E1210, a new broad-spectrum antifungal, suppresses Candida albicans hyphal growth through inhibition of glycosylphosphatidylinositol biosynthesis

Abstract

Continued research toward the development of new antifungals that act via inhibition of glycosylphosphatidylinositol (GPI) biosynthesis led to the design of E1210. In this study, we assessed the selectivity of the inhibitory activity of E1210 against Candida albicans GWT1 (Orf19.6884) protein, Aspergillus fumigatus GWT1 (AFUA_1G14870) protein, and human PIG-W protein, which can catalyze the inositol acylation of GPI early in the GPI biosynthesis pathway, and then we assessed the effects of E1210 on key C. albicans virulence factors. E1210 inhibited the inositol acylation activity of C. albicans Gwt1p and A. fumigatus Gwt1p with 50% inhibitory concentrations (IC(50)s) of 0.3 to 0.6 μM but had no inhibitory activity against human Pig-Wp even at concentrations as high as 100 μM. To confirm the inhibition of fungal GPI biosynthesis, expression of ALS1 protein, a GPI-anchored protein, on the surfaces of C. albicans cells treated with E1210 was studied and shown to be significantly lower than that on untreated cells. However, the ALS1 protein levels in the crude extract and the RHO1 protein levels on the cell surface were found to be almost the same. Furthermore, E1210 inhibited germ tube formation, adherence to polystyrene surfaces, and biofilm formation of C. albicans at concentrations above its MIC. These results suggested that E1210 selectively inhibited inositol acylation of fungus-specific GPI which would be catalyzed by Gwt1p, leading to the inhibition of GPI-anchored protein maturation, and also that E1210 suppressed the expression of some important virulence factors of C. albicans, through its GPI biosynthesis inhibition.

Fig 1

Autoradiograph of TLC separation of GPI intermediates produced from UDP-[¹⁴C]GlcNAc in the presence of E1210. The membranes of the S. cerevisiae GWT1-disrupted cells overexpressing C. albicans GWT1 (A), A. fumigatus GWT1 (B), and human PIG-W (C) were assayed for the inositol acylation of GPI in the presence of E1210, fluconazole (FLCZ), amphotericin B (AMPH), and micafungin (MCFG). (D) PI-PLC treatment of radiolabeled lipids produced by membranes of S. cerevisiae overexpressing CaGWT1 and of S. cerevisiae overexpressing ScGWT1. These images were trimmed from representative autoradiographs of the TLC plates derived from three independent experiments.

Fig 2

Effects of E1210 and other antifungals on the expression of Als1 protein on the surface of C. albicans IFM49971 cells. The relative amounts of Als1 protein on the cell surface of C. albicans treated with each compound in MOPS–RPMI 1640 medium for 1 h were determined on filter plates using ELISAs. Each data point represents the mean ± standard error of the mean (SEM) (n = 6). Asterisks indicate a P value of <0.05 compared to control at 1 h, as determined by the Dunnett multiple-comparison test.

Fig 3

Effects of E1210 on the Als1 protein level from crude extracts of C. albicans IFM49971 cells. The C. albicans cells treated with E1210 for 1 h were disrupted by mixing with glass beads, and the Als1 protein levels from crude extracts were determined utilizing ELISA and standardized using the protein concentration from crude extracts. Each data point represents the mean ± SEM (n = 3) from experiments performed in duplicate.

Fig 4

Effects of E1210 on the Rho1 protein levels on the surface of C. albicans IFM49971 cells. The relative amounts of Rho1 protein on the cell surface of C. albicans treated with each compound in MOPS–RPMI 1640 medium for 1 h were determined on filter plates using ELISAs. Each data point represents the mean ± SEM (n = 6).

Fig 5

Effects of E1210 and other antifungals on the adherence of C. albicans IFM49971 cells to polystyrene surfaces. C. albicans cells were incubated in the presence of each compound for 1 h in MOPS–RPMI 1640, and cells that adhered to polystyrene surfaces were stained using crystal violet. Each data point represents the mean ± SEM from three independent plates assayed in duplicate. Symbols: ●, E1210; ○, fluconazole; △, micafungin; □, amphotericin B.

Fig 6

Effects of E1210 and other antifungals on germ tube formation in C. albicans IFM49971 cells. C. albicans cells were incubated in the presence of each compound for 4 h in MOPS–RPMI 1640, and cells that converted from yeast form to the hyphal form and adhered to polystyrene surfaces were stained with crystal violet. Each data point represents the mean ± SEM from three independent plates assayed in duplicate. Symbols: ●, E1210; ○, fluconazole; △, micafungin; □, amphotericin B.

Fig 7

Microscopic images of C. albicans cells after treatment with E1210 and other antifungals for 4 h. Cells were left untreated or were treated with 0.5×MIC, 1×MIC, and the highest concentration tested of each compound for 4 h. The magnification of all images is ×200. Abbreviations: FLCZ, fluconazole; MCFG, micafungin; AMPH, amphotericin B.

Fig 8

Effects of E1210 on germ tube formation in C. albicans IFM49971 cells in various media. The C. albicans cells were incubated in the presence of E1210 for 4 h, and cells that converted from yeast form to hyphal form and adhered to polystyrene surfaces were stained with crystal violet. Each data point represents the mean ± SEM from three independent plates assayed in duplicate. Symbols: ●, MOPS–RPMI 1640; ○, Spider medium; ■, Lee's medium; □, YPD broth supplemented with 10% FCS; ▴, yeast nitrogen base without amino acids but supplemented with 2.5 mM GlcNAc.

Fig 9

Microscopic images of C. albicans IFM49971 cells after treatment with E1210 for 4 h in hypha-inducing media. Images of cells untreated or treated at 4×MIC of E1210 in each medium are shown. The magnification of all images is ×200. Abbreviations: YPD + FCS, YPD broth supplemented with 10% FCS; YNB + GlcNAc, yeast nitrogen base without amino acids but supplemented with 2.5 mM GlcNAc.

Fig 10

Microscopic images of C. albicans colonies growing in YPS agar for 48 h. (A) Control; (B) 0.001 μg/ml E1210; (C) 0.002 μg/ml E1210; (D) 0.008 μg/ml E1210. Colonies were photographed at ×40.

Fig 11

Effects of E1210 and other antifungals on the density of C. albicans IFM49971 biofilms after incubation for 24 h. The biofilm density was quantified using safranin staining. Each data point represents the mean ± SEM from experiments on three independent plates assayed in duplicate. Symbols: ●, E1210; ○, fluconazole; △, micafungin; □, amphotericin B.

Fig 12

Scanning electron micrographs of C. albicans IFM49971 biofilms in plastic slide wells after incubation for 24 h with either medium (A) or 0.008 μg E1210 per ml (B). Bar, 10 μm.