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A putative P-type ATPase, Apt1, is involved in stress tolerance and virulence in Cryptococcus neoformans.

ABSTRACT: The export of virulence factors, such as the capsule polysaccharide, to the cell surface is a critical aspect of the pathogenicity of Cryptococcus neoformans. A view of capsule export via exocytosis and extracellular vesicles is emerging, but the molecular mechanisms underlying virulence factor transport pathways remain to be established. In this study, we characterized the APT1 gene, which encodes a predicted integral membrane P-type ATPase belonging to the type IV, Drs2 family of aminophospholipid translocases (flippases) (APTs). APTs maintain the phospholipid asymmetry that is critical in membrane fusion events for trafficking and in establishing cell polarity. Deletion of the APT1 gene resulted in phenotypes consistent with similar roles in C. neoformans. These included altered actin distribution, increased sensitivity to stress conditions (oxidative and nitrosative stress) and to trafficking inhibitors, such as brefeldin A and monensin, a reduction in exported acid phosphatase activity, and hypersensitivity to the antifungal drugs amphotericin B, fluconazole, and cinnamycin. However, there was no difference in growth, capsule size, or melanin production between the wild type and the apt1 mutant strains at either 30 degrees C or 37 degrees C. Despite the absence of an influence on these major virulence factors, Apt1 was required for survival during interactions with macrophages, and apt1 mutants exhibited attenuated virulence in a mouse inhalation model of cryptococcosis. Therefore, Apt1 contributes to virulence and the stress response in C. neoformans through apparent functions in membrane fusion and trafficking that do not influence the deposition of major virulence factors, such as capsule and melanin, outside the cell.

SUBMITTER: Hu G

PROVIDER: S-EPMC2805298 | biostudies-other | 2010 Jan

REPOSITORIES: biostudies-other

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A putative P-type ATPase, Apt1, is involved in stress tolerance and virulence in Cryptococcus neoformans.

Hu Guanggan G Kronstad James W JW

Eukaryotic cell 20091130 1

The export of virulence factors, such as the capsule polysaccharide, to the cell surface is a critical aspect of the pathogenicity of Cryptococcus neoformans. A view of capsule export via exocytosis and extracellular vesicles is emerging, but the molecular mechanisms underlying virulence factor transport pathways remain to be established. In this study, we characterized the APT1 gene, which encodes a predicted integral membrane P-type ATPase belonging to the type IV, Drs2 family of aminophosphol ...[more]

PMID: 19949048

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Project description:Cryptococcus neoformans kills 200,000 people worldwide each year. After inhalation, this environmental yeast proliferates either extracellularly or within host macrophages. Under conditions of immunocompromise, cryptococci disseminate from the lungs to the brain, causing a deadly meningoencephalitis that is 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 that remains understudied in C. neoformans is the Mediator complex. In other organisms, this complex promotes transcription of specific genes by increasing assembly of the RNA polymerase II preinitiation complex. We focused on the Kinase Module of Mediator, which consists of cyclin C (Ssn801), cyclin-dependent kinase 8 (Cdk8), Med12, and Med13. This module provides important inhibitory control of Mediator complex assembly and activity. Using transcriptomics, we discovered that Cdk8 and Ssn801 together regulate cryptococcal functions such as the ability to grow on acetate and the response to oxidative stress, both of which were experimentally validated. Deletion of CDK8 yielded altered mitochondrial morphology and the dysregulation of genes involved in oxidation-reduction processes. This strain exhibited increased susceptibility to oxidative stress, resulting in an inability of mutant cells to proliferate within phagocytes, decreased lung burdens, and attenuated virulence in vivo These findings increase our understanding of cryptococcal adaptation to the host environment and its regulation of oxidative stress resistance and virulence.IMPORTANCECryptococcus neoformans is a fungal pathogen that primarily affects severely immunocompromised patients, resulting in 200,000 deaths every year. This yeast occurs in the environment and can establish disease upon inhalation into the lungs of a mammalian host. In this harsh environment it must survive engulfment by host phagocytes, including the oxidative stresses it experiences inside them. To adapt to these challenging conditions, C. neoformans deploys a variety of regulatory proteins to alter gene expression levels and enhance its ability to survive. We have elucidated the role of a protein complex that regulates the cryptococcal response to oxidative stress, survival within phagocytes, and ability to cause disease. These findings are important because they advance our understanding of cryptococcal disease, which we hope will help in the efforts to control this devastating infection.

Project description:Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i) interesting physiological property of C. neoformans regarding its amino acid uptake system; (ii) an important aspect of virulence, which is the need for amino acid permeases during thermal and oxidative stress resistance and, hence, host invasion and colonization; and (iii) provides a convenient prototype for antifungal development, which are the amino acid permeases Aap4/Aap5 and their inhibitor.

Dataset Information

A putative P-type ATPase, Apt1, is involved in stress tolerance and virulence in Cryptococcus neoformans.

Publications

A putative P-type ATPase, Apt1, is involved in stress tolerance and virulence in Cryptococcus neoformans.

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