Team:Paris/Addressing overview3

From 2009.igem.org

(Difference between revisions)
(→A. ClyA)
 
(45 intermediate revisions not shown)
Line 1: Line 1:
{{Template:Paris2009}}
{{Template:Paris2009}}
{{Template:Paris2009_menu3}}
{{Template:Paris2009_menu3}}
-
== Overview ==
 
-
* [[Team:Paris/Addressing_overview2#Overview |Introduction ]]
 
-
* [[Team:Paris/Addressing_overview3#Overview |A. Clya ]]
 
-
* [[Team:Paris/Addressing_overview4#Overview |B. OmpA ]]
 
-
* [[Team:Paris/Addressing_overview2_strategy#Overview |C. Our strategy]]
 
-
* [[Team:Paris/Addressing_overview_Construction#bottom |D. Construction]]
 
-
<html>
 
-
</div>
 
-
<div id="paris_content_boxtop">
 
-
</div>
 
-
<div id="paris_content">
 
-
</html>
 
-
==A. ClyA==
 
 +
<span/ id="1">
 +
==Addressing the message in the outer membrane : ClyA==
 +
<html>
 +
<style type="text/css">
 +
#left-side {
 +
    position: absolute;
 +
    height: 23px;
 +
    width: 30px;
 +
    top: 0px;
 +
    left: 160px;
 +
    margin-top:10px;
 +
    padding-top: 7px;
 +
    background: url(https://static.igem.org/mediawiki/2009/1/1b/Left_menu_pari.png);
 +
    z-index:4;
 +
}
-
We work on the cell-cell communication using vesicle, but in a first time we need to adress a signal on outer membrane. So we have to find something which can go from the cytoplasm to vesicle, can be exported a protein signal through the vesicle and finally adressing a signal when its fuse with the target cells.
+
#middle-side {
 +
    height: 25px;
 +
    width: 380px;
 +
    position: absolute;
 +
    top: 0px;
 +
    left: 170px;
 +
    margin-top:10px;
 +
    padding-top: 5px;
 +
    background: #dadada;
 +
    z-index:5;
 +
}
-
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 persiplasm 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]]]). But when there are too many ClyA, overproducing strains suggested that ClyA could accumulate in the periplasmic space[[http://www.ncbi.nlm.nih.gov/pubmed/14532000|[3]]].
+
#right-side {
 +
    position: absolute;
 +
    height: 23px;
 +
    width: 30px;
 +
    margin-top:10px;
 +
    padding-top: 7px;
 +
    top: 0px;
 +
    left: 530px;
 +
    background: url(https://static.igem.org/mediawiki/2009/4/40/Right_menu_paris.png);
 +
    z-index:4;
 +
}
 +
a.menu_sub {
 +
    padding-left: 7px;
 +
    padding-right: 7px;
 +
}
-
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]]]
+
a.menu_sub_active {
-
[[Image:Clya_structure.jpg|150px|left]] [[Image:Clya_structure2.jpg|100px|right]][[Image:ClyA.jpg|150px|center]]  [ajouter une photo de cly A tt seul]
+
    padding-left: 7px;
 +
    padding-right: 7px;
 +
    color:#b0310e;
 +
    font-weight:bold;
 +
}
 +
</style>
 +
<div id="left-side"></div>
 +
<div id="middle-side"><center>
 +
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|
 +
<a class="menu_sub_active" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|
 +
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|
 +
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|
 +
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a>
 +
</center>
 +
</div>
 +
<div id="right-side"></div>
 +
</html>
 +
We work on the cell-cell communication using vesicle:
 +
<br>
 +
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 <sup>[[Team:Paris/Addressing_overview3#References|[3]]]</sup>. ClyA is thus expressed on bacteria and OMVs surface. Moreover, when ClyA is overproduced, it is accumulated into the periplasmic space<sup>[[Team:Paris/Addressing_overview3#References|[3]]]</sup>.
-
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.  
+
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.<sup>[[Team:Paris/Addressing_overview3#References|[2]]]</sup>
 +
[[Image:Clya_simple.jpg|ClyA subunit|150px|left]] [[Image:Clya_structure.jpg|ClyA assembled|150px|right]]  [[Image:ClyA.jpg|ClyA are assembling in outer membrane of a host cell|150px|center]]
-
In some article, it’s fused to GFP in order to observed the vesicle, 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. And it's this information which we will exploit for our project. In fact we will fuse ClyA to RFP in our case in order to observe if our ClyA are in our vesicle, in a first time then we can think that this system couple to fec system can transduct a signal from outer membran to the cytoplasm.[[http://www.ncbi.nlm.nih.gov/pubmed/18511069|[1]]] <font color="green"><---- voir si c'est pas de trop car je l'ai déjà mis dans our strategie, peut etre qu'il vaut mieux faire un lien direct dans our strategy??</font>
 
-
'''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.
+
Some article argue that E.Coli K12 use this ClyA to lyse other cell (specially mamalian cell or eurcaryote cell<sup>[[Team:Paris/Addressing_overview3#References|[3]]]</sup>). But E. coli cells expressing clyA do not lyse each other.
-
-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.
 
 +
Kim et al. have successfully fused clyA to GFP in order to observe vesicles <sup>[[Team:Paris/Addressing_overview3#References|[1]]]</sup>, 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].
-
'''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.
 
 +
{{Template:Paris2009_guided|Addressing_overview2#bottom|Addressing_overview4#bottom}}
-
'''INTERESTING QUOTATIONS:'''
+
<html>
-
 
+
</div>
-
- "unfused ClyA accumulated in the cytoplasm, periplasm and OMV fractions."[[http://www.ncbi.nlm.nih.gov/pubmed/18511069|[1]]
+
<div id="paris_content_boxtop">
-
 
+
</div>
-
+
<div id="paris_content">
-
 
+
</html>
-
-"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]]]
+
-
 
+
-
 
+
-
'''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
+
====References====
-
- Westermark, M. & Uhlin, B.E. Silencing and Activation of ClyA Cytotoxin Expression in Escherichia coli
+
<ol class="references">
 +
<li>[[Team:Paris/Addressing_overview3#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/18511069 18511069]</li>
 +
<li>[[Team:Paris/Addressing_overview3#1| ^]]Muller, M. & Ban, N. The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism Nature (4 June 2009) 459, 726-730. [http://www.ncbi.nlm.nih.gov/pubmed/19421192 19421192]</li>
 +
<li>[[Team:Paris/Addressing_overview3#1| ^]]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. [http://www.ncbi.nlm.nih.gov/pubmed/14532000 14532000]</li>
 +
</ol>

Latest revision as of 02:49, 22 October 2009


Addressing the message in the outer membrane : ClyA

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 subunit
ClyA assembled
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[3]). 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.


Open book.gif

← Previous - Next →


References

  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/18511069 18511069]
  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. [http://www.ncbi.nlm.nih.gov/pubmed/19421192 19421192]
  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. [http://www.ncbi.nlm.nih.gov/pubmed/14532000 14532000]

Retrieved from "http://2009.igem.org/Team:Paris/Addressing_overview3"