Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin

Abstract

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.

Klebsiellaspp. as Nosocomial Pathogens: Epidemiology, Taxonomy, Typing Methods, and Pathogenicity Factors

“…As in many enterobacteria, and as has been especially well studied in E. coli, other factors have also been demonstrated in Klebsiella spp. Although the production of cytotoxins (102,127,149,150,229), enterotoxins (95,(122)(123)(124)(125)148) and hemolysin (4-6, 17) has been sporadically described, these features probably play a rather minor role in Klebsiella.…”

In vitro transfer of Ent plasmid from Escherichia coli to Shigella dysenteriae and Salmonella

“…The results described in this paper show that an Although there is no direct evidence for this so far, it may be demonstrated by rigorous epidemiological studies. Reports of the isolation of LT-like toxin from Salmonella [10], Klebsiella pneumoniae [1 1] and Enterobacter cloacae [11], and of ST-like toxin from Yersinia enterocolitica [12], Klebsiella pneumoniae [ 13], Enterobacter cloacae [ 14] and Sh. dysenteriae [15] support the possibility of transfer of E. coli Ent plasmids.…”

Cross-neutralization of heat-stable enterotoxin activity of enterotoxigenic Escherichia coli and of Yersinia enterocolitica

“…Enterotoxigenic Escherichia coli produces two kinds of enterotoxin, heat-labile (LT) and heat-stable (ST). ST-like enterotoxin is reported to be produced by other bacteria, such as Yersinia enterocolitica [1 ], Klebsiella pneumoniae [2] and Enterobacter cloacae [3]. Previously, we suggested an immunological relatedness between E. coli ST and Y. enterocolitica ST by demonstrating the neutralization of crude E. coli ST by anti-Y, enterocolitica ST. As a confirmation, we demonstrate in this paper the cross-neutralization of purified ST preparations of E. coli and Y. enterocolitica.…”