Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans
Introduction
Ergosterol is a major sterol component found mainly in fungal membrane (Kristan and Rižner, 2012). Recent investigations have focused not only on the function of ergosterol as a structurally rigid component of the plasma membrane, but also as a key regulator of complex aspects of cell physiology (Koselny et al., 2018, Rodrigues, 2018). Besides acting as an important component of cellular membranes, the sterols regulate diverse biologic processes by affecting signal transduction, cytoskeleton organization, cell growth, vesicle transport and virulence (Dufourc, 2008, Hannich et al., 2011, Kristan and Rižner, 2012, Parks and Casey, 1995). Recently, ergosterol was defined as an immunologically active molecule that influences innate immune activation and macrophage pyroptosis (Koselny et al., 2018).
The absence of ergosterol enhances the membrane fluidity, which is responsible for increasing diffusion of various drugs. Polyenes and azoles are two antifungal classes that impair ergosterol function in the fungal cell membrane. The first drug group, exemplified by amphotericin B, directly targets ergosterol in the membrane, and causes cell injury by multiple means, including physically depleting membrane ergosterol, forming pores causing leakage of intracellular cations, and inducing the accumulation of reactive oxygen species (ROS) within the cell (Mesa-Arango et al., 2014). In contrast, azoles directly inhibit Erg11/CYP51, an intermediate enzyme of sterol biosynthesis. The ergosterol biosynthetic pathway enzymes have attracted many studies to identify new drug targets; however, a serious concern is that some of them are also shared with cholesterol biosynthesis pathways in mammalian cells (Daum et al., 1998, Espenshade and Hughes, 2007, Kristan and Rižner, 2012).
Ergosterol biosynthesis and its regulation have been studied in most detail in the yeast model Saccharomyces cerevisiae (Dupont et al., 2011, Gaber et al., 1989, Hu et al., 2017, Jacquier and Schneiter, 2012, Parks and Casey, 1995, Servouse and Karst, 1986). In contrast, less is known about this process in fungal pathogens such as C. neoformans (Chang et al., 2009, Kim et al., 1975, Nes et al., 2009, Toh-e et al., 2017). The terminal steps of sterol production in S. cerevisiae occur in the endoplasmic reticulum (ER). Afterwards, ergosterol molecules are transferred as steryl-esters to different subcellular compartments such as the plasma membrane and endomembrane system of peroxisomes, mitochondria, vacuoles and the ER where they are found as free sterols (Alvarez et al., 2007, Jacquier and Schneiter, 2012, Parks and Casey, 1995). The metabolic conversion between sterols and fatty acids and their transport through the cell is still not completely defined for many fungi. Sterols are closely associated with unsaturated fatty acids and distributed asymmetrically in plasma membrane microdomains known as lipid “rafts” and packed in a liquid-ordered state, which is responsible for maintaining the membrane micro fluid state (Dufourc, 2008, Wachtler and Balasubramanian, 2006). Previous studies revealed the presence of sterol-rich domains in the bud tips of C. neoformans (Nichols et al., 2004), suggesting that lipid “rafts” may regulate fungal morphology, and also may play a role in pathogenicity by exposing virulence factors on membrane (Farnoud et al., 2015).
Cryptococcosis is a disease that affects primarily immunocompromised hosts, mainly occurring in the setting of HIV/AIDS, chemotherapy and transplant patients. Globally, it is estimated that 181,000 patients die of cryptococcal meningitis each year, with most cases occurring in resource-limited regions of the world with concomitant high incidences of HIV infection (Rajasingham et al., 2017). Cryptococcus neoformans is one of the related species that causes cryptococcosis, a disease initiated by the inhalation of an infectious propagule, and most often resulting in an asymptomatic pulmonary infection. However, in the setting of immunosuppression, this opportunistic fungal pathogen can disseminate to the central nervous system leading to lethal meningoencephalitis (reviewed in (Maziarz and Perfect, 2016)).
The Erg6 enzyme catalyzes steps of the ergosterol synthesis pathway that are unique for sterol biosynthesis in fungi, plants and some protozoa (Weete et al., 2010). C. neoformans ERG6 encodes a 24-C-methyltransferase that converts zymosterol into fecosterol, the first step in the ergosterol pathway that diverges from cholesterol biosynthesis (Nes et al., 2009). Erg6 also acts in the alternative pathway of ergosterol biosynthesis to convert lanosterol to eburicol (Nes et al., 2009). Mutants with loss-of-function in ERG6 have been studied in other fungi, mainly ascomycetes, such as S. cerevisiae (Gaber et al., 1989), Candida albicans (Jensen-Pergakes et al., 1998), Candida lusitaniae (Young et al., 2003), Candida glabrata (Vandeputte et al., 2007) and Kluyveromyces lactis (Konecna et al., 2016). Although ERG6 is not an essential gene for those microorganisms, its absence resulted in a variety of compromised phenotypes related to membrane permeability and fluidity, alterations in the susceptibility to different antifungal compounds, impairment in cell wall integrity, and accumulation of sterol intermediate compounds (Gaber et al., 1989, Jensen-Pergakes et al., 1998, Konecna et al., 2016, Vandeputte et al., 2007, Young et al., 2003).
Although the role of Erg6 on membrane properties and its impact on fungal physiology has been widely characterized in ascomycetes, its role in basidiomycetes and diverse fungal pathogens is less well explored. Recently, Toh-e et al. constructed a selection of C. neoformans erg mutants to assess the effect of ergosterol defects on the cellular response to treatment with antifungal drugs targeting cell wall processes (Toh-e et al., 2017). This study builds upon prior work to more fully examine the involvement of C. neoformans Erg6 in ergosterol homeostasis, membrane composition and pathogenesis. Finally, based on the fact the membranes of fungi and humans are fundamentally different in the sterol content, and that ergosterol is such an important structural and active molecule related to several fungal biological process, we propose that fungal-specific components of sterol synthesis, such as Erg6, may offer unique insight into mechanisms of adaptive environmental responses and potential targets for antimicrobial development.
Section snippets
1. Media, cell line and strains
The H99 strain of C. neoformans var. grubii serotype A was used as the wild type strain and also to generate the erg6Δ mutant and the reconstituted strains erg6Δ + ERG6 (corresponding to CNAG_03819) (Table S1). The strains were kept at −80 °C and freshly streaked for single colonies every week on YPD agar plates (1% yeast extract, 2% bacto peptone, and 2% dextrose [pH 5.6]). The cells were incubated at 30 °C for 48 h unless noted otherwise. The murine macrophage line J774A.1 was maintained at
In silico analysis of ergosterol biosynthetic pathway in C. neoformans
Ergosterol synthesis is a result of a cascade with over 20 enzymatic reactions, which starts with the reaction of two acetyl-CoA molecules catalyzed by the enzyme acetoacetyl-CoA thiolase, encoded by ERG10. The difference between the cholesterol and ergosterol pathways starts with the conversion of zymosterol to fecosterol by Erg6 (Daum et al., 1998). After fecosterol formation, it is converted into episterol, which generates ergosta-5,7,24(28)-trienol, ergosta-5,7,24(28)-trienol, which is
Discussion
Plasma membranes are formed by a combination of different lipids, sterol and proteins, that can combine in domains establishing its physical and chemical properties (Ermakova and Zuev, 2017). The presence of ergosterol in the membrane influences the structural organization that maintains its dynamic function as the cellular homeostasis (Alvarez et al., 2007).
Mutants defective in enzymes related to sterol biosynthesis have been used to study the ergosterol pathway and understanding the function
Credit authorship contribution statement
Fabiana Freire M. Oliveira: Formal analysis, Investigation, Writing - original draft. Hugo Costa Paes: Methodology, Investigation, Writing - review & editing. Luísa Defranco F. Peconick: Investigation. Fernanda L. Fonseca: Formal analysis, Investigation, Writing - review & editing. Clara Luna Freitas Marina: Formal analysis, Investigation, Writing - review & editing. Anamélia Lorenzetti Bocca: Methodology, Resources, Funding acquisition. Mauricio Homem-de-Mello: Methodology, Formal analysis,
Acknowledgments
We thank Dr. Susana Frases for help with DLS and zeta potential measurements. This work was sponsored by the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq) Grant (GENOPROT 559572/2009-3) and Fundação de Apoio à Pesquida do Distrito Federal, Brazil (FAP-DF) (PRONEX 193.000.569/2009 and 193.001.533/2016) to MSSF and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES). JAA is supported by a National Institutes of Health, United States (NIH)
References (101)
- et al.
Ergosterol biosynthesis pathway in Aspergillus fumigatus
Steroids
(2008) - et al.
Extracellular vesicles of human pathogenic fungi
Curr. Opin. Microbiol.
(2019) - et al.
Decrease of H2O2 plasma membrane permeability during adaptation to H2O2 in Saccharomyces cerevisiae
J. Biol. Chem.
(2004) - et al.
Monoclonal antibody based ELISAs for cryptococcal polysaccharide
J. Immunol. Methods
(1992) - et al.
Nature of sterols affects plasma membrane behavior and yeast survival during dehydration
Biochim. Biophys. Acta – Biomembr.
(2011) - et al.
Effect of ergosterol on the fungal membrane properties. All-atom and coarse-grained molecular dynamics study
Chem. Phys. Lipids
(2017) - et al.
A simple method for the isolation and purification of total lipides from animal tissues
J. Biol. Chem.
(1957) - et al.
H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae
Biochim. Biophys. Acta – Biomembr.
(2008) - et al.
Structural and functional properties of the Trichosporon asahii glucuronoxylomannan
Fungal Genet. Biol.
(2009) - et al.
Mechanisms of sterol uptake and transport in yeast
J. Steroid Biochem. Mol. Biol.
(2012)
Yeast mutant requiring only a sterol as growth supplement
Biochem. Biophys. Res. Commun.
Isolation of pleiotropic yeast mutants requiring ergosterol for growth
Biochem. Biophys. Res. Commun.
Resistance to amphotericin B associated with defective sterol Δ8→7 isomerase in a Cryptococcus neoformans strain from an AIDS patient
FEMS Microbiol. Lett.
An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers
Biochem. Biophys. Res. Commun.
Steroid-transforming enzymes in fungi
J. Steroid Biochem. Mol. Biol.
Cryptococcosis
Infect. Dis. Clin. N Am.
Yeast sterol C24-methyltransferase: Role of highly conserved tyrosine-81 in catalytic competence studied by site-directed mutagenesis and thermodynamic analysis
Arch. Biochem. Biophys.
Sterol 24-C-methyltransferase: An enzymatic target for the disruption of ergosterol biosynthesis and homeostasis in Cryptococcus neoformans
Arch. Biochem. Biophys.
Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis
Lancet Infect. Dis.
Cell wall-linked cryptococcal phospholipase B1 is a source of secreted enzyme and a determinant of cell wall integrity
J. Biol. Chem.
Synthesis, in vitro antifungal activity and mechanism of action of four sterol hydrazone analogues against the dimorphic fungus Paracoccidioides brasiliensis
Steroids
Yeast lipid rafts? An emerging view
Trends Cell Biol.
Fungal extracellular vesicles: modulating host–pathogen interactions by both the fungus and the host
Microbes Infect.
Role of Candida albicans transcription factor Upc2p in drug resistance and sterol metabolism
Eukaryot. Cell
Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes
Cell. Microbiol.
Sterol-rich plasma membrane domains in fungi
Eukaryot. Cell
Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages
Sci. Rep.
Transport of newly synthesized sterol to the sterol-enriched plasma membrane occurs via nonvesicular equilibration
Biochemistry
Extracellular vesicles from the dermatophyte trichophyton interdigitalemodulate macrophage and keratinocyte functions
Front. Immunol.
Δ 24 -sterol methyltransferase plays an important role in the growth and development of Sporothrix schenckii and Sporothrix brasiliensis
Front. Microbiol.
Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi
Nat. Rev. Microbiol.
Conservation of the sterol regulatory element-binding protein pathway and its pathobiological importance in cryptococcus neoformans
Eukaryot. Cell
Posaconazole exhibits in vitro and in vivo synergistic antifungal activity with caspofungin or FK506 against Candida albicans
PLoS ONE
Cell biology: Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi
Science (80-.)
Stress, drugs, and evolution: The role of cellular signaling in fungal drug resistance
Eukaryot. Cell
Superoxide dismutase influences the virulence of Cryptococcus neoformans by affecting growth within macrophages
Infect. Immun.
Calcineurin is essential for survival during membrane stress in Candida albicans
EMBO J.
Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae
Yeast
Extracellular vesicles in fungi: composition and functions
Curr. Top. Microbiol. Immunol.
Role of phospholipases in fungal fitness, pathogenicity, and drug development – lessons from Cryptococcus neoformans
Front. Microbiol.
Sterols and membrane dynamics
J. Chem. Biol.
Regulation of sterol synthesis in Eukaryotes
Annu. Rev. Genet.
Raft-like Membrane Domains in Pathogenic Microorganisms, Current Topics in Membranes
Gain-of-function mutations in UPC2 are a frequent cause of ERG11 upregulation in azole-resistant clinical isolates of Candida albicans
Eukaryot. Cell
Role for chitin and chitooligomers in the capsular architecture of cryptococcus neoformans∇
Eukaryot. Cell
Capsule of Cryptococcus neoformans grows by enlargement of polysaccharide molecules
Proc. Natl. Acad. Sci. U.S.A.
The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol
Mol. Cell. Biol.
Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis
Proc. Natl. Acad. Sci. U.S.A.
Nanovesicles from malassezia sympodialis and host exosomes induce cytokine responses – novel mechanisms for host-microbe interactions in atopic eczema
PLoS One
Cited by (21)
Resistance mechanisms of Saccharomyces cerevisiae against silver nanoparticles with different sizes and coatings
2024, Food and Chemical ToxicologyMedia optimization of antimicrobial activity production and beta-glucan content of endophytic fungi Xylaria sp. BCC 1067
2022, Biotechnology ReportsCitation Excerpt :Recently, the ∆erg6 strain has been found to increase susceptibility to cytochalasin treatment and display defective actin depolymerization and aberrant accumulation of sterol intermediates [19]. However, the ∆erg6 strain shows resistance to commercial drugs including Amphotericin B (AmB) and nystatin, and higher susceptibility to azoles, lovastatin, and fenpropimorph [63, 66]. Guan et al. [67] also indicated that the ∆pdr5 strain is also resistant to polyene drugs such as Amphotericin B.
Metabolic engineering of Saccharomyces cerevisiae for gram-scale diosgenin production
2022, Metabolic EngineeringCitation Excerpt :However, ERG6 deletion resulted in ergosterol depletion from the membrane, damaging its fluidity. The most serious problem caused by the knockout of ERG6 in yeast is that the efficiency of genetic transformation diminishes sharply (Gaber et al., 1989), while the null mutant cells also became more sensitive to osmotic and oxidative stress (Oliveira et al., 2020). All these disadvantages are not conducive to the development of a cell factory for the gram-scale production of DSG based on high-cell-density fermentation.
The steroidal alkaloids α-tomatine and tomatidine: Panorama of their mode of action and pharmacological properties
2021, SteroidsCitation Excerpt :Moreover, the authors identified two nonsynonymous mutations in the erg6 gene (D249G and G132D) responsible for TD resistance [25], thus reinforcing the assumption that TD effectively inhibits ergosterol biosynthesis, and selectively targets the C-24 sterol methyltransferase Erg6, in agreement with a previous report [24]. The Erg6 inhibitory capacity of TD could be exploited further because Erg6 plays an active role in other fungi, such as in the virulence of the AIDS-associated fungal pathogen Cryptococcus neoformans [26]. The enzyme is also found in parasites, such as (i) the amoeba Naegleria fowleri, which can cause severe brain infection (primary amebic meningoencephalitis) in humans [27], (ii) the soilborne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), an important lethal disease of banana [28], and (iii) the protozoan tropical parasite Trypanosoma cruzi responsible for the Chagas disease.
- 1
L.F and M.S.S.F. share senior authorship of this article.