Sophorolipid Biosurfactant Can Control Cutaneous Dermatophytosis Caused by Trichophyton mentagrophytes

Abstract

Trichophyton mentagrophytes, a zoophilic species, is one of the most frequently isolated dermatophytes in many parts of the world. This study investigated the efficacy of a sophorolipid (SL-YS3) produced by Rhodotorula babjevae YS3 against dermatophytosis caused by T. mentagrophytes. SL-YS3 was characterized by gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography, coupled with electrospray mass spectrometry (UPLC-ESI-MS). SL-YS3 comprised of six different fatty acids as the hydrophobic components of constituent congeners and sophorose as the hydrophilic component. Inhibitory effects of purified SL-YS3 against hyphal growth was found to be 85% at a 2 mg ml^-1 concentration, and MIC was 1 mg ml^-1. Microscopic examination with scanning electron microscopy (SEM), atomic force microscopy, and confocal laser scanning microscopy (CLSM) revealed that SL-YS3 exerts its effect by disrupting cell membrane integrity causing cell death. SL-YS3 was also effective in reducing the biofilms formed by T. mentagrophytes, which was observed spectrophotometrically with crystal-violet staining and further validated with SEM and CLSM studies of treated biofilms. In vivo studies in a mouse model of cutaneous dermatophytosis involving macroscopic observations, percent culture recovery from skin samples, and histopathological studies showed that SL-YS3 could effectively cure the infected mice after 21 days of topical treatment. Terbinafine (TRB) was used as a standard drug in the experiments. We demonstrate, for the first time, the antidermatophytic activity of a sophorolipid biosurfactant. The findings are suggestive that SL-YS3 can be formulated as a novel antifungal compound to treat cutaneous mycoses caused by T. mentagrophytes.

Keywords: Trichophyton mentagrophytes; antibiofilm activity; biosurfactant; dermatophytosis; sophorolipid; topical application; ultramicroscopy.

FIGURE 1

Gas chromatography profiles (TIC) of the fatty acid methyl esters (FAMEs) of the (A) standard sophorolipid (SL-S) and (B) the sophorolipid produced by Rhodotorula babjevae YS3 (SL-YS3).

FIGURE 2

Mass spectra of the fatty acids present in the constituent congeners derivatized to fatty acid methyl esters (FAMEs) (A) commercially available sophorolipid 1′,4′′-Sophorolactone 6′,6′′-diacetate (SL-S) (B) sophorolipid produced by R. babjevae YS3 (SL-YS3) as identified by GC–MS analysis.

FIGURE 3

The glycan part of the biosurfactant produced by R. babjevae YS3 (SL-YS3) is sophorose. (A) UPLC chromatogram (TIC) of the sugar component of the sophorolipid under study (SL-YS3, black) in comparison with that of the standard sophorolipid (SL-S, red). The single peak with the same retention time (Rt) indicates the same sugar composition of SL-YS3 and SL-S. The positive ESI-MS spectra of (B) SL-S and (C) SL-YS3 exhibited sodiated adducts of glucose monomer [M + Na] + and dimer [2M + Na-H₂O] + at m/z 203 and m/z 365 respectively.

FIGURE 4

Antifungal activity of sophorolipid produced by R. babjevae YS3 (SL-YS3) against Trichophyton mentagrophytes in vitro. (A) Dose-dependent effect of purified SL-YS3, sophorolipid standard 1, 4′′-sophorolactone 6′,6′′-diacetate (SL-S), terbinafine (TRB) against spores of T. mentgrophytes as determined according to CLSI guidelines (M38 A-2). (B) Inhibition of mycelial growth of T. mentagrophytes by purified SL-YS3. Data are percentage of the mean of triplicates with respect to control. Error bars represent standard deviation. Different letters within each concentration indicate significantly different values as per ANOVA-LSD.

FIGURE 5

Microscopic observation of the effect of the sophorolipid produced by R. babjevae YS3 (SL-YS3) on mycelial integrity of T. mentagrophytes. (A) Scanning electron micrographs of the mycelial damage induced by SL-YS3 and terbinafine (TRB) treatment. Magnification = 5000, scale bar = 2 μm. (B) Atomic force microscopic images to visualize the topographical changes induced by SL-YS3 and TRB treatment (1 mg ml^–1) in comparison to the untreated control mycelia. Images were acquired after 24 h of treatment. Total scanning area for the images are 100 μm × 100 μm. (C) Confocal microscopic images to determine the uptake of propidium iodide (PI) in the mycelia treated with SL-YS3 and TRB at a concentration of 1 mg ml^–1 (w/v) as compared to the untreated mycelia. Scale bar = 25 μm.

FIGURE 6

Dispersal of pre-formed biofilms of T. mentagrophytes by the sophorolipid produced by R. babjevae YS3 (SL-YS3), sophorolipid standard 1, 4′′-sophorolactone 6′,6′′-diacetate (SL-S), and terbinafine (TRB). Data are percentage of the mean of triplicates with respect to control. Error bars represent standard deviation. Different letters within each concentration indicate significantly different values as per ANOVA-LSD.

FIGURE 7

Scanning electron microscopic (SEM) images of the effect of sophorolipid produced by R. babjevae YS3 and terbinafine (TRB) on mature biofilms of T. mentagrophytes. Scale bar = 20 μm (2000), 2 μm (5000).

FIGURE 8

Confocal laser scanning microscopic (CLSM) images of the effect of sophorolipid produced by R. babjevae YS3 and terbinafine (TRB) on mature biofilms of T. mentagrophytes. Scale bar = 25 μm.

FIGURE 9

Therapeutic efficacy of sophorolipid produced by R. babjevae YS3 (SL-YS3) on experimentally induced dermatophytosis in mice infected with T. mentagrophytes. (A) Macroscopic observation of the infected sites of the mice treated with 1 mg ml^–1 (w/v) SL-YS3 and terbinafine (TRB) at the end of 21 days of treatment in comparison to the uninfected and infected, untreated control. (B) Culture recovery (%) of T. mentagrophytes from skin samples collected at different intervals of treatment period (0, 7, 14, 21 days) and cultured to determine the therapeutic efficacy in eradication of the fungus. Error bars represent standard deviation. Different letters indicate significantly different values as per ANOVA-LSD. (C) Histopathology of skin samples collected from sophorolipid (SL-YS3) and terbinafine (TRB) treated groups, stained with hematoxylin & eosin (H&E) and Periodic acid- Schiff (PAS) stains. SG, sweat glands; Ep, epidermis; HF, hair follicle; BV, blood vessel; CT, collagenase tissue; IC, inflammatory cells. Magnification = 10 ×, Scale bar = 100 μm.

FIGURE 10

Schematic depicting the mechanism of action of SL-YS3 on mycelial membrane of T. mentagrophytes. Owing to the amphiphilic nature, SL-YS3 might alter the permeability of the cell membrane of T. mentagrophytes, interfering with the ion transport leading to cell membrane rupture and cell death.