FIG 2.

Effect of cysteine on toxin production in different C. difficile strains. (A) Cytotoxicity assays on Vero cells. Twofold serial dilutions of intracellular bacterial crude extracts were performed, and the dilutions were added to a 96-well plate of confluent Vero cells. The toxin titer corresponds to the lowest dilution of C. difficile crude extracts required for >50% cell rounding. Cytotoxicity results are presented as the ratio of the toxin titers of bacterial cells grown in the presence of cysteine (PYC) to those of bacterial cells grown in the absence of cysteine (PY). (B) TcdA dot blot analysis. The crude extracts of C. difficile strains (200 ng for strains 630Δerm, M7404, and M7404 complemented with pDLL17-tcdC and 20 ng for strain VPI 10463) were probed with anti-TcdA antibodies as described in Materials and Methods. The results presented are representative of crude extracts tested from at least three independent experiments. (C) Transcript levels of the tcdR, tcdA, and tcdB genes in strain 630Δerm grown in the presence or absence of cysteine. Results are presented as the ratio of the mRNA level (arbitrary units) of each gene in bacterial cells grown in the presence of cysteine (PYC) to that of each gene in cells grown in the absence (PY) of cysteine. The results are the averages from at least three independent experiments, and error bars are the standard deviations from the mean values. The statistical analysis was performed by using a t test (tcdA and tcdR) or a Mann-Whitney test (tcdB).