Characterization of hybrid toxins produced in Escherichia coli by assembly of A and B polypeptides from type I and type II heat-labile enterotoxins

Abstract

The genes encoding the individual A and B polypeptides of the type I enterotoxin LTp-I and type II enterotoxins LT-HIa and LT-IIb were cloned and tested for complementation in Escherichia coli. Each gene encoding an A polypeptide was cloned into pACYC184, and each gene encoding a B polypeptide was cloned into the compatible plasmid Bluescript KS+. In addition, operon fusions representing all combinations of A and B genes were constructed in Bluescript KS+. Extracts from strains of E. coli expressing each combination of A and B genes, either from compatible plasmids or from operon fusions, were tested for immunoreactive holotoxin by radioimmunoassays and for toxicity by Y1 adrenal cell assays. Biologically active holotoxin was detected in each case, but the toxicity of extracts containing the hybrid toxins was usually less than that of extracts containing the wild-type holotoxins. The ganglioside-binding activity of each holotoxin was tested, and in each case, the B polypeptide determined the ganglioside-binding specificity. The A and B polypeptides of the type II heat-labile enterotoxins were also shown to form holotoxin in vitro without exposure to denaturing conditions, in contrast to the polypeptides of the type I enterotoxins that failed to form holotoxin in vitro under comparable conditions. These findings suggest that type I and type II enterotoxins have conserved structural features that permit their A and B polypeptides to form hybrid holotoxins, although the B polypeptides of the type I and type II enterotoxins have very little amino acid sequence homology.The heat-labile enterotoxins of Escherichia coli and Vibrio cholerae constitute a family of proteins that are related in structure and function (22,33). They are classified into two serogroups (9,21,(37)(38)(39). Antisera against the enterotoxins in serogroup I do not neutralize the toxins in serogroup II, and vice versa. Serogroup I includes cholera toxin (CT) and the E. coli type I heat-labile enterotoxins (LT-I). LTh-I and LTp-I are antigenically cross-reacting variants of LT-I produced by E. coli strains from humans and pigs, respectively (6, 23). Serogroup II includes the E. coli type II heat-labile enterotoxins (LT-II), with antigenic variants designated LT-

Ganglioside Structure Dictates Signal Transduction by Cholera Toxin and Association with Caveolae-like Membrane Domains in Polarized Epithelia

“…Sulfo-NHS-biotin ( Pierce Chemical Co. , Rockford, IL) was used to label purified CT and LTIIb as previously described ( Lencer et al, 1992 ). Antibodies used included polyclonal rabbit antiserum against CT A and B subunits ( Lencer et al, 1995 b ) and LTIIb holotoxin ( Connell and Holmes, 1992 ) and monoclonal antibodies raised against CT B and A subunits ( Holmes and Twiddy, 1983 ) and LTIIb B and A subunits (as described in Holmes and Twiddy, 1983 ). Monoclonal antibody 11E5 is specific for the B subunit of LtIIb, and 3C4 is specific for the A subunit of LTIIb.…”

Heterogeneity of detergent-insoluble membranes from human intestine containing caveolin-1 and ganglioside GM1

“…Polyclonal rabbit antisera against CT and LTIIb were previously described (16,24). Recombinant LTIIb was prepared and purified as previously described (8,37). All secondary antibodies and chemical reagents were from Sigma Chemical (St. Louis, MO) unless otherwise specified.…”

Endochitinase Is Transported to the Extracellular Milieu by theeps-Encoded General Secretory Pathway ofVibrio cholerae

“…Preparation of culture supernatants and periplasmic extracts. Culture supernatants and periplasmic extracts of bacterial cultures having an optical density at 600 nm of between 4.1 and 5.6 for late-log-phase cultures and between 4.5 and 5.4 for stationary-phase cultures were obtained as previously described (9,11). GM 1 ELISA for CT. Methods to measure CT in samples using ganglioside GM 1 -dependent enzyme-linked immunosorbent assay (ELISA) and a rabbit anti-CT antiserum were previously described (11).…”