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Chapter 66 : Immunochemical Analysis of Outer Membrane Vesicles

Authors: Jürgen H. Helbig1, Esteban Fernandez-Moreira2, Paul Christian Lück3, Enno Jacobs4, Michele S. Swanson5
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Affiliations: 1: Medical Faculty TU Dresden, Institute of Medical Microbiology and Hygiene, D-01307 Dresden, Germany; 2: University of Michigan Medical School, Department of Microbiology and Immunology, Ann Arbor, MI 48109; 3: Medical Faculty TU Dresden, Institute of Medical Microbiology and Hygiene, D-01307 Dresden, Germany; 4: Medical Faculty TU Dresden, Institute of Medical Microbiology and Hygiene, D-01307 Dresden, Germany; 5: University of Michigan Medical School, Department of Microbiology and Immunology, Ann Arbor, MI 48109
Content Type: Monograph
Publication Year: 2006

Category: Bacterial Pathogenesis

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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

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Enzyme-Linked Immunosorbent Assay
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Immunochemical Analysis of Legionella pneumophila Outer Membrane Vesicles
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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
/content/10.1128/9781555815660.ch66.ch66fig01
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.
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10.1128/9781555815660/f0271-01.gif
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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/content/10.1128/9781555815660.ch66.ch66fig02
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.
10.1128/9781555815660/f0272-01_thmb.gif
10.1128/9781555815660/f0272-01.gif
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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References

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1. Derre, I., and, R. R. Isberg. 2004. Legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system. Infect. Immun. 72: 3048— 3053.
2. Fernandez-Moreira, E.,, J. H. Helbig, and, M. S. Swanson. 2006. Membrane vesicles shed by Legionella pneumophila inhibit fusion of phago-somes with lysosomes. Infect. Immun. 74 (In press).
3. Flesher, A. R.,, S. Ito,, B. J. Mansheim, and, D. L. Kasper. 1979. The cell envelope of the Legionnaires’ disease bacterium. Ann. Intern. Med. 90: 628— 630.
4. Garduno, R. A.,, E. Garduno, and, P. S. Hoffman. 1998. Surface-associated Hsp60 chaperonin of Legionella pneumophila mediates invasion in HeLa cell model. Infect. Immun. 66: 4602— 4610.
5. Helbig, J. H.,, S. Bernander,, M. Castellani Pastoris,, J. Etienne,, V. Gaia,, S. Lauwers,, D. Lindsay,, P. C. Lück,, T. Marques,, S. Mentula,, M. F. Peeters,, C. Pelaz,, M. Struelens,, S. A. Uldum,, G. Wewalka, and, T. G. Harrison. 2002. Pan-European study on culture-proven Legionnaires’ disease: distribution of Legionella pneumophila serogroups and monoclonal subgroups. Eur. J. Clin. Microbiol Infect. Dis. 21: 710— 716.
6. Helbig, J. H.,, B. König,, H. Knospe,, B. Bubert,, C. Yu,, P. C. Lück,, A. Riboldi-Tunnicliffe,, R. Hilgenfeld,, E. Jacobs,, J. Hacker, and, G. Fischer. 2003. The PPIase active site of Legionella pneumophila Mip protein is involved in the infection of eukaryotic host cells. Biol. Chem. 384: 125— 137.
7. Helbig, J. H.,, J. B. Kurtz,, M. Castellani Pastroris,, C. Pelaz, and, P. C. Lück. 1997. The antigenic lipopolysaccharide components of Le-gionella pneumophila recognized by monoclonal antibodies: possibilities and limitations for division of the species into serogroups. J. Clin. Microbiol. 35: 2841— 2845.
8. Helbig, J. H.,, P. C. Lück,, Y. A. Knirel,, W. Witz-leb, and, U. Zähringer. 1995. Molecular characterization of a virulence-associated epitope on the lipopolysaccharide of Legionella pneumophila sero-group 1. Epidemiol. Infect. 115: 71— 78.
9. Lynch, D.,, N. Fieser,, K. Glöggler,, V. Forsbach-Birk, and, R. Marre. 2003. The response regulator LetA regulates the stationary-phase stress response in Legionella pneumophila and is required for efficient infection of Acanthamoeba castellanii. FEMS Microbiol. Lett. 219: 241— 248.
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. 165— 169. 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: 4137— 4141.

Tables

Immunochemical Analysis of Legionella pneumophila Outer Membrane Vesicles
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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
/content/10.1128/9781555815660.ch66.ch66tab01
TABLE 1
MAbs used for immunochemical analysis of OM vesicles
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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