Separation and Purification of Enterotoxins From a Strain Escherichia Coli Pathogenic for Pigs

Mitchell, I. de G.; Tame, M. J.; Kenworthy, R.

doi:10.1099/00222615-7-4-439

Cited by 13 publications

(13 citation statements)

References 12 publications

(10 reference statements)

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“…In fact K. pneumoniae ST is heat-stable, methanolsoluble, mercaptoethanol-sensitive, active in the SMA, but inactive in the 18-h ileal loop assay. The pH pattern is also similar to that described for STa toxins (25,26). Furthermore, the intestinal mucosa (27).…”

Section: St Heat-stable Enterotoxinsupporting

confidence: 61%

Characteristics and Mechanism of Action of a Heat-Stable Enterotoxin Produced by Klebsiella pneumoniae from Infants with Secretory Diarrhea

et al. 1989

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ABSTRACT. Escherichia coli heat-stable enterotoxinsIt is now becoming clear that ST enterotoxins are elaborated (ST) are classified into STa and STb according to their not only by E. coli strains but also by other bacteria. We recently physicochemical and biologic characteristics. STa induces described the physicochemical properties of an E. coli ST-like diarrhea, activating the guanylate cyclase-cGMP system. enterotoxin produced by Citrobacter freundii and found that it ST-like enterotoxins can be produced by bacteria other cross-reacted with MAb raised against pure E. coli ST (7). Klipthan E. coli, including Klebsiellapneumoniae. A Klebsiella stein et al. have investigated the biochemical (8) and immuno-ST has previously been shown to share some chemical and logic (9) properties of an ST produced by Klebsiella pneumoniae immunologic characteristics with E. coli ST. Aiming to and found it similar to the ST produced by E. coli. define better the nature of Klebsiella ST, we have screened Aiming to define better the nature and the characteristics 3f 237 children with diarrhea and 179 controls for ST-pro-Klebsiella ST, we have systematically searched for Klebsiella in ducing Klebsiella, using the SMA. We detected 26 Kleb-the stools of children with diarrhea. Two SMA-positive strains siella strains from patients, two of which were positive in of K. pneumoniae have been isolated from two infants with the SMA, and 36 from controls, all negative for ST. A diarrhea. In this report, we describe our studies of the physicopartial purification was performed using an acetone precip-chemical characteristics, the mechanism of action, and the lack itation followed by ultrafiltration and gel filtration tech-of cross-reactivity of K. pneumoniae enterotoxic activity with niques. Klebsiella toxin was heat-stable, methanol-soluble, anti-E. coli ST MAb. sensitive to mercaptoethanol, active at acid pH values, but not at pH >8. The time course of Klebsiella toxin in the SMA resembled that of E. coli STa. Klebsiella ST caused MATERIALS AND METHODS

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Section: St Heat-stable Enterotoxinsupporting

confidence: 61%

Characteristics and Mechanism of Action of a Heat-Stable Enterotoxin Produced by Klebsiella pneumoniae from Infants with Secretory Diarrhea

et al. 1989

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“…There was an insufficient quantity of the G-50 eluate of the UM-2 filtrate, and therefore the G-25 eluate of this material was examined. Heat treatment, 100 C for 30 min, had no effect on the activity of the retentate G-50 eluate but it did reduce, but not abolish, the activity of the G-25 eluate of the filtrate. Acid treatment, consisting of lowering the pH to 1 for 4 h by the addition of concentrated HCI, and incubation with either Pronase for 24 h or trypsin for 3 h, did not reduce the enterotoxic activity of the purified fractions.…”

Section: Resultsmentioning

confidence: 85%

“…E. coli enterotoxins are aerobic growth, either stationary or agitated (6,14,18,19,25,26,30,36). We are unaware of any published information concerning the effect of anaerobic culture conditions on E. coli enterotoxin production; in our laboratory, we find that anaerobic growth yields E. coli ST only (C. S. Lee, and F. A. Klipstein, unpublished observations).…”

Section: Resultsmentioning

confidence: 90%

“…Whether the equally potent protein peak that was present in the void volume of the G-50 eluate of the UM-2 filtrate represents another separate enterotoxin or, as is probably more likely, a polymer of the larger-sized toxin is unknown. Evidence obtained by Mitchell et al (30), who also used UF and GF separation techniques, suggests that an E. coli enterotoxin with a molecular weight in the range of 6,000 fragments to produce a smaller-sized toxin.…”

Section: Resultsmentioning

confidence: 99%

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Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin

Klipstein¹,

Engert²

1976

Infect Immun

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The enterotoxic material in cell-free growth preparations of Klebsiella pneumoniae serotype 5 was purified by sequential ultrafiltration and gel filtration (GF) procedures and the fractions were assayed for enterotoxic activity by determining their ability to induce in vivo net water secretion in the rat jejunum. Whole-cell lysates were inactive. Anaerobic broth culture conditions yielded a 10-fold increase in toxin production over aerobic conditions. Enterotoxic activity was absent in the UM-10 retentate of the broth filtrate but present in both the retentate and filtrate of the UM-2 membrane. GF of the two UM-2 ultrafiltration fractions through a Sephadex G-25 column yielded an active eluate, whose potency was increased by 10or 200-fold, in or adjacent to the void volume. When subsequently passed through a G-50 column, these pools eluted at a K,. of between 0.4 and 0.6 and were further increased in potency by twoor fivefold. A second equally potent fraction was also recovered in the void volume of the G-50 eluate of the UM-2 filtrate; this may represent a polymer. Progressive purification by GF was associated with an increased protein and decreased carbohydrate content of the most active fractions. The most active G-50 eluate of the UM-2 retentate had a minimal effective enterotoxic dose of 5 mg/ml and that of the filtrate was less than 0.1 ,ug/ml. Heating the active GF eluates to 100 C for 30 min did not abolish enterotoxic activity and lowering the pH to 1 or incubation with either Pronase or trypsin had no effect on activity. These observations indicate that K. pneumoniae heat-stable enterotoxin is probably a single toxin with an apparent molecular weight in the range of5,000. The elution characteristics during GF as well as the chemical composition of the most purified enterotoxin fractions indicate that the toxin is not associated with endotoxin.

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“…A recent report describes the use of a defined medium containing salts, glucose, and vitamins for the production and purification of porcine LT (14). Since in LT purification studies it may be advantageous to start with preparations containing as little interfering proteins as possible, the effect of elimination of large-molecular-weight substances from CAYE-2 medium was studied.…”

Section: Downloaded Frommentioning

confidence: 99%

Factors influencing heat-labile Escherichia coli enterotoxin activity

Mundell¹,

Anselmo²,

Wishnow³

1976

Infect Immun

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In this study, conditions for production, detection, and storage of heat-labile Escherichia coli enterotoxin (LT) in culture filtrates from E. coli H-10407 were defined by using the adrenal tumor cell assay system. An enriched medium containing 0.6% yeast extract, 2% Casamino Acids, and 0.25% glucose buffered at pH 8.5 produced the highest LT activity of the various test media. In E. coli strain H-10407, LT activity was markedly decreased if the initial pH of the culture media was reduced to pH 7.5 or less. In contrast to E. coli P-263, if strain H-10407 was grown in the presence of mitomycin C there was no increase in LT production. Crude-culture filtrates containing LT can be stored at 4°C for several days without an appreciable loss of activity; however, for long-term storage lyophilization or freezing at -70°C is recommended.

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Separation and Purification of Enterotoxins From a Strain Escherichia Coli Pathogenic for Pigs

Cited by 13 publications

References 12 publications

Characteristics and Mechanism of Action of a Heat-Stable Enterotoxin Produced by Klebsiella pneumoniae from Infants with Secretory Diarrhea

Characteristics and Mechanism of Action of a Heat-Stable Enterotoxin Produced by Klebsiella pneumoniae from Infants with Secretory Diarrhea

Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin

Factors influencing heat-labile Escherichia coli enterotoxin activity

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