Simple method of purification of Escherichia coli heat-labile enterotoxin and cholera toxin using immobilized galactose

Uesaka, Yuri; Otsuka, Yoko; Wang, Qisheng; Yamasaki, Shinji; Yamaoka, Junichi; Kurazono, Hisao; Takeda, Yoshifumi

doi:10.1006/mpat.1994.1007

Cited by 81 publications

(52 citation statements)

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“…Purification of LT. LT was extracted from E. coli C600, a derivative of E. coli K-12 containing the EWD299 plasmid (6), and purified by one-step chromatography with an immobilized D-galactose column, using the protocol described by Uesaka et al (43).…”

Section: Methodsmentioning

confidence: 99%

EnterotoxigenicEscherichia coliModulates Host Intestinal Cell Membrane Asymmetry and Metabolic Activity

et al. 2009

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Enterotoxigenic Escherichia coli (ETEC) is a common cause of travelers' and postweaning diarrhea in humans and swine, respectively. The extent to which ETEC damages host cells is unclear. Experiments are presented that probe the ability of porcine ETEC isolates to induce apoptosis and cell death in porcine intestinal epithelial cells. Quantification of host phosphatidylserine exposure following ETEC infection suggested that ETEC induced changes in plasma membrane asymmetry, independent of the expression of the heat-labile enterotoxin. Significant host cell death was not observed. ETEC infection also caused a drastic inhibition of host esterase activity, as measured by calcein fluorescence. While ETEC infection resulted in activation of host caspase 3, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling of DNA double-strand breakage, indicative of late stages of apoptosis, was not observed. Camptothecin-induced apoptosis markedly increased subsequent ETEC adherence. Transfer of cell-free supernatants from apoptotic cells to bacterial inocula prior to infection of naïve cells increased the transcriptional activity of the regulatory region upstream of the K88ac operon and promoted subsequent adherence to host cells.Enterotoxigenic Escherichia coli (ETEC) causes childhood mortality and travelers' diarrhea in humans (36) and postweaning diarrhea in pigs (32). ETEC adherence to intestinal cells is mediated by proteinaceous, species-specific colonization factors (25). ETEC induces water and electrolyte loss from the intestine due to the activities of several enterotoxins, including the heat-labile enterotoxin (LT), the heat-stable toxins (ST), and the enteroaggregative E. coli heat-stable enterotoxin 1 (32).Bacterial pathogens often induce host cell apoptosis to promote their survival and dissemination (16,30). While several E. coli serotypes (1, 4, 9, 15) and toxins (3, 19) have been shown to induce apoptosis of epithelial cells, the extent to which ETEC damages host cells is unclear. The prototypic ETEC isolate H10407 was cytotoxic to but failed to induce apoptosis in the macrophage cell line J774 (as measured by DNA fragmentation) (24). The extent to which LT induces apoptosis has also received considerable attention due to interest in the receptor-binding subunit (LT-B) as a mucosal adjuvant. LT-B induces apoptosis of CD8 ϩ but not CD4 ϩ T cells (37). Some studies indicate that binding of LT-B to GM1 may be sufficient for triggering apoptosis of CD8 ϩ T cells (33), while others suggest that modified LT-B constructs (e.g., H57S) still able to bind GM-1 may fail to trigger apoptosis (14). Yet other studies suggest that LT-mediated apoptosis requires the ADP-ribosylation activity of the LT-A subunit (41).Overall, the roles of ETEC and LT in mediating apoptosis have not been defined clearly and remain somewhat controversial. The goals of this study were therefore to quantify the ability of ETEC to damage porcine intestinal epithelial cells, to clarify the role of LT in these processes, and to me...

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Section: Methodsmentioning

confidence: 99%

EnterotoxigenicEscherichia coliModulates Host Intestinal Cell Membrane Asymmetry and Metabolic Activity

et al. 2009

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“…Cells were harvested by centrifugation (6,000 ϫ g, 10 min at 4°C), washed in 10 ml of TEN (50 mM Tris, pH 7.4, 1 mM EDTA, 0.2 M NaCl), resuspended in 25 ml of TEN containing 1 mg/ml polymyxin B, and incubated at 37°C for 10 min. This extract was applied to a galactose affinity resin (Pierce) at room temperature, washed with 20 ml of TEN, and eluted with 1 M galactose in TEN (26). Pooled fractions were dialyzed extensively against HBSS and stored at Ϫ80°C.…”

Section: Methodsmentioning

confidence: 99%

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells

Lencer¹,

Constable²,

Moe³

et al. 1997

Journal of Biological Chemistry

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Cholera and Escherichia coli heat-labile toxins (CT and LT) require proteolysis of a peptide loop connecting two major domains of their enzymatic A subunits for maximal activity (termed "nicking"). To test whether host intestinal epithelial cells may supply the necessary protease, recombinant rCT and rLT and a protease-resistant mutant CTR192H were prepared. Toxin action was assessed as a Cl ؊ secretory response (Isc) elicited from monolayers of polarized human epithelial T84 cells. When applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 A/cm 2 ). Isc was reduced 2-fold, however, when toxins were applied to basolateral membranes. Pretreatment of wild type toxins with trypsin in vitro restored the "basolateral" secretory responses to "apical" levels. Toxin entry into T84 cells via apical but not basolateral membranes led to nicking of the A subunit by a serine-type protease. T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when applied to apical membranes. Thus, T84 cells express a serine-type protease(s) fully sufficient for activating the A subunits of CT and LT. The protease, however, is only accessible for activation when the toxin enters the cell via the apical membrane.

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“…Recombinant CT was prepared by cultivating B. choshinensis bearing pNCMO2-CTB-CTA at 30°C for 3 days [26] and purified from the culture supernatant by affinity chromatography using D-galactose immobilized agarose (Pierce Chemicals, Rockford, IL) [27]. For construction of recombinant CT-A and CT-B, a His-tag sequence derived from the pET28 vector (Novagen, Madison, WI) was introduced in front of each CT subunit gene (resulting in pNCMO2 his-CTA and pNCMO2 his-CTB, respectively).…”

Section: Brevibacillus Choshinensismentioning

confidence: 99%

Cholera toxin activates dendritic cells through dependence on GM1‐ganglioside which is mediated by NF‐κB translocation

et al. 2003

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Cholera toxin (CT) is a potent adjuvant; however, the mechanism for its ability to enhance mucosal immunity has not been fully elucidated. We report here that CT exerts its adjuvant properties by signaling through the GM1 ganglioside receptor. When ganglioside-defective mice were given the antigen (Ag) ovalbumin (OVA) with CT by the oral route, CT failed to support either OVA-specific antibody or CD4 + T cell responses. In vitro treatment of murine bone marrow-derived dendritic cells (DC) with CT induced full maturation as evidenced by upregulation of the costimulatory molecules, as well as by an enhanced ability to effectively present OVA for Ag-specific T cell responses. On the other hand, ganglioside-defective DC failed to differentiate to full function as Ag-presenting cells in response to CT. Since ganglioside-defective DC showed a mature phenotype after stimulation with lipopolysaccharide (LPS), the effects of CT on DC was independent of signal transduction through adjuvant receptor for LPS, the Toll-like receptor 4. Furthermore, CT also induced nuclear translocation of nuclear factor (NF)-‹ B in DC in a GM1-dependent fashion. These results highlight gangliosides expressed by DC for recognition of the non-self protein bacterial enterotoxin, which employ a unique signaling pathway to induce both innate and adaptive immunity.

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Simple method of purification of Escherichia coli heat-labile enterotoxin and cholera toxin using immobilized galactose

Cited by 81 publications

References 0 publications

EnterotoxigenicEscherichia coliModulates Host Intestinal Cell Membrane Asymmetry and Metabolic Activity

EnterotoxigenicEscherichia coliModulates Host Intestinal Cell Membrane Asymmetry and Metabolic Activity

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells

Cholera toxin activates dendritic cells through dependence on GM1‐ganglioside which is mediated by NF‐κB translocation

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