Morphological Effect of the NewAntifungal Agent ME1111 on HyphalGrowth of Trichophyton mentagrophytes,Determined by Scanning andTransmission Electron Microscopy

Yayoi Nishiyama,a Sho Takahata,b Shigeru Abea

Teikyo University Institute of Medical Mycology, Tokyo, Japana; Meiji Seika Pharma Co., Ltd., Tokyo, Japanb

ABSTRACT The effects of ME1111, a novel antifungal agent, on the hyphal morphol-ogy and ultrastructure of Trichophyton mentagrophytes were investigated by usingscanning and transmission electron microscopy. Structural changes, such as pit for-mation and/or depression of the cell surface, and degeneration of intracellular or-ganelles and plasmolysis were observed after treatment with ME1111. Our resultssuggest that the inhibition of energy production by ME1111 affects the integrity andfunction of cellular membranes, leading to fungal cell death.

KEYWORDS antifungal agents, electron microscopy, mode of action, morphology

Anew class of antifungal agent, ME1111 [2-(3, 5-dimethyl-1H-pyrazol-1-yl)-5-methyl-phenol] is being developed as a topical agent for onychomycosis. It is highly active

in vitro and in vivo against Trichophyton rubrum and Trichophyton mentagrophytes andshows excellent human nail permeability (1–4). A previous study on its mechanism ofaction revealed that the molecular target of ME1111 was succinate dehydrogenase(complex II) in the mitochondrial electron transport system (5). However, its effects onthe morphology and ultrastructure of hyphal cells are poorly understood. Electronmicroscopy appeared to be the most suitable approach for a better understanding ofthe essential events involved in the antidermatophytic action of ME1111. In this study,we investigated the effect of ME1111 on the ultrastructure of T. mentagrophytes grownin a liquid medium by scanning electron microscopy (SEM) and transmission electronmicroscopy (TEM).

T. mentagrophytes TIMM 2789 was grown in RPMI 1640 medium and Sabourauddextrose broth (SDB) for study with SEM and TEM, respectively. The MIC of ME1111against this strain was 0.5 �g/ml in RPMI 1640 medium and 0.25 �g/ml in SDB, basedon the broth microdilution method (CLSI document M38-A2) (6). Conidia of T. menta-

Received 8 June 2016 Returned formodification 16 July 2016 Accepted 23October 2016

Accepted manuscript posted online 31October 2016

Citation Nishiyama Y, Takahata S, Abe S. 2017.Morphological effect of the new antifungalagent ME1111 on hyphal growth ofTrichophyton mentagrophytes, determined byscanning and transmission electronmicroscopy. Antimicrob Agents Chemother61:e01195-16. https://doi.org/10.1128/AAC.01195-16.

Copyright © 2016 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Yayoi Nishiyama,ynishiya@main.teikyo-u.ac.jp.

FIG 1 SEM photographs of untreated control T. mentagrophytes hyphae grown for 4 h. Lower (A) andhigher (B) magnification of hyphal cells. Bar � 1 �m.

MECHANISMS OF ACTION:PHYSIOLOGICAL EFFECTS

crossm

January 2017 Volume 61 Issue 1 e01195-16 aac.asm.org 1Antimicrobial Agents and Chemotherapy

on January 9, 2017 by TE

IKY

O U

NIV

http://aac.asm.org/

Dow

nloaded from

grophytes were inoculated into liquid medium (2 � 105 conidia/ml) and incubated for16 h at 35°C. When most of the conidia began to germinate, sub-MIC (1/4 MIC) and MICdoses 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 samplesas described previously (7, 8).

The characteristic morphology of untreated T. mentagrophytes hyphae revealed bySEM is shown in Fig. 1. The cells were composed of elongated and blanched hyphaethat were 1.0 to 1.5 �m in width and had smooth surfaces. In contrast, when cultureswere treated with ME1111, hyphal growth was inhibited in a dose-dependent andtime-dependent manner, and various morphological alterations were observed. Aftertreatment 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 occasionallyobserved (Fig. 2B). The depression observed on the cell surface indicates a possible lossof cytoplasmic volume. A decreasing trend of dry weight of fungal hyphae was alsoshown after treatment with 1/4 MIC of ME1111 for 4 and 24 h in another test (data notshown). These morphological alterations were more extensive at higher drug concen-trations and after exposure to the MIC dose of ME1111 for 24 h: hyphal elongation wascompletely 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 ofchanges in intracellular osmotic pressure, suggesting that ME1111 mainly affects thepermeability of the cell membrane.

The ultrastructural appearance of the thin-sectioned control hyphal cells grown for24 h is shown in Fig. 4. Cells were delimited by a cell wall of about 150 to 200 nmthickness, and their cell membranes were closely attached to the walls. The typicalfeatures of several major organelles, such as nuclei, mitochondria, vacuoles, andendoplasmic reticula, were visible in the cytoplasm.

No remarkable ultrastructural changes were found in the cell after 4 h of treatmentwith 1/4 MIC and MIC doses of ME1111. However, after 8 h of treatment with either the1/4 MIC or MIC of the drug, disorder of the cytoplasmic membrane, degeneration of cellorganelles, and cytoplasm vacuolation were observed (Fig. 5). After 24 h of treatmentwith the MIC of ME1111, the degenerated cytoplasm appeared contracted, and the cellmembrane was completely separated from the cell wall, i.e., plasmolysis (Fig. 6).

FIG 2 SEM photographs of T. mentagrophytes cells treated with 1/4 MIC (0.12 �g/ml) of ME1111 for 4 h(A) and 24 h (B). Bar � 1 �m.

FIG 3 SEM photographs of T. mentagrophytes cells treated with MIC (0.5 �g/ml) of ME1111 for 24 h. Noteshort necrotic hyphae (A) and outflow of cellular content (B). Bar � 1 �m.

Nishiyama et al. Antimicrobial Agents and Chemotherapy

January 2017 Volume 61 Issue 1 e01195-16 aac.asm.org 2

on January 9, 2017 by TE

IKY

O U

NIV

http://aac.asm.org/

Dow

nloaded from

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

The antifungal agents currently available for the treatment of onychomycosis arelimited to terbinafine, itraconazole, ciclopirox, amorolfine, efinaconazole, and tava-borole. The mechanisms of action of these antifungals can be classified into inhibitionof ergosterol biosynthesis, chelation of polyvalent cations, inhibition of aminoacyl tRNAsynthetase, and interaction with microtubules (11). In regard to the influence of theseantifungal agents on the morphology of the hyphal cells of T. mentagrophytes, asanalyzed by electron microscopy, only a few cases have been reported (12–17). SEMand/or TEM analysis of all drugs studied, mostly ergosterol synthesis inhibitors, revealedcommon cellular changes, including the thickening of the hyphal cell wall and accu-mulation of electron-dense granules in the cell wall (12–17). The reported granularstructures are speculated to be agglomerates of intermediates of sterol metabolismthat accumulated during the process of ergosterol synthesis inhibition (18, 19). Theseformations are thought to impair the structure and function of the cell membrane andinhibit hyphal growth. The present study with TEM did not show changes such as cellwall thickening and accumulation of granules in hyphal cells; instead, different changes

FIG 5 TEM photographs of a hyphal cell treated with MIC (0.25 �g/ml) (A) and 1/4 MIC (0.06 �g/ml) (B)of ME1111 for 8 h. Bar � 1 �m.

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 �m.

Morphological Changes in T. mentagrophytes by ME1111 Antimicrobial Agents and Chemotherapy

January 2017 Volume 61 Issue 1 e01195-16 aac.asm.org 3

on January 9, 2017 by TE

IKY

O U

NIV

http://aac.asm.org/

Dow

nloaded from

such as plasmolysis, contraction of the cytoplasm, disintegration of organelles, andfragmentation and disappearance of the cell membrane were observed after treatmentwith ME1111.

In this study, we demonstrated the morphological and ultrastructural changes ofhyphal cells of T. mentagrophytes treated with sub-MIC and MIC doses of ME1111. Thestudy results strongly support the mechanism of action of ME1111, and we suggest thatME1111 elicits its antifungal activity through the following processes: (i) discontinua-tion of ATP production by succinate dehydrogenase inhibition, (ii) impairment of theATP-dependent active transport system at the cell membrane and vacuole, and (iii)disintegration of the cell and organelle membranes and the subsequent cell lysisassociated with deterioration of the intercellular environment.

ACKNOWLEDGMENTSWe thank Yayoi Hasumi for excellent technical assistance.This study was financially supported in part by Meiji Seika Pharma Co., Ltd.Sho Takahata is a full-time employee of Meiji Seika Pharma Co., Ltd.We alone are responsible for the content and writing of this paper.

REFERENCES1. Ghannoum M, Isham N, Long L. 2015. In vitro antifungal activity of

ME1111, a new topical agent for onychomycosis, against clinical isolatesof dermatophytes. Antimicrob Agents Chemother 59:5154 –5158.https://doi.org/10.1128/AAC.00992-15.

2. Tabata Y, Takai-Masuda N, Kubota N, Takahata S, Ohyama M, Kaneda K,Iida M, Maebashi K. 2016. Characterization of antifungal activity and nailpenetration of ME1111, a new antifungal agent for topical treatment ofonychomycosis. Antimicrob Agents Chemother 60:1035–1039. https://doi.org/10.1128/AAC.01739-15.

3. Ghannoum M, Chaturvedi V, Diekema D, Ostrosky-Zeichner L, Rennie R,Walsh T, Wengenack N, Fothergill A, Wiederhold N. 2016. Multilabora-tory evaluation of in vitro antifungal susceptibility testing of dermato-phytes for ME1111. J Clin Microbiol 54:662– 665. https://doi.org/10.1128/JCM.03019-15.

4. Long L, Hager C, Ghannoum M. 2016. Evaluation of the efficacy ofME1111 in the topical treatment of dermatophytosis in guinea pigmodel. Antimicrob Agents Chemother 60:2343–2345. https://doi.org/10.1128/AAC.03073-15.

5. Takahata S, Kubota N, Takei-Masuda N, Yamada T, Maeda M, AlshahmiMM, Abe S, Tabata Y, Maebashi K. 2016. Mechanism of action of ME1111,a novel antifungal agent for topical treatment of onychomycosis. Anti-microb Agents Chemother 60:873– 880. https://doi.org/10.1128/AAC.01790-15.

6. Clinical and Laboratory Standards Institute. 2008. Reference method forbroth dilution antifungal susceptibility testing of filamentous fungi;approved standard—2nd ed. CLSI document M38-A2. Clinical Labora-tory Standards Institute, Wayne, PA.

7. Nishiyama Y, Hasumi Y, Ueda K, Uchida K, Yamaguchi H. 2005. Effects ofmicafungin on the morphology of Aspergillus fumigatus. J Electron Mi-crosc (Tokyo) 54:67–77. https://doi.org/10.1093/jmicro/dfh100.

8. Nishiyama Y, Uchida K, Yamaguchi H. 2002. Morphological changes ofCandida albicans induced by micafungin (FK463), a water-solubleechinocandin-like lipopeptide. J Electron Microsc (Tokyo) 51:247–255.https://doi.org/10.1093/jmicro/51.4.247.

9. Cecchini G. 2003. Function and structure of complex II of the respira-tory chain. Annu Rev Biochem 72:77–109. https://doi.org/10.1146/annurev.biochem.72.121801.161700.

10. Hagerhall C. 1997. Succinate: quinone oxidoreductases: variations on aconserved theme. Biochim Biophys Acta 1320:107–141. https://doi.org/10.1016/S0005-2728(97)00019-4.

11. Rosen T, Stein Gold L. 2016. Antifungal drugs for onychomycosis: effi-cacy, safety, and mechanisms of action. Semin Cutan Med Surg 35(Suppl3):S51–S55. https://doi.org/10.12788/j.sder.2016.009.

12. Nishiyama Y, Asagi Y, Hiratani T, Yamaguchi H, Yamada N, Osumi M.1992. Morphological changes associated with growth inhibition ofTrichophyton mentagrophytes by amorolfine. Clin Exp Dermatol 17(Suppl1):13–17.

FIG 6 TEM photographs of a hyphal cell treated with MIC (0.25 �g/ml) of ME1111 for 24 h showinghyphae with plasmolysis (A) and a degenerated cell (B). Bar � 1 �m.

Nishiyama et al. Antimicrobial Agents and Chemotherapy

January 2017 Volume 61 Issue 1 e01195-16 aac.asm.org 4

on January 9, 2017 by TE

IKY

O U

NIV

http://aac.asm.org/

Dow

nloaded from

13. Nishiyama Y, Asagi Y, Hiratani T, Yamaguchi H, Yamada N, Osumi M.1991. Ultrastructural changes induced by terbinafine, a new antifungalagent, in Trichophyton mentagrophytes. Jpn J Med Mycol 32:165–175.https://doi.org/10.3314/jjmm.32.165.

14. Nishiyama Y, Asagi Y, Hiratani T, Yamaguchi H, Yamada N, Osumi M.1991. Effect of amorolfine, a new antifungal agent, on the ultrastructureof Trichophyton mentagrophytes. Jpn J Med Mycol 32:227–237.

15. Nishiyama Y, Maebashi K, Asagi Y, Hiratani T, Yamaguchi H, Yamada N,Taki A, Ji XR, Osumi M. 1991. Effect of SS717 on the ultrastructure ofTrichophyton mentagrophytes as observed by scanning and transmissionelectron microscopy. Jpn J Med Mycol 32:43–54. https://doi.org/10.3314/jjmm.32.43.

16. Ohmi T, Sakumoto S, Niwano Y, Kanai K, Tanaka T, Suzuki T, Uchida M,Yamaguchi H. 1992. Effect of NND-318 on the ultrastructure of Tricho-

phyton mentagrophytes as observed by scanning and transmission elec-tron microscopy. Jpn J Med Mycol 33:339 –348. https://doi.org/10.3314/jjmm.33.339.

17. Tatsumi Y, Nagashima M, Shibanushi T, Iwata A, Kangawa Y, Inui F, SiuWJ, Pillai R, Nishiyama Y. 2013. Mechanism of action of efinaconazole, anovel triazole antifungal agent. Antimicrob Agents Chemother 57:2405–2409. https://doi.org/10.1128/AAC.02063-12.

18. Vanden Bossche H. 1988. Mode of action of pyridine, pyrimidine andazole antifungals, p79 –119. In Berg D, Plempel M (ed), Sterol biosynthe-sis inhibitors, pharmaceutical and agrochemical aspects. Ellis Horwood,Chichester, England.

19. Vanden Bossche H, Marichal P. 1992. Azole antifungals: mode of action,p 25– 40. In Yamaguchi H, Kobayashi GS, Takahashi H (ed), Recentprogress in antifungal chemotherapy. Marcel Dekker, New York, NY.

Morphological Changes in T. mentagrophytes by ME1111 Antimicrobial Agents and Chemotherapy

January 2017 Volume 61 Issue 1 e01195-16 aac.asm.org 5

on January 9, 2017 by TE

IKY

O U

NIV

http://aac.asm.org/

Dow

nloaded from