Characterization of a partially purified methanol-soluble heat-stable Escherichia coli enterotoxin in infant mice

Abstract

While studying the involvement of cyclic adenosine 3',5 '-monophosphate (cAMP) in the fluid secretion caused by heat-stable enterotoxin (ST) from Escherichia coli P16 in infant mice, it was noted that the culture filtrate containing ST also contained large amounts of cAMP. The present paper details attempts to obtain a cAMP-free ST preparation. The organisms were grown in a defined medium, and the heated culture filtrate was concentrated by reverse osmosis. After methanol extraction of the filtrate, which removed 80% of the nonactive solids, the methanol-soluble ST was further purified by gel filtration through a Sephadex G-10 column. The first fraction recovered after gel chromatography contained ST with a negligible amount of cAMP. Treatment with methanol did not adversely affect the enterotoxic activity. Certain parameters of the infant mouse model have been investigated, and using our ST preparation it has been found that animals remain responsive up to 15 days of age with an optimum assay time of 2 h after toxin challenge.

Production of heat-stable, methanol-soluble enterotoxin by Yersinia enterocolitica

“…When assayed in suckling mice, Y. enterocolitica enterotoxin is highly resistant to heating. We observed no reduction in enterotoxic activity after 30 min at 100 or 1210C, unlike E. coli ST, which is inactivated after 30 min at 1210C (12). Yersinia enterotoxin is also heat stable (1000C for 15 min) when assayed in the rabbit leal loop assay (13).…”

“…Y. enterocolitica enterotoxin and E. coli ST both resist low pH. Although enterotoxic activity of E. coli ST is not affected by acid, it is destroyed by exposure to pH 11 for 4 h at 370C (5,12). Under similar conditions, Y. enterocolitica A2627 crude toxin was unaffected by pH 1 through pH 11. Kohler (8) and Mullan et al (12) found that E. coli ST could be extracted from culture supernatants using methanol without losing its heat and pH stability and enterotoxic activity in suckling mice.…”

“…All seven strains were enterotoxigenic in the suckling mouse model, and three of five isolates tested produced keratoconjunctivitis in the guinea pig eye model (Sereny test). Enterotoxin was detected in broth supernatant fluid after 12 h of incubation at 250C. The toxin was not inactivated by exposure to 121'C for 30 min or by storage at 4 or -40'C for at least 5 months.…”

Purification and partial characterization of heat-stable enterotoxin of enterotoxigenic Escherichia coli

“…Recently, Alderete and Robertson (1) reported the purification of ST from porcine strains of enterotoxigenic E. coli and some data on its properties. Mullan et al (15) also reported the partial purification of ST and demonstrated the existence of two types of ST molecules (3). Moreover, Field et al (9) studied the mode of action of a purified preparation of ST and found that cyclic guanosine 5'-monophosphate was involved in its action.…”

“…Culture supernatants were collected by centrifugation at 15,000 x g for 20 min. Protamine sulfate (50 mg/100 ml) was added in the cold, and the resulting precipitates were discarded after centrifugation at 15,000 x g for 15 min. The supernatant was filtered through an Amincon PM-10 membrane, and the filtrate was concentrated by using a rotary evaporator.…”