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Chapter 66 : Immunochemical Analysis of Outer Membrane Vesicles

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Immunochemical Analysis of Outer Membrane Vesicles, Page 1 of 2

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Abstract:

This chapter presents the description of the components of outer membrane (OM) vesicles produced by . The composition of purified vesicles was compared by immumoblot analysis and enzyme-linked immunosorbent assay using the panel of monoclonal antibodies (MAbs) listed. Additional components of the vesicles included the chaperone Hsp60, the amoebal uptake protein (Aup), and the Mip PPIase protein which is necessary for full virulence. The authors assumed that lipid A remains anchored to intravesical structural proteins, whereas carbohydrate moieties of lipopolysaccharide (LPS) are altered, either before or after vesicle formation. The LPS-enriched vesicles are composed of several structural as well as virulence-associated proteins of , e.g., Mip, Aup, and Hsp60. The detection of particular gene products inside the OM vesicles would be one approach to investigate how OM vesicles contribute to pathogenesis during phagosome formation and maturation, as well as during biofilm establishment and persistence. In addition to growth phase-dependent regulation, the authors demonstrated that the LPS content of OM vesicles varies between serotypes.

Citation: H. Helbig J, Fernandez-Moreira E, Christian Lück P, Jacobs E, S. Swanson M. 2006. Immunochemical Analysis of Outer Membrane Vesicles, p 269-273. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch66

Key Concept Ranking

Enzyme-Linked Immunosorbent Assay
0.47727516
Outer Membrane Proteins
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Sodium Dodecyl Sulfate
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0.47727516
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Figures

Image of FIGURE 1
FIGURE 1

Immunoblot analysis of Lp02 vesicles obtained at different growth phases. ( and ) vesicles released at postexponential phase culture densities of optical density at 600 nm of (OD) 3.5 () and 4.2 (). () Vesicles prepared from later postexponential phase cultures characterized by their decrease from the peak OD. The KDO equivalent of the vesicle solution was adjusted to 0.2 mg of KDO per 1 ml of solution for and . The concentration in amounted to 1.5 mg of KDO per 1 ml of solution.

Citation: H. Helbig J, Fernandez-Moreira E, Christian Lück P, Jacobs E, S. Swanson M. 2006. Immunochemical Analysis of Outer Membrane Vesicles, p 269-273. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch66
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Image of FIGURE 2
FIGURE 2

Immunoblot analysis of vesicles prepared from MAb 3/1-negative strains cultured to the postexponential growth phase indicated by OD of 3.5. () Strain Görlitz 6543 (serogroup 1, monoclonal subgroup Bellingham); () strain L270 (serogroup 3); () strain L138 (serogroup 6). The LPS-specific MAbs are indicated.

Citation: H. Helbig J, Fernandez-Moreira E, Christian Lück P, Jacobs E, S. Swanson M. 2006. Immunochemical Analysis of Outer Membrane Vesicles, p 269-273. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch66
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Download as Powerpoint

References

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10. Steudel, C.,, J. H. Helbig, and, P. C. Lück. 2002. Characterization of a 16 kilodalton species-specific protein of Legionella pneumophila promoting uptake in amoebae, p. 165169. In R. Marre (ed.), Legionella. American Society for Microbiol-ogy, Washington, D.C.
11. Zou, C. H.,, Y. A. Knirel,, J. H. Helbig,, U. Zähringer, and, C. S. Mintz. 1999. Molecular cloning and characterization of a locus responsi ble for O-acetylation of the O polysaccharide of Legionella pneumophila serogroup 1 lipopolysac- charide. J. Bacteriol. 181:41374141.

Tables

Generic image for table
TABLE 1

MAbs used for immunochemical analysis of OM vesicles

Citation: H. Helbig J, Fernandez-Moreira E, Christian Lück P, Jacobs E, S. Swanson M. 2006. Immunochemical Analysis of Outer Membrane Vesicles, p 269-273. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch66

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