Investigating the structure-activity relationship of Escherichia coli heat labile enterotoxin (LT) by site-directed mutagenesis.

摘要

Heat-labile enterotoxin of enterotoxigenic Escherichia coli and cholera toxin of Vibrio cholerae are structurally, functionally, and immunologically homologous proteins. In addition to their role as the main virulence factors responsible for diarrheal diseases, they also function as mucosal adjuvants for co-administered antigens. Numerous attempts have been made to dissociate the toxicity of these two molecules from their adjuvant properties. Unfortunately, different observations reported by various groups using a variety of animal models with different antigens, different mutants of LT or CT, and different routes of immunization have complicated rather than clarified that understanding. The objective of this thesis was to investigate the structure-activity relationship of LT using various classes of LT mutants in order to better understand the underlying mechanisms of LT-mediated adjuvanticity with respect to toxicity and other biological functions. Mutations at each of the three functional domains of LT, namely, the active site, the protease site, and the ER retention signal sequences, were constructed and characterized. Each LT mutant was examined for cytotoxicity in the Y-1 Adrenal Tumor Cell assay, enterotoxicity in the Patent Mouse model, the ability to induce cAMP accumulation in a non-polarized cell system, and the ability to function as an adjuvant when administered either intranasally or orally as assessed by induction of antigen-specific humoral and cell-mediated immune responses.;It is obvious that LT and CT have significant potential to facilitate the development of entirely new classes of vaccines for mucosal delivery. The results presented in this thesis make it clear that different mutants of LT have different properties, which vary depending upon the nature of the mutation and the route of delivery. Specifically, those mutants that retain the ability to induce cAMP elicit quantitatively and qualitatively different responses than do those mutants that lack this enzymatic function. The link between induction of cAMP and enterotoxicity must also be addressed. With the adjuvant effective dose of native LT and any of the mutants of LT in humans as yet unknown, it may be possible to establish a reasonable therapeutic window for use, in humans, of LT mutants that retain some level of residual enterotoxicity.