Morphological Effect of the New Antifungal Agent ME1111 on Hyphal Growth of Trichophyton mentagrophytes, Determined by Scanning and Transmission Electron Microscopy

ABSTRACT

The effects of ME1111, a novel antifungal agent, on the hyphal morphology and ultrastructure of Trichophyton mentagrophytes were investigated by using scanning and transmission electron microscopy. Structural changes, such as pit formation and/or depression of the cell surface, and degeneration of intracellular organelles and plasmolysis were observed after treatment with ME1111. Our results suggest that the inhibition of energy production by ME1111 affects the integrity and function of cellular membranes, leading to fungal cell death.

TEXT

A new class of antifungal agent, ME1111 [2-(3, 5-dimethyl-1H-pyrazol-1-yl)-5-methylphenol] is being developed as a topical agent for onychomycosis. It is highly active in vitro and in vivo against Trichophyton rubrum and Trichophyton mentagrophytes and shows excellent human nail permeability (1–4). A previous study on its mechanism of action revealed that the molecular target of ME1111 was succinate dehydrogenase (complex II) in the mitochondrial electron transport system (5). However, its effects on the morphology and ultrastructure of hyphal cells are poorly understood. Electron microscopy appeared to be the most suitable approach for a better understanding of the essential events involved in the antidermatophytic action of ME1111. In this study, we investigated the effect of ME1111 on the ultrastructure of T. mentagrophytes grown in a liquid medium by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

T. mentagrophytes TIMM 2789 was grown in RPMI 1640 medium and Sabouraud dextrose broth (SDB) for study with SEM and TEM, respectively. The MIC of ME1111 against this strain was 0.5 μg/ml in RPMI 1640 medium and 0.25 μg/ml in SDB, based on the broth microdilution method (CLSI document M38-A2) (6). Conidia of T. mentagrophytes were inoculated into liquid medium (2 × 10⁵ conidia/ml) and incubated for 16 h at 35°C. When most of the conidia began to germinate, sub-MIC (1/4 MIC) and MIC doses of ME1111 were added to the culture broth. After 4, 8, and 24 h of incubation, hyphal cells were collected by centrifugation and prepared for SEM and TEM samples as described previously (7, 8).

The characteristic morphology of untreated T. mentagrophytes hyphae revealed by SEM is shown in Fig. 1. The cells were composed of elongated and blanched hyphae that were 1.0 to 1.5 μm in width and had smooth surfaces. In contrast, when cultures were treated with ME1111, hyphal growth was inhibited in a dose-dependent and time-dependent manner, and various morphological alterations were observed. After treatment with 1/4 MIC of ME1111 for 4 h, a small pit was formed on the cell surface (Fig. 2A). After 24 h of incubation, collapsed and deflated hyphae were occasionally observed (Fig. 2B). The depression observed on the cell surface indicates a possible loss of cytoplasmic volume. A decreasing trend of dry weight of fungal hyphae was also shown after treatment with 1/4 MIC of ME1111 for 4 and 24 h in another test (data not shown). These morphological alterations were more extensive at higher drug concentrations and after exposure to the MIC dose of ME1111 for 24 h: hyphal elongation was completely inhibited, and short necrotic cells and hyphal disruption were observed (Fig. 3A and B). Collapse and distortion of hyphae were considered to be the results of changes in intracellular osmotic pressure, suggesting that ME1111 mainly affects the permeability of the cell membrane.

FIG 1.

SEM photographs of untreated control T. mentagrophytes hyphae grown for 4 h. Lower (A) and higher (B) magnification of hyphal cells. Bar = 1 μm.

FIG 2.

SEM photographs of T. mentagrophytes cells treated with 1/4 MIC (0.12 μg/ml) of ME1111 for 4 h (A) and 24 h (B). Bar = 1 μm.

FIG 3.

SEM photographs of T. mentagrophytes cells treated with MIC (0.5 μg/ml) of ME1111 for 24 h. Note short necrotic hyphae (A) and outflow of cellular content (B). Bar = 1 μm.

The ultrastructural appearance of the thin-sectioned control hyphal cells grown for 24 h is shown in Fig. 4. Cells were delimited by a cell wall of about 150 to 200 nm thickness, and their cell membranes were closely attached to the walls. The typical features of several major organelles, such as nuclei, mitochondria, vacuoles, and endoplasmic reticula, were visible in the cytoplasm.

FIG 4.

TEM photograph of untreated control T. mentagrophytes cells grown for 24 h. CW, cell wall; CM, cell membrane; M, mitochondria; V, vacuole; ER, endoplasmic reticulum. Bar = 1 μm.

No remarkable ultrastructural changes were found in the cell after 4 h of treatment with 1/4 MIC and MIC doses of ME1111. However, after 8 h of treatment with either the 1/4 MIC or MIC of the drug, disorder of the cytoplasmic membrane, degeneration of cell organelles, and cytoplasm vacuolation were observed (Fig. 5). After 24 h of treatment with the MIC of ME1111, the degenerated cytoplasm appeared contracted, and the cell membrane was completely separated from the cell wall, i.e., plasmolysis (Fig. 6).

FIG 5.

TEM photographs of a hyphal cell treated with MIC (0.25 μg/ml) (A) and 1/4 MIC (0.06 μg/ml) (B) of ME1111 for 8 h. Bar = 1 μm.

FIG 6.

TEM photographs of a hyphal cell treated with MIC (0.25 μg/ml) of ME1111 for 24 h showing hyphae with plasmolysis (A) and a degenerated cell (B). Bar = 1 μm.

Succinate dehydrogenase is one of the constitutive enzymes of the electron transport system and of the citric acid cycle. This enzyme is involved in the production of ATP, which is the fuel essential to most cellular activities (9, 10). After ATP synthesis terminates because of its inhibition by ME1111 in hyphal cells, normal cellular activities are expected to be impaired, thus leading to various cellular dysfunctions. Indeed, a decrease in osmotic pressure in hyphal cells was confirmed after the observation of plasmolysis by TEM. This suggests that ME1111 impairs the active transport system at the cell membrane or vacuolar membrane, which requires ATP. Moreover, we speculate that when the transport of water or intracellular materials is blocked, organelle and cell membranes disintegrate because of the damage caused by cell dehydration or by concentrated salt solution and by the deterioration of the intracellular environment, which leads to cell death.

The antifungal agents currently available for the treatment of onychomycosis are limited to terbinafine, itraconazole, ciclopirox, amorolfine, efinaconazole, and tavaborole. The mechanisms of action of these antifungals can be classified into inhibition of ergosterol biosynthesis, chelation of polyvalent cations, inhibition of aminoacyl tRNA synthetase, and interaction with microtubules (11). In regard to the influence of these antifungal agents on the morphology of the hyphal cells of T. mentagrophytes, as analyzed by electron microscopy, only a few cases have been reported (12–17). SEM and/or TEM analysis of all drugs studied, mostly ergosterol synthesis inhibitors, revealed common cellular changes, including the thickening of the hyphal cell wall and accumulation of electron-dense granules in the cell wall (12–17). The reported granular structures are speculated to be agglomerates of intermediates of sterol metabolism that accumulated during the process of ergosterol synthesis inhibition (18, 19). These formations are thought to impair the structure and function of the cell membrane and inhibit hyphal growth. The present study with TEM did not show changes such as cell wall thickening and accumulation of granules in hyphal cells; instead, different changes such as plasmolysis, contraction of the cytoplasm, disintegration of organelles, and fragmentation and disappearance of the cell membrane were observed after treatment with ME1111.

In this study, we demonstrated the morphological and ultrastructural changes of hyphal cells of T. mentagrophytes treated with sub-MIC and MIC doses of ME1111. The study results strongly support the mechanism of action of ME1111, and we suggest that ME1111 elicits its antifungal activity through the following processes: (i) discontinuation of ATP production by succinate dehydrogenase inhibition, (ii) impairment of the ATP-dependent active transport system at the cell membrane and vacuole, and (iii) disintegration of the cell and organelle membranes and the subsequent cell lysis associated with deterioration of the intercellular environment.