Capric acid secreted by S. boulardii inhibits C. albicans filamentous growth, adhesion and biofilm formation

Abstract

Candidiasis are life-threatening systemic fungal diseases, especially of gastro intestinal track, skin and mucous membranes lining various body cavities like the nostrils, the mouth, the lips, the eyelids, the ears or the genital area. Due to increasing resistance of candidiasis to existing drugs, it is very important to look for new strategies helping the treatment of such fungal diseases. One promising strategy is the use of the probiotic microorganisms, which when administered in adequate amounts confer a health benefit. Such a probiotic microorganism is yeast Saccharomyces boulardii, a close relative of baker yeast. Saccharomyces boulardii cells and their extract affect the virulence factors of the important human fungal pathogen C. albicans, its hyphae formation, adhesion and biofilm development. Extract prepared from S. boulardii culture filtrate was fractionated and GC-MS analysis showed that the active fraction contained, apart from 2-phenylethanol, caproic, caprylic and capric acid whose presence was confirmed by ESI-MS analysis. Biological activity was tested on C. albicans using extract and pure identified compounds. Our study demonstrated that this probiotic yeast secretes into the medium active compounds reducing candidal virulence factors. The chief compound inhibiting filamentous C. albicans growth comparably to S. boulardii extract was capric acid, which is thus responsible for inhibition of hyphae formation. It also reduced candidal adhesion and biofilm formation, though three times less than the extract, which thus contains other factors suppressing C. albicans adherence. The expression profile of selected genes associated with C. albicans virulence by real-time PCR showed a reduced expression of HWP1, INO1 and CSH1 genes in C. albicans cells treated with capric acid and S. boulardii extract. Hence capric acid secreted by S. boulardii is responsible for inhibition of C. albicans filamentation and partially also adhesion and biofilm formation.

Conflict of interest statement

Competing Interests: This work was partly supported by a grant from Biocodex, France, which initiated research on Sacchromyces boulardii. However the funding ended long before the authors were able to identify the active compound described in the paper. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. GC-MS analysis of active fraction separated from S. boulardii extract.

Peak at 3.98 min corresponds to presence of caproic acid (C6:0), peak at 5.13 min to 2-phenylethanol, peak at 5.32 min to caprylic acid (C8:0), peak at 6.64 min to capric acid (C10:0).

Figure 2. ESI-MS pattern of active fraction showing ions at m/z 115.076, 143.107 and 171.138.

Figure 3. UPLC chromatograms of 2-phenylethanol and fatty acids as compared with S. boulardii extract monitored at 220 nm.

Solid line represents 96 µg of S. boulardii extract, dashed line 5.1 µg of 2-phenylethanol, dotted line 17.4 µg of caproic acid (C6:0), dash-dot-dot line 21.4 µg of caprylic acid (C8:0), dash-dot line 25.8 µg of capric acid (C10:0).

Figure 4. C. albicans morphology incubated for 2 h (left panel) and 24 h (right panel) in RPMI-1640 at 37°C in the presence of the compounds.

Control sample contained 1% methanol, other samples contained 160 µg/ml of S. boulardii extract, 17.9 µg/ml of caprylic acid (C8:0), 45.3 µg/ml of capric acid (C10:0) or 4.2 µg/ml of 2-phenylethanol. MIX – mixture of all the compounds at concentrations indicated above. Bar, 10 µm.

Figure 5. Effect of the compounds on C. albicans adhesion.

(A) control sample with 1% methanol, (B) 160 µg/ml of S. boulardii extract, (C) 37.8 µg/ml of caproic acid (C6:0), (D) 17.9 µg/ml of caprylic acid (C8:0), (E) 45.3 µg/ml of capric acid (C10:0), (F) 4.2 µg/ml of 2-phenylethanol, (G) mixture of all these compounds at concentrations indicated above. Statistical analysis was performed using paired Student t-test. P values<0.05 were considered significant. Double stars 0.001<P<0.01, triple stars P<0.001.

Figure 6. Relative expression of 3 mRNAs of C. albicans cells treated with 45.3 µg/ml of capric acid (C10:0) and 160 µg/ml of S. boulardii extract.

Statistical analysis was performed using one sample Student t-test. P values<0.05 were considered significant. Single stars denote 0.01<P<0.05, double stars 0.001<P<0.01, triple stars P<0.001.