Biological and immunological characterization of a cloned cholera toxin-like enterotoxin from Salmonella typhimurium

Abstract

A chromosomal DNA fragment, encoding an enterotoxin gene of Salmonella typhimurium Q1, was cloned into bacteriophage EMBL3 and plasmid vector pBR322. The recombinant clones lambda B8 and pC1 were identified using a synthetic oligonucleotide probe made to the B subunit region of the cholera toxin gene (ctx). Cell lysates of Escherichia coli VCS257 [lambda B8] induced fluid secretion in rabbit intestinal loops, while lysates of E. coli DH5 alpha [pC1] failed to elicit an enterotoxic response in this model. Both lysates and partially purified preparations elongated Chinese hamster ovary (CHO) cells, elevated cellular cAMP and PGE2, and bound to ganglioside GM1. The biological activity associated with the cloned enterotoxin was neutralized by monospecific antiserum to cholera toxin (CT). Immunoblots of pC1 and lambda B8 lysates probed with anti-CT, exhibited a 30 kDa protein similar to that of pJM17, which carried the ctx gene. Under non-dissociating conditions, anti-CT immunoblots of the same lysates revealed two proteins, one corresponding in size to the holotoxin and the other to CT-A. When analysed by DNA-directed protein synthesis in vitro, both pC1 and lambda B8 DNA expressed two unique proteins (30 and 11 kDa) similar to that of pJM17.