Cyclic Adenosine Monophosphate and Alteration of Chinese Hamster Ovary Cell Morphology: a Rapid, Sensitive In Vitro Assay for the Enterotoxins of Vibrio cholerae and Escherichia coli

Abstract

The major limitation to our understanding of the clinical importance of enterotoxigenic Escherichia coli in diarrheal illness has been the lack of a simple rapid assay for the enterotoxin produced by certain E. coli. On the basis of the activation of adenylate cyclase by heat-labile enterotoxin of E. coli (LT) and by cholera toxin (CT) in intestinal and other tissues, cultured Chinese hamster ovary (CHO) cells with known morphological responses to dibutyryl cyclic adenosine 5'-monophosphate (AMP) were exposed to these enterotoxins. Crude culture filtrates of LT-producing E. coli and CT stimulated cyclic AMP accumulation and cell elongation in CHO cells. The similarity of time course, concentration dependence, and potentiation by phosphodiesterase inhibitors suggested cyclic AMP mediation of the morphological change. Heat inactivated CT and LT in this system. Choleragenoid inhibited CT; antiserum against CT inhibited both enterotoxin effects. In contrast to culture filtrates of 16 strains of E. coli known to produce LT, culture filtrates from 13 E. coli that do not produce LT did not alter CHO cell morphology. The morphological change is a simple, specific assay for these enterotoxins and detect 3 x 10-17 mol of CT or a 1: 250 dilution of crude culture filtrate of LT-producing E. coli 334.

The occurrence of colonisation factor (CF) in enterotoxigenic Escherichia coli

“…They had been isolated from faecal specimens of patients with diarrhoea in 14 different countries and belonged to 41 different O : H serogroups in 19 different recognised O groups. All the strains were shown to produce either heat stable enterotoxin (ST) or heat-labile enterotoxin (LT), or both, by means of the suckling mouse test for ST [4] and the Y1 [5] and CHO [6] cell tissue culture tests for LT (Table 1).…”

Salmonella typhimurium enterotoxin epitopes shared among bacteria

“…One of the E. coli strains that exhibited a 29-kDa protein antigen was negative for the production of both LT-I and heat-stable (STa) enterotoxins, while the other produced only STa. We were unable to detect a Stn-like toxin from these E. coli by biological assays, such as elongation of Chinese hamster ovary (CHO) cells [10]. This might be explained by the low amount of this enterotoxin being synthesized or by evolutionary loss of biological potency of the protein.…”

Enterotoxins from gram-negative bacteria relevant for veterinary medicine

“…To synthesize labelled proteins, tritiated amino acids were added to the cell-free mixture which contained plasmid DNA. The synthesized product was positive in the Chinese hamster ovary cell assay (Guerrant et al, 1974). The effect could be antagonized by antiserum against purified cholera toxin (anticholeragen) and by heterologous antitoxin prepared in rabbits against semipurified toxin from the enterotoxigenic E. coli strain P263 (Dorner et al, 1976).…”

“…Marked morphological changes of the cell (rounding) are observed and evaluated. An analogous method works with Chinese hamster ovary cells (Guerrant et al, 1974). In this case elongation of the cells is evaluated.…”