Destination and Specific Impact of Different Bile Acids in the Intestinal Pathogen Clostridioides difficile

Abstract

The anaerobic bacterium Clostridioides difficile represents one of the most problematic pathogens, especially in hospitals. Dysbiosis has been proven to largely reduce colonization resistance against this intestinal pathogen. The beneficial effect of the microbiota is closely associated with the metabolic activity of intestinal microbes such as the ability to transform primary bile acids into secondary ones. However, the basis and the molecular action of bile acids (BAs) on the pathogen are not well understood. We stressed the pathogen with the four most abundant human bile acids: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA). Thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM), and electron microscopy (EM) were employed to track the enrichment and destination of bile acids in the bacterial cell. TLC not only revealed a strong accumulation of LCA in C. difficile, but also indicated changes in the composition of membrane lipids in BA-treated cells. Furthermore, morphological changes induced by BAs were determined, most pronounced in the virtually complete loss of flagella in LCA-stressed cells and a flagella reduction after DCA and CDCA challenge. Quantification of both, protein and RNA of the main flagella component FliC proved the decrease in flagella to originate from a change in gene expression on transcriptional level. Notably, the loss of flagella provoked by LCA did not reduce adhesion ability of C. difficile to Caco-2 cells. Most remarkably, extracellular toxin A levels in the presence of BAs showed a similar pattern as flagella expression. That is, CA did not affect toxin expression, whereas lower secretion of toxin A was determined in cells stressed with LCA, DCA or CDCA. In summary, the various BAs were shown to differentially modify virulence determinants, such as flagella expression, host cell adhesion and toxin synthesis. Our results indicate differences of BAs in cellular localization and impact on membrane composition, which could be a reason of their diverse effects. This study is a starting point in the elucidation of the molecular mechanisms underlying the differences in BA action, which in turn can be vital regarding the outcome of a C. difficile infection.

Keywords: Clostridioides difficile; adhesion; bile acids; flagella; toxin A.

FIGURE 1

Flagella abundance. (A) Immunofluorescence (IF) of C. difficile challenged with the four different bile acids (CA, cholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; LCA, lithocholic acid) and untreated cells co-cultivated on Caco-2 cells. IF staining: green – anti-C. difficile antibody, red – Rhodamine-phalloidine and blue – DAPI, scale bar = 5 μm. (B) Scanning electron micrographs of exponential phase C. difficile cells challenged with BAs prior to co-cultivation with Caco-2 cells, scale bar = 1 μm.

FIGURE 2

Flagella protein expression. (A) Western Blot analysis of FliC and FliD expression in untreated cells and in cells stressed with the four different BAs with a Coomassie-stained gel in the upper left corner that was run as a loading control in parallel. Right (top): Average quantification of signal intensities of FliC as well as FliD bands of three replicates normalized to untreated condition. (B) Transcriptional profiling of fliC expression by Northern Blotting. Left (top): Methylene blue staining of RNA gel serving as a loading control. Left (bottom): Exemplary fliC-specific Northern Blot. Right: Three biological replicates were analyzed and normalized to the control condition. Standard deviation is indicated and values significantly different by a Student’s t-test compared to untreated cells are marked (***p-value <0.001).

FIGURE 3

Co-cultivation of bile acid stressed C. difficile with Caco-2 cells. (A) Scanning electron micrographs of co-cultivation of Caco-2 cells with untreated C. difficile and C. difficile challenged with four different bile acids (CA, cholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; LCA, lithocholic acid), scale bar = 5 μm. Red squares indicate zoomed in area shown below. (B) Adhesion assay after co-cultivation of Caco-2 cells with bile acid stressed C. difficile. Colony forming units of three independent experiments with three technical replicates each were determined and the number of adherent bacteria per Caco-2 cell was visualized. Standard deviation is indicated and values significantly different by a Student’s t-test compared to untreated cells are marked (***p-value <0.001).

FIGURE 4

Morphology of bile acid stressed C. difficile in stationary phase. (A) Scanning electron micrographs of untreated C. difficile (untreated) and C. difficile challenged with four different bile acids (CA, cholic acid, DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; LCA, lithocholic acid). Scale bar = 1 μm. Circles in DCA micrograph indicate “swellings.” (B) Average length of bacterial cells is given in μm. (C) Scanning electron micrographs of co-cultivated untreated C. difficile and C. difficile challenged with four different bile acids (CA, DCA, CDCA, and LCA) with Caco-2 cells, scale bar = 2 μm. (D) Adhesion assay after co-cultivation of Caco-2 cells with bile acid stressed C. difficile from stationary phase. Colony forming units of 3 independent experiments with three technical replicates each were determined and the number of adherent bacteria per Caco-2 cell was visualized. Standard deviation is indicated and values significantly different by a Student’s t-test compared to untreated cells are marked (***p-value <0.001).

FIGURE 5

Western Blot analysis of secreted toxin A. (A) C. difficile strain 630 (left) and strain R20291 (right) were cultivated for 72 h in the presence of four different bile acids (CA, cholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; LCA, lithocholic acid) and without any stress conditions (untreated). Secreted proteins were extracted from cultivation supernatant and an amount of 150 μg was separated via SDS-PAGE (loading control in the bottom) to subsequently detect toxin A by Western Blot analysis (top). (B) Three biological replicates were analyzed and related to the untreated condition. Standard deviations are indicated.

FIGURE 6

Thin layer chromatography (TLC) of C. difficile lipid extracts. (A) Lipids of exponentially growing cells and (B) stationary phase cells under control conditions (untreated) and challenged with different bile acids (CA, cholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; LCA, lithocholic acid) were extracted and separated via TLC. Hydrophobic species were stained with phosphomolybdic acid. References of the four bile acids were separated on the right side of each TLC plate. Two additional biological replicates for exponentially growing cells and stationary phase cells are provided Supplementary Figure 3.

FIGURE 7

Putative localization of various BAs in the C. difficile cell. Left: Transmission electron micrographs of C. difficile challenged with the four different bile acids (CA, cholic acid, DCA, deoxycholic acid, CDCA, chenodeoxycholic acid, LCA, lithocholic acid) and of untreated cells, all in exponential growth phase. Morphological changes are highlighted as white arrows. Scale bars, top 1 μm and bottom 200 nm. Middle: Scanning electron micrographs of C. difficile challenged with four different bile acids (CA, DCA, CDCA, and LCA) and of untreated cells, scale bar = 1 μm. Right top and center: Immunofluorescence (IF) of C. difficile challenged with BAs and stained with mouse anti-CA antibody (green), mouse anti-CDCA antibody (green), rabbit anti-DCA antibody (green), rabbit anti-LCA antibody (green) as well as DAPI (blue) and rabbit anti-C. difficile antibody (purple). Due to the same antibody host (rabbit), LCA samples were not counterstained with anti-C. difficile but with Nile red (red) and DCA challenged cells were only co-stained with DAPI. Scale bars = 5 μm. Right bottom: IF of TEM sections of C. difficile challenged with BAs and stained with anti-CA (green) and counterstained with anti-C. difficile (purple) and anti-LCA (green) counterstained with Nile red (red). An IF staining of TEM sections of DCA, CDCA and untreated samples was not possible. Scale bars = 5 μm.