Team:Paris/Addressing overview3

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==Adressing the message in the outer membrane : ClyA==
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==Addressing the message in the outer membrane : ClyA==
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We work on the cell-cell communication using vesicle. To begin we need to adress a signal to the outer membrane, so we have to find something which can go from the cytoplasm to the outer membrane (using a protein signal). Thus these protein have to be into vesicles to allow the transmission of the message.
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We work on the cell-cell communication using vesicle:
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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.
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In this direction ClyA (the collicin A of ''E.Coli'') seems to have an interesting ways to succeed. Actually, ClyA was found to have a high expression into OMVs, thanks to the type I pathway. It’s also one of the proteins that can be transported to the outer membrane. [[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]]).  
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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>.
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However there are some inconvenients to use this protein. The major one is that ClyA is an alpha-PFT (Pore Forming Toxins). 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 the permeability of membrane barrier and, in another way, 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.
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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>
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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]]]
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[[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]]
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[[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]]
 
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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.
 
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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.
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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]
 
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'''AVANTAGE'''
 
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- ClyA can be used to co-localize fully functional heterologous proteins directly in bacterial OMVs
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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].
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-We can fuse GFP to the C or N term of Cly A, to track OMVs easily.
 
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-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.
 
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-Cly A confers vesicle binding to and invasion of host cells.[[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]]]
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{{Template:Paris2009_guided|Addressing_overview2#bottom|Addressing_overview4#bottom}}
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-ClyA was significantly enriched in OMVs relative to other lumenal and membrane bound OMV proteins.
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'''DRAWBACK'''
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====References====
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-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.
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<ol class="references">
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<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>
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<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>
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'''INTERESTING QUOTATIONS:'''
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<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>
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- "unfused ClyA accumulated in the cytoplasm, periplasm and OMV fractions."[[http://www.ncbi.nlm.nih.gov/pubmed/18511069|[1]]]
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-"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]]]
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'''Source:'''
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[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
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[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 (4 June 2009) 459, 726-730  
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[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 (October 2003), 115,25-35
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[http://www.ncbi.nlm.nih.gov/pubmed/10383763 4-] Oscarsson, J. & Uhlin, B.E. Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli Molecular Microbiology (1999) 32(6), 1226–1238
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[http://jb.asm.org/cgi/content/abstract/182/22/6347 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
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{{Template:Paris2009_guided|Addressing_overview2#bottom|Addressing_overview4#bottom}}
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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.


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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]

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