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. 2023 Dec 15;24(24):17519.
doi: 10.3390/ijms242417519.

Combating Black Fungus: Using Allicin as a Potent Antifungal Agent against Mucorales

Christina Schier  1   2 , Martin C H Gruhlke  3   4 , Georg Reucher  2 , Alan J Slusarenko  1   3 , Lothar Rink  2
Affiliations

Combating Black Fungus: Using Allicin as a Potent Antifungal Agent against Mucorales

Christina Schier et al. Int J Mol Sci. .

Abstract

Invasive fungal (IF) diseases are a leading global cause of mortality, particularly among immunocompromised individuals. The SARS-CoV-2 pandemic further exacerbated this scenario, intensifying comorbid IF infections such as mucormycoses of the nasopharynx. In the work reported here, it is shown that zygomycetes, significant contributors to mycoses, are sensitive to the natural product allicin. Inhibition of Mucorales fungi by allicin in solution and by allicin vapor was demonstrated. Mathematical modeling showed that the efficacy of allicin vapor is comparable to direct contact with the commercially available antifungal agent amphotericin B (ampB). Furthermore, the study revealed a synergistic interaction between allicin and the non-volatile ampB. The toxicity of allicin solution to human cell lines was evaluated and it was found that the half maximal effective concentration (EC50) of allicin was 25-72 times higher in the cell lines as compared to the fungal spores. Fungal allicin sensitivity depends on the spore concentration, as demonstrated in a drop test. This study shows the potential of allicin, a sulfur-containing defense compound from garlic, to combat zygomycete fungi. The findings underscore allicin's promise for applications in infections of the nasopharynx via inhalation, suggesting a novel therapeutic avenue against challenging fungal infections.

Keywords: COVID-19; allicin; amphotericin B; antimycotic; glutathione reductase; mucormycosis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Macroscopic and microscopic growth characteristics of (A) Rhizopus stolonifer and (B) Mucor racemosus. Growth was studied on Czapek agar (CZA), malt extract agar (MEA), potato dextrose agar (PDA), Sabouraud glucose agar (SGA) and oatmeal agar (OA). Spore suspension (20 µL 5 × 105 spores per mL) was pipetted three times onto different solid media and incubated at 22 °C for 40 h. Surface growth and colony reverse were documented photographically. Fungal reproductive structures were examined using transmitted light microscopy (light microscope DMRBE, Leica, Wetzlar, Germany).
Figure 2
Figure 2
Drop test showing allicin sensitivity of Mucorales strains. 20 µL of serial diluted spore suspensions of (A) Rhizopus stolonifer and (B) Mucor racemosus (stock: 5 × 105 spores per mL) were plated onto PDA medium containing allicin at the concentrations indicated. Double distilled water (H2Odd) was used as a control. Growth was documented after 24 h of incubation at 22 °C. For a better visualisation of growth, the contrast of the photos was increased.
Figure 3
Figure 3
Allicin inhibits spore germination in a concentration-dependent manner. Spore containing agar was prepared by adding spore suspension to 20 mL PDA medium (50 °C) to obtain a final concentration of 105 spores per mL. Triplicates were made for each fungus. R. stolonifer was incubated for 24 h and M. racemosus for 48 h at 22 °C. (A) For agar diffusion tests, three holes (Ø = 0.6 cm) were punched into the agar after solidification and filled with 40 µL of 10 mM, 20 mM aqueous allicin solution or H2Odd, respectively. (B) n = 3, error bars show standard deviation. Same letters indicate no significant difference (p > 0.05) in a One-Way ANOVA with Holm–Šidák method. (C) For vapour treatments, H2Odd, 96% ethanol or aqueous allicin solution (25 mM resp. 50 mM) was applied to the Petri dish lid after agar solidification. (D) n = 3, error bars show standard deviation. Same letters indicate no significant difference (p > 0.05) in a One-Way ANOVA with Holm–Šidák method.
Figure 4
Figure 4
Allicin inhibits spore germination more effectively than amphotericin B. (A) Fungal spore suspension was mixed with sample solution. Spore germination of R. stolonifer was studied microscopically after 24 h, that of M. racemosus after 48 h. The ratio of germinated spores to total spore number was determined (H2Odd or 1% DMSO was set to 100% spore germination). The mean values of three biological replicates with three technical replicates each are shown. Sample solution concentration was plotted logarithmically. Error bars represent the standard deviation. (B) n = 9, sample comprises three biological replicates with three technical replicas each, error bars show standard deviation. Significant differences between species in a Student’s t-test are marked by asterisks (p < 0.001 = ***).
Figure 5
Figure 5
Visualization of different models for the deposition of allicin via the gas phase. The realistic distribution in an inverted inhibition zone test according to [38] (top center) was simplified by assuming an equal allicin distribution across the inhibition zone (ciz, bottom center). Equal distribution across the entire agar (left) and closely confined distribution directly over the droplet (right) correspond to the boundary concentrations clo and cup in the calculations.
Figure 6
Figure 6
Comparison of GR activity and cellular glutathione content of R. stolonifer and M. racemosus. (A) Mean glutathione reductase activity and (B) cellular glutathione in fungal cell lysates were measured in a glutathione reductase recycling assay (Section 4.11 and Section 4.12). Protein content in cell lysates was determined using a Bradford assay (Section 4.10). GR activity was standardized to protein content. n = 9, sample comprises three biological replicates with three technical replicas each, error bars show standard deviation. Significant differences in a Student’s t-test are marked by asterisks (p < 0.001 = ***).
Figure 7
Figure 7
Allicin enhances the antifungal activity of amphotericin B. Allicin was placed in a 96 well plate along with ampB and 50 µL spore suspension was added. Final concentrations of the sample solutions per well are given. Spore germination of R. stolonifer was studied microscopically after 24 h, that of M. racemosus after 48 h. The ratio of germinated spores to total spore number was determined (Spore germination without antifungal agent was set as 100% spore germination. Mean values of three biological replicates are shown. Inhibition of spore germination is represented by color: red—inhibition, green—no inhibition.
Figure 8
Figure 8
Dose-dependent toxicity of allicin on human epithelial cell lines. A549 (A) and WISH (B) cell lines were incubated with 100 µL allicin-containing media (20 µM–2500 µM) for 1 h. The medium was changed and 20 µL MTT (5 mg/mL) was added. Cells were incubated at 37 °C with 5% CO2. MTT-containing medium was removed and formazan was dissolved with dimethyl sulfoxide (DMSO). The absorbance at 570 nm (reference: 690 nm) was measured (SpectraMax i3x Multi-Mode microplate reader, Molecular Devices, San José, CA, USA). n = 3–4, mean values are shown, error bars show standard deviation.
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