Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans

https://doi.org/10.1016/j.fgb.2020.103368Get rights and content

Highlights

  • Erg6 is required for Cryptococcus neoformans high temperature growth.

  • ERG6 deletion impairs susceptibility to stress and antifungal agents.

  • Sterol membrane composition affects virulence of human fungal pathogen.

Abstract

Ergosterol is the most important membrane sterol in fungal cells and a component not found in the membranes of human cells. We identified the ERG6 gene in the AIDS-associated fungal pathogen, Cryptococcus neoformans, encoding the sterol C-24 methyltransferase of fungal ergosterol biosynthesis. In this work, we have explored its relationship with high-temperature growth and virulence of C. neoformans by the construction of a loss-of-function mutant. In contrast to other genes involved in ergosterol biosynthesis, C. neoformans ERG6 is not essential for growth under permissive conditions in vitro. However, the erg6 mutant displayed impaired thermotolerance and increased susceptibility to osmotic and oxidative stress, as well as to different antifungal drugs. Total lipid analysis demonstrated a decrease in the erg6Δ strain membrane ergosterol content. In addition, this mutant strain was avirulent in an invertebrate model of C. neoformans infection. C. neoformans Erg6 was cyto-localized in the endoplasmic reticulum and Golgi complex. Our results demonstrate that Erg6 is crucial for growth at high temperature and virulence, likely due to its effects on C. neoformans membrane integrity and dynamics. These pathogen-focused investigations into ergosterol biosynthetic pathway components reinforce the multiple roles of ergosterol in the response of diverse fungal species to alterations in the environment, especially that of the infected host. These studies open perspectives to understand the participation of ergosterol in mechanism of resistance to azole and polyene drugs. Observed synergistic growth defects with co-inhibition of Erg6 and other components of the ergosterol biosynthesis pathway suggests novel approaches to treatment in human fungal infections.

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)

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