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Infection and Immunity, May 2007, p. 2208-2213, Vol. 75, No. 5
0019-9567/07/$08.00+0     doi:10.1128/IAI.01829-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Carina Gonçalves,1 Vincent Vachon,2 Jean-Louis Schwartz,2 and J. Daniel Dubreuil1*

Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Quebec J2S 7C6,1 Groupe d'Étude des Protéines Membranaires, Université de Montréal, Montreal, Quebec H3C 3J7, Canada2

Received 17 November 2006/ Returned for modification 2 January 2007/ Accepted 12 February 2007

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 membrane-permeabilizing 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.

* Corresponding author. Mailing address: Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Sicotte Street, Saint-Hyacinthe, Quebec J2S 7C6, Canada. Phone: (450) 773-8521, ext. 18433. Fax: (450) 778-8108. E-mail: daniel.dubreuil{at}umontreal.ca

{triangledown} Published ahead of print on 16 February 2007.

Editor: J. F. Urban, Jr.

Infection and Immunity, May 2007, p. 2208-2213, Vol. 75, No. 5
0019-9567/07/$08.00+0     doi:10.1128/IAI.01829-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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