Characterization of hybrid toxins produced in Escherichia coli by assembly of A and B polypeptides from type I and type II heat-labile enterotoxins.
The genes encoding the individual A and B polypeptides of the type I enterotoxin LTp-I and type II enterotoxins LT-IIa 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.
Connell
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TD
Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814.
Holmes
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RK
D001427:Bacterial Toxins / Q000633:toxicity*; D003001:Cloning, Molecular; D004768:Enterotoxins / Q000633:toxicity*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005732:Gangliosides / Q000378:metabolism; D005816:Genetic Complementation Test; D006027:Glycosides / Q000138:chemical synthesis / Q000378:metabolism / Q000633:toxicity; D066298:In Vitro Techniques; D010957:Plasmids; D011863:Radioimmunoassay; D011995:Recombination, Genetic; D014315:Triterpenes / Q000138:chemical synthesis / Q000378:metabolism / Q000633:toxicity
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D005732:Gangliosides; D006027:Glycosides; D014315:Triterpenes; C001883:stichoposide; C027538:heat-labile enterotoxin, E coli