Team:Warsaw/Stage3/Design

From 2010.igem.org

Example Tabs

BactoDHL design

The construct

AraC
R0080

B0032

K177010

B0032

K177026

B0030
GFP
E0040

B0010

B0012

J23102

B0032

K177010

B0032

K177026

B0030
GFP
E0040

B0010

B0012

The first step of our continuous search for an efficient intracellular protein delivery system began with creating a construct consisting of harmoniously acting elements. Two different sets of regulatory sequences provide a high-level expression of Invasin (INV; K177010) and Listreriolysin (LLO; K177026), proteins encoded by primary-function genes of the invasiveness operon.

Invasin (INV) plays a key role in the initiation of Yersinia enterocolytica and Yersinia pseudotuberculosis infection. Through interaction with a beta1-integrin receptor present on the surface of eucaryotic cell membranes it triggers a signal-transduction pathway leading to internalisation of the whole bacterium in the endocytosis-dependent manner. The strong affinity of invasin to it’s receptor results in highly selective binding to the target molecule. Mammalian cells depleted of beta1-integrin cannot be infected.

The presence of invasin on the surface of BactoDHL results in completion of stage one of the protein delivery scheme – selective binding and internalization.

Left: Pore formed by 12 molecules of hemolysin;
Right: Structure of hemolysin which is close related to listeriolysin.

In terms of endocytosis-dependent protein delivery route endosomal escape is essential for preventing the cargo from being degraded by lysosomal enzymes. Listeriolysin (LLO) had already been proven to be sufficient to allow escape of Bacillus subtilis and Escherichia coli from the phagocytic vaquole, which explains the use of the protein in construction of BactoDHL. LLO often has also been used as a key-role protein determining live vaccine vectors performance.

Left: Structural model of the LLO monomer from: P. Schnupf , D.A. Portnoy Listeriolysin O: a phagosome-specific lysin, Microbes and infection (2007) 1176-1187

Listeriolysin is a member of a widespread cholesterol-dependent pore-forming cytolysins family (CDCs). The first step of the process eventually resulting in BactoDHL’s phagosomal escape involves binding of monomeric listeriolysin molecules to lipid bilayer containing cholesterol. The binding induces conformational change that subsequently leads to the formation of a prepores oligomeric structures (consisting of 33-50 monomers) converting into large (maximum 350A-diameter) pores. This severely disturbs the stability of endosomal membrane and causes it’s rupture.

LLO is a phagosome-specyfic lysin. The acidic pH is necessary for it’s full hemolytic activity. Neutral pH of cytosol causes premature unfolding of TMH domains responsible for aqueous pore formation. This mechanism prevents Listeria spp from killing the host cell and losing the intracellular environment. In case of BactoDHL it guarantees the lowest possible level of cytotoxicity, incomparable to this involved with the use of any other protein from CDCs family.

Regulatory elements:

Two kinds of strong, constitutive promoters were used in development of the constructs: AraC-regulated promoter (R0080) and J23102, fully synthetic promoter, slightly weaker than the arabinose promoter. Double terminator (B0010+B0012) guarantees the stability of polycistronic RNA as a product of transcription. The translational-level regulatory elements are Shine-Dalgarno sequences (B0032) of a medium strength in relation to B0034. GFP as a reporter allows the tracking of gene expression levels and BactoDHL’s localization.