Analyses of mutant analogs of Escherichia coli heat-labile enterotoxin (LT), a close relative of cholera toxin (CT), have continued. Several residues have been identified by site-directed mutagenic analyses as playing an essential catalytic role in the ADP-ribosyltransferase activity of these toxins. Substitutions at these positions appear to allow retention of native conformation. These mutations, in conjunction with other previously described substitutions may be a suitable target for the generation of, multiply substituted mutant proteins that are devoid of toxic/enzymatic activity and that retain native conformation. The wild-type LT and mutant analogs are also being used as delivery systems for heterologous antigens and peptides, particularly those from Chlamydia trachomatis, to exploit the effectiveness of LT in potentiating mucosal immune responses. We have also continued to investigate the mechanism(s) by which these toxins are internalized and gain access to intracellular G protein substrates. Studies using various metabolic inhibitors indicate some interesting differences in response to LT and CT among various target cell lines or types. The data also indicate that microtubule-mediated processes are not involved in toxin trafficking and indeed enhance their cytotonic/cytotoxic effects under some circumstances. Other studies indicate that occupation of a non-acidified endocytic compartment is involved in the relevant intoxication pathway. The data collectively are consistent with an internalization pathway that involves the trans-Golgi region or another late endosomal compartment that bypasses lysosomal or other acidified endocytic vacuoles.
