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

Abstract

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...

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles

“…It remains to be demonstrated whether our model for OMV cargo selection in P. gingivalis can be extended to other species, which may explain previous reports showing that certain toxins are enriched in the OMV compared with the OM in diverse pathogenic species (5,16,35,36,38). Proper OMV cargo selection could be critical for pathogenesis, and therefore our results identify new opportunities for intervention against pathogenic bacteria.…”

“…Other factors could contribute to the assembly of OM microdomains and therefore influence protein cargo selection. For example, it has been proposed that the lipid composition is different in OM and OMV (35,36). In addition, OM compartmentalization could be coupled to LPS and/or OM protein translocation processes (44).…”

Origin of life: LUCA and extracellular membrane vesicles (EMVs)

“…During the growth the OM 'blebs' outwards and pinches off, forming vesicles (20-250 nm) that are spherical portions of OM with lumenal periplasmic content. Thus, the composition of OMVs reflects components of the OM and periplasm, for example, soluble proteins, integral membrane proteins, lipoproteins and glycolipids (Beveridge 1999;Horstman & Kuehn 2000;Schooling et al 2009). OMVs are potent virulence factors of pathogenic diderm bacteria.…”

Predatory activity of Myxococcus xanthus outer-membrane vesicles and properties of their hydrolase cargo

“…For instance, phosphatases (hydrolases that liberate inorganic phosphate from organophosphate molecules) are frequently found in OMVs (e.g. Bomberger et al, 2009;Horstman & Kuehn, 2000;Pierson et al, 2011). OMVs can lyse next to Gram-positive cells, releasing their contents next to a target cell.…”