Background

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 N 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

References

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.