Exp Mol Med.  1999 Jun;31(2):101-107.

Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin

• Park EJ
• Chang JH
• Kim JS
• Chung SI
• Yum JS

Abstract

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.

Keyword

mutant LT (mLT); ADP-ribosyltransferase activity; cAMP; immunogenicity; secretory IgA (sIgA)

MeSH Terms

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