Antifungal activity of phlorotannins against dermatophytes and yeasts: approaches to the mechanism of action and influence on Candida albicans virulence factor

Abstract

In the last few decades, fungal infections, particularly nosocomial, increased all around the world. This increment stimulated the search for new antifungal agents, especially those derived from nature. Among natural products, those from marine sources have gained prominence in the last years. Purified phlorotannins extracts from three brown seaweeds (Cystoseira nodicaulis (Withering) M. Roberts, Cystoseira usneoides (Linnaeus) M. Roberts and Fucus spiralis Linnaeus) were screened for their antifungal activity against human pathogenic yeast and filamentous fungi. The purified phlorotannins extracts from the studied seaweeds displayed fungistatic and fungicidal activity against yeast and dermatophytes, respectively, pointing to their interest as anti-dermatophyte agent. C. albicans ATCC 10231 was the most susceptible among yeast, while Epidermophyton floccosum and Trichophyton rubrum were the most susceptible among dermatophytes. Since the antifungal mechanism constitutes an important strategy for limiting the emergence of resistance to the commercially available agents, the mechanism of action of purified phlorotannins extracts was approached. C. nodicaulis and C. usneoides seem to act by affecting the ergosterol composition of the cell membrane of yeast and dermatophyte, respectively. F. spiralis influenced the dermatophyte cell wall composition by reducing the levels of chitin. Phlorotannins also seem to affect the respiratory chain function, as all of the studied species significantly increased the activity of mitochondrial dehydrogenases and increased the incorporation of rhodamine 123 by yeast cells. Phlorotannins from F. spiralis inhibited the dimorphic transition of Candida albicans, leading to the formation of pseudohyphae with diminished capacity to adhere to epithelial cells. This finding is associated with a decrease of C. albicans virulence and capacity to invade host cells and can be potentially interesting for combined antifungal therapy, namely for the control of invasive candidiasis.

Figure 1. Effect of purified phlorotannins extracts from F. spiralis in the dimorphic transition of C. albicans ATCC 10231 (Untreated control cells - A1; cells treated with extract at MIC/32 – B1), in the adherence of the yeast to the epithelial cells (Untreated control cells – A2; cells treated with extract at MIC/32 - B2) and in the germ tube formation (C).

Arrows show a constriction resulting from an incomplete budding, where the bud remains attached to the mother cell, originating pseudohyphae. Levels of magnification are as shown: Bars, 50 µm (A1 and B1) and 250 µm (A2 and B2). Results are expressed as mean (±SD) of three independent assays. * P<0.05, ** P<0.01, ***P<0.001 (C).

Figure 2. Ergosterol concentration in C. albicans ATCC 10231 (A) and T. rubrum FF5 (B) cells treated with purified phlorotannins extracts (1/2 to 1/8 of the MIC), determined by HPLC-DAD (detection wavelength 280 nm).

Results are expressed as mean (±SD) of three independent assays. * P<0.05, ** P<0.01, ***P<0.001.

Figure 3. Mitochondrial activity of C. albicans ATCC 10231 cells treated with different concentrations of phlorotannins purified extracts.

Results are expressed as the percent change of MTT reduction using the nontreated cells as control (mean (±SD) of three independent assays performed in duplicate). For concentrations lower than MIC/1024 the mitochondrial activity was similar to the untreated cells. Arrows show the formazan salts produced by C. albicans mitochondria. *P<0.05, **P<0.01, ***P<0.001.

Figure 4. Percentage of Rhodamine 123 fluorescence of C. albicans ATCC 10231 cells treated with different concentrations of purified phlorotannins extracts, relative to control.

For concentrations lower than MIC/1024 the percentage of fluorescence was similar to the untreated cells. Results are expressed as mean (±SD) of three independent assays performed in duplicate. * P<0.05, ** P<0.01, ***P<0.001.