Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin.
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.
Park
E J
EJ
Mogam Biotechnology Research Institute, Kyonggido, Korea.
Chang
J H
JH
Kim
J S
JS
Chung
S I
SI
Yum
J S
JS
D019943:Amino Acid Substitution; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000633:toxicity*; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004768:Enterotoxins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000633:toxicity*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005260:Female; D007082:Ileum / Q000378:metabolism; D018928:Immunity, Mucosal; D007071:Immunoglobulin A, Secretory / Q000097:blood; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000633:toxicity
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D007071:Immunoglobulin A, Secretory; D011994:Recombinant Proteins; C027538:heat-labile enterotoxin, E coli; D000242:Cyclic AMP; D011065:Poly(ADP-ribose) Polymerases