Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.
Amino Acid Substitution ; Animal ; Bacterial Toxins/toxicity* ; Bacterial Toxins/metabolism ; Bacterial Toxins/immunology* ; Bacterial Toxins/genetics ; CHO Cells ; Cyclic AMP/metabolism ; Enterotoxins/toxicity* ; Enterotoxins/metabolism ; Enterotoxins/immunology* ; Enterotoxins/genetics ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli*/metabolism ; Escherichia coli*/genetics ; Female ; Hamsters ; IgA, Secretory/blood ; Ileum/metabolism ; Immunity, Mucosal ; Mice ; Mice, Inbred BALB C ; Mutagenesis, Site-Directed ; NAD+ ADP-Ribosyltransferase/metabolism ; Recombinant Proteins/toxicity ; Recombinant Proteins/metabolism ; Recombinant Proteins/immunology ; Recombinant Proteins/chemistry