FIG 5.
Importance of Asn26 for the holin function of TcdE142. (A) The expression of mutant forms of TcdE142 was induced with 50 ng/ml ATc for 3 h. After induction, 10-fold serial dilutions were prepared, and 5 μl of the dilutions was spotted for overnight growth at 37°C. (B) Membrane proteins prepared from C. difficile strains expressing various TcdE142 constructs were separated by SDS-PAGE and probed with anti-TcdE antibodies and anti-ATPase antibodies. The mutant forms of TcdE142 and TcdE WT along with TcdE140 were induced with 10 ng/ml ATc for 3 h, the culture supernatants were collected, and the toxin titers were determined by an ELISA. Data are expressed as the means ± standard errors from three replicate samples. Student's t test was used for statistical analysis for comparing the effects of TcdE142N26A, TcdE142N26V, and TcdE142N26Q to those of TcdE142. Results of statistical analyses performed to compare the effects of all test samples to the effect of the vector alone were significant (P < 0.001).