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Adressing the message in the outer membrane : ClyA

We work on the cell-cell communication using vesicle:
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 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."[[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[[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.[[2]]

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.

In some article, it’s fused to GFP in order to observed the vesicle[[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 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.[[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[[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."[[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."[[2]]

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