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J Biol Chem, Vol. 275, Issue 17, 12489-12496, April 28, 2000
From the Duke University Medical Center, Department of
Biochemistry, Durham, North Carolina 27710
Escherichia coli and other
Gram-negative bacteria produce outer membrane vesicles during normal
growth. Vesicles may contribute to bacterial pathogenicity by serving
as vehicles for toxins to encounter host cells. Enterotoxigenic
E. coli (ETEC) vesicles were isolated from culture
supernatants and purified on velocity gradients, thereby removing any
soluble proteins and contaminants from the crude preparation. Vesicle
protein profiles were similar but not identical to outer membranes and
differed between strains. Most vesicle proteins were resistant to
dissociation, suggesting they were integral or internal. Thin layer
chromatography revealed that major outer membrane lipid components are
present in vesicles. Cytoplasmic membranes and cytosol were absent in
vesicles; however, alkaline phosphatase and AcrA, periplasmic
residents, were localized to vesicles. In addition, physiologically
active heat-labile enterotoxin (LT) was associated with ETEC vesicles.
LT activity correlated directly with the gradient peak of vesicles,
suggesting specific association, but could be removed from vesicles
under dissociating conditions. Further analysis revealed that LT is
enriched in vesicles and is located both inside and on the exterior of
vesicles. The distinct protein composition of ETEC vesicles and their
ability to carry toxin may contribute to the pathogenicity of ETEC strains.
Enterotoxigenic Escherichia coli Secretes Active
Heat-labile Enterotoxin via Outer Membrane Vesicles*
*
This work was supported by a Burroughs Wellcome Career Award
(to M. J. K.) and National Institutes of Health Training Grant GM-07184 (to A. L. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Duke University
Medical Center, Dept. of Biochemistry, Box 3711, Durham, NC 27710. Tel.: 919-684-2545; Fax: 919-684-8885; E-mail:
mkuehn@biochem.duke.edu.
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