The ERG6 gene is crucial for the biosynthesis of ergosterol, a key component of yeast cell membranes. Our study examines the impact of ERG6 gene deletion on the membrane composition and physicochemical properties of the pathogenic yeast Candida glabrata. Specifically, we investigated changes in selected sterol content, phospholipid composition, transmembrane potential, and PDR16 gene activity. Sterol levels were measured using high-performance liquid chromatography, the phospholipid profile was analysed via thin-layer chromatography, transmembrane potential was assessed with fluorescence spectroscopy, and gene expression levels were determined by quantitative PCR. Our findings revealed a depletion of ergosterol, increased zymosterol and eburicol content, an increased phosphatidylcholine and a reduced phosphatidylethanolamine content in the Δerg6 strain compared to the wt. Additionally, the Δerg6 strain exhibited membrane hyperpolarization without changes in PDR16 expression. Furthermore, the Δerg6 strain showed increased sensitivity to the antifungals myriocin and aureobasidine A. These results suggest that ERG6 gene deletion leads to significant alterations in membrane composition and may activates an alternative ergosterol synthesis pathway in the C. glabrata Δerg6 deletion mutant.

Centre for Biosciences SAS Institute of Biochemistry and Genetics of Animals SAS Bratislava Slovakia

Department of Microbiology and Virology Faculty of Natural Sciences Comenius University Bratislava Bratislava Slovakia

Department of Nuclear Physics and Biophysics Faculty of Mathematics Physics and Informatics Comenius University Bratislava Bratislava Slovakia

Institute of Physics Charles University Prague Czechia

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Prasad Rajendra, ed. Candida Albicans Cellular and Molecular Biology. 2nd ed. Springer International Publishing; 2017.

Hull, C. M., Parker, J. E., Bader, O., Weig, M., Gross, U., & Warrilow, A. G. S., et al. (2012). Facultative sterol uptake in an ergosterol-deficient clinical isolate of candida glabrata harboring a missense mutation in ERG11 and exhibiting cross-resistance to azoles and amphotericin B. Antimicrobial Agents and Chemotherapy, 56(8), 4223–4232. 10.1128/aac.06253-11. PubMed PMC

Stead, D. A., Walker, J., Holcombe, L., Gibbs, S. R. S., Yin, Z., & Selway, L., et al. (2010). Impact of the transcriptional regulator, Ace2, on the Candida glabrata secretome. Proteomics, 10(2), 212–223. 10.1002/pmic.200800706. PubMed

Herman, P., Vecer, J., Opekarova, M., Vesela, P., Jancikova, I., & Zahumensky, J., et al. (2015). Depolarization affects the lateral microdomain structure of yeast plasma membrane. The FEBS Journal, 282(3), 419–434. 10.1111/febs.13156. PubMed

Toth Hervay, N., Goffa, E., Svrbicka, A., Simova, Z., Griac, P., & Jancikova, I., et al. (2015). Deletion of the PDR16 gene influences the plasma membrane properties of the yeast Kluyveromyces lactis. Canadian Journal of Microbiology, 61(4), 273–279. 10.1139/cjm-2014-0627. PubMed

Healey, K. R., Ortigosa, C. J., Shor, E., & Perlin, D. S. (2016). Genetic drivers of multidrug resistance in Candida glabrata. Frontiers in Microbiology, 7(DEC), 1–9. 10.3389/fmicb.2016.01995. PubMed PMC

Alexander, B. D., Johnson, M. D., Pfeiffer, C. D., Jiménez-Ortigosa, C., Catania, J., & Booker, R., et al. (2013). Increasing echinocandin resistance in candida glabrata: Clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations. Clinical Infectious Diseases, 56(12), 1724–1732. 10.1093/cid/cit136. PubMed PMC

Dellière, S., Healey, K., Gits-Muselli, M., Carrara, B., Barbaro, A., & Guigue, N., et al. (2016). Fluconazole and echinocandin resistance of Candida glabrata correlates better with antifungal drug exposure rather than with MSH2 mutator genotype in a French cohort of patients harboring low rates of resistance. Frontiers in Microbiology, 7, 1–9. 10.3389/fmicb.2016.02038. PubMed PMC

Oliveira, F. F. M., Paes, H. C., Peconick, L. D. F., Fonseca, F. L., Marina, C. L. F., & Bocca, A. L., et al. (2020). Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans. Fungal Genetics and Biology, 140, 103368. 10.1016/j.fgb.2020.103368. PubMed PMC

Geber, A., Hitchcock, C. A., Swartz, J. E., Pullen, F. S., Marsden, K. E., & Kwon-Chung, K. J., et al. (1995). Deletion of the Candida glabrata ERG3 and ERG11 genes: Effect on cell viability, cell growth, sterol composition, and antifungal susceptibility. Antimicrobial Agents and Chemotherapy, 39(12), 2708–2717. 10.1128/AAC.39.12.2708. PubMed PMC

Munn, A. L., Heese-Peck, A., Stevenson, B. J., Pichler, H., & Riezman, H. (1999). Specific sterols required for the internalization step of endocytosis in yeast. Molecular Biology of the Cell, 10(11), 3943–3957. 10.1128/AAC.39.12.2708. PubMed PMC

Parks, L. W., Crowley, J. H., Leak, F. W., Smith, S. J., & Tomeo, M. E. (1999). Use of sterol mutants as probes for sterol functions in the yeast, Saccharomyces cerevisiae. Critical Reviews in Biochemistry and Molecular Biology, 34(6), 399–404. 10.1080/10409239991209381. PubMed

Heese-Peck, A., Pichler, H., Zanolari, B., Watanabe, R., Daum, G., & Riezman, H. (2002). Multiple functions of sterols in yeast endocytosis. Molecular Biology of the Cell, 13, 4100–4109. 10.1091/mbc.e02-04-0186. PubMed PMC

Mukhopadhyay, K., Kohli, A., & Prasad, R. (2002). Drug susceptibilities of yeast cells are affected by membrane lipid composition. Antimicrobial Agents and Chemotherapy, 46(12), 3695–3705. 10.1128/AAC.46.12.3695-3705.2002. PubMed PMC

Young, L. Y., Hull, C. M., & Heitman, J. (2003). Disruption of ergosterol biosynthesis confers resistance to amphotericin B in Candida lusitaniae. Antimicrobial Agents and Chemotherapy, 47(9), 2717–2724. 10.1128/AAC.47.9.2717-2724.2003. PubMed PMC

Sanglard, D., Ischer, F., Parkinson, T., Falconer, D., & Bille, J. (2003). Candida albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrobial Agents and Chemotherapy, 47(8), 2404–2412. 10.1128/AAC.47.8.2404-2412.2003. PubMed PMC

Konečná, A., Toth Hervay, N., Valachovič, M., & Gbelska, Y. (2016). ERG6 gene deletion modifies Kluyveromyces lactis susceptibility to various growth inhibitors. Yeast, 33, 621–632. 10.1002/yea.3212. PubMed

Konečná, A., Hervay, N. T., Benčová, A., Morvová, M., Šikurová, L., & Jancikova, I., et al. (2018). Erg6 gene is essential for stress adaptation in Kluyveromyces lactis. FEMS Microbiology Letters, 365(23), 1–8. 10.1093/femsle/fny265. PubMed

Jensen-Pergakes, K. L., Kennedy, M. A., Lees, N. D., Barbuch, R., Koegel, C., & Bard, M. (1998). Sequencing, disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) gene: Drug susceptibility studies in erg6 mutants. Antimicrobial Agents and Chemotherapy, 42(5), 1160–1167. 10.1128/aac.42.5.1160. PubMed PMC

Vandeputte, P., Tronchin, G., Larcher, G., Ernoult, E., Bergès, T., & Chabasse, D., et al. (2008). A nonsense mutation in the ERG6 gene leads to reduced susceptibility to polyenes in a clinical isolate of Candida glabrata. Antimicrobial Agents and Chemotherapy, 52(10), 3701–3709. 10.1128/AAC.00423-08. PubMed PMC

Pasrija, R., Panwar, S. L., & Prasad, R. (2008). Multidrug transporters CaCdr1p and CaMdr1p of Candida albicans display different lipid specificities: Both ergosterol and sphingolipids are essential for targeting of CaCdr1p to membrane rafts. Antimicrobial Agents and Chemotherapy, 52(2), 694–704. 10.1128/AAC.00861-07. PubMed PMC

Xie, J., Rybak, J. M., Martin-Vicente, A., Guruceaga, X., Thorn, H. I., & Nywening, A. V., et al. (2024). The sterol C-24 methyltransferase encoding gene, erg6, is essential for viability of Aspergillus species. Nature Communications, 15(1), 1–13. 10.1038/s41467-024-48767-3. PubMed PMC

Rollin-Pinheiro, R., Bayona-Pacheco, B., Domingos, L. T. S., Curvelo, J. AdaR., de Castro, G. M. M., & Barreto-Bergter, E., et al. (2021). Sphingolipid inhibitors as an alternative to treat candidiasis caused by fluconazole-resistant strains. Pathogens, 10(7), 856. 10.3390/pathogens10070856. PubMed PMC

Hu, C., Zhou, M., Wang, W., Sun, X., Yarden, O., & Li, S. (2018). Abnormal ergosterol biosynthesis activates transcriptional responses to antifungal azoles. Frontiers in Microbiology, 9, 1–14. 10.3389/fmicb.2018.00009. PubMed PMC

Cassilly, C. D., & Reynolds, T. B. (2018). PS, it’s complicated: The roles of phosphatidylserine and phosphatidylethanolamine in the pathogenesis of Candida albicans and other microbial pathogens. Journal of Fungi, 4(1), 28. 10.3390/jof4010028. PubMed PMC

Carman, G. M., & Han, G. S. (2011). Regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae. Annual Review of Biochemistry, 80, 859–883. 10.1146/annurev-biochem-060409-092229. PubMed PMC

Flis, V. V., Fankl, A., Ramprecht, C., Zellnig, G., Leitner, E., & Hermetter, A., et al. (2015). Phosphatidylcholine supply to peroxisomes of the yeast Saccharomyces cerevisiae. PLoS ONE, 10(8), 140080. 10.1371/journal.pone.0135084. PubMed PMC

Henderson, C. M., & Block, D. E. (2014). Examining the role of membrane lipid composition in determining the ethanol tolerance of Saccharomyces cerevisiae. Applied and Environmental Microbiology, 80(10), 2966–2972. 10.1128/AEM.04151-13. PubMed PMC

Schneiter, R., Brügger, B., Sandhoff, R., Zellnig, G., Leber, A., & Lampl, M., et al. (1999). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane. Journal of Cell Biology, 146(4), 741–754. 10.1083/jcb.146.4.741. PubMed PMC

Young, S. A., Mina, J. G., Denny, P. W., & Smith, T. K. (2012). Sphingolipid and ceramide homeostasis: Potential therapeutic targets. Biochemistry Research International, 2012(1), 248135. 10.1155/2012/248135. PubMed PMC

Simons, K., & Ikonen, E. (1997). Functional rafts in cell membranes. Nature, 387(6633), 569–572. 10.1038/42408. PubMed

Bankaitis, V. A., Mousley, C. J., & Schaaf, G. (2010). The Sec14-superfamily and mechanisms for crosstalk between lipid metabolism and lipid signaling. Trends in Biochemical Sciences, 35(3), 150–160. 10.1016/j.tibs.2009.10.008. PubMed PMC

Saidane, S., Weber, S., De Deken, X., St-Germain, G., & Raymond, M. (2006). PDR16-mediated azole resistance in Candida albicans. Molecular Microbiology, 60(6), 1546–1562. 10.1111/j.1365-2958.2006.05196.x. PubMed

Znaidi, S., De Deken, X., Weber, S., Rigby, T., Nantel, A., & Raymond, M. (2007). The zinc cluster transcription factor Tac1p regulates PDR16 expression in Candida albicans. Molecular Microbiology, 66(2), 440–452. 10.1111/j.1365-2958.2007.05931.x. PubMed

Culaková, H., Dzugasová, V., Perzelová, J., Gbelská, Y., & Subík, J. (2013). Mutation of the CgPDR16 gene attenuates azole tolerance and biofilm production in pathogenic Candida glabrata. Yeast, 30, 403–414. 10.1002/yea.2978. PubMed

Van Den Hazel, H. B., Pichler, H., Do Valle Matta, M. A., Leitner, E., Goffeau, A., & Daum, G. (1999). PDR16 and PDR17, two homologous genes of Saccharomyces cerevisiae, affect lipid biosynthesis and resistance to multiple drugs. Journal of Biological Chemistry, 274(4), 1934–1941. 10.1074/jbc.274.4.1934. PubMed

Šimová, Z., Poloncová, K., Tahotná, D., Holič, R., Hapala, I., & Smith, A. R., et al. (2013). The yeast Saccharomyces cerevisiae Pdr16p restricts changes in ergosterol biosynthesis caused by the presence. Yeast, 30, 229–241. 10.1002/yea.2956. PubMed

Goffa, E., Balazfyova, Z., Toth Hervay, N., Simova, Z., Balazova, M., & Griac, P., et al. (2014). Isolation and functional analysis of the KlPDR16 gene. FEMS Yeast Research, 14(2), 337–345. 10.1111/1567-1364.12102. PubMed

Zahumenský, J., Jančíková, I., Drietomská, A., Švenkrtová, A., Hlaváček, O., & Hendrych, T., et al. (2017). Yeast Tok1p channel is a major contributor to membrane potential maintenance under chemical stress. Biochimica et Biophysica Acta - Biomembranes, 1859(10), 1974–1985. 10.1016/j.bbamem.2017.06.019. PubMed

Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 25(4), 402–408. 10.1006/meth.2001.1262. PubMed

Dupont, S., Fleurat-Lessard, P., Cruz, R. G., Lafarge, C., Grangeteau, C., & Yahou, F., et al. (2021). Antioxidant properties of ergosterol and its role in yeast resistance to oxidation. Antioxidants, 10(7), 1024. https://www.mdpi.com/2076-3921/10/7/1024, 10.3390/antiox10071024. PubMed PMC

Gaber, R. F., Copple, D. M., Kennedy, B. K., Vidal, M., & Bard, M. (1989). The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol. Molecular and Cellular Biology, 9(8), 3447–3456. 10.1128/mcb.9.8.3447-3456.1989. PubMed PMC

Johnston, E. J., Moses, T., & Rosser, S. J. (2020). The wide-ranging phenotypes of ergosterol biosynthesis mutants, and implications for microbial cell factories. Yeast, 37(1), 27–44. 10.1002/yea.3452. PubMed

Jordá, T., & Puig, S. (2020). Regulation of ergosterol biosynthesis in saccharomyces cerevisiae. Genes, 11(7), 1–18. 10.3390/genes11070795. PubMed PMC

Abe, F., & Hiraki, T. (2009). Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae. Biochimica et Biophysica Acta - Biomembranes, 1788(3), 743–752. 10.1016/j.bbamem.2008.12.002. PubMed

Guan, X. L., Souza, C. M., Pichler, H., Dewhurst, G., Schaad, O., & Kajiwara, K., et al. (2009). Functional interactions between sphingolipids and sterols in biological membranes regulating cell physiology. Molecular Biology of the Cell, 20, 2083–2095. 10.1091/mbc.e08-11-1126. PubMed PMC

Elias, D., Toth Hervay, N., Jacko, J., Morvova, M., Valachovic, M., & Gbelska, Y. (2022). Erg6p is essential for antifungal drug resistance, plasma membrane properties and cell wall integrity in Candida glabrata. FEMS Yeast Research, 21(1), 1–9. 10.1093/femsyr/foac045. PubMed

Li, Q. Q., Tsai, H. F., Mandal, A., Walker, B. A., Noble, J. A., & Fukuda, Y., et al. (2018). Sterol uptake and sterol biosynthesis act coordinately to mediate antifungal resistance in Candida glabrata under azole and hypoxic stress. Molecular Medicine Reports, 17(5), 6585–6597. 10.3892/mmr.2018.8716. PubMed PMC

Weete, J. D., Abril, M., & Blackwell, M. (2010). Phylogenetic distribution of fungal sterols. PLoS ONE, 5(5), 3–8. 10.1371/journal.pone.0010899. PubMed PMC

Derkacz, D., & Krasowska, A. (2023). Alterations in the level of ergosterol in candida albicans’ plasma membrane correspond with changes in virulence and result in triggering diversed inflammatory response. International Journal of Molecular Sciences, 24(4), 3966. 10.3390/ijms24043966. PubMed PMC

Ahmad, S., Joseph, L., Parker, J. E., Asadzadeh, M., & Kelly, S. L. (2019). ERG6 and ERG2 are major targets conferring reduced susceptibility to amphotericin B in clinical Candida glabrata isolates in Kuwait. Antimicrobial Agents and Chemotherapy, 63(2), 1–12. 10.1128/AAC.01900-18. PubMed PMC

Jin X., Luan X., Xie F., Chang W., Lou H. (2023). Erg6 acts as a downstream effector of the transcription factor Flo8 to regulate biofilm formation in Candida albicans. Microbiology Spectrum, 11(3). 10.1128/spectrum.00393-23 PubMed PMC

Liao, H., Li, Q., Chen, Y., Tang, J., Mou, B., & Lu, F., et al. (2024). Genome-wide identification of resistance genes and response mechanism analysis of key gene knockout strain to catechol in Saccharomyces cerevisiae. Frontier in Microbiology, 15, 1–14. 10.3389/fmicb.2024.1364425. PubMed PMC

Subden, R. E., Safe, L., Morris, D. C., Brown, R. G., & Safe, S. (1977). Eburicol, lichesterol, ergosterol, and obtusifoliol from polyene antibiotic resistant mutants of Candida albicans. Canadian Journal of Microbiology, 23(6), 751–754. 10.1139/m77-111. PubMed

Alcazar-Fuoli, L., Mellado, E., Garcia-Effron, G., Lopez, J. F., Grimalt, J. O., & Cuenca-Estrella, J. M., et al. (2008). Ergosterol biosynthesis pathway in Aspergillus fumigatus. Steroids, 73(3), 339–347. 10.1016/j.steroids.2007.11.005. PubMed

Zhou, W., Lepesheva, G. I., Waterman, M. R., & Nes, W. D. (2006). Mechanistic analysis of a multiple product sterol methyltransferase implicated in ergosterol biosynthesis in Trypanosoma brucei. Journal of Biological Chemistry, 281(10), 6290–6296. 10.1074/jbc.M511749200. PubMed

Ansari, S., & Prasad, R. (1993). Effect of miconazole on the structure and function of plasma membrane of Candida albicans. FEMS Microbiology Letters, 114(1), 93–98. 10.1111/j.1574-6968.1993.tb06556.x. PubMed

Calzada E., Onguka O., Claypool S. M. (2016). Phosphatidylethanolamine metabolism in health and disease. International Review of Cell and Molecular Biology (Vol. 321, pp. 29–88). Elsevier Inc. Available from: 10.1016/bs.ircmb.2015.10.001. PubMed PMC

Perczyk, P., Wójcik, A., Wydro, P., & Broniatowski, M. (2020). The role of phospholipid composition and ergosterol presence in the adaptation of fungal membranes to harsh environmental conditions–membrane modeling study. Biochimica et Biophysica Acta - Biomembranes [Internet], 1862(2), 183136. 10.1016/j.bbamem.2019.183136. PubMed

Qi, Y., Liu, H., Yu, J., Chen, X., & Liu, L. (2017). Med15B regulates acid stress response and tolerance in Candida glabrata by altering membrane lipid composition. Applied and Environmental Microbiology, 83(18), e01128–17. 10.1111/j.1574-6968.1993.tb06556.x. PubMed PMC

Geddes, C. D. Reviews in Fluorescence. 2010/. New York, NY: Springer Science+Business Media, LLC, 2012. Print.

Jasińska A., Różalska S., Rusetskaya V., Słaba M., Bernat P. (2022). Microplastic-induced oxidative stress in metolachlor-degrading filamentous fungus Trichoderma harzianum. International Journal of Molecular Sciences, 23(21). 10.3390/ijms232112978 PubMed PMC

Loffler, J., Einsele, H., Hebart, H., Schumacher, U., Hrastnik, C., & Daum, G. (2000). Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains. FEMS Microbiology Letters, 185(1), 59–63. 10.1111/j.1574-6968.2000.tb09040.x. PubMed

Mishra, N. N., Prasad, T., Sharma, N., & Gupta, D. K. (2008). Membrane fluidity and lipid composition of fluconazole resistant and susceptible strains of Candida albicans isolated from diabetic patients. Brazilian Journal of Microbiology, 39(2), 219–225. 10.1590/S1517-83822008000200004. PubMed PMC

Kodedová, M., & Sychrová, H. (2015). Changes in the sterol composition of the plasma membrane affect membrane potential, salt tolerance and the activity of multidrug resistance pumps in Saccharomyces cerevisiae. PLoS ONE, 10(9), 1–19. 10.1371/journal.pone.0139306. PubMed PMC

Gupta, S. S., Ton, V. K., Beaudry, V., Rulli, S., Cunningham, K., & Rao, R. (2003). Antifungal activity of amiodarone is mediated by disruption of calcium homeostasis. Journal of Biological Chemistry, 278(31), 28831–28839. 10.1074/jbc.M303300200. PubMed

Al Aboody, M. S., & Mickymaray, S. (2020). Anti-fungal efficacy and mechanisms of flavonoids. Antibiotics, 9(2), 45. 10.3390/antibiotics9020045. PubMed PMC

Bhattacharya, S., Esquivel, B. D., & White, T. C. (2018). Overexpression or deletion of ergosterol biosynthesis genes alters doubling time, response to stress agents, and drug susceptibility in Saccharomyces cerevisiae. MBio, 9(4), 1–14. 10.1128/mBio.01291-18. PubMed PMC

Navarro-Rodríguez, P., Martin-Vicente, A., López-Fernández, L., Guarro, J., & Capilla, J. (2020). Expression of ERG11 and efflux pump genes CDR1, CDR2 and SNQ2 in voriconazole susceptible and resistant Candida glabrata strains. Medical Mycology, 58(1), 30–38. 10.1093/mmy/myz014. PubMed

Silva, S., Negri, M., Henriques, M., Oliveira, R., Williams, D. W., & Azeredo, J. (2012). Candida glabrata, Candida parapsilosis and Candida tropicalis: Biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiology Reviews, 36(2), 288–305. 10.1111/j.1574-6976.2011.00278.x. PubMed

Suchodolski, J., & Krasowska, A. (2019). Plasma membrane potential of candida albicans measured by Di-4-ANEPPS fluorescence depends on growth phase and regulatory factors. Microorganisms, 7(4), 110. 10.3390/microorganisms7040110. PubMed PMC