Fig 1.
Extracellular concentration of toxin A (TcdA) excreted by C. difficile in CAM supplemented with different sugar derivatives, sugar alcohols and L-lactate.
Shown are concentrations of TcdA in ng/mgCDW in the supernatant of C. difficile 630Δerm grown in casamino acids medium (CAM, reference) and CAM supplemented with different sugar derivatives, sugar alcohols and L-lactate. The TcdA concentrations were determined by ELISA after 48 h of cultivation. All TcdA concentrations measured after cultivations in media with different additives were significantly altered compared to that determined in the control medium CAM without any additions (p-value < 0.01).
Fig 2.
Glucose concentrations in the supernatant of the different cultivations of C. difficile 630Δerm.
Shown are the glucose concentrations of the cultivations in CAM (no glucose addition) and CDMM (addition of 2 g/L glucose) in the growth medium (med), in the samples taken in the middle of the exponential phase (½ ODmax, exp) and at the beginning of the stationary phase (ODmax, stat). The concentrations were analyzed by an enzyme assay, results are based on four independent cultivations. *: not detected.
Fig 3.
Metabolic data of C. difficile 630Δerm in CAM and CDMM.
Shown are the extracellular metabolites in heatmaps as log2 fold changes of the different amino acids (A) or the fermentation products (B) of the cultivations in CDMM (2 g/L glucose) compared to CAM (no glucose) in the exponential (exp) and the stationary phase (stat). Grey squares indicate not detected metabolites (* not in CDMM, ** not in CAM, *** neither in CDMM nor CAM). The amino acids were analyzed by HPLC-FLD and the fermentation products by GC-MS. The intracellular metabolites were shown as scatterplots from the detected metabolites in the exponential (C) and the stationary phase (D) of cultivations in CAM and CDMM. Known metabolites significantly altered between the two conditions (p-value < 0.05) are labeled in orange. All experiments are based on four independent cultivations.
Fig 4.
Overview of adaptation of reductive and oxidative pathways in C. difficile by 2 g/L glucose.
Shown are data of transcriptome and metabolome analyses in the exponential (½ ODmax, left side) and stationary phase (ODmax, right side) comparing data from CDMM-cultures (2 g/L Glc) with CAM-cultures (without Glc). Pathway boxes represent the transcriptomic data, generally upregulated pathways are shown in red, while downregulated pathways are blue (-1 > log2 fold change > 1, p-value < 0.05). Metabolomic data are represented by the metabolite names and the pathway names inside the boxes, generally increased concentrations are shown in bold and red, while decreased concentrations are italics and blue (fold change > 1.5). Products of Stickland reactions are carboxylic acids with the same length of the corresponding amino acid in the reductive path and one carbon atom shorter than the corresponding amino acid (n-1) in the oxidative path.
Fig 5.
Scatterplots of the intracellular and extracellular metabolome analysis of C. difficile grown in absence and presence of L-lactate.
Shown are the detected intracellular metabolites of the exponential (A) and the stationary phase (B) samples as well as the detected extracellular metabolites of the exponential (C) and the stationary phase (D) samples of C. difficile 630Δerm cultivated in casamino acids medium without (CAM) or supplemented with 500 mg/L L-lactate (CAM+L). The majority of metabolite concentrations remained unchanged (p-value < 0.05) for the two tested conditions. Metabolites detected only in one condition were shown in boxes. All experiments are based on four independent cultivations.
Table 1.
Determination of D- and L-lactate in the culture supernatant of C. difficile.
Table 2.
Differently expressed genes in C. difficile cultivated in absence and presence of L-lactate.
Fig 6.
Influence of L-Lactate on NAD+/NADH ratio and intra- and extracellular toxin concentration.
Shown are the NAD+/NADH ratio (A), the intracellular (B) and the secreted (C) toxin A (grey) and B (blue) concentrations in CAM (dark color) and CAM+L (light color). All experiments are based on four independent cultivations. Single values of toxin A and B and of NAD+ and NADH are shown in S5 File. *: significant difference in TcdA concentration between the values of CAM and CAM+L (p-value < 0.05), Φ no toxin detected.