Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores
Cd630 spores were incubated with 0.2% Tc, 50mM glycine and different concentrations of EGTA (A). Cd630 spores were incubated with the indicated combinations of 0.2% Tc, 50 mM glycine, 1 mM CaCl2, and 50 μM EGTA (B). EGTA (1mM) pretreated Cd630 spores and non-treated Cd630 spores were incubated with the indicated combinations of chelex-treated germinants, Tc (0.2%) and glycine (50mM) (C). Activation of SleC was assessed by western blot. Cd630 spores were incubated 37°C for 15 minutes with 1% Tc and the indicated combinations of 50mM glycine and 1mM EGTA (D). Germination was tracked by loss of OD at 37°C over the course of one hour. Germination assays were performed in triplicate. Germination assays and western blots are representative of 3 independent spore preps. Error bars are mean plus or minus SD.