Team:Paris/Addressing overview clya

From 2009.igem.org

(Difference between revisions)
(→Overview)
(→B. ClyA)
 
(8 intermediate revisions not shown)
Line 1: Line 1:
-
<span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview#top | Adressing]] > [[Team:Paris/Addressing_overview#bottom | Overview]] > [[Team:Paris/Addressing_overview_clya#bottom | ClyA ]]
+
<span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview#top | Adressing]] > [[Team:Paris/Addressing_overview#bottom | Export & ClyA]] > [[Team:Paris/Addressing_overview_clya#bottom | ClyA ]]
{{Template:Paris2009}}
{{Template:Paris2009}}
{{Template:Paris2009_menu3}}
{{Template:Paris2009_menu3}}
Line 15: Line 15:
<div id="paris_content">
<div id="paris_content">
</html>
</html>
-
===B. ClyA===
+
===ClyA===
-
We work on the cell-cell communication using vesicle. that’s why we have to find something to track our vesicle, but also send a protein (or anything witch create a signal) in this vesicle, in order to make a signal transduction. ClyA seems to have an interesting ways to success in these functions. Let's see more about it and found why.
+
We work on the cell-cell communication using vesicle. That’s why we have to find something to track our vesicle, but also put a signal peptide (or anything which can create a signal) in this vesicle, in order to make a signal transduction when the vesicle fusion with his target cell. So ClyA seems to have an interesting ways to success in these functions, in fact ClyA in E.Coli in high expression in OMV, and it’s one of the proteins that can pass the cytoplasm to the persiplasm and integrated to vesicle.
-
====B.1. Let's introduce it====
+
However there are some inconvenient 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.
 +
[[Image:ClyA.jpg|150px|center]]
 +
So some article show that E.Coli K12 using this ClyA to “destruct” other bacteria or eucaryot cell. But this virulence was not show in same strain.  
-
[[Image:ClyA.jpg|150px|right]] ClyA was found in pathogenic and non-pathogenic <i>E. Coli</i> strains, in <i>Salmonella enterica serovars Typhi</i> and in <i>Paratyphi A</i>.
+
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.
-
+
-
It’s an alpha-PFT for Pore Forming Toxins. PFTs are potent virulence factors class starting in a soluble from to a outer membrane-integrated pore. They exhibit their toxic effect either by membrane permeability barrier destruction or by toxic components delivery through the pores. PFTs can be subdivided into two classes; &alpha;-PFTs and &beta;-PFTs, depending on the suspected mode of membrane integration, either by &alpha;-helical or &beta;-sheet elements.
+
-
Moreover the ClyA gene in <i>E. Coli K-12 </i> is silenced in non-pathogenic strains by nucleoid protein H-NS which bind clyA sequences in the promoter region. We decided to work on this protein 'cause of its hight expression in OMV.
+
'''AVANTAGE'''
 +
 
 +
- Cly A 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, then we could be easily tracked during vesicle interaction other cells, since once it’s fused OMV become highly fluorescent.
 +
 
 +
-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.
 +
 
 +
-ClyA was significantly enriched in OMVs relative to other lumenal and membrane bound OMV proteins.
 +
 
 +
 
 +
'''DRAWBACK'''
 +
 
 +
-Cly A is cytolitic and a PFTàso it’s virulent; it does target cell lysis by forming a pore or by delivery of toxic components through the pores. But we find only that ClyA is virulent for mammalian cell or erythrocytes, and it’s a strong interaction with cholesterol which is in mammalian cell membrane. For the virulence in bacteria cell we think that it’s not because there is no cholesterol in the bacteria membrane.  
 +
 
 +
 
 +
'''INTERESTING QUOTATIONS:'''
 +
 
 +
- unfused ClyA accumulated in the cytoplasm, periplasm and OMV fractions
 +
 
 +
 +
-"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."
-
The ClyA water-soluble form (monomer form) is a bundle of four major a-helices (aA, aB, aC and aF). Monomer form have :
 
-
* a small surface exposed
 
-
* a hydrophobic B-hairpin at the head end of the structure located in a predicted transmembrane region witch have an important role in membrane association and trigger the conformational switch.
 
-
* the N and C terminal at the tail end.
 
 +
'''Source:'''
-
For the lipid-associated ClyA (promoter form), it's organized as a three-helix bundle (aB, aC and aF) and form an oligomeric pore complex composed of either 8 or 13 ClyA subunits.
+
-Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66
-
In the assembled pore, all helices (except alpha-G) contribute to protomer–protomer contacts. Each helix of the twisted three-helix bundle contributes to both interfaces. A network of 25 hydrogen bonds and 13 salt bridges indicates tight and specific promoters association. These specific interfaces do not exist in the soluble ClyA monomer, indicating that conversion of ClyA to the protomeric form has to precede oligomerization. '''So, monomeric Cly A switchs to protomeric form when B-hairpin are associated in the membrane.'''
+
-Muller, M. & Ban, N. The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism Nature 459, 726-730 (4 June 2009)
-
====B.2. ClyA pore formation====
 
-
ClyA is inserted into the outer membrane as inactive monomers. Then they are released and embedded in OMVs, at this point they are converted into an active form. During the host fusion of the OMV the functional ClyA pore is inserted, and then, deliver the membrane active virulence factors into the host cell. So '''ClyA don’t lysis the bacteria which produce it.'''
+
- Westermark, M. & Uhlin, B.E.  Silencing and Activation of ClyA Cytotoxin Expression in Escherichia coli
-
the step are as follow:
+
- Oscarsson, J. & Uhlin, B.E. Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli
-
* The amino-terminal helix (aA) of each protomer is protruding into an iris-like arrangement forming the constriction at the top of the pore.
+
-
* Closing of the pore will lead to compaction and buckling of the amphipathic helices. As a result, these helices will be pushed upwards wedging, rather then ‘punching’, a hole in the membrane.
+
-
*Once formed, the pore opening is maintained by the iris-like interlocking of the amphipathic helices.
+

Latest revision as of 21:37, 14 October 2009

iGEM > Paris > Adressing > Export & ClyA > ClyA

Overview

ClyA

We work on the cell-cell communication using vesicle. That’s why we have to find something to track our vesicle, but also put a signal peptide (or anything which can create a signal) in this vesicle, in order to make a signal transduction when the vesicle fusion with his target cell. So ClyA seems to have an interesting ways to success in these functions, in fact ClyA in E.Coli in high expression in OMV, and it’s one of the proteins that can pass the cytoplasm to the persiplasm and integrated to vesicle.

However there are some inconvenient 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.

ClyA.jpg

So some article show that E.Coli K12 using this ClyA to “destruct” other bacteria or eucaryot cell. But this virulence was not show in same strain.

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.


AVANTAGE

- Cly A 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, then we could be easily tracked during vesicle interaction other cells, since once it’s fused OMV become highly fluorescent.

-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.

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


DRAWBACK

-Cly A is cytolitic and a PFTàso it’s virulent; it does target cell lysis by forming a pore or by delivery of toxic components through the pores. But we find only that ClyA is virulent for mammalian cell or erythrocytes, and it’s a strong interaction with cholesterol which is in mammalian cell membrane. For the virulence in bacteria cell we think that it’s not because there is no cholesterol in the bacteria membrane.


INTERESTING QUOTATIONS:

- unfused ClyA accumulated in the cytoplasm, periplasm and OMV fractions


-"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."


Source:

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


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


- Westermark, M. & Uhlin, B.E. Silencing and Activation of ClyA Cytotoxin Expression in Escherichia coli


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

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