Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157:H7 - PubMed (original) (raw)

Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157:H7

G L Kolling et al. Appl Environ Microbiol. 1999 May.

Abstract

Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for beta-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.

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Figures

FIG. 1

FIG. 1

Cells and vesicles of E. coli O157:H7. (A) Ultrathin sections show vesicles associated with a whole cell. The inset is an enlargement of the enclosed area and clearly shows a vesicle membrane bilayer (arrow). Bar = 50 nm. (B) Negatively stained vesicle preparations demonstrate the uniform size and morphology of vesicles. The arrowheads indicate representative individual vesicles. Note that the vesicles appear to contain electron-dense material. Bar = 250 nm.

FIG. 2

FIG. 2

SDS-PAGE protein profiles of whole cells (WC), OMPs, and vesicles (MV) in a 10% polyacrylamide gel stained with silver stain (samples are from strain E. coli O157:H7 ATCC 43895). Each lane contains 25 μg of the total protein. Samples were not heat treated prior to loading. Molecular masses (in kilodaltons) are indicated on the left.

FIG. 3

FIG. 3

Immunoblot of E. coli O157:H7 whole cells (WC), OMPs, and vesicles (MV). The blot was probed with polyclonal anti-E. coli antibody. Each lane contains 25 μg of total protein. A 30-kDa protein was highly immunoreactive in the vesicle sample. Molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 4

FIG. 4

SDS-PAGE profiles of vesicles isolated from various E. coli O157:H7 strains. Each lane contains 15 μg of total protein. Gels were silver stained. Samples were not heat treated prior to loading. Lanes: 1, VDH5; 2, H8302; 3, B19261; 4, DEC3D; 5, ATCC 33694. Molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 5

FIG. 5

Agarose gel analysis of PCR products produced with E. coli O157:H7 vesicle-associated DNA. (A) Profiles for stx1 (614 bp) and stx2 (779 bp). Primers for stx1 and stx2 were used with samples in lanes 1 to 5 and 6 to 10, respectively. Samples in each lane were as follows: lanes 1 and 6, intact vesicles; lanes 2 and 7, intact vesicles treated with DNase; lanes 3 and 8, lysed vesicles treated with DNase; lanes 4 and 9, whole cells; and lanes 5 and 10, negative control (PCR cocktail, no template DNA). The lack of fragments in lanes 3 and 8 and the presence of fragments in lanes 2 and 7 indicate that DNA is located in the vesicles. Moreover, the results indicate that DNase treatment was sufficient to digest vesicle-associated DNA. (B) PCR products of eae (863 bp) and uidA (922 bp) primers. Primers for eae and uidA were used with samples in lanes 1 to 3 and lanes 4 to 6, respectively. Lanes: 1 and 4, intact vesicles (treated with DNase); 2 and 5, whole cells; 3 and 6, negative control (PCR cocktail, no template DNA). Molecular size standards (in kilobases) are indicated on the left.

FIG. 6

FIG. 6

Immunoblots of Shiga toxin association with E. coli O157:H7 vesicles. The blot was probed with pooled monoclonal antibody against Stx1 and Stx2. Lanes; 1, DEC3D; 2, B4516; 3, H8247; 4, H8302; 5, ATCC 33694; 6, ATCC 43895; 7, B19261; 8, 93-111; 9, DEC8B; 10, VDH5. Note the absence of bands in lane 5 (non-O157, non-Stx-producing isolate) and lane 10 (O157, non-Stx-producing isolate). An arrow indicates the major protein immunologically reactive to pooled monoclonal Stx1 and Stx2 antibody.

FIG. 7

FIG. 7

Immunoblot demonstrating that Shiga toxins are located inside vesicles. Intact and lysed vesicles (from E. coli O157:H7) were treated with proteinase K to determine whether Stx is located inside the vesicles. The blot was probed with pooled monoclonal antibody against Stx1 and Stx2. Lanes: 1, intact vesicles treated with proteinase K; 2, lysed vesicles treated with proteinase K; 3, intact vesicles; 4, lysed vesicles. Presence of a band in lane 1 (arrow) and no band in lane 2 indicates that Stx was protected from hydrolysis by virtue of its location within the vesicle (lane 1). Prestained molecular mass markers (in kilodaltons) are indicated on the left.

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