Team:Paris/Transduction overview fusion

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Fusion: Jun/Fos and AIDA


Jun and Fos

Fos and Jun, the protein products of the nuclear proto-oncogenes c-fos and c-jun, associate preferentially to form a heterodimer. Both Fos and Jun contain a single leucine zipper region. Previous studies [1],[2] have shown that the leucine zippers of Fos and Jun are necessary and sufficient to mediate preferential heterodimer formation and that Jun : Fos heterodimers have higher stability than Jun homodimers


In our project we would like to be sure that vesicles are going to recognize target bacteria. In this direction we decided to use the Jun and Fos recognition system. The problem was that Jun is able to form an homodimer and an heterodimer with Fos, so the specific interaction between vesicles and receiver cell is not specific. An article demonstrated that 2 mutations in the leucine-zipper allow the Jun/Fos dimerisation but abolished the Jun/Jun dimer formation [3].


AIDA

The cell envelope of gram-negative bacteria consists of two membranes, the cytoplasmic or inner membrane and the outer membrane. Transport of proteins across the inner membrane in most cases follows the general secretory pathway (GSP) [4]. Therefore, in gram-negative bacteria, proteins end up in the periplasm. To translocate proteins to the outer surface or into the supernatant, gram-negative bacteria have developed several distinct mechanisms. In contrast to the secretory systems that require a variety of specialized accessory proteins that, often in combination with the GSP, are responsible for the export of one or several passenger proteins into the supernatant, the autotransporter protein family members carry the export signal and machinery within a single polypeptide chain. The adhesin-involved-in-diffuse-adherence (AIDA) autotransporter has been identified as a virulence factor of the enteropathogenic Escherichia coli strain 2787 [5] and predicted to be a member of the autotransporter protein family [6]

This AIDA autotransporter is using to translocate Jun and Fos to the outer membrane of bacteria (Jun for the donnor, Fos for the receiver).




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Fusion: g3p

What is the g3p and how could it be a key part in the vesicles-bacteria fusion ?

Description of g3p

Infection of Escherichia coli by filamentous bacteriophages as M13, fd, f1, is mediated by the phage gene 3 protein (g3p or pIII). This protein of 406 amino acid residues, has a signal peptide, two N-terminal domains and one C-terminal domain, separated by two flexible glycin-rich linkers. All three domains are indispensable for phage infectivity.
g3p could be also found in phage helper like M13KO7 [10]

  • The signal peptide (1-18aa) address the protein to the cell membrane before being cleaved. We deleted it because we fusione g3p to OmpA-Linker (BBa_K103996).
  • The first N-terminal domain (N1) binds to the bacterial periplasmatic domain of TolA ([http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG11007 TolAIII]), receptor presumably at the inner face of the outer membrane [9].
  • The second N-terminal domain (N2) gives recognition of the host cell by binding the F-pilus on the surface of E. coli. F-pilus is encode by the F episome of male E. coli, and is the primary receptor of the host cell [12].
  • In fact, N1 and N2 interact with each other to form a blocked di-domain (N1G1N2). The binding of N2 to the tip of the bacterial F-pilus releases N1, which becomes free to interact with its receptor TolA (TolAIII) [8][11].
  • The C terminus (CT) of g3p anchors the g3p in the phage coat by interacting with phage coat protein 6, at the tip of the phage. Its seem that phages are released from the bacterial membrane by a two-step mechanism involving a short C-terminal fragment of g3p [7].
  • N1, N2 and N3 domain are linked by flexible glycin-rich domains (G1 and G2). G1 is composed of four tandem copies of the sequence Glu-Gly-Gly-Gly-Ser. In a recent study it has been showed that it may have an active role in F-pilus-dependent infection.
  • Fusion of peptides or proteins to the N-terminus of intact g3p does not compromise infectivity of the phage, but insertion of polypeptides between N2 and N3 appear to reduce the infectivity.

Our use

The viral protein known as g3p is naturally exposed at the surface of the filamentous bacteriophage which enable it to get in the bacteria. The M13 phage has a high affinity for E.coli, and if we could place its g3p on the surface of the vesicles it could activate the fusion with the Outer membrane of the targeted bacteria.

To be sure to target the receiving bacteria, we separe the donnor from the receiver with the criterium of the presence or not of pilli, because the g3p need a pillus to start its incorporation process. So the donnor would be pillus negative and the receiver pillus positive.

OmpA-Linker is the second protein required because it is a protein that target any protein that is fuse to it to the surface of the Outer membrane, consequently we fuse G3P with OmpA-Linker.


Fusion: SNAREs

SNARE proteins are a large protein superfamily consisting of more than 60 members in yeast and mammalian cells.


The primary role of SNARE proteins is to mediate vesicle fusion, that is, the exocytosis of cellular transport vesicles with the cell membrane at the porosome or with a target compartment (such as a lysosome).


SNAREs can be divided into two categories: vesicle-SNAREs (or v-SNAREs), which are incorporated into the membranes of transport vesicles during budding, and target-SNAREs (or t-SNAREs), which are located in the membranes of target compartments.


The core of any functionnal SNARE complex is composed by four α-helices provided by the synaptobrevin (for one helix) by the syntaxin (for another helix) and by two SNAP-25 ( for the last two helices) :


Synaptobrevin : is a small integral membrane protein of secretory vesicles with molecular weight of 18 kilodalton (kDa) that is part of the vesicle-associated membrane protein (VAMP) family


Syntaxin : Syntaxin 1A was initially identified as a 35 kDa protein in the plasma membrane of amacrine cells [13], as a subunit of Ca2+ channels [14],[15] and as a synaptotagmin-binding protein [16]. Since these initial reports, the function of syntaxin as a central component in the synaptic vesicle membrane fusion machinery has been well established.


SNAP 25 : SNAP-25 is a membrane bound protein anchored to the cytosolic face of membranes via palmitoyl side chains in the middle of the molecule. SNAP-25 is a protein contributing two α-helices in the formation of the exocytotic fusion complex in neurons where it assembles with syntaxin-1 and synaptobrevin.



Exocytosis-machinery.jpg


Legend : Molecular machinery driving vesicle fusion in neuromediator release. The core SNARE complex is formed by four α-helices contributed by synaptobrevin, syntaxin and two SNAP-25. Synaptotagmin serves as a calcium sensor and regulates intimately the SNARE zipping



Using SNAREs in Bacteria : not so easy ...

Our first idea was to use SNAREs to perform the fusion between vesicles and target bacteria. The main problem is that the 3D structure of the SNARE complex is crucial for the fusion to proceed [16], [17], [18]. As SNAREs don't exist in bacteria we have to clone these genes (into bacteria) and we have to merge the SNAREs protein with a bacterial protein which is localized in the outer membrane (to allow the localization of SNARE protein to the surface of cell). In this direction we weren't sure to obtain the correct conformation of both v- (for the donnor) and t- (for the receiver) SNAREs after their exportation to the bacterial membrane, so we weren't sure that this mechanism will perform. We decided to focus our effort on the Jun/Fos strategy.






References

  1. ^Kouzarides, T. and E. Ziff. 1988. The role of the leucine zipper in the fos-jun interaction. Nature 336: 646-656. [http://www.ncbi.nlm.nih.gov/pubmed/2974122 2974122]
  2. ^Gentz, R., F.J. Rauscher III, C. Abate, and T. Curran. 1989. Parallel association of Fos and Jun leucine zippers juxtaposes DNA-binding domains. Science 243:16951699.[http://www.ncbi.nlm.nih.gov/pubmed/2494702 2494702]
  3. ^ Tod Smeal, Peter Angel, Jennifer Meek, and Michael Karin 1989. Different requirements for formation of Jun: Jun and Jun : Fos complexes. GENES & DEVELOPMENT 3:2091-2100. [http://www.ncbi.nlm.nih.gov/pubmed/2516828 2516828]
  4. ^Benz, I., and M. A. Schmidt. 1989. Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli. Infect. Immun. 57:1506–1511. [http://www.ncbi.nlm.nih.gov/pubmed/2565291 2565291]
  5. ^Murphy, C., W. Prinz, M. Pohlschroder, A. Derman, and J. Beckwith. 1995. Essential features of the pathway for protein translocation across the Escherichia coli cytoplasmic membrane. Cold Spring Harbor Symp. Quant. Biol. 60:277–283. [http://www.ncbi.nlm.nih.gov/pubmed/8824401 8824401]
  6. ^Klauser, T., J. Pohlner, and T. F. Meyer. 1990. Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation- dependent outer membrane translocation. EMBO J. 9:1991–1999. [http://www.ncbi.nlm.nih.gov/pubmed/2189728 2189728]
  7. ^Jef D.Boeke & Peter Model. A prokaryotic membrane anchor sequence: carboxyl terminus of bacteriophage f1 gene III protein retains it in the membrane. 1982.[http://www.ncbi.nlm.nih.gov/pubmed/6291030 6291030]
  8. ^Chatellier J & Riechmann L. Interdomain interactions within the gene 3 protein of filamentous phage. 1999.[http://www.ncbi.nlm.nih.gov/pubmed/10606756 10606756]
  9. ^ Lubkowski J & Wlodawer A. Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA. 1999.[http://www.ncbi.nlm.nih.gov/pubmed/10404600 10404600]
  10. ^Baek H & Cha S. An improved helper phage system for efficient isolation of specific antibody molecules in phage display. 2002.[http://www.ncbi.nlm.nih.gov/pubmed/11861923 11861923]
  11. ^ Karlsson F & Malmborg-Hager AC. The mechanism of bacterial infection by filamentous phages involves molecular interactions between TolA and phage protein 3 domains. 2003.[http://www.ncbi.nlm.nih.gov/pubmed/12670988 12670988]
  12. ^Caro LG, Schnös M. The attachment of the male-specific bacteriophage F1 to sensitive strains of Escherichia coli. 1966.[http://www.ncbi.nlm.nih.gov/pubmed/5338586 5338586]
  13. ^Barnstable C. J.Hofstein R.,Akagawa K. A marker of early amacrine cell development in rat retina.(1985) Brain Res 352:286–290.[http://www.ncbi.nlm.nih.gov/pubmed/3896407 3896407]
  14. ^Inoue A.Obata K.Akagawa K.(1992)Cloning and sequence analysis of cDNA for a neuronal cell membrane antigen, HPC-1 J. Biol. Chem. 267:10613–10619. [http://www.ncbi.nlm.nih.gov/pubmed/1587842 1587842]
  15. ^Yoshida A.,Oho C.,Omori A.,Kuwahara R.,Ito T.,Takahashi M.(1992)HPC-1 is associated with synaptotagmin and omega-conotoxin receptor J. Biol. Chem. 267:24925–24928. [http://www.ncbi.nlm.nih.gov/pubmed/1334074 1334074]
  16. ^Bennett M. K.,Calakos N.,Scheller R. H(1992). Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257:255–259. [http://www.ncbi.nlm.nih.gov/pubmed/1321498 1321498]Hu C, Ahmed M, Melia TJ, Söllner TH, Mayer T, Rothman JE. Fusion of cells by flipped SNAREs. Science. 2003 Jun 13;300(5626):1745-9. [http://www.ncbi.nlm.nih.gov/pubmed/12805548 12805548]
  17. ^Waters MG, Hughson FM. Membrane tethering and fusion in the secretory and endocytic pathways. Traffic. 2000 Aug;1(8):588-97. [http://www.ncbi.nlm.nih.gov/pubmed/11208146 11208146]
  18. ^Giraudo CG, Garcia-Diaz A, Eng WS, Chen Y, Hendrickson WA, Melia TJ, Rothman JE. Alternative zippering as an on-off switch for SNARE-mediated fusion. Science. 2009 Jan 23;323(5913):512-6. [http://www.ncbi.nlm.nih.gov/pubmed/19164750 19164750]

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