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The Project Background Design Parts Submitted to the Registry Notebook The Team Outreach & Human Practices


Protein Structure of ClyA with labeled N and C Terminus 3

Outer membrane vesicles (OMVs) are spherical proteoliposomes secreted by gram-negative bacteria and range in size from 50-200nm. They are made when the outer membrane discharges during bacterial growth and largely contain phosopholipids and periplasmic proteins.1 One peculiar characteristic of OMVs is their inclination to exclude certain periplasmic proteins and select others.6 One such selected protein is Cytolysin A, ClyA, a surface hemolytic protein found in Escherichia coli. The structure of ClyA includes four α-helices with a β-hairpin hydrophobic head, which is localized in the plasma membrane. The C terminus is exposed on the surface of the vesicle, and, via genetic engineering, other proteins can be attached to it.4,7 The attachments negate the hemolytic activity of ClyA4 and open the potential to create numerous signaling possibilities on OMV surfaces.

Because OMVs are naturally enriched in the ClyA surface protein4, we decided to create a variety of ClyA fusion constructs. Previous work involving OMVs and ClyA constructs involved the expression of a single ClyA fusion on the surface of an OMV such as ClyA-GFP and ClyA-scFv (ClyA-single-chain antibody fragment) as a proof of concept4. Our goals were to determine the number of proteins we could co-express on OMV surfaces and to discern if a dilution effect occurred as the number of ClyA fusion constructs increased. We set out to create multiple ClyA-fluorescent protein fusions, co-express them in E. coli such that they are localized on OMV surfaces, and quantify surface protein expression.

Model of Vesicle Biogenesis 5

In functional ClyA fusion constructs, proteins attached to ClyA retain their original properties, thus allowing us to utilize the characteristics of multiple protein fusions on the surfaces of OMVs 4. For example, fused GFP would produce a fluorescent signal, and a fused single-chain antibody fragment would bind to surface antigens of interest on another cell, thus allowing us to monitor OMV-cell interactions in vitro. Such observations may illuminate whether OMVs aid gram-negative bacteria by serving as virulence factors and may show how host cells receive OMVs.4

OMVs have great potential as a multifunctional platform because they are easily produced en masse via simple genetic mutations in E. coli, easily purified by ultracentrifugation, non-toxic to mammalian cells, and inexpensive to produce.2


1. Beveridge, T. J. (1999). Structures of gram-negative cell walls and their derived membrane vesicles. J. Bacteriol. 181, 4725–4733.

2. Chen, D. J., Osterrieder, N., Metzger, S. M., Buckles, E., Doody, A. M., DeLisa, M. P., & Putnam, D. (2010). Delivery of foreign antigens by engineered outer membrane vesicle vaccines. Proc Natl Acad Sci U S A. 107, 3099-3104.

3. Eifler, N., Vetsch, M., Gregorini, M., Ringler, P., Chami, M., Philippsen, A. et al. (2006). Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state. EMBO J. 25, 2652–2661.

4. Kim, J. Y., Doody, A. M., Chen, D. J., Cremona, G. H., Shuler, M. L., Putnam, D., & DeLisa, M. P. (2008). Engineered Bacterial Outer Membrane Vesicles with Enhanced Functionality. J. Mol. Biol. 380, 51–66.

5. Kuehn, M. J., & Kesty, N. C. (2005). Bacterial outer membrane vesicles and the host-pathogen interaction. Genes Dev. 19, 2645-2655.

6. Wai, S. N., Lindmark, B., Soderblom, T., Takade, A., Westermark, M., Oscarsson, J. et al. (2003). Vesicle mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell. 115, 25–35.

7. Wallace, A. J., Stillman, T. J., Atkins, A., Jamieson, S. J., Bullough, P. A., Green, J. & Artymiuk, P. J. (2000). E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy. Cell. 100, 265–276.

8. Wilchek, M., & Bayer, E. A. (1988). The Avidin-Biotin Complex in Bioanalytical Applications. Anal. Biochem. 171, 1–32.