Release of outer membrane fragments from normally growing Escherichia coli

Hoekstra, Dick; Vanderlaan, Jw; Leij, Lfmh de; Witholt, Bernard

doi:10.1016/0005-2736(76)90058-4

Cited by 242 publications

(179 citation statements)

References 27 publications

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“…LT Secreted by the GSP Is Vesicle-associated-Previous work has demonstrated that LT secreted by ETEC is associated with vesicles (21,22,40,41). To investigate whether this association of LT with vesicles was simply because of a lack of GSP expression, we wanted to determine whether LT secreted by the GSP is soluble or associated with vesicles.…”

Section: Resultsmentioning

confidence: 99%

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Bacterial Surface Association of Heat-labile Enterotoxin through Lipopolysaccharide after Secretion via the General Secretory Pathway

Horstman

Kuehn

2002

Journal of Biological Chemistry

135

164

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Heat-labile enterotoxin (LT) is an important virulence factor expressed by enterotoxigenic Escherichia coli.The route of LT secretion through the outer membrane and the cellular and extracellular localization of secreted LT were examined. Using a fluorescently labeled receptor, LT was found to be specifically secreted onto the surface of wild type enterotoxigenic Escherichia coli. The main terminal branch of the general secretory pathway (GSP) was necessary and sufficient to localize LT to the bacterial surface in a K-12 strain. LT is a heteromeric toxin, and we determined that its cell surface localization was mediated by the its B subunit independent of an intact G M1 ganglioside binding site and that LT binds lipopolysaccharide and G M1 concurrently. The majority of LT secreted into the culture supernatant by the GSP in E. coli associated with vesicles. Only a mutation in hns, not overexpression of the GSP or LT, caused an increase in vesicle yield, supporting a specific vesicle formation machinery regulated by the nucleoidassociated protein HNS. We propose a model in which LT is secreted by the GSP across the outer membrane, secreted LT binds lipopolysaccharide via a G M1 -independent binding region on its B subunit, and LT on the surface of released outer membrane vesicles interacts with host cell receptors, leading to intoxication. These data explain a novel mechanism of vesicle-mediated receptor-dependent delivery of a bacterial toxin into a host cell. Enterotoxigenic Escherichia coli (ETEC)1 is an important pathogen responsible for traveler's diarrhea and Ͼ700,000 childhood deaths annually because of diarrhea in third world countries (1-4). ETEC produces two toxins implicated in the etiology of diarrhea, heat stabile toxin and heat-labile enterotoxin (LT) (1, 5). LT, which is encoded on a relatively uncharacterized 60-kb virulence plasmid (1), is one of a group of AB 5 toxins (6, 7) that also includes Shiga toxin, pertussis toxin, and cholera toxin (CT). These heteromeric toxins consist of a catalytic A subunit (LTA) and a pentamer of receptor-binding B subunits (LTB) (8, 9). In addition to structural homology, LT shares 80% sequence homology with the Vibrio cholerae toxin CT (10, 11). The ring-shaped B pentamer of both LT and CT mediates binding to the host epithelial receptor G M1 (12)(13)(14). LT is more promiscuous than CT in that it can also bind other receptors containing a terminal galactose (13). After binding, the receptor/toxin complex is internalized, and LTA is trafficked to the cytosol (13,15,16). Upon entry into the cytosol, the catalytic subunit constitutively activates adenylate cyclase, resulting in water and electrolyte efflux from the host cells (17).One major difference between the CT and LT lies in the fact that although CT is secreted from the cell, LT reportedly remains periplasmic (5, 18 -20). Some studies have also found LT to be associated with membranes extracellularly (3,21,22). Despite the equivalent activity that CT and LT exhibit in bioassays, disease caused by ETEC is much...

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Section: Resultsmentioning

confidence: 99%

“…These observations were explained by the reports that LT was associated with ETEC vesicles (21,22,40,41). Most Gram-negative bacteria shed portions of their outer membrane into the cell culture supernatant (41)(42)(43)(44)(45)(46)(47)(48)(49). These spherical outer membrane fragments termed "vesicles" are composed of LPS, lipids, proteins, and toxins.…”

mentioning

confidence: 94%

Bacterial Surface Association of Heat-labile Enterotoxin through Lipopolysaccharide after Secretion via the General Secretory Pathway

Horstman

Kuehn

2002

Journal of Biological Chemistry

135

164

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“…A biochemical mechanism to explain this observation has not been identified, as no known outer membrane transport machinery exists for LT. One possibility is that LT is secreted via vesicles, as proposed by Wai et al (21). Membrane vesicles, or MVs, have been broadly defined as spherical fragments of the bacterial membrane and are produced by a wide variety of [20][21][22][23][24][25][26][27][28]. Vesicles have been proposed to play a role in several virulence mechanisms: periplasmic enzyme delivery (24,25,27,(29)(30)(31)(32) DNA transport (24,28,33), bacterial adherence (23), and evasion of the immune system (22, 34).…”

Section: Enterotoxigenic Escherichia Coli (Etec)mentioning

confidence: 99%

Enterotoxigenic Escherichia coli Secretes Active Heat-labile Enterotoxin via Outer Membrane Vesicles

Horstman

Kuehn

2000

Journal of Biological Chemistry

362

418

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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 (ETEC)1 is an important pathogen responsible for traveler's diarrhea and causes more than 700,000 childhood deaths due to diarrhea per year in third-world countries (1-4). ETEC produce several toxins, including the heat labile enterotoxin (LT), which disrupts electrolyte balance in the gut endothelium (2,5,6). LT is an AB 5 toxin that binds Gal␤1,3GalNAc␤1(NeuAc␣2,3),4Gal␤1,4Glc ceramide (G M1 ) ganglioside on epithelial cells via its B subunit (7). Once internalized by the epithelial cell, the enzymatic A subunit catalyzes the ADP-ribosylation of the G s␣ subunit in the adenylate cyclase pathway leading to an increase in cAMP (2, 8 -10). Elevated cAMP levels cause chloride efflux and, thereby, diarrhea. Despite intimate knowledge of its structure and function (1, 11), the mode of LT secretion from ETEC remains unclear.LT shares more than 80% sequence homology with another AB 5 toxin, Vibrio cholerae toxin, CT (2). Purified CT and LT exhibit equivalent activity in bioassays; however, disease caused by V. cholerae is more severe than that caused by ETEC (1). This suggests V. cholerae-and ETEC-mediated toxicity may be partially dependent upon the efficiency of toxin secretion (2). The signal sequences of the A and B subunits from both LT and CT are cleaved upon entrance into the periplasmic space after transport across the cytoplasmic membrane (11-13). The similarities stop in the periplasm, however; soluble CT is secreted from the cell, whereas soluble LT is reported to remain in the periplasm (3, 6). E. coli transformed with a CT-expressing plasmid does not efficiently secrete CT, whereas V. cholerae will secrete LT e...

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“…Although this study attempted to assess the binding of anti-LPS mAbs to whole cells, release of membrane fragments from cells must also be considered. Indeed, variations in growth conditions could alter the extent and nature of the excretion of such fragments (Hoekstra et al, 1976).…”

Section: Discussionmentioning

confidence: 99%

Monoclonal antibodies as probes for detecting lipopolysaccharide expression on Escherichia coli from different growth conditions

Nelson

Bathgate²,

Poxton³

1991

Journal of General Microbiology

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Release of outer membrane fragments from normally growing Escherichia coli

Cited by 242 publications

References 27 publications

Bacterial Surface Association of Heat-labile Enterotoxin through Lipopolysaccharide after Secretion via the General Secretory Pathway

Bacterial Surface Association of Heat-labile Enterotoxin through Lipopolysaccharide after Secretion via the General Secretory Pathway

Enterotoxigenic Escherichia coli Secretes Active Heat-labile Enterotoxin via Outer Membrane Vesicles

Monoclonal antibodies as probes for detecting lipopolysaccharide expression on Escherichia coli from different growth conditions

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