Article Figures & Data
Figures
- 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.
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.
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.
SDS-PAGE profiles of vesicles isolated from variousE. 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.
Agarose gel analysis of PCR products produced withE. coli O157:H7 vesicle-associated DNA. (A) Profiles forstx1 (614 bp) and stx2 (779 bp). Primers forstx1 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 ofeae (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.
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.
Immunoblot demonstrating that Shiga toxins are located inside vesicles. Intact and lysed vesicles (from E. coliO157: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.
Tables
- Table 1.
Summary of E. coli isolates
Strain Serotype Stx production Source DEC8B O111:H8 Stx1, Stx2 R. Wilsona DEC3D O157:H7 Stx1, Stx2 R. Wilson 43895 O157:H7 Stx1, Stx2 American Type Culture Collection 33694 NDd Negative American Type Culture Collection 93-111 O157:H7 Stx1, Stx2 P. Fives-Taylorb VDH5 O157:H7 Negative Vermont Department of Health 4516 O157:H7 Stx1, Stx2 D. G. Whitec 19261 O157:H7 Stx1, Stx2 D. G. White 8247 O157:H7 Stx1, Stx2 D. G. White 8302 O157:H7 Stx1, Stx2 D. G. White JM109 ND Negative Promega
