Role of receptor binding in toxicity, immunogenicity, and adjuvanticity of Escherichia coli heat-labile enterotoxin

Abstract

The role of receptor binding in the toxicity, immunogenicity, and adjuvanticity of the heat-labile enterotoxin of Escherichia coli (LT) was examined by comparing native LT and LT(G33D), a B-subunit receptor binding mutant, with respect to the ability to bind to galactose and to GM1, toxicity on mouse Y-1 adrenal tumor cells, the ability to stimulate adenylate cyclase in Caco-2 cells, enterotoxicity in the patent mouse model, and oral immunogenicity and adjuvanticity. In contrast to native LT, LT(G33D) was unable to bind to the galactosyl moiety of Sepharose 4B or GM1 but did retain the lectin-like ability to bind to immobilized galactose on 6% agarose beads. LT(G33D) had no enterotoxicity in the patent mouse model but exhibited residual toxicity on mouse Y-1 adrenal tumor cells and had an ability equivalent to that of native LT to stimulate adenylate cyclase in Caco-2 cells (5,000 versus 6,900 pmol per mg of protein). In addition, LT(G33D) was unable to serve as an effective oral adjuvant for induction of immunoglobulin G or A directed against a coadministered antigen. Furthermore, LT(G33D) elicited negligible serum and mucosal antibody responses against itself. These data indicate that the toxicity, immunogenicity, and oral adjuvanticity of LT are dependent upon binding of the B subunit to ganglioside GM1.

Safety and immunogenicity of bacterial and tobacco plant cell line derived recombinant native and mutantEscherichia coliheat-labile toxin in chickens

“…In addition, these data also suggest that SLT102 cell extracts form both LT-B subunit pentamers and AB5 pentamers at an estimated ratio of 20:1. The lack of LTK63/LTB enzymatic activity in the Y1 bioassay is consistent with previous results in other expression and animal model systems in which LTK63 was shown to be 1000-fold to 10,000-fold less toxic than wild-type LT (Guidry et al, 1993;Pizza et al, 1994;Del Guidice et al, 1998). Additionally, the absence of LTK63/LTB enzymatic activity in vitro is unlikely due to the cell extraction and processing method.…”

“…Wild-type LT and LT-B recombinant subunit proteins purified from E. coli (kindly provided by J. Clements, Tulane University New Orleans, LA, USA) were used as reference antigens in enzyme-linked immunosorbent assays (ELISAs) or used as an immunogen source for rabbit antisera production. For bird studies, wild-type recombinant LT was purified from the E. coli strain DH5a transformed with pCS96 as previously described (Guidry et al, 1993;Uesaka et al, 1994).…”

“…Serial two-fold dilutions of SLT102 test samples were transferred to cell plates and incubated for overnight at 378C. The end-point titre of LT toxin is the amount of protein required to obtain 50% cytotoxicity (cell death) as previously described (Guidry et al, 1993).…”

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“…This is in apparent contradiction to the blood group protection hypothesis, however, it is not clear if the capability to stimulate cAMP production is equivalent to toxicity. In this respect, the study by Guidry et al is of significant interest, who demonstrated that an LT variant lacking GM1-binding properties (LT-G33D) was non-toxic in vivo, while retaining the capability to stimulate intracellular cAMP levels in vitro in Caco-2 intestinal-epithelial cells [71]. Similar studies using CT-G33D have also been undertaken [72].…”

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“…Both LT and CT also function as mucosal adjuvants, stimulating antibody production against codelivered antigens. The ganglioside-binding activity of the LT-B pentamer is required both for its mucosal immunogenicity and for the adjuvanticity of the holotoxin (Guidry et al, 1997).…”