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Food Microbiology

Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli O157:H7

Glynis L. Kolling, Karl R. Matthews
Glynis L. Kolling
Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901
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Karl R. Matthews
Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901
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DOI: 10.1128/AEM.65.5.1843-1848.1999
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  • Fig. 1.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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

  • Figures
  • Table 1.

    Summary of E. coli isolates

    StrainSerotypeStx productionSource
    DEC8BO111:H8Stx1, Stx2R. Wilsona
    DEC3DO157:H7Stx1, Stx2R. Wilson
    43895O157:H7Stx1, Stx2American Type Culture Collection
    33694NDdNegativeAmerican Type Culture Collection
    93-111O157:H7Stx1, Stx2P. Fives-Taylorb
    VDH5O157:H7NegativeVermont Department of Health
    4516O157:H7Stx1, Stx2D. G. Whitec
    19261O157:H7Stx1, Stx2D. G. White
    8247O157:H7Stx1, Stx2D. G. White
    8302O157:H7Stx1, Stx2D. G. White
    JM109NDNegativePromega
    • ↵a Pennsylvania State University, University Park, Pa.

    • ↵b University of Vermont, Burlington, Vt.

    • ↵c North Dakota State University, Fargo, N.D.

    • ↵d ND, not determined.

  • Table 2.

    DNA content associated with vesicles from E. coli O157:H7 isolates

    StrainSerotypeAmt (ng) of DNA/30 μg of vesicle protein (avg ± SE)a
    IntactLysedb
    ATCC 43895O157:H71.29 ± 0.432.54 ± 0.18
    8302O157:H73.77 ± 1.76.93 ± 1.1
    ATCC 33694None0.347 ± 0.241.51 ± 0.16
    • ↵a n = 3.

    • ↵b Vesicles lysed with GES reagent.

  • Table 3.

    Transfer of genetic material by vesicles isolated from E. coli O157:H7 to E. coli JM109

    E. coli JM109 cell stateNo. of colonies positive/no. tested for target gene:
    eaestx1stx2
    Competent cells0/54/5NDa
    Noncompetent cells3/55/55/5
    • ↵a ND, not determined.

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Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli O157:H7
Glynis L. Kolling, Karl R. Matthews
Applied and Environmental Microbiology May 1999, 65 (5) 1843-1848; DOI: 10.1128/AEM.65.5.1843-1848.1999

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Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli O157:H7
Glynis L. Kolling, Karl R. Matthews
Applied and Environmental Microbiology May 1999, 65 (5) 1843-1848; DOI: 10.1128/AEM.65.5.1843-1848.1999
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KEYWORDS

Bacterial Toxins
Escherichia coli O157
Genes, Bacterial

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