FIG. 1.
Three mutations impair the secretion of LT from ETEC. (A) Representative immunoblots of TCA-precipitated total culture samples, adjusted for CFU, showing induced expression of wild-type LT (WT), LT[L25E], LT[Q3K], and LT[E11K] in strain jf570. Blots were probed with cross-reactive anti-CT antibody. (B) Strains expressing wild-type (WT) and the indicated mutant toxins were fractionated to isolate cell-free supernatant (Sup't) and periplasm (Pp). Each fraction was tested for toxin levels by GM1 ELISA, with wild-type levels set to 100%. Supernatant levels were normalized to CFU, and periplasm levels were normalized to alkaline phosphatase activity. *, P < 0.01; **, P < 0.005; ***, P < 10−6 compared to wild-type (n ≥ 3); n.d., not determined. For some experiments, cultures were grown in the presence of a protease inhibitor cocktail (+ prot. inh.) (n ≥ 2). Inset: representative immunoblot showing levels of wild-type LT (WT) and LT[L25E] secreted from jf570. Cell-free supernatant fractions were precipitated with TCA, and samples were adjusted for CFU. Blots were probed with cross-reactive anti-CT antibody.