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

Abstract

Abbreviations: ETEC, enterotoxigenic Escherichia coli; LT, Escherichia c o l i h e a t -l a b i l e enterotoxin; CT, cholera toxin; GM 1, monosialoganglioside; ELISA, enzyme-linked immunosorbent assay AbstractEscherichia 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; Ser 6 3 to Ty r 6 3 in LTS63Y and Glu 11 0 , G l u 11 2 were deleted in LT 11 0 / 112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB 5 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 m LTs 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 Ser 63 to Ty r 6 3 or deletion of Glu 11 0 and Glu 11 2 eliminate the toxicity of LT without a change of AB 5 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.

Oral Administration of Recombinant Cholera Toxin Subunit B Inhibits IL-12-Mediated Murine Experimental (Trinitrobenzene Sulfonic Acid) Colitis

“…In contrast, the B subunit, the part of the molecule that binds to the cell surface via G M1 (or other gangliosides) and facilitates entry of the holotoxin into the cell, has an as yet an uncertain adjuvant effect. This arises from the fact that the adjuvant activity of the A subunit is largely retained in modified toxins in which the ribosyltransferase activity has been greatly attenuated by site-specific mutagenesis (33,34). Thus, many earlier studies conducted with purified B subunit are suspect since purified B subunit may contain sufficient amounts of A subunit to reflect A subunit effects rather than B subunit effects (8,9).…”

The mucosal adjuvanticity of two nontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes

“…These mutant LTs were shown to be immunogenic in a previous study and induce high titers of LT-specific antibodies following IG and IN immunization (Park et al, 1999). In the present study, the mutant LTs showed no ADP-ribosyltransferase activity, but retained adjuvant activity.…”

“…We previously showed that both mutant LTs were immunogenic following either IG or IN administration, and that antibody titers induced by IN immunization were slightly higher than those induced by IG immunization (Park et al, 1999). In the present study, the abilities of mutant LTs to act as mucosal adjuvants were assessed by IG immunization in mice.…”

“…These observations suggest that mutations at key positions in the active site may affect ribosyltransferase activity, and that the specific amino acid used to replace wild-type residues may affect binding affinities for NAD or substrates. We constructed two novel LT mutants by point mutation of serine at amino acid position 63 to tyrosine (LTS63Y), and double deletion of glutamic acid at positions 110 and 112 (LT∆110/112) (Park et al, 1999). Modification of these residues, which are located at the putative ADPribosyltransferase active center, has been shown to inactivate ADP-ribosyltransferase activity and enterotoxicity.…”

Cell-Free Systems Enable the Production of AB5 Toxins for Diagnostic Applications

“…Thus, the infected intestinal cell secretes chloride and water, leading to typical symptoms of diarrhea. In vitro assays confirmed the activation of the adenylate cyclase and showed that Chinese hamster ovary (CHO) target cells were elongated after intoxication with Ctx and LT, suggesting morphological changes attributed to the increased cAMP level [10,11].…”