Interplay between Lactobacillus rhamnosus GG and Candida and the involvement of exopolysaccharides

Abstract

A number of clinical studies have shown protective effects of lactobacilli against Candida species in the gastrointestinal tract, the urogenital tract and the oral cavity, while others did not show clear effects. Evidence on the mode of action of lactobacilli against Candida is also still lacking. In this study, the anti-Candida activity of the model probiotic strain Lactobacillus rhamnosus GG was explored in different assays to determine molecular interactions. We found that L. rhamnosus GG was able to interfere with Candida growth, morphogenesis and adhesion. These three aspects of Candida's physiology are all crucial to its opportunistic pathogenesis. In follow-up assays, we compared the activity of L. rhamnosus GG wild-type with its exopolysaccharide (EPS)-deficient mutant and purified EPS to evaluate the involvement of this outer carbohydrate layer. Our data demonstrate that purified EPS can both interfere with hyphal formation and adhesion to epithelial cells, which indicates that EPS is part of a combined molecular mechanism underlying the antihyphal and anti-adhesion mechanisms of L. rhamnosus GG.

Figure 1

Inhibition of growth and hyphal induction of Candida albicans. (A) Well‐diffusion and spot assays of C. albicans in the presence of wild‐type L. rhamnosus GG (10⁸ CFU ml⁻¹), EPS mutant (EPS MT) (10⁸ CFU ml⁻¹), CFS, isolated EPS (2 mg ml⁻¹) and a 2% L‐lactic acid solution (n=4). Hexetidine (0.1%), miconazole (8 μg ml⁻¹) were used as positive controls and water as negative control. (B‐C) Hyphal induction of C. albicans (10⁶ cells ml⁻¹) during co‐incubation with LGG (10⁸ CFU ml⁻¹), its EPS mutant (EPS MT) (10⁸ CFU ml⁻¹) or isolated EPS. Asterisks indicate p‐values below 0.05 compared to C. albicans solely. (D) Evolution of the optical density of C. albicans and C. glabrata cultures in presence of EPS in different concentrations and a 2% L‐lactic acid solution (n=3). Hexetidine (0.1%), miconazole (8 μg ml⁻¹) were used as positive controls and water as negative control.

Figure 2

Inhibition of Candida adherence to epithelial cells. (A) Percentage of Candida albicans, C. glabrata, Lactobacillus rhamnosus GG WT and EPS mutant (EPS MT) adhered to VK2/E6E7 monolayers after 1 h of incubation. (B) Percentage of C. albicans, L. rhamnosus GG and EPS mutant adhered to Calu‐3 monolayers after 1 h of incubation. (C, E) Percentage of C. albicans or C. glabrata adhered to VK2/E6E7 monolayers when added in competition with L. rhamnosus GG WT, EPS mutant or L. rhamnosus GG EPS. (D, F) Percentage of C. albicans or C. glabrata adhered to Calu‐3 monolayers when added in competition with L. rhamnosus GG WT, EPS mutant or L. rhamnosus GG EPS. The results were normalized to adherence of C. albicans solely. In all tests, C. albicans and C. glabrata were used at an initial concentration of 10⁶ CFU, L. rhamnosus GG WT and EPS mutant at 10⁸ CFU, and purified EPS was added to a final concentration of 200 μg ml⁻¹. The percentages represent mean values of at least three biological repeats. Asterisks indicate p‐values below 0.05 when conditions were compared to each other.