Team:Paris/Addressing overview3

From 2009.igem.org

(Difference between revisions)

Revision as of 21:52, 21 October 2009

Menu accordéon avec jQuery

Adressing the message in the outer membrane : ClyA

Main | ClyA| OmpA| Our strategy| Construction

We work on the cell-cell communication using vesicle:
In this part we look into adressing a protein into the sender outer membrane that could be incoporated into outer membrane vesicles (OMVs). This protein would then be able to transmit a message after the fusion of OMVs with a receiver cell.

In this direction ClyA (the cytolysine A of E.Coli) seems to be a good candidate. ClyA is one of the proteins that has been previously detected into OMVs and is known to be specificly exported to the outer membrane [[3]]. ClyA is thus expressed on bacteria and OMVs surface. Moreover, when ClyA is overproduced, it is accumulated into the periplasmic space [[3]].

However there is an inconvenient to use this protein. ClyA is an alpha-Pore Forming Toxin (PFT). PFT are widely distributed proteins which form lesions in biological membranes. They exhibit their toxic effect in different manner. The first one is that ClyA allows the destruction of membrane permeability barrier. Furthermore, the toxic effect of ClyA could be explain by its capacity to deliver toxic component after the assembly of 8 or 13 of its 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.[[2]]

ClyA are assembling in outer membrane of a host cell

Some article argue that E.Coli K12 use this ClyA to lyse other cell (specially mamalian cell or eurcaryote cell). But E. coli cells expressing clyA do not lyse each other.

Kim et al. have successfully fused clyA to GFP in order to observe vesicles [[1]], so we know that we can try to fuse clyA to a protein domain that would induce a signal transduction into the receiver cell. To see how we want to exploite clyA properties see our strategy.

Source:

1-Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66

2-Muller, M. & Ban, N. The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism Nature (4 June 2009) 459, 726-730

3-Wai, S.N. & Lindmark, B. Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin Cell (October 2003), 115,25-35

4- Oscarsson, J. & Uhlin, B.E. Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli Molecular Microbiology (1999) 32(6), 1226–1238

5- Westermark, M. & Uhlin, B.E. Silencing and Activation of ClyA Cytotoxin Expression in Escherichia coli Journal of bacteriology,(Nov. 2000), Vol. 182, No. 22 6347–6357

← Previous - Next →

@@ Line 68: / Line 68: @@
 We work on the cell-cell communication using vesicle:
 <br>
-To begin we need to adress a signal to the outer membrane, then exported into the outer membrane vesicles (OMVs). And finally, this protein will be able to transmit a message via the properties to fusion with the outer membrane of the target cell.
+In this part we look into adressing a protein into the sender outer membrane that could be incoporated into outer membrane vesicles (OMVs). This protein would then be able to transmit a message after the fusion of OMVs with a receiver cell.
-In this direction ClyA (the cytolysine A of E.Coli) seems to have an interesting ways to correspond. Actually, ClyA is one of the proteins that has a high expression into OMVs, thanks to the type I pathway. This "pathways is 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]]]. Thanks to this mechanism : ClyA is expressed on bacteria and OMVs surface. Moreover, when ClyA is overproduced, it could be accumulated into the periplasmic space[[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]].
+In this direction ClyA (the cytolysine A of E.Coli) seems to be a good candidate. ClyA is one of the proteins that has been previously detected into OMVs and is known to be specificly exported to the outer membrane [[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]]. ClyA is thus expressed on bacteria and OMVs surface. Moreover, when ClyA is overproduced, it is accumulated into 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]]]
+However there is an inconvenient to use this protein. ClyA is an alpha-Pore Forming Toxin (PFT). PFT are widely distributed proteins which form lesions in biological membranes. They exhibit their toxic effect in different manner. The first one is that ClyA allows the destruction of membrane permeability barrier. Furthermore, the toxic effect of ClyA could be explain by its capacity to deliver toxic component after the assembly of 8 or 13 of its 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]]]
 [[Image:Clya_simple.jpg|ClyA subunit|150px|left]] [[Image:Clya_structure2.jpg|ClyA assembled|100px|right]][[Image:ClyA.jpg|ClyA are assembling in outer membrane of a host cell|150px|center]]  [[Image:Clya_structure.jpg|ClyA assembled|150px|center]]
@@ Line 79: / Line 80: @@
-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.
+Some article argue that E.Coli K12 use this ClyA to lyse other cell (specially mamalian cell or eurcaryote cell). But E. coli cells expressing clyA do not lyse each other.
-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 [https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#Overview 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."[[http://www.ncbi.nlm.nih.gov/pubmed/18511069|[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."[[http://www.ncbi.nlm.nih.gov/pubmed/19421192|[2]]]
+Kim et al. have successfully fused clyA to GFP in order to observe vesicles [[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]]], so we know that we can try to fuse clyA to a protein domain that would induce a signal transduction into the receiver cell. To see how we want to exploite clyA properties see [https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#Overview our strategy].