The Escherichia coli Enterotoxin STb Permeabilizes Piglet Jejunal Brush Border Membrane Vesicles

Abstract

The membrane-permeabilizing ability of the Escherichia coli enterotoxin STb was evaluated using brush border membrane vesicles isolated from piglet jejunum and a membrane-potential-sensitive fluorescent probe, 3,3-dipropylthiadicarbocyanine iodide. A strong membrane potential was generated by the efflux of K ؉ ions from the vesicles in the presence of the potassium ionophore valinomycin. Under these conditions, preincubation of the vesicles with STb efficiently depolarized the membrane in a dose-dependent and saturable manner. This activity was independent of pH, however, at least between pH 5.5 and 8.0. On the other hand, in the absence of valinomycin, STb had no significant influence on the measured fluorescence levels, indicating that it was unable to modify the ionic selectivity of the intact membrane. In agreement with the fact that the integrity of the disulfide bridges of STb is known to be essential for its biological activity, a reduced and alkylated form of the toxin was unable to depolarize the membrane in the presence of valinomycin. Furthermore, two previously described poorly active STb mutants, M42S and K22A-K23A, showed no membranepermeabilizing capacity. These results demonstrate for the first time that STb can permeabilize its target membrane and suggest that it does so by forming nonspecific pores.

Animal EnterotoxigenicEscherichia coli

“…It can do so by forming nonspecific pores confirming previous trypan blue and PLB studies (473, 488). An electrophysiological study using planar lipid bilayers (PLB) where the receptor for STb was reconstituted into large unilamellar vesicles made of PE osmotically fused to PE:PC:cholesterol (7:2:1) showed resolved channels currents (489).…”

Escherichia coli virulence factors

“…All STaP-, STaH-, STb-and EAST1-encoding genes are plasmidlocated (Gyles et al, 1974;Mainil et al, 1992Mainil et al, , 1998bMainil, 2002;Nataro, 2005;Weintraub, 2007;Gyles and Fairbrother, 2010). STaH, STaP, and probably EAST1 (based on structural similarity), exert their toxicity via activation of an intra-cellular cascade (Sears and Kaper, 1996;Kaper et al, 2004;Gyles and Fairbrother, 2010), while STb seems to act as a non-specific pore-forming toxin (Gonç alves et al, 2007;Dubreuil, 2008). If newborn calves, lambs, piglets and babies in developing countries are sensitive to the action of STaP and/or STaH that can also be involved in traveller's diarrhoea, the in vivo roles of STb and EAST1 are still controversial (Nataro and Kaper, 1998;Nagy and Fekete, 1999;Kaper et al, 2004;Dubreuil, 2008;Gyles and Fairbrother, 2010;Konno et al, 2011).…”

Major virulence factors of enterotoxigenic Escherichia coli in pigs

“…Once inside the cell, STb induces nonspecific pore formation in brush border vesicles from pig jejunal tissue. These pores may account for the secretion of electrolytes observed in STb-provoked colibacillosis (Goncalves et al 2007). In fact, although STb is often associated with pig diarrhea, up to recently, it has been difficult to experimentally reproduce a profuse diarrheal disease with a bacterial strain producing only the STb toxin.…”