Δ(24)-Sterol Methyltransferase Plays an Important Role in the Growth and Development of Sporothrix schenckii and Sporothrix brasiliensis

Abstract

Inhibition of Δ(24)-sterol methyltransferase (24-SMT) in Sporothrix schenckii sensu stricto and Sporothrix brasiliensis was investigated in vitro. The effects on fungal growth and sterol composition of the 24-SMT inhibitor 22-hydrazone-imidazolin-2-yl-chol-5-ene-3β-ol (H3) were compared to those of itraconazole. MIC and MFC analysis showed that H3 was more effective than itraconazole against both species in both their filamentous and yeast forms. H3 showed fungistatic activity in a time-kill assay, with inhibitory activity stronger than that of itraconazole. GC analysis of cell sterol composition showed that sterols present in control cells (ergosterol and precursors) were completely replaced by 14α-methylated sterols after H3 exposure. Itraconazole only partially inhibited ergosterol synthesis but completely arrested synthesis of other sterols found in control cells, promoting accumulation of nine 14α-methyl sterols. Based on these results, we propose a schematic model of sterol biosynthesis pathways in S. schenckii and S. brasiliensis. Effects on cell morphology due to 24-SMT inhibition by H3 as analyzed by SEM and TEM included irregular cell shape, reduced cytoplasmic electron-density, and reduced thickness of the microfibrillar cell wall layer. Moreover, 24-SMT inhibition by H3 promoted mitochondrial disturbance, as demonstrated by alterations in MitoTracker(®) Red CMXRos fluorescence intensity evaluated by flow cytometry. When used in conjunction with itraconazole, H3 enhanced the effectiveness of itraconazole against all tested strains, reducing at least half (or more) the MIC values of itraconazole. In addition, cytotoxicity assays revealed that H3 was more selective toward these fungi than was itraconazole. Thus, 24-SMT inhibition by H3 was an effective antifungal strategy against S. schenckii and S. brasiliensis. Inhibition of the methylation reaction catalyzed by 24-SMT has a strong antiproliferative effect via disruption of ergosterol homeostasis, suggesting that this enzyme is a promising target for novel antifungal therapies against sporotrichosis, either as sole treatments or in combination with itraconazole.

Keywords: Sporothrix brasiliensis; Sporothrix schenckii; antifungal activity; sterol biosynthesis; Δ24-sterol methyltransferase.

FIGURE 1

Molecular structure of the Δ²⁴-sterol methyltransferase (24-SMT) inhibitor H3.

FIGURE 2

Time-kill plots showing activity of the 24-SMT inhibitor H3 in comparison with that of itraconazole against the yeast forms of Sporothrix schenckii ATCC MYA 4821 and S. brasiliensis ATCC MYA 4823.

FIGURE 3

Ultrastructural analysis of Sporothrix schenckii ATCC MYA 4821 yeast cells treated with 0.125 mg/L H3 (D–F) or 0.25 mg/L itraconazole (G–I) (MIC/2) compared to untreated cells (A–C). Scanning electron microscopy (A,D,G) and transmission electron microscopy (B,C,E,F,H,I) images show the presence of: m, mitochondria (B); n, nucleus (B); G, Golgi complex (H); inner cell wall (ICW); microfibrillar cell wall layer (ML). The arrowhead in (I) indicates a groove in the cell wall structure. (J) Cell diameter analysis by calculation of Feret diameters. (K) Analysis of ICW and ML thickness as measured in TEM images. ^∗∗p < 0.001; ^∗∗∗p < 0.0001. Scale bars: 5 μm (A,D,G); 0.5 μm (B,E,H); 0.2 μm (C,F,I).

FIGURE 4

Ultrastructural analysis of Sporothrix brasiliensis ATCC MYA 4823 cells after exposure to 0.03 mg/L H3 (D–F) or 0.25 mg/L itraconazole (G–I) compared to untreated cells (A–C). Scanning electron microscopy (A,D,G) and transmission electron microscopy (B,C,E,F,H,I) images show the presence of: m, mitochondria (B); n, nucleus (B,H); v, vacuole (B); inner cell wall (ICW); microfibrillar cell wall layer (ML). Arrowheads indicate small vesicles associated with the plasma membrane (I). (J) Cell diameter analysis by calculation of Feret diameters. (K) Analysis of ICW and ML thickness as measured in TEM images. ^∗p < 0.05, ^∗∗p < 0.001, ^∗∗∗p < 0.0001. Scale bars: 5 μm (A,D,G); 0.5 μm (B,E,H); 0.2 μm (C,F,I).

FIGURE 5

Mitochondrial activity after treatment of Sporothrix schenckii ATCC MYA 4821 and S. brasiliensis ATCC MYA 4823 yeast cells with H3 or itraconazole. Cells untreated or treated for 96 h with sub-inhibitory concentrations of H3 or itraconazole were stained with MitoTracker Red CMXRos, after which fluorescence intensity was analyzed by flow cytometry. H3 exposure induced a statistically significant fluorescence intensity increase (S. schenckii 4821) or decrease (S. brasiliensis 4823), reflecting mitochondrial disturbance (^∗p < 0.05).

FIGURE 6

Representative pathway of sterol biosynthesis in Sporothrix schenckii and Sporothrix brasiliensis, and sites of action of the studied inhibitors. Sterols identified on Table 2 are shown in this figure: (1) 14α-methyl-cholesta-8-en-3-one, (2) ergosterol, (3) 14α-methyl-ergosta-8,24(24’)-dien-3β-ol, (4) ergosta-5,7,22,24-tetra-en-3β-ol, (5) ergosta-5,7,24(24′)-trien-3β-ol, (6) 5-dehydro-episterol, (7) 4,14-dimethyl-ergosta-5,7,24(24’)-trien-3β-ol, (8) stigmasterol, (9) obtusifoliol, (10) lanosta-8,24-dien-3-one, (11) lanosterol, (12) 24-methylene-lanosta-8-en-3-one, (13) eburicol, (14) 24-ethyl-lanosta-8,22-dien-3β-ol, (15) 24(E)-ethylidenelanost-8-en-3β -ol, (16) 24(Z)-ethylidenelanost-8-en-3β-ol, (17) 4,4-dimethyl-ergosta-8,14,24(24′)-trien-3β-ol, (18) 4,4′-dimethyl-ergosta-8,24(24′)-dien-3β-ol, (19) 4-methyl-ergosta-8,24(24′)-dien-3β-ol, (20) fecosterol, (21) 4,4-dimethyl-ergosta-8,14,24-trien-3β-ol, (22) 4,4-dimethyl-ergosta-8,24-dien-3β-ol, (23) 4-methyl-ergosta-8,24-dien-3β-ol, and (24) zymosterol. Thick lines indicate the main pathways (pathway A or B) from lanosterol to ergosterol. Dashed arrows show accumulation of intermediate sterols by action of the sterol biosynthesis inhibitors itraconazole and H3 (pathways C, D, E). Arrows marked with an ‘X’ indicate inhibitory interactions of the sterol hydrazone H3 and itraconazole with Δ²⁴-sterol methyl transferase (24-SMT₁) and C14α-demethylase, respectively. 24-SMR, Δ²⁴-sterol methyl reductase.