Donald C. Robertson

Development of a competitive enzyme-linked immunosorbent assay (ELISA) for Escherichia coli heat-stable enterotoxin (STa).

A sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the low molecular weight heat-stable enterotoxin (STa) in culture supernatant fluids of enterotoxigenic Escherichia coli (ETEC). Competitive inhibition was observed between STa in solution and a glutaraldehyde-coupled STa-human serum albumin (HSA) conjugate bound to microtiter wells when antiserum raised against a glutaraldehyde-coupled STa-bovine serum albumin (BSA) conjugate was used as detecting antibody. No competition was observed with conjugates prepared using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or dimethyl suberimidate and antisera raised against each conjugate. A biotin/avidin system increased the sensitivity of the assay such that 133 pg/ml of purified STa can be detected in less than 4 h. The assay was used to detect and quantify STa in culture supernatant fluids from human, porcine, and bovine ETEC isolates. No cross-reactivity was observed with the heat-labile enterotoxin (LT) or the form of ST with biological activity only in piglets (STb). Results from the quantitative STa ELISA showed good correlation (0.87) with the suckling mouse bioassay and a previously described radioimmunoassay. The quantitative assay was modified to reduce the total incubation time to less than 2 h. The qualitative STa ELISA provides a rapid and sensitive assay for clinical isolates of ETEC and should facilitate epidemiological studies on the incidence of STa-producing ETEC.

Inhibition of Escherichia coli heat-stable enterotoxin effects on intestinal guanylate cyclase and fluid secretion by quinacrine

Enterotoxigenic Escherichia coli may produce a heat-stable enterotoxin (ST) that causes diarrheal disease in humans and in animals ST activates particulate guanylate cyclase in intestinal mucosal cells and causes intestinal fluid secretion. In this study, we examined the effects of quinacrine on ST activation of guanylate cyclase and ST-mediated intestinal fluid secretion. Quinacrine significantly reduced ST activation of particulate guanylate cyclase in rat intestinal tissue. Additionally, quinacrine reduced ST-mediated fluid secretion in a rat intestinal loop assay (P less than 0.05). In the suckling mouse model, subcutaneous quinacrine (0.1 mumole/mouse) reduced ST-induced fluid secretion at a submaximally effective dose of the toxin, but it did not reduce ST-mediated fluid secretion at a near maximally effective dose. Quinacrine (0.1 mumole/mouse) did not significantly reduce intestinal fluid secretion induced by the analog of cyclic GMP, 8-bromo cyclic GMP. However, at a higher concentration of quinacrine (1 mumole/mouse), significant inhibition of 8-bromo cyclic GMP-induced secretion was observed. Inhibition by the antimalarial agent quinacrine of ST-induced fluid secretion, by a block prior to guanylate cyclase activation, suggests a possible role for a phospholipase early in the sequence of events of ST activation of guanylate cyclase. The results suggest that ST may activate membrane phospholipases prior to ST activation of guanylate cyclase.

High Affinity Receptor for Heat-Stable Enterotoxins (STa) on Rat Intestinal Epithelial Cells

Studies on the association of heat-stable enterotoxin (ST) produced by enterotoxigenic E. coli (ETEC) strain 431 with isolated rat intestinal epithelial cells and brush border membranes indicated that STas bind to a single class of specific high affinity receptor. Specific binding of radioiodinated ST (specific activity 600 to 900 Ci/mmole) was temperature dependent and reached a maximum between 5 and 10 minutes which correlated with increased activity of particulate guanylate cyclase. The rate of dissociation of 431 ST from either intestinal cells or brush border membranes was significantly less than the rate of association. The number of ST-specific receptors on rat intestinal cells determined by Scatchard analysis was 20,478± 6,185 per cell with an affinity constant (Ka) of 6.25 × 1011 L/M. The affinity constant determined for brush border membranes (Ka = 9.4 × 1011 L/M) agreed with the value obtained with whole cells. Binding experiments with 125I-431 ST and brush border membranes showed that purified STas from ETEC strains 667 (class 1 porcine enteropathogen), B-41 (bovine enteropathogen), and human strains 213 C2 (Mexico) and 153961–2 (Dacca, Bangladesh) exhibited similar patterns of competitive inhibition as homologous ST produced by strain 431 (class 2 enteropathogen). Further, purified LT (strain 286 C2) did not inhibit binding of 431 ST. Digestion of brush border membranes showed the ST receptor to be resistant to lipases and glycosidases but somewhat sensitive to proteases. The putative ST binding protein radiolabeled with 125I-431 ST was solubilized with sodium deoxycholate. One major radioactive band was detected by SDS-polyacrylamide gel electrophoresis followed by radioautography.

Studies on the Mechanism of Action of the Escherichia Coli Heat-Stable Enterotoxin (STa)

Rat intestinal cells and brush border membranes have been shown to contain a particulate guanylate cyclase which can be stimulated by E. coli heat-stable enterotoxin (ST). The sequence of events prior to activation of guanylate cyclase are unknown, however, anti-inflammatory agents have been able to block secretory activity of ST (Greenberg, et al., Infect. Immun. 29:908–913, 1980) which suggested that activation of guanylate cyclase occurs via metabolism of arachidonic acid with formation of prostaglandins or other oxidized intermediates. Treatment of either intact rat enterocytes or brush border membranes with ST caused a dose dependent accumulation of cGMP. Evidence for the release and conversion of arachidonic acid to an intermediate of prostaglandin synthesis or hydroxy-derivative was not obtained by: 1) following the rate and amounts of [3H]-arachidonate released from prelabeled cells incubated in the presence and absence of ST; 2) use of specific inhibitors of the cyclooxygenase and lipooxygenase pathways and 3) the fact that 02 was not required for activation of guanylate cyclase by ST.