Team:Paris/Addressing overview2

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<span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview#top | Adressing]] > [[Team:Paris/Addressing_overview#bottom | Clya]]
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<span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview#top | Adressing]] > [[Team:Paris/Addressing_overview#bottom | ClyA]]
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== Overview ==
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* [[Team:Paris/Addressing_overview2#Overview |Clya ]]
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* [[Team:Paris/Addressing_overview2_strategy#Overview |C. Our strategy]]
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==Addressing the message in the outer membrane : Main==
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* [[Team:Paris/Addressing_overview2_Construction#Overview |D. Construction]]
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==ClyA==
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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.
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|
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<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a>
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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.
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Bacterial pathogens display proteins on their surface that may interact with their hosts in order to mount successful infections. Although the primary function of the peptidoglycan is to provide a physical barrier for protection against both mechanical and osmotic stresses, it also serves as a scaffold to anchor external structures such as the outer cell membrane in ''Escherichia Coli''. Over the past 20 years, it has become apparent that Gram-negative bacteria have evolved a variety of mechanism by which proteins are displayed on the cell surface (Tat and Sec transporter for example, cf the section "to the periplasm, export system" for more information about them).
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[[Image:ClyA.jpg|150px|center]]
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Using these transport system, lot of protein are localized on the outer membrane, and one of the major protein of the bacterial membrane is OmpA. There is also an other protein, ClyA, but less known than OmpA which can be exported from cytoplasm to outer membrane, then by vesiculation interact with their host.
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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.  
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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.
 
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'''AVANTAGE'''
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===ClyA===
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- Cly A can be used to co-localize fully functional heterologous proteins directly in bacterial OMVs
 
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-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.
 
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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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Pore-forming toxins (PFTs) are a class of potent virulence factors that convert from a soluble form to a membrane-integrated pore. They exhibit their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores.
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-Cly A confers vesicle binding to and invasion of host cells.
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Cytolysin A (ClyA, also known as HlyE), a PFT, is a cytolytic α-helical toxin responsible for the haemolytic phenotype of several ''E. coli''. <sup>[[Team:Paris/Addressing_overview2#References|[1]]]</sup>.
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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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Why did we decided to use ClyA?
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-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.  
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Althought the toxic effect of Cly A, using it is an interesting way to adress protein to the external membrane, because ClyA contain the signal peptide required to be exported form the cytoplasm to the outer membrane. In addition, it has been shown that certain membrane and/or soluble periplasmic proteins are enriched in vesicles while others are preferentially excluded. The majority of these enriched proteins happen to be virulence factors including cytolysin A of ''E. coli'' <sup>[[Team:Paris/Addressing_overview2#References|[2]]]</sup>..
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'''INTERESTING QUOTATIONS:'''
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Previous studies demonstrated that genetic fusions between ClyA of ''E. coli'' and reporter proteins such as Bla and GFP were translocated across the cytoplasmic membrane <sup>[[Team:Paris/Addressing_overview2#References|[3]]]</sup>.,<sup>[[Team:Paris/Addressing_overview2#References|[4]]]</sup>. and that localization was independent of the position (N or C terminus) of ClyA in the fusion protein. Moreover a recent article <sup>[[Team:Paris/Addressing_overview2#References|[5]]]</sup>. demonstrated that fusions to the C terminus of ClyA allow the transport of protein closer to the surface of outer membrane and to extend them into the extracellular environment.
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<span/ id="6">
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- unfused ClyA accumulated in the cytoplasm, periplasm and OMV fractions
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===OmpA===
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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."
 
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OmpA is a major protein of the outer membrane of ''E.Coli''. Moreover Kesty and co-workers demonstrated that OmpA is an outer-membrane component of native vesicles <sup>[[Team:Paris/Addressing_overview2#References|[6]]]</sup>.. In this direction, our strategy is to fuse our protein of interest to OmpA to allow its transport to the outer membrane and to increase the probability of its integration into vesicles.
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We finally decided to focus our efforts on the ClyA strategy because of the previous work that was done on this protein especially the "proof" that it's possible to merge our protein of interest with ClyA without inhibiting both activities.
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'''Source:'''
 
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-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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-Muller, M. & Ban, N. The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism Nature 459, 726-730 (4 June 2009)
 
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====References====
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<ol class="references">
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<li> [[Team:Paris/Addressing_overview2#1 | ^]] The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism, M.Mueller & N.Ban. 2009 - [http://www.ncbi.nlm.nih.gov/pubmed/19421192 | 19421192 ]</li>
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<li> [[Team:Paris/Addressing_overview2#1 | ^]]S.N. Wai, B.Lindmark, T.Soderblom, A.Takade, M.Westermark, J.Oscarsson, et al. Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin, 2003, Cell, 115, 25–35. [http://www.ncbi.nlm.nih.gov/pubmed/14532000 14532000]</li>
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<li> [[Team:Paris/Addressing_overview2#1 | ^]]F.J del Castillo, F. Moreno. and I.del Castillo. Secretion of the Escherichia coli K-12 SheA hemolysin is independent of its cytolytic activity, 2001, FEMS Microbiol.Lett. 204, 281–285. [http://www.ncbi.nlm.nih.gov/pubmed/11731136 11731136]</li>
 +
<li> [[Team:Paris/Addressing_overview2#1 | ^]]J.E.Galen, L.Zhao, M.Chinchilla, J.Y.Wang,M.F.Pasetti, J.Green and M.M. Levine. Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA, 2004, Infect. Immun. 72, 7096–7106.[http://www.ncbi.nlm.nih.gov/pubmed/15557633 15557633]</li>
 +
<li> [[Team:Paris/Addressing_overview2#1 | ^]] J.Y. Kim, A.M. Doody, D. J. Chen, G.H. Cremona, M.L. Shuler, D.Putnam,and M.P. DeLisa.Engineered. Bacterial Outer Membrane Vesicles with Enhanced Functionality, 2008, J. Mol. Biol. 380, 51–66. [http://www.ncbi.nlm.nih.gov/pubmed/18511069 18511069]</li>
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<li> [[Team:Paris/Addressing_overview2#6 | ^]]N.C.Kesty, M.J.Kuehn. Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles, 2004, J Biol Chem. 279(3):2069-76.[http://www.ncbi.nlm.nih.gov/pubmed/14578354 14578354]</li>
 +
</ol>
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- Westermark, M. & Uhlin, B.E.  Silencing and Activation of ClyA Cytotoxin Expression in Escherichia coli
 
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- Oscarsson, J. & Uhlin, B.E. Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli
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Latest revision as of 01:36, 22 October 2009

iGEM > Paris > Adressing > ClyA


Contents

Addressing the message in the outer membrane : Main

Bacterial pathogens display proteins on their surface that may interact with their hosts in order to mount successful infections. Although the primary function of the peptidoglycan is to provide a physical barrier for protection against both mechanical and osmotic stresses, it also serves as a scaffold to anchor external structures such as the outer cell membrane in Escherichia Coli. Over the past 20 years, it has become apparent that Gram-negative bacteria have evolved a variety of mechanism by which proteins are displayed on the cell surface (Tat and Sec transporter for example, cf the section "to the periplasm, export system" for more information about them). Using these transport system, lot of protein are localized on the outer membrane, and one of the major protein of the bacterial membrane is OmpA. There is also an other protein, ClyA, but less known than OmpA which can be exported from cytoplasm to outer membrane, then by vesiculation interact with their host.


ClyA

Pore-forming toxins (PFTs) are a class of potent virulence factors that convert from a soluble form to a membrane-integrated pore. They exhibit their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores.

Cytolysin A (ClyA, also known as HlyE), a PFT, is a cytolytic α-helical toxin responsible for the haemolytic phenotype of several E. coli. [1].


Why did we decided to use ClyA?

Althought the toxic effect of Cly A, using it is an interesting way to adress protein to the external membrane, because ClyA contain the signal peptide required to be exported form the cytoplasm to the outer membrane. In addition, it has been shown that certain membrane and/or soluble periplasmic proteins are enriched in vesicles while others are preferentially excluded. The majority of these enriched proteins happen to be virulence factors including cytolysin A of E. coli [2]..


Previous studies demonstrated that genetic fusions between ClyA of E. coli and reporter proteins such as Bla and GFP were translocated across the cytoplasmic membrane [3].,[4]. and that localization was independent of the position (N or C terminus) of ClyA in the fusion protein. Moreover a recent article [5]. demonstrated that fusions to the C terminus of ClyA allow the transport of protein closer to the surface of outer membrane and to extend them into the extracellular environment.

OmpA

OmpA is a major protein of the outer membrane of E.Coli. Moreover Kesty and co-workers demonstrated that OmpA is an outer-membrane component of native vesicles [6].. In this direction, our strategy is to fuse our protein of interest to OmpA to allow its transport to the outer membrane and to increase the probability of its integration into vesicles. We finally decided to focus our efforts on the ClyA strategy because of the previous work that was done on this protein especially the "proof" that it's possible to merge our protein of interest with ClyA without inhibiting both activities.



References

  1. ^ The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism, M.Mueller & N.Ban. 2009 - | 19421192
  2. ^S.N. Wai, B.Lindmark, T.Soderblom, A.Takade, M.Westermark, J.Oscarsson, et al. Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin, 2003, Cell, 115, 25–35. 14532000
  3. ^F.J del Castillo, F. Moreno. and I.del Castillo. Secretion of the Escherichia coli K-12 SheA hemolysin is independent of its cytolytic activity, 2001, FEMS Microbiol.Lett. 204, 281–285. 11731136
  4. ^J.E.Galen, L.Zhao, M.Chinchilla, J.Y.Wang,M.F.Pasetti, J.Green and M.M. Levine. Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA, 2004, Infect. Immun. 72, 7096–7106.15557633
  5. ^ J.Y. Kim, A.M. Doody, D. J. Chen, G.H. Cremona, M.L. Shuler, D.Putnam,and M.P. DeLisa.Engineered. Bacterial Outer Membrane Vesicles with Enhanced Functionality, 2008, J. Mol. Biol. 380, 51–66. 18511069
  6. ^N.C.Kesty, M.J.Kuehn. Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles, 2004, J Biol Chem. 279(3):2069-76.14578354


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