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 λB8 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[λB8] induced fluid secretion in rabbit intestinal loops, while lysates of E. coli DH5α[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 PGE 2 , and bound to ganglioside G M1 . The biological activity associated with the cloned enterotoxin was neutralized by monospecific antiserum to cholera toxin (CT). Immunoblots of pC1 and λB8 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 λB8 DNA expressed two unique proteins (30 and 11 kDa) similar to that of pJM17.