung to the brain, where it causes fatal disease. These findings significantly advance our understanding of a serious disease.

Project description:The acquisition of iron and the maintenance of iron homeostasis are important aspects of virulence for the pathogenic fungus Cryptococcus neoformans In this study, we characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis and virulence in C. neoformans Monothiol glutaredoxins are important regulators of iron homeostasis because of their conserved roles in [2Fe-2S] cluster sensing and trafficking. We initially identified Grx4 as a binding partner of Cir1, a master regulator of iron-responsive genes and virulence factor elaboration in C. neoformans We confirmed that Grx4 binds Cir1 and demonstrated that iron repletion promotes the relocalization of Grx4 from the nucleus to the cytoplasm. We also found that a grx4 mutant lacking the GRX domain displayed iron-related phenotypes similar to those of a cir1Δ mutant, including poor growth upon iron deprivation. Importantly, the grx4 mutant was avirulent in mice, a phenotype consistent with observed defects in the key virulence determinants, capsule and melanin, and poor growth at 37°C. A comparative transcriptome analysis of the grx4 mutant and the WT strain under low-iron and iron-replete conditions confirmed a central role for Grx4 in iron homeostasis. Dysregulation of iron-related metabolism was consistent with grx4 mutant phenotypes related to oxidative stress, mitochondrial function, and DNA repair. Overall, the phenotypes of the grx4 mutant lacking the GRX domain and the transcriptome sequencing (RNA-Seq) analysis of the mutant support the hypothesis that Grx4 functions as an iron sensor, in part through an interaction with Cir1, to extensively regulate iron homeostasis.IMPORTANCE Fungal pathogens cause life-threatening diseases in humans, particularly in immunocompromised people, and there is a tremendous need for a greater understanding of pathogenesis to support new therapies. One prominent fungal pathogen, Cryptococcus neoformans, causes meningitis in people suffering from HIV/AIDS. In the present study, we focused on characterizing mechanisms by which C. neoformans senses iron availability because iron is both a signal and a key nutrient for proliferation of the pathogen in vertebrate hosts. Specifically, we characterized a monothiol glutaredoxin protein, Grx4, that functions as a sensor of iron availability and interacts with regulatory factors to control the ability of C. neoformans to cause disease. Grx4 regulates key virulence factors, and a mutant is unable to cause disease in a mouse model of cryptococcosis. Overall, our study provides new insights into nutrient sensing and the role of iron in the pathogenesis of fungal diseases.

Dataset Information

Ribosomal Protein L40e Fused With a Ubiquitin Moiety Is Essential for the Vegetative Growth, Morphological Homeostasis, Cell Cycle Progression, and Pathogenicity of Cryptococcus neoformans.

Publications

Ribosomal Protein L40e Fused With a Ubiquitin Moiety Is Essential for the Vegetative Growth, Morphological Homeostasis, Cell Cycle Progression, and Pathogenicity of <i>Cryptococcus neoformans</i>.

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