Expression of Lanosterol 14-Demethylase (ERG11) Gene of Three-Drug Combinations in Candida albicans

Document Type : Research Paper


1 Department of Microbiology, Yasooj Branch, Islamic Azad University, Yasooj, Iran

2 Department of Biology, Gachsaran Branch, Islamic Azad University, Gachsaran, Iran



Patients with impaired immunity are at particular risk of infections with Candida albicans. Antifungal drugs such as azoles commonly used for candidiasis treatment, but drug resistance is one of the most common problems for public health. The aim of this study was to evaluate the expression of lanosterol 14-demethylase (ERG11) gene for three-drug combinations in C. albicans. Disk diffusion and broth microdilution susceptibility tests were employed to evaluate the synergic effects of amphotericin B, fluconazole, voriconazole, ketoconazole and itraconazole. Quantification of ERG11 gene expression was carried out in C. albicans treated with three-drug combinations of fluconazole/ ketoconazole/ voriconazole and fluconazole/ ketoconazole/ itraconazole. Three-drug combinations revealed synergistic and partial synergistic effect for all tested isolates (FIC index range of 0.27-0.77). The expression levels of ERG11 were down-regulated by three-drug combination of fluconazole/ ketoconazole/ voriconazole treatment. Fluconazole synergizes with ketoconazole and voriconazole in three-drug combination against C. albicans by targeting of the ERG11 gene.


[1] Richardson JP, Moyes DL. Adaptive immune responses to Candida albicans infection. Virulence (2015) 6 (4): 327-337.
[2] Khodavandi A, Alizadeh F.Antifungal Agents and Their Mechanism of Action. 1st ed., Islamic Azad University. Iran (2017).
[3] Song JL, Harry JB, Eastman RT, Oliver BG, White TC. The Candida albicans lanosterol 14-alpha-demethylase (ERG11) gene promoter is maximally induced after prolonged growth with antifungal drugs. Agents Chemother. (2004) 48 (4): 1136-1144.
[4] Gray KC, Palacios DS, Dailey I, Endo MM, Uno BE, Wilcock BC, Burke MD. Amphotericin primarily kills yeast by simply binding ergosterol. Natl. Acad. Sci. USA (2012) 109 (7): 2234-2239.
[5] Mast N, Zheng W, Stout CD, Pikuleva IA. Antifungal azoles: structural insights into undesired tight binding to cholesterol-metabolizing CYP46A1. Pharmacol. (2013) 84 (1): 86-94.
[6] Li X, Hou Y, Yue L, Liu S, Duc J, Sun S. Potential targets for antifungal drug discovery based on growth and virulence in Candida albicans. Antimicrob. Agents Chemother. (2015) 59 (10): 5885-5891.
[7] Alizadeh F, Khodavandi A, Esfandyari S, Nouripour-Sisakht S. Analysis of ergosterol and gene expression profiles of sterol Δ5,6-desaturase (ERG3) and lanosterol 14α-demethylase (ERG11) in Candida albicans treated with carvacrol. Herbmed. Pharmacol. (2018) 7 (2): 79-87.
[8] Odds FC, Brown AJP, Gow NAR. Antifungal agents: mechanisms of action. TRENDS Microbiol. (2003) 11(6): 272-27
[9] Johnson MD, Perfect JR. Use of antifungal combination therapy: agents, order, and timing. Fungal Infect. Rep. (2010) 4 (2): 87-95.
[10] Chau AS, Gurnani M, Hawkinson R, Laverdiere M, Cacciapuoti A, McNicholas PM. Inactivation of sterol Delta5,6-desaturase attenuates virulence in Candida albicans. Agents Chemother. (2005) 49 (9): 3646-3651.
[11] Dupont S, Lemetais G, Ferreira T, Cayot P, Gervais P, Beney L. Ergosterol biosynthesis: a fungal pathway for life on land? Evolution (2012) 66 (9): 2961-2968.
[12] Scorzoni et al. Antifungal therapy: new advances in the understanding and treatment of mycosis. Microbiol. (2017) 8: 36.
[13] Lv QZ, Yan L, Jiang YY. The synthesis, regulation, and functions of sterols in Candida albicans: Well-known but still lots to learn. Virulence (2016) 7 (6): 649-659.
[14] Ostrosky-Zeichner L. Combination antifungal therapy: a critical review of the evidence. Microbiol. Infect. (2008)14 (Suppl 4): 65-70.
[15] Pianalto KM, Alspaugh JA. New horizons in antifungal therapy. J Fungi (Basel) (2016) 2 (4). pii: E26.
[16] Alizadeh F, Khodavandi A, Zalakian S. Quantitation of ergosterol content and gene expression profile of ERG11 gene in fluconazole-resistant Candida albicans. Med. Mycol. (2017) 3 (1): 13-19.
[17] CLSI (Clinical and Laboratory Standards Institute). Method for antifungal disk diffusion susceptibility testing of yeasts. Approved standard M44-A2. 2nd ed. Wayne, PA: Clinical and Laboratory Standards Institute (2009).
[18] CLSI (Clinical and Laboratory Standards Institute). Reference method for broth dilution antifungal susceptibility testing of yeasts; Approved standard M27-A3. 3rd ed. Wayne, PA: Clinical and Laboratory Standards Institute (2008).
[19] Khodavandi A, Alizadeh F, Aala F, Sekawi Z, Chong PP. In vitro investigation of antifungal activity of allicin alone and in combination with azoles against Candida Mycopathologia (2010) 169 (4): 287-95.
[20] Khodavandi A, Alizadeh Aghai Vanda N, Karimi G, Chong PP. Possible mechanisms of the antifungal activity of fluconazole in combination with terbinafine against Candida albicans. Biol. (2014) 52 (12): 1505-1509.
[21] Khodavandi et al. Comparison between allicin and fluconazole in Candida albicans biofilm inhibition and in suppression of HWP1 gene expression. Phytomedicine (2011) 19 (1): 56-63.
[22] Chamilos G, Kontoyiannis DP. The rationale of combination antifungal therapy in severely immunocompromised patients: empiricism versus evidence-based medicine. Opin. Infect. Dis. (2006) 19 (4): 380-385.
[23] Hatipoglu N, Hatipoglu H. Combination antifungal therapy for invasive fungal infections in children and adults. Expert Rev. Anti. Infect. Ther. (2013) 11 (5): 523-535.
[24] Carrillo-Muñoz AJ, Finquelievich J, Tur-Tur C, Eraso E, Jauregizar N, Quindós G, Giusiano G. Combination antifungal therapy: a strategy for the management of invasive fungal infections. Esp. Quimioter. (2014) 27(3): 141-158.
[25] Cuenca-Estrella M. Combinations of antifungal agents in therapy–what value are they? Antimicrob. Chemother. (2004) 54 (5): 854-869.
[26] Chen et al. ASDCD: antifungal synergistic drug combination database. PLoS One (2014) 9(1): e86499.
[27] Ghannoum MA, Fu Y, Ibrahim AS, Mortara LA, Shafiq MC, Edwards Jr JE, Criddle RS. In vitro determination of optimal antifungal combinations against Cryptococcus neoformans and Candida albicans. Antimicrob. Agents Chemother. (1995) 39 (11): 2459-2465.
[28] Sanati H, Ramos CF, Bayer AS, Ghannoum MA. Combination therapy with amphotericin B and fluconazole against invasive candidiasis in neutropenic-mouse and infective-endocarditis rabbit models. Agents Chemother. (1997) 41 (6): 1345-1348.
[29] Paterson PJ, McWhinney PHM, Potter M, Kibbler CC, Prentice HG. The combination of oral amphotericin B with azoles prevents the emergence of resistant Candida species in neutropenic patients. J. Haematol. (2001) 112 (1): 175-180.
[30] Rex et al. A randomized and blinded multicenter trial of high-dose fluconazole plus placebo versus fluconazole plus amphotericin B as therapy for candidemia and its consequences in nonneutropenic subjects. Infect. Dis. (2003)36(10): 1221-1228.
[31] Odds FC. Fluconazole plus amphotericin b combinations are not contraindicated and may add benefit for the treatment of candidemia. Infect. Dis. (2003) 36: 1229-1231.
[32] Johnson MD, MacDougall C, Ostrosky-Zeichner L, Perfect JR, Rex JH. Combination antifungal therapy. Agents Chemother. (2004) 48 (3): 693-715.
[33] Scheid LA, Mario DA, Kubiça TF, Santurio JM, Alves SH. In vitro activities of antifungal agents alone and in combination against fluconazole-susceptible and -resistant strains of Candida dubliniensis. Braz J. Infect. Dis. (2012) 16 (1): 78-81
[34] Shojaei et al. Effects of several common antifungal drugs (clotrimazole, miconazole, fluconazole) alone and in combination with amphotericin B on Candida species isolated from chronic candidal vulvovaginitis. J Fasa Univ. Med. Sci (2014) 4 (3): 327-334.
[35] Cui J, Ren B, Tong Y, Dai H, Zhang L. Synergistic combinations of antifungals and anti-virulence agents to fight against Candida albicans. Virulence (2015) 6 (4): 362-371.
[36] Borecká-Melkusová S, Moran GP, Sullivan DJ, Kucharíková S, Chorvát D Jr, Bujdáková H. The expression of genes involved in the ergosterol biosynthesis pathway in Candida albicans and Candida dubliniensis biofilms exposed to fluconazole. Mycoses (2009) 52 (2): 118-128.
[37] Ahmad A, Wani MY, Khan A, Manzoor N, Molepo J. Synergistic interactions of eugenol-tosylate and its congeners with fluconazole against Candida albicans. PLoS One (2015) 10 (12): e0145053.
[38] Ghannoum MA, Rice LB. Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin. Microbiol. Rev. (1999) 12 (4): 501-517.
[39] Khodavandi A, Alizadeh F, Abraheh Z. Comparison of ERG11 gene expression profiles of Candida albicans treated with Thymus vulgaris extracts alone and in combination with Mentha Biol. J. Microorganism (2018) 7 (25): 87-99.