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Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans.

ABSTRACT:

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Cryptococcus neoformans is a human fungal pathogen and a major cause of fungal meningitis in immunocompromised individuals. Treatment options for cryptococcosis are limited. Of the two major antifungal drug classes, azoles are active against C. neoformans but exert a fungistatic effect, necessitating long treatment regimens and leaving open an avenue for emergence of azole resistance. Drugs of the echinocandin class, which target the glucan synthase and are fungicidal against a number of other fungal pathogens, such as Candida species, are ineffective against C. neoformans Despite the sensitivity of the target enzyme to the drug, the reasons for the innate resistance of C. neoformans to echinocandins remain unknown. To understand the mechanism of echinocandin resistance in C. neoformans, we screened gene disruption and gene deletion libraries for mutants sensitive to the echinocandin-class drug caspofungin and identified a mutation of CDC50, which encodes the ?-subunit of membrane lipid flippase. We found that the Cdc50 protein localized to membranes and that its absence led to plasma membrane defects and enhanced caspofungin penetration into the cell, potentially explaining the increased caspofungin sensitivity. Loss of CDC50 also led to hypersensitivity to the azole-class drug fluconazole. Interestingly, in addition to functioning in drug resistance, CDC50 was also essential for fungal resistance to macrophage killing and for virulence in a murine model of cryptococcosis. Furthermore, the surface of cdc50? cells contained increased levels of phosphatidylserine, which has been proposed to act as a macrophage recognition signal. Together, these results reveal a previously unappreciated role of membrane lipid flippase in C. neoformans drug resistance and virulence.

Importance

Cryptococcus neoformans is a fungal pathogen that is the most common cause of fungal meningitis, causing over 620,000 deaths annually. The treatment options for cryptococcosis are very limited. The most commonly used drugs are either fungistatic (azoles) or highly toxic (amphotericin B). Echinocandins are the newest fungicidal drug class that works well in treating candidiasis and aspergillosis, yet they are ineffective in treating cryptococcosis. In this study, we showed that the regulatory subunit of the lipid translocase (flippase), a protein that regulates the asymmetrical orientation of membrane lipids, is required for C. neoformans resistance to caspofungin, as well as for virulence during infection. This discovery identifies lipid flippase as a potential C. neoformans drug target, which plays an important role in the innate resistance of C. neoformans to echinocandins and in fungal virulence.

SUBMITTER: Huang W

PROVIDER: S-EPMC4959666 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans.

Huang Wei W Liao Guojian G Baker Gregory M GM Wang Yina Y Lau Richard R Paderu Padmaja P Perlin David S DS Xue Chaoyang C

mBio 20160510 3

<h4>Unlabelled</h4>Cryptococcus neoformans is a human fungal pathogen and a major cause of fungal meningitis in immunocompromised individuals. Treatment options for cryptococcosis are limited. Of the two major antifungal drug classes, azoles are active against C. neoformans but exert a fungistatic effect, necessitating long treatment regimens and leaving open an avenue for emergence of azole resistance. Drugs of the echinocandin class, which target the glucan synthase and are fungicidal against ...[more]

PMID: 27165800

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Project description:Echinocandins show fungicidal activity against common invasive mycoses but are ineffective against cryptococcosis. The underlying mechanism for echinocandin resistance in Cryptococcus neoformans remains poorly understood but has been shown to involve Cdc50, the regulatory subunit of lipid flippase. In a forward genetic screen for cdc50? suppressor mutations that are caspofungin resistant, we identified Crm1 (caspofungin resistant mutation 1), a homolog of mechanosensitive channel proteins, and showed that crm1? restored caspofungin resistance in cdc50? cells. Caspofungin-treated cdc50? cells exhibited abnormally high intracellular calcium levels ([Ca2+]c) and heightened activation of the calcineurin pathway. Deletion of CRM1 in the cdc50? background normalized the abnormally high [Ca2+]c. Cdc50 interacts with Crm1 to maintain cellular calcium homeostasis. Analysis of chitin/chitosan content showed that deleting CRM1 reversed the decreased chitosan production of cdc50? cells. Together, these results demonstrate that Cdc50 and Crm1 regulation of the calcineurin pathway and cytoplasmic calcium homeostasis may underlie caspofungin resistance in C. neoformans IMPORTANCE Cryptococcus neoformans is the leading cause of fungal meningitis, accounting for ?15% of HIV/AIDS-related deaths, but treatment options for cryptococcosis are limited. Echinocandins are the newest fungicidal drug class introduced but are ineffective in treating cryptococcosis. Our previous study identified the lipid flippase subunit Cdc50 as a contributor to echinocandin resistance in C. neoformans Here, we further elucidated the mechanism of Cdc50-mediated caspofungin drug resistance. We discovered that Cdc50 interacts with the mechanosensitive calcium channel protein Crm1 to regulate calcium homeostasis and caspofungin resistance via calcium/calcineurin signaling. These results provide novel insights into echinocandin resistance in this pathogen, which may lead to new treatment options, as well as inform echinocandin resistance mechanisms in other fungal organisms and, hence, advance our understanding of modes of antifungal drug susceptibility and resistance.

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.IMPORTANCE Cryptococcus 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.

Project description:Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

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.

Dataset Information

Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans.

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Importance

Publications

Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans.

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