Team:Paris/Addressing overview3

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Overview

• Introduction
• A. Clya
• B. OmpA
• C. Our strategy
• D. Construction

A. ClyA

We work on the cell-cell communication using vesicle, but in a first time we need to adress a signal to the outer membrane. So we have to find something which can go from the cytoplasm to outer membrane , can be exported a protein signal through the vesicle and finally adressing a signal when its fuse with the target cells.

So ClyA seems to have an interesting ways to success in these functions, in fact ClyA in E.Coli is in high expression in OMVs, and it’s one of the proteins that can pass the cytoplasm to the outer membrane and integrated to vesicle easily utilizing the type I pathway( this "pathways are one- step mechanisms by which the secreted proteins cross directly from the cytoplasm to the bacterial surface."[[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]]). Thus ClyA is express on bacteria and OMVs surface , however when ClyA is overproduced, it could accumulate in the periplasmic space[[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]]

However there are some inconvenient using this protein because ClyA is an alpha-PFT for Pore Forming Toxins. PFTs are potent virulence factors class starting in a soluble form to an outer membrane-integrated pore. They exhibit their toxic effect either by membrane permeability barrier destruction or by toxic components delivery through the pores which forming by several assembly 8 or 13 ClyA subunits. PFTs can be subdivided into two classes; α-PFTs and β-PFTs, depending on the suspected mode of membrane integration, either by α-helical or β-sheet elements.[[http://www.ncbi.nlm.nih.gov/pubmed/19421192 [2]]]

So some article show that E.Coli K12 using this ClyA to lyse other cell (specially mamalian cell or eurcaryote cell). But this virulence was not show in same strain.

In some article, it’s fused to GFP in order to observed the vesicle[[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]]], so we think the fusion of ClyA with a peptide signal can induct the receptor when the vesicle fusion to its cell target liberate the Cly A in the target cell, or when ClyA on the OMVs interact with outer membrane receptor of the receiver cell. And it's this information which we will exploit for our strategy

AVANTAGE

- ClyA can be used to co-localize fully functional heterologous proteins directly in bacterial OMVs

-We can fuse GFP to the C or N term of Cly A, to track OMVs easily.

-ClyA is capable of co-localizing a variety of structurally diverse fusion partners to the surface of E. coli and their released vesicles, but only when the periplasmic disulfide bond-forming machinery was present ,it’s makes OMVs an ideal structure to transport hydrophobic compounds like membrane proteins into the host.

-Cly A confers vesicle binding to and invasion of host cells.[[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]]]

-ClyA was significantly enriched in OMVs relative to other lumenal and membrane bound OMV proteins.

DRAWBACK

-Cly A is a alpha-PFT; it can form pore in cell target. But we find ClyA is virulent for mammalian cell or erythrocytes only[[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]], because of its strong interaction with choloesterol which constitute mammalian cell membrane. For the virulence in bacteria cell we think that it’s not possible because there is no cholesterol in the bacteria membrane.

INTERESTING QUOTATIONS:

- "unfused ClyA accumulated in the cytoplasm, periplasm and OMV fractions."[1]

-"It may also be possible to use this molecule as a model system to develop predictive rules that will aid in understanding of molecular events that govern related cellular processes such as membrane fusion of cellular compartments and viral membrane fusion."[2]

Source:

[[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1-]]]Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66

[[http://www.ncbi.nlm.nih.gov/pubmed/19421192 [2-]]]Muller, M. & Ban, N. The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism Nature 459, 726-730 (4 June 2009)

[[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3-]]]Wai, S.N. & Lindmark, B. Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin Cell 115,25-35, October 3, 2003

- Oscarsson, J. & Uhlin, B.E. Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli