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Cerezyme treatment effect on the amount of GlcCer in cultures of Cn. Mass spectroscopy was used to analyze amount of GlcCer of wild-type H99 or Δgcs1 mutant strain treated with different concentrations of Cerezyme for 1 hour at 37°C normalized to phosphate (Pi). Enzyme treatment hydrolyzed GlcCer in a dose-dependent manner. *, P<0.05 by Student t test, WT 100 or 200 versus untreated (−).
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Cryptococcus neoformans (Cn) is a significant human pathogen that, despite current treatments, continues to have a high morbidity rate especially in sub-Saharan Africa. The need for more tolerable and specific therapies has been clearly shown. In the search for novel drug targets, the gene for glucosylceramide synthase (GCS1) was deleted in Cn, res...
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Citations
... Both D4-Cer and D4,D8-Cer are known to be converted to D4-GlcCer (GlcCer(d18:1/ 18:0 h)) and D4,D8-GlcCer (GlcCer(d18:2/18:0 h)) by the Gcs1 in Golgi [19]. Upon production, these GlcCer structures are exported by the vesicular trafficking pathways to the plasma membrane, and cell wall or are secreted to extracellular space in vesicles [8,70,71]. ...
Sphingolipids (SLs) are essential to fungal survival and represent a major class of structural and signaling lipids. Unique SL structures and their biosynthetic enzymes in filamentous fungi make them an ideal drug target. Several studies have contributed towards the functional characterization of specific SL metabolism genes, which have been complemented by advanced lipidomics methods which allow accurate identification and quantification of lipid structures and pathway mapping. These studies have provided a better understanding of SL biosynthesis, degradation and regulation networks in filamentous fungi, which are discussed and elaborated here.
... GlcCer, a cell surface molecule in C. neoformans, is essential for fungal growth in the extracellular environment of the host (Fan et al., 2005). The deletion of Glucosylceramide synthase1 (GCS1) in C. neoformans results in the loss of pathogenicity in mouse models (Rhome et al., 2011). A C. neoformans mutant strain lacking GlcCer cannot grow in vitro at neutral or alkaline pH but grows normally at an acidic pH. ...
Sphingolipids are a class of membrane lipids that serve as vital structural and signaling bioactive molecules in organisms ranging from yeast to animals. Recent studies have emphasized the importance of sphingolipids as signaling molecules in the development and pathogenicity of microbial pathogens including bacteria, fungi, and viruses. In particular, sphingolipids play key roles in regulating the delicate balance between microbes and hosts during microbial pathogenesis. Some pathogens, such as bacteria and viruses, harness host sphingolipids to promote development and infection, whereas sphingolipids from both the host and pathogen are involved in fungus–host interactions. Moreover, a regulatory role for sphingolipids has been described, but their effects on host physiology and metabolism remain to be elucidated. Here, we summarize the current state of knowledge about the roles of sphingolipids in pathogenesis and interactions with host factors, including how sphingolipids modify pathogen and host metabolism with a focus on pathogenesis regulators and relevant metabolic enzymes. In addition, we discuss emerging perspectives on targeting sphingolipids that function in host–microbe interactions as new therapeutic strategies for infectious diseases.
... Cerezyme, an enzyme that metabolizes GlcCer, promoted deficiencies in C. neoformans that were similar to those observed in the Δgcs1 strain, including membrane integrity defects, and reduced its ability to grow in physiological CO 2 atmosphere. Treatment with Cerezyme prolonged the survival of intranasally infected mice [43]. More recently, a new class of a synthetic drug able to reduce synthesis of fungal GlcCer was reported by Mor and colleagues [40]. ...
... The anti-GlcCer reactivity was concentrated at the C. neoformans cell wall and distinctively enriched at budding sites. Interestingly, localization of GlcCer at the cell surface was considerably higher under neutral pH and high CO 2 , conditions regularly found by yeasts of C. neoformans during infection [43]. Furthermore, incubation of C. neoformans with anti-GlcCer resulted in inhibition of cell budding and fungal growth in vitro [30]. ...
Purpose of Review
Cryptococcus spp. are responsible for life-threatening infections in humans causing mortality rates of 70% in developing countries. Antifungal therapy to combat cryptococcosis is based on the combination of amphotericin B, azoles, and 5-flucytosine. However, treatment failure is frequently triggered by antifungal resistance, drug-drug interactions, and toxicity. New alternatives to prevent cryptococcosis are imperative. Here, we discuss the roles of lipids in the immunological control of the disease caused by Cryptococcus spp.
Recent Findings
Recently, remarkable advances on immunology of fungal infections have been made and a number of studies indicated the potential of vaccine formulations to combat cryptococcosis. New formulations exploiting virulence regulators and genetically modified attenuated strains have been tested. In this context, lipids have emerged as virulence regulators and immunogens to be explored.
Summary
Glucosylceramide (GlcCer), sterylglycosides (SGs), and lipid-containing extracellular vesicles have been recently tested in vaccine formulations and their anticryptococcal efficacy was confirmed in vivo. Together, the data discussed here encourage the use of fungal lipids in anticryptococcal vaccinal strategies.
... Monoclonal antibodies specific to C. neoformans GlcCer provide protection against C. neoformans infection, likely due to downregulation of the inflammatory response (52). Moreover, treatment with Cerezyme, a recombinant enzyme that metabolizes GlcCer, hydrolyzes fungal GlcCer in growing C. neoformans cultures, disrupts membrane integrity, and exhibits in vivo efficacy in an inhalation murine model of cryptococcosis (53). Notably, sphingolipid biosynthesis is also important for the host. ...
Antifungal Drugs: The Current Armamentarium and Development of New Agents, Page 1 of 2
Abstract
Disease and death caused by fungal infections have transitioned from a rare curiosity to a major global health problem. Because the vast majority of fungi capable of causing life-threatening infections target individuals with impaired immunity, recent decades have witnessed a stark rise in fungal infections due to medical interventions such as chemotherapy for cancer and immunosuppression for organ transplantation and due to the prevalence of HIV infection ( 1 ). Pathogenic fungi are the causative agents of billions of infections annually, with ∼1.5 million attributable mortalities ( 2 ). The public health burden is comparable to that observed with more notable infectious diseases such as tuberculosis and malaria, yet the impact of fungal infections on human health has been largely overlooked ( 3 ).
... Interestingly, deletion of gcs1 in C. neoformans results in a dramatic growth defect at neutral/alkaline pH, 5% CO 2 and 37°C [15], suggesting that GlcCer may be important in fungi to promote alkaline tolerance [60]. When exposed to a neutral/alkaline environment, C. neoformans cells do concentrate GlcCer at the plasma membrane/cell wall/bud necks without upregulating its synthesis [61]. This phenomenon was observed not only during in vitro growth, but also in cells recovered ex vivo from murine infection model [61]. ...
... When exposed to a neutral/alkaline environment, C. neoformans cells do concentrate GlcCer at the plasma membrane/cell wall/bud necks without upregulating its synthesis [61]. This phenomenon was observed not only during in vitro growth, but also in cells recovered ex vivo from murine infection model [61]. This redistribution of GlcCer at fungal membranes under these physiological environments (neutral/alkaline pH) may be important for proper function of specific transmembrane transporters [62], which may be particularly important for pro moting fungal cell replication at neutral and alkaline pH. ...
... However, whether this effect is due directly to the inhibition of fungal GCS1 or to an off-target effect is not known. In C. neoformans, for instance, these inhibitors have no significant effect against C. neoformans GCS1 [61]. ...
Invasive fungal infections have significantly increased in the last few decades. Three classes of drugs are commonly used to treat these infections: polyenes, azoles and echinocandins. Unfortunately each of these drugs has drawbacks; polyenes are toxic, resistance against azoles is emerging and echinocandins have narrow spectrum of activity. Thus, the development of new antifungals is urgently needed. In this context, fungal sphingolipids have emerged as a potential target for new antifungals, because their biosynthesis in fungi is structurally different than in mammals. Besides, some fungal sphingolipids play an important role in the regulation of virulence in a variety of fungi. This review aims to highlight the diverse strategies that could be used to block the synthesis or/and function of fungal sphingolipids.
... Some of these compounds may be involved in the interaction with bacterial associates (Benoit et al. 2015). In particular glycosphingolipids such as ceramide monohexosides (CMHs) may play unexpected roles in fungal cell envelopes, as recently proposed in several articles (Nimrichter and Rodrigues 2011;Rhome et al. 2011;Guimarães et al. 2014). These compounds usually contain glucose or galactose end-groups, with β-glycosidic linkages to the primary alcohol of an N-acyl sphingoid base (ceramide). ...
... These compounds usually contain glucose or galactose end-groups, with β-glycosidic linkages to the primary alcohol of an N-acyl sphingoid base (ceramide). They can be, to a certain extent, exposed on fungal surfaces (Barreto-Bergter, Sassaki and de Souza 2011; Rhome et al. 2011;Calixto et al. 2016), and have predicted roles in fungal immunogenicity and lipid raft architecture, besides the regulation of fungal growth, differentiation and virulence (Da Silva et al. 2004;Nimrichter and Rodrigues 2011;Zhu et al. 2014). ...
... As the attachment of bacterial cells to fungal surfaces depends on surface-expressed molecules, we here studied if CMHs might constitute 'anchors' for B. terrae BS001 at fungal surfaces. Indeed, CMH molecules can be localized at fungal cell envelopes as minor components, as shown by immunogold and other antibody assays (Rodrigues et al. 2000;Da Silva et al. 2004;Rhome et al. 2011). Rodrigues et al. (2000) provided initial evidence for the tenet that CMH is transported by vesicles from the plasma membrane towards the cell wall, being deposited locally in the latter. ...
Burkholderia terrae BS001 has previously been proposed to be a ‘generalist’ associate of soil fungi, of which we largely ignore its strategies of interaction. Here, we studied the chemotactic behavior of B. terrae BS001 towards Lyophyllum sp. strain Karsten and Trichoderma asperellum 302 and the role of fungal surface molecules in their physical interaction with the bacteria. To assess the involvement of the type 3 secretion system (T3SS), wild-type strain BS001 and T3SS mutant strain BS001-ΔsctD were used in the experiments. First, the two fungi showed divergent behavior when confronted with B. terrae BS001 on soil extract agar medium. Lyophyllum sp. strain Karsten revealed slow growth towards the bacterium, whereas T. asperellum 302 grew avidly over it. Both on soil extract and M9 agar, B. terrae BS001 and BS001-ΔsctD moved chemotactically towards the hyphae of both fungi, with a stronger response to Lyophyllum sp. strain Karsten than to T. asperellum 302. The presence of a progressively increasing glycerol level in the M9 agar enhanced the level of movement. Different oxalic acid concentrations exerted varied effects, with a significantly raised chemotactic response at lower, and a subdued response at higher concentrations. Testing of the adherence of B. terrae BS001 and BS001-ΔsctD to Lyophyllum sp. strain Karsten and to cell envelope extracted ceramide monohexosides (CMH) revealed that CMH in both conidia and hyphae could bind strain BS001 cells. As BS001-ΔsctD adhered significantly less to the CMH than BS001, the T3SS was presumed to have a role in the interaction. In contrast, such adherence was not detected with T. asperellum 302. Thus, B. terrae BS001 shows a behavior characterized by swimming towards Lyophyllum sp. strain Karsten and T. asperellum 302 and attachment to the CMH moiety in the cell envelope of the former fungus.
... Our laboratory recently reported the characterization of a Cryptococcus mutant, Δsgl1, which accumulated the glycolipid, sterylglycoside, and led to complete mice survival when applied as a vaccine prior to infection. In addition, antibodies against glucosylceramide (GlcCer), a glycosphingolipid primarily localized in the cell membrane of C. neoformans [13], have been shown to inhibit the growth and division of C. neoformans [14]. Another glycolipid, galactosylceramide (GalCer), has been shown to activate the natural killer cells and increase the immune response induced by malaria vaccines [15]. ...
... As positive control, and anti-GlcCer IgM antibody was used (F09) (data not shown). This IgM produces an OD of 0.5 at 450 nm when 50 microliters of 1:64 dilution of 1 mg/ml is used in the ELISA [13]. As negative controls, the secondary IgM (or IgG) antibodies were used alone. ...
... Administration of GlcCer resulted in anti-GlcCer antibody production. Mechanistic studies have revealed that GlcCer is involved in the regulation of fungal cell replication in environments characterized by neutral/alkaline environment [13,17,29]. C. neoformans mutants that lack GlcCer cannot progress through the cell cycle in neutral/alkaline pH and lose their ability to establish virulence in the mouse model [17,30,31]. ...
Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.
... In Candida albicans, Rim101 regulates the pH responsive pathway [43] involved in their transition from acidic to alkaline, which in turn stimulates a switch from yeast to filamentous form. Documented evidence of increase in GlcCer on the cell surface at alkaline pH in vitro [44] and also during infection, together with previous reports of GlcCer being associated with the ability to grow in alkaline pH, indicates therefore that this sphingolipid may play an important role in cryptococcal pH responsive pathway. In addition to PKA pathway being involved in tolerating increased pH in Cryptococcus, a rim101 mutant is hyper susceptible to not only elevated pH but also to iron deprivation [45]. ...
Background:
The sphingolipid glucosylceramide (GlcCer) and factors involved in the fungal GlcCer pathways were shown earlier to be an integral part of fungal virulence, especially in fungal replication at 37 °C, in neutral/alkaline pH and 5 % CO2 environments (e.g. alveolar spaces). Two mutants, ∆gcs 1 lacking glucosylceramide synthase 1 gene (GCS1) which catalyzes the formation of sphingolipid GlcCer from the C9-methyl ceramide and ∆smt1 lacking sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position nine of the sphingosine backbone of ceramide, of this pathway were attenuated in virulence and have a growth defect at the above-mentioned conditions. These mutants with either no or structurally modified GlcCer located on the cell-membrane have reduced membrane rigidity, which may have altered not only the physical location of membrane proteins but also their expression, as the pathogen's mode of adaptation to changing need. Importantly, pathogens are known to adapt themselves to the changing host environments by altering their patterns of gene expression.
Results:
By transcriptional analysis of gene expression, we identified six genes whose expression was changed from their wild-type counterpart grown in the same conditions, i.e. they became either down regulated or up regulated in these two mutants. The microarray data was validated by real-time PCR, which confirmed their fold change in gene expression. All the six genes we identified, viz siderochrome-iron transporter (CNAG_02083), monosaccharide transporter (CNAG_05340), glucose transporter (CNAG_03772), membrane protein (CNAG_03912), membrane transport protein (CNAG_00539), and sugar transporter (CNAG_06963), are membrane-localized and have significantly altered gene expression levels. Therefore, we hypothesize that these genes function either independently or in tandem with a structurally modified cell wall/plasma membrane resulting from the modifications of the GlcCer pathway and thus possibly disrupt transmembrane signaling complex, which in turn contributes to cryptococcal osmotic, pH, ion homeostasis and its pathobiology.
Conclusion:
Six genes identified from gene expression microarrays by gene set enrichment analysis and validated by RT-PCR, are membrane located and associated with the growth defect at neutral-alkaline pH due to the absence and or presence of a structurally modified GlcCer. They may be involved in the transmembrane signaling network in Cryptococcus neoformans, and therefore the pathobiology of the fungus in these conditions.
... GlcCer regulates fungal cell replication in environments of neutral or alkaline pH (5,11). When fungal cells lacking GlcCer are exposed to neutral/alkaline pH, they cannot progress through the cell cycle, and cytokinesis does not occur (5,9,12). ...
Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N'-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N'-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM.
Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.
Copyright © 2015 Mor et al.
... Confocal imaging was performed as described previously. 21 Briefly, cells were grown at pH 4.0 for the specified amount of time and fixed using 2.5 mL of 5x fixation reagent (46 mL of 0.5 M potassium phosphate and 54 mL of formaldehyde) per 10 mL of culture. After 2 hours of shaking, the cells were centrifuged at 1700 g for 5 minutes, resuspended in 1x fixation reagent and left in a shaker incubator at room temperature overnight. ...
Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans.
