LGG mediates protection against <i>C</i>. <i>albicans</i> infection in TR146 monolayer.

<p>LDH activity in response to <i>C</i>. <i>albicans</i> infection was quantified in cell culture supernatant 6 h (A) and 2 h (B) post infection. n = 3 ***<i>p</i><0.001.</p

Preincubation of TR146 cells with LGG impairs major virulence attributes of <i>C</i>. <i>albicans</i>.

<p>(A-C) Monolayers of TR146 cells were preincubated with PBS or LGG for 12 h. In some conditions LGG was then removed by rinsing the cells with PBS (LGG off) and/or glucose was added to the medium (5 mg/ml). Then, epithelial cells were infected with <i>C</i>. <i>albicans</i> cells. (A) Adhesion of <i>C</i>. <i>albicans</i> to epithelial cells was analyzed 1 h post infection. Invasiveness (B) and hyphal growth (C) was measured 3 h post infection. (D) LDH release by TR146 cells was quantified 6 h post infection. (A-D) n = 3 *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

<i>L</i>. <i>rhamnosus</i> GG (LGG) protects against <i>C</i>. <i>albicans</i> infection (adapted from [29]).

<p>(A) <i>C</i>. <i>albicans</i>-induced release of lactate dehydrogenase (LDH). Semi-thin sections of <i>C</i>. <i>albicans</i>-infected RHOEs pretreated either with PBS (B) or LGG (C). Images are representative for three individual experiments. Scale bars equal 100 μm. Supernatants of RHOEs were further analyzed for cytokines. Content of interleukin-8 (IL8; D), granulocyte macrophage colony-stimulating factor (GM-CSF; E) and IL1α (F) was quantified. n = 3–7 *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

<i>C</i>. <i>albicans</i> viability in response to LGG.

<p>(A and B adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184438#pone.0184438.ref029" target="_blank">29</a>]). (A and B) FUN1-staining of <i>C</i>. <i>albicans</i>, grown in absence (A) or presence of LGG (B). Fluorescence staining was performed after 24 h of co-culture. Images are representative of three individual experiments. Scale bars equal 10 μm. Live fungal cells are indicated by green fluorescence and a well-defined vacuole structure. Red fluorescence inside these vacuoles indicates metabolic activity (red arrows). Yellowish fluorescence indicates dead fungi (yellow arrows). LGG stains green (white arrow). (C and D) Transmission electron microscopy of <i>C</i>. <i>albicans</i>. <i>C</i>. <i>albicans</i> was grown for 24 h in absence (C) or presence (D) of LGG. Cell organelles were counted in 25 cells per condition and experiment. Nuclei are highlighted in brown, organelles in blue. Micrographs are representative for three individual experiments. Scale bars equal 0.2 μm. (E) Metabolic activity of <i>C</i>. <i>albicans</i> grown in absence or presence of LGG, Amphotericin B (Ampho B; 0.1 μg/ml) served as control. (C, D and E) n = 3 *<i>p</i><0.05.</p

Impact of LGG on <i>C</i>. <i>albicans</i> ergosterol content and glucose concentration in cell culture supernatants.

<p>(A) Extracts of <i>C</i>. <i>albicans</i> grown on TR146 monolayers pretreated with either PBS or LGG were analyzed for their ergosterol content. (B) Exemplary selected ion chromatogram of one sample, where LGG-preincubated cells were infected with <i>C</i>. <i>albicans</i>, containing cholestane (<i>m/z</i> 217), cholesterol (<i>m/z</i> 368) from TR146 cells and ergosterol (<i>m/z</i> 363) from <i>C</i>. <i>albicans</i>. (C) Monolayers of TR146 cells were preincubated with PBS or LGG 12 h prior to infection with <i>C</i>. <i>albicans</i> cells. Amount of glucose was evaluated 6 h post infection. (D) Monolayers of TR146 cells were preincubated with PBS or LGG 12 h prior to infection. Directly before infecting epithelial cells with <i>C</i>. <i>albicans</i>, glucose was added to the experiment (5 mg/ml). LDH activity was quantified 6 h post infection. n = 3 *<i>p</i><0.05, ***<i>p</i><0.001.</p

Regulation of <i>C</i>. <i>albicans</i> gene expression in response to LGG.

<p>Monolayers of TR146 cells were preincubated with PBS or LGG for 12 h. Then, epithelial cells were infected for 6 h with <i>C</i>. <i>albicans</i> cells. For analysis of mRNA expression this experiment was performed three times. (A) Fold changes of mRNA expression in <i>C</i>. <i>albicans</i> in response to LGG. (B) Gene ontology enrichment of metabolic pathways in <i>C</i>. <i>albicans</i> in response to LGG. Corresponding sets of up- and down-regulated genes were mapped with the „GO term finder” at CGD to biological processes. X-fold enrichment is calculated as ratio of percentages of the cluster frequency of tested gene set and the cluster frequency of genomic background. * <i>FOX2</i>, <i>ICL1</i> and <i>MLS1</i> are associated with fatty acid catabolism and glyoxylate cycle. n = 3.</p

CFU of LGG and <i>C</i>. <i>albicans</i> used for different experiments.

<p>CFU of LGG and <i>C</i>. <i>albicans</i> used for different experiments.</p

Influence of experimental supernatants on LDH activity.

<p>Influence of experimental supernatants on LDH activity.</p