scholarly journals The Vacuolar Ca2+ Exchanger Vcx1 Is Involved in Calcineurin-Dependent Ca2+ Tolerance and Virulence in Cryptococcus neoformans

2010 ◽  
Vol 9 (11) ◽  
pp. 1798-1805 ◽  
Author(s):  
Lívia Kmetzsch ◽  
Charley Christian Staats ◽  
Elisa Simon ◽  
Fernanda L. Fonseca ◽  
Débora L. de Oliveira ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Cyclosporine A ◽  
Capsular Polysaccharide ◽  
Cytosolic Calcium ◽  
Content Type ◽  
Human Body Temperature ◽  
Life Threatening ◽  
High Concentrations ◽  
Capsule Size ◽  
Virulence Attribute

ABSTRACT Cryptococcus neoformans is an encapsulated yeast that causes a life-threatening meningoencephalitis in immunocompromised individuals. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this pathogen. This trait is controlled in part by the Ca2+-calcineurin pathway, which senses and utilizes cytosolic calcium for signaling. In the present study, the identification of the C. neoformans gene VCX1, which encodes a vacuolar calcium exchanger, is reported. The VCX1 knockout results in hypersensitivity to the calcineurin inhibitor cyclosporine A at 35°C, but not at 30°C. Furthermore, high concentrations of CaCl2 lead to growth inhibition of the vcx1 mutant strain only in the presence of cyclosporine A, indicating that Vcx1 acts in parallel with calcineurin. The loss of VCX1 does not influence cell wall integrity or capsule size but decreases secretion of the major capsular polysaccharide glucuronoxylomannan (GXM) in culture supernatants.Vcx1 also influences C. neoformans phagocytosis by murine macrophages and is required for full virulence in mice. Analysis of cellular distribution by confocal microscopy confirmed the vacuolar localization of Vcx1 in C. neoformans cells.

scholarly journals Dectin-2-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Suresh Ambati ◽  
Emma C. Ellis ◽  
Jianfeng Lin ◽  
Xiaorong Lin ◽  
Zachary A. Lewis ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Aspergillus Fumigatus ◽  
Effective Dose ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Antifungal Drugs ◽  
Extracellular Matrices ◽  
Content Type ◽  
Order Of Magnitude ◽  
Life Threatening

ABSTRACT Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2’s mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases. IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

scholarly journals Pleiotropic Roles of the Msi1-Like Protein Msl1 in Cryptococcus neoformans

2012 ◽  
Vol 11 (12) ◽  
pp. 1482-1495 ◽  
Author(s):  
Dong-Hoon Yang ◽  
Shinae Maeng ◽  
Anna K. Strain ◽  
Anna Floyd ◽  
Kirsten Nielsen ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Stress Responses ◽  
Fungal Pathogens ◽  
Independent Manner ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
Human Fungal Pathogen ◽  
Assembly Factor ◽  
Life Threatening ◽  
Stress Related Genes

ABSTRACT Msi1-like (MSIL) proteins contain WD40 motifs and have a pleiotropic cellular function as negative regulators of the Ras/cyclic AMP (cAMP) pathway and components of chromatin assembly factor 1 (CAF-1), yet they have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans , which causes life-threatening meningoencephalitis in humans. Notably, Msl1 plays pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners largely independent of Ras. Msl1 negatively controls antioxidant melanin production and sexual differentiation, and this was repressed by the inhibition of the cAMP-signaling pathway. In contrast, Msl1 controls thermotolerance, diverse stress responses, and antifungal drug resistance in a Ras/cAMP-independent manner. Cac2, which is the second CAF-1 component, appears to play both redundant and distinct functions compared to the functions of Msl1. Msl1 is required for the full virulence of C. neoformans . Transcriptome analysis identified a group of Msl1-regulated genes, which include stress-related genes such as HSP12 and HSP78 . In conclusion, this study demonstrates pleiotropic roles of Msl1 in the human fungal pathogen C. neoformans , providing insight into a potential novel antifungal therapeutic target.

scholarly journals EDTA Inhibits Biofilm Formation, Extracellular Vesicular Secretion, and Shedding of the Capsular Polysaccharide Glucuronoxylomannan by Cryptococcus neoformans

2012 ◽  
Vol 78 (22) ◽  
pp. 7977-7984 ◽  
Author(s):  
Emma J. Robertson ◽  
Julie M. Wolf ◽  
Arturo Casadevall
Keyword(s):  
Cryptococcus Neoformans ◽  
Biofilm Formation ◽  
Capsular Polysaccharide ◽  
Inhibitory Effect ◽  
Antifungal Drugs ◽  
Biofilm Growth ◽  
Content Type ◽  
Sublethal Concentrations ◽  
Vesicular Secretion

ABSTRACTThe fungal pathogenCryptococcus neoformanscan grow as a biofilm on a range of synthetic and prosthetic materials. Cryptococcal biofilm formation can complicate the placement of shunts used to relieve increased intracranial pressure in cryptococcal meningitis and can serve as a nidus for chronic infection. Biofilms are generally advantageous to pathogensin vivo, as they can confer resistance to antimicrobial compounds, including fluconazole and voriconazole in the case ofC. neoformans. EDTA can inhibit biofilm formation by several microbes and enhances the susceptibility of biofilms to antifungal drugs. In this study, we evaluated the effect of sublethal concentrations of EDTA on the growth of cryptococcal biofilms. EDTA inhibited biofilm growth byC. neoformans, and the inhibition could be reversed by the addition of magnesium or calcium, implying that the inhibitory effect was by divalent cation starvation. EDTA also reduced the amount of the capsular polysaccharide glucuronoxylomannan shed into the biofilm matrix and decreased vesicular secretion from the cell, thus providing a potential mechanism for the inhibitory effect of this cation-chelating compound. Our data imply that the growth ofC. neoformansbiofilms requires the presence of divalent metals in the growth medium and suggest that cations are required for the export of materials needed for biofilm formation, possibly including extracellular vesicles.

scholarly journals Cryptococcus neoformans Requires the ESCRT Protein Vps23 for Iron Acquisition from Heme, for Capsule Formation, and for Virulence

2012 ◽  
Vol 81 (1) ◽  
pp. 292-302 ◽  
Author(s):  
Guanggan Hu ◽  
Mélissa Caza ◽  
Brigitte Cadieux ◽  
Vivienne Chan ◽  
Victor Liu ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Virulence Factor ◽  
Fungal Pathogens ◽  
Capsular Polysaccharide ◽  
Iron Acquisition ◽  
Growth Defect ◽  
Capsule Formation ◽  
Content Type ◽  
Dna Insertion ◽  
Essential Nutrient

Iron availability is a key regulator of virulence factor elaboration inCryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that attack humans. In this study, we mutagenizedC. neoformansbyAgrobacterium-mediated T-DNA insertion and screened for mutants with reduced growth on heme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomalsortingcomplexrequired fortransport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect in uptake via endocytosis or misregulation of iron acquisition from heme. Remarkably,vps23mutants were also defective in the elaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression ofVps23resulted in cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in thevps23mutant in a mouse model of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role for trafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection between iron and virulence factor deployment inC. neoformans.

scholarly journals Cryptococcosis in the era of AIDS--100 years after the discovery of Cryptococcus neoformans.

1995 ◽  
Vol 8 (4) ◽  
pp. 515-548 ◽  
Author(s):  
T G Mitchell ◽  
J R Perfect
Keyword(s):  
T Cells ◽  
Cryptococcus Neoformans ◽  
Capsular Polysaccharide ◽  
Cell Mediated Immunity ◽  
Murine Models ◽  
Effector Cells ◽  
Distinctive Features ◽  
Treatment And Prevention ◽  
Aids Pandemic ◽  
Life Threatening

Although Cryptococcus neoformans and cryptococcosis have existed for several millennia, a century has passed since the discovery of this encapsulated yeast and its devastating disease. With the advent of the AIDS pandemic, cryptococcal meningitis has emerged as a leading cause of infectious morbidity and mortality and a frequently life-threatening opportunistic mycosis among patients with AIDS. Both basic and clinical research have accelerated in the 1990s, and this review attempts to highlight some of these advances. The discussion covers recent findings, current concepts, controversies, and unresolved issues related to the ecology and genetics of C. neoformans; the surface structure of the yeast; and the mechanisms of host defense. Regarding cell-mediated immunity, CD4+ T cells are crucial for successful resistance, but CD8+ T cells may also participate significantly in the cytokine-mediated activation of anticryptococcal effector cells. In addition to cell-mediated immunity, monoclonal antibodies to the major capsular polysaccharide, the glucuronoxylomannan, offer some protection in murine models of cryptococcosis. Clinical concepts are presented that relate to the distinctive features of cryptococcosis in patients with AIDS and the diagnosis, treatment, and prevention of cryptococcosis in AIDS patients.

scholarly journals Monoclonal Antibodies Specific for Immunorecessive Epitopes of Glucuronoxylomannan, the Major Capsular Polysaccharide of Cryptococcus neoformans, Reduce Serotype Bias in an Immunoassay for Cryptococcal Antigen

2011 ◽  
Vol 18 (8) ◽  
pp. 1292-1296 ◽  
Author(s):  
Ann Percival ◽  
Peter Thorkildson ◽  
Thomas R. Kozel
Keyword(s):  
Monoclonal Antibodies ◽  
Cryptococcus Neoformans ◽  
Capsular Polysaccharide ◽  
Polyclonal Antibodies ◽  
Sub Saharan Africa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Screening Strategies ◽  
Sub Saharan ◽  
High Degree

ABSTRACTImmunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide ofCryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to removeO-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population.

scholarly journals Methamphetamine Enhances Cryptococcus neoformans Pulmonary Infection and Dissemination to the Brain

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Dhavan Patel ◽  
Gunjan M. Desai ◽  
Susana Frases ◽  
Radames J. B. Cordero ◽  
Carlos M. DeLeon-Rodriguez ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Respiratory Tract ◽  
Cryptococcus Neoformans ◽  
Biofilm Formation ◽  
Pulmonary Infection ◽  
Capsular Polysaccharide ◽  
Content Type ◽  
The Impact ◽  
The Brain

ABSTRACTMethamphetamine (METH) is a major addictive drug of abuse in the United States and worldwide, and its use is linked to HIV acquisition. The encapsulated fungusCryptococcus neoformansis the most common cause of fungal meningitis in patients with AIDS. In addition to functioning as a central nervous system stimulant, METH has diverse effects on host immunity. Using a systemic mouse model of infection andin vitroassays in order to critically assess the impact of METH onC. neoformanspathogenesis, we demonstrate that METH stimulates fungal adhesion, glucuronoxylomannan (GXM) release, and biofilm formation in the lungs. Interestingly, structural analysis of the capsular polysaccharide of METH-exposed cryptococci revealed that METH alters the carbohydrate composition of this virulence factor, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Additionally, we show that METH promotesC. neoformansdissemination from the respiratory tract into the brain parenchyma. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms.IMPORTANCEMethamphetamine (METH) is a major health threat to our society, as it adversely changes people’s behavior, as well as increases the risk for the acquisition of diverse infectious diseases, particularly those that enter through the respiratory tract or skin. This report investigates the effects of METH use on pulmonary infection by the AIDS-related fungusCryptococcus neoformans. This drug of abuse stimulates colonization and biofilm formation in the lungs, followed by dissemination of the fungus to the central nervous system. Notably,C. neoformansmodifies its capsular polysaccharide after METH exposure, highlighting the fungus’s ability to adapt to environmental stimuli, a possible explanation for its pathogenesis. The findings may translate into new knowledge and development of therapeutic and public health strategies to deal with the devastating complications of METH abuse.

scholarly journals Antibody-Mediated Protection in Murine Cryptococcus neoformans Infection Is Associated with Pleotrophic Effects on Cytokine and Leukocyte Responses

2002 ◽  
Vol 70 (3) ◽  
pp. 1571-1580 ◽  
Author(s):  
Marta Feldmesser ◽  
Aron Mednick ◽  
Arturo Casadevall
Keyword(s):  
Immune Response ◽  
Cryptococcus Neoformans ◽  
Capsular Polysaccharide ◽  
Granulomatous Inflammation ◽  
Surface Expression ◽  
Interleukin 4 ◽  
Significant Difference ◽  
Predominant Cell Type ◽  
Immunosuppressed Patients ◽  
Life Threatening

ABSTRACT Cryptococcus neoformans, an encapsulated yeast, is a common cause of life-threatening meningoencephalitis in immunosuppressed patients. We previously observed that administration of a monoclonal antibody (MAb) to the capsular polysaccharide to mice with pulmonary infection prolonged survival and enhanced granulomatous inflammation without reducing lung CFU. To understand the mechanism of MAb action, we studied leukocyte recruitment and cytokine profiles in lungs of A/JCr mice. B lymphocytes were the predominant cell type in lung infiltrates, comprising 15 to 30% of the leukocytes. Despite alterations in histological appearance, fluorescence-activated cell sorter analysis revealed no significant difference in total numbers of lung leukocytes in MAb-treated mice and controls. Differences in the immune response to C. neoformans between MAb-treated mice and controls included (i) an increase in the percentage of granulocytes among lung leukocytes on day 14, (ii) higher macrophage surface expression of CD86 on day 28, (iii) larger amounts of IL-10 in lung homogenates at day 7, (iv) a trend toward smaller amounts of gamma interferon mRNA and protein on day 7, and (v) a smaller increase in the levels of interleukin-4 mRNA and protein on day 7. Hence, the immune responses to C. neoformans infection in the presence and absence of specific antibody were qualitatively similar, and antibody administration was associated with several subtle quantitative differences in immune response parameters that could translate into enhanced survival. MAb may function partly by down-regulating the inflammatory response and reducing host damage. Our findings demonstrate unexpected complexity in the interaction between specific MAb and other components of the host immune response.

scholarly journals Identification of a Cryptococcus neoformans Cytochrome P450 Lanosterol 14α-Demethylase (Erg11) Residue Critical for Differential Susceptibility between Fluconazole/Voriconazole and Itraconazole/Posaconazole

2011 ◽  
Vol 56 (3) ◽  
pp. 1162-1169 ◽  
Author(s):  
Edward Sionov ◽  
Yun C. Chang ◽  
H. Martin Garraffo ◽  
Michael A. Dolan ◽  
Mahmoud A. Ghannoum ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Missense Mutation ◽  
Treated Patient ◽  
Differential Susceptibility ◽  
Missense Mutations ◽  
Gene Encoding ◽  
Content Type ◽  
High Concentrations ◽  
Increased Susceptibility

ABSTRACTCryptococcus neoformansstrains resistant to azoles due to mutations causing alterations in theERG11gene, encoding lanosterol 14α-demethylase, have rarely been reported. In this study, we have characterized aC. neoformansserotype A strain that is resistant to high concentrations of fluconazole (FLC). This strain, which was isolated from an FLC-treated patient, contained five missense mutations in theERG11gene compared to the sequence of reference strain H99. Molecular manipulations of theERG11gene coupled with susceptibility to triazole revealed that a single missense mutation resulting in the replacement of tyrosine by phenylalanine at amino acid 145 was sufficient to cause the high FLC resistance of the strain. Importantly, this newly identified point mutation in theERG11gene ofC. neoformansafforded resistance to voriconazole (VRC) but increased susceptibility to itraconazole (ITC) and posaconazole (PSC), which are structurally similar to each other but distinct from FLC/VRC. Thein vitrosusceptibility/resistance of the strains with or without the missense mutation was reflected in the therapeutic efficacy of FLC versus ITC in the animals infected with the strains. This study shows the importance of the Y145F alteration of Erg11 inC. neoformansfor manifestation of differential susceptibility toward different triazoles. It underscores the necessity ofin vitrosusceptibility testing for each FLC-resistantC. neoformansclinical isolate against different groups of azoles in order to assist patient management.

scholarly journals A Unique Chromosomal Rearrangement in the Cryptococcus neoformans var. grubii Type Strain Enhances Key Phenotypes Associated with Virulence

mBio ◽  
2012 ◽  
Vol 3 (2) ◽  
Author(s):  
Carl A. Morrow ◽  
I. Russel Lee ◽  
Eve W. L. Chow ◽  
Kate L. Ormerod ◽  
Anita Goldinger ◽  
...  
Keyword(s):  
Body Temperature ◽  
Cryptococcus Neoformans ◽  
Human Body ◽  
Fungal Pathogen ◽  
Type Strain ◽  
Clinical Sample ◽  
Disease Patient ◽  
Sub Saharan Africa ◽  
Content Type ◽  
Human Body Temperature

ABSTRACTThe accumulation of genomic structural variation between closely related populations over time can lead to reproductive isolation and speciation. The fungal pathogenCryptococcusis thought to have recently diversified, forming a species complex containing members with distinct morphologies, distributions, and pathologies of infection. We have investigated structural changes in genomic architecture such as inversions and translocations that distinguish the most pathogenic variety,Cryptococcus neoformansvar.grubii, from the less clinically prevalentCryptococcus neoformansvar.neoformansandCryptococcus gattii. Synteny analysis between the genomes of the threeCryptococcusspecies/varieties (strains H99, JEC21, and R265) reveals thatC. neoformansvar.grubiipossesses surprisingly few unique genomic rearrangements. All but one are relatively small and are shared by all molecular subtypes ofC. neoformansvar.grubii. In contrast, the large translocation peculiar to theC. neoformansvar.grubiitype strain is found in all tested subcultures from multiple laboratories, suggesting that it has possessed this rearrangement since its isolation from a human clinical sample. Furthermore, we find that the translocation directly disrupts two genes. The first of these encodes a novel protein involved in metabolism of glucose at human body temperature and affects intracellular levels of trehalose. The second encodes a homeodomain-containing transcription factor that modulates melanin production. Both mutations would be predicted to increase pathogenicity; however, when recreated in an alternate genetic background, these mutations do not affect virulence in animal models. The type strain ofC. neoformansvar.grubiiin which the majority of molecular studies have been performed is therefore atypical for carbon metabolism and key virulence attributes.IMPORTANCEThe fungal pathogenCryptococcusis a major cause of mortality among the immunocompromised population, primarily in AIDS patients of sub-Saharan Africa. Most research into the particular variety ofCryptococcusresponsible for the vast majority of infections,Cryptococcus neoformansvar.grubii, is performed using the type strain isolated in 1978 from a Hodgkin’s disease patient from North Carolina. We have determined that this particular isolate contains a chromosomal translocation that directly interrupts two genes, which all descendants of this strain from various research laboratories appear to possess. Disruption of these two genes affects multiple virulence factors ofCryptococcus, particularly the ability to grow at human body temperature, which could have wide-ranging implications for molecular genetic studies and virulence assays using this important strain.

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