Revision as of 02:10, 28 October 2010

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

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 amplify others.⁶ One such amplified 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.

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 cheap 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.

@@ Line 13: / Line 13: @@
 =Background=
 [[Image:Clya.jpg|right|frame|Protein Structure of ClyA with labeled N and C Terminus <sup>3</sup>]]
-Within the medical field, there is great interest in developing targeted delivery of drugs to specific regions of the body.  Previous attempts have included sustained release formulations, biodegradable capsules and direct injection to the targeted tissue or region. One very promising proposal is the use of Outer Membrane Vesicles, OMVs, as a transmission method of various proteins and lipids to mammalian cells.<sup>1</sup> OMVs are spherical proteoliposomes secreted by gram-negative bacteria and range in size from 50-200 nm. They are constituted of the outer membrane discharged during bacterial growth and largely contain phosopholipids and periplasmic proteins.<sup>1</sup> One peculiar observation of OMVs is their inclination to exclude certain periplasmic proteins and amplify others.<sup>6</sup> One such amplified protein is Cytolysin A, ClyA, a surface hemolytic protein found in Escherichia coli.<sup>4</sup> The structure of ClyA is four α-helices with a β-hairpin hydrophobic head which localizes in the plasma membrane and a C and N terminus exposed on the surface of the vesicle.<sup>7</sup> Through genetic engineering, other proteins can be attached to either the N or C terminus of ClyA. The attachments negate the hemolytic activity of ClyA<sup>4</sup> and open the potential to create myriad signaling possibilities on the surface of OMVs.
+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.<sup>1</sup> One peculiar characteristic of OMVs is their inclination to exclude certain periplasmic proteins and amplify others.<sup>6</sup> One such amplified 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.<sup>4,7</sup>  The attachments negate the hemolytic activity of ClyA4 and open the potential to create numerous signaling possibilities on OMV surfaces.
-<br><br>
-[[Image:Vesicle.jpg|300px|thumb|left|Model of Vesicle Biogenesis<sup>5</sup>]]
+Because OMVs are naturally enriched in the ClyA surface protein<sup>4</sup>, 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 concept<sup>4</sup>.  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.
-OMVs present a strong opportunity to create an efficient transport system for tailored drug delivery. However, there is very little scientific knowledge about the movement of OMVs through the body or their intracellular interactions. We propose a tracking system of OMVs through the use of ClyA to observe their behavior and uptake in mammalian cells. We believe this can be done by displaying multiple ClyA fusion proteins on single OMVs. One ClyA fusion protein will present an antibody fragment to target specific host cells, and a second ClyA fusion protein will be attached, through biotin-streptavidin interaction, to a contrast agent for imaging. Since the chemical contrast agent cannot be attached to ClyA through genetic engineering, the high affinity biotin-streptavidin interaction provides an alternative stable link.<sup>8</sup> The benefits of OMVs over present target delivery methods are that the vesicles are easily produced en mass through simple genetic mutations in E. coli, easily purified by ultracentrifugation, non-toxic to mammalian cells, and cheap to produce.<sup>2</sup> Importantly, we aim for this tracking system to shed light on the feasibility of an OMV drug delivery system adapted for any target antigen in a patient. We are currently developing the ClyA fusion proteins and the necessary components for important proofs of concept.
+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 <sup>4</sup>.  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.<sup>4</sup>
+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 cheap to produce.<sup>2</sup>
 === References ===

Team:Cornell/Project/Background

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Revision as of 02:10, 28 October 2010

Background

References