Team:Warsaw/Project/invasion

Entrance of bacteria into eukaryotic cells

The main aim of our project is to make, using synthetic biology methods, Escherichia coli capable of entering mammalian cells. We've decided to use proteins used in the same manner by species like Listeria sp. or Yersinia sp.

Theoretical basis

Many bacterial species are capable of entering mammalian cells. One of the crucial proteins for this process is invasin. It is capable of selective interaction with integrins, which are present on external eukaryotic membrane. This triggers signalling cascade, indispensable to start endocytosis [9]. This interaction occurs via the invasin N-terminal domain, which is capable of promoting effective endocytosis of bacteria that synthesize invasin by cells normally unable to undertake phagocytosis [3]. The other protein which is involved in process of entering mammalian cells is internalin, which can be found on the surface of Listeria monocytogenes. Internalin is selecively interacting with cadherins found on the external eukaryotic membrane.

Details

Invasion process

Invasion process has to be driven by 2 proteins: invasin from Yersinia sp. or InternalinA from Listeria monocytogenes. In a presence of listeriolysin from Listeria monocytogenes both proteins should effectively bind to surface of mammalian cells and trigger internalisation of whole bacteria. We would like to test both invasin and InternalinA and test which one is the most effective.

Listeriolysin will be fused with secretion system because, in the opposite to invasin and internalinA, it doesn't contain its own secretion signal. Listeriolysin is also important for the next step of invasion - escape from the endosome.

Regulation

Our invasion module is controlled by a bistable switch, composed by 2 promoters and 2 repressors. Invasion proteins are under control of bacteriophage promoter (pR) that is repressed by cI protein. The second part of the operon is gene cI_ts that encodes for thermosensitive version of the cI repressor protein from phage λ. It is under the control of lactose promoter (Plac). This λ system of promoters and regulatory genes creates two mutually exclusive negative feedback loops and is dubbed the bistable switch [19]. It has two states depending on conditions within the cell. cI protein binds the PR promoter inhibiting its activity and expression of the invasion operon genes. cI_ts protein is inactivated by high temperature 42°C what allows the expression of invasion operon that codes for lacI. lacI stops gene expression from the Plac promoter ( the expression of cI protein). IPTG that binds and inactivates lacI can be used to switch on the expression from Plac promoter. Another way to control the operon is in trans expression of cI or lacI genes. Activated invasion operon causes internalisation of bacteria by mammalian cells. Expression of green fluorescence protein allows observation of the process using fluorescence microscopy.

Fig 2. The overview of the invasion operon. It is based on a lacI/cI bistable switch. After thermal activation (42°C) the expression of genes that enable invasion of mammalian cells is activated. The invasion operon is composed of lacI – lactose operon repressor, llo – hemolysin from Listeria monocytogenes, inv – invasin from Yersinia sp., phoQ/phoP two component system detecting endosomal conditions from Salmonella typhimurium, GFP – Green Fluorescent Protein from Aqueora victoria. Proteins are fused with LVA sequence which provides quick proteolytic degradation.

The invasion operon includes lacI– lactose operon repressor, llo – hemolysin from Listeria monocytogenes (listeriolysin), inv – invasin from Yersinia sp., PhoP/PhoQ two component system that detects endosomal conditions from Salmonella typhimurium, GFP – green fluorescent protein. (Fig.2.). These genes are under the control of bacteriophage promoter (PR) that is repressed by cI protein. The second part of the operon is gene cI_ts that encodes for thermosensitive version of the cI repressor protein from phage λ. It is under the control of lactose promoter (Plac). This λ system of promoters and regulatory genes creates two mutually exclusive negative feedback loops and is dubbed the bistable switch [19]. It has two states depending on conditions within the cell. cI protein binds the PR promoter inhibiting its activity and expression of the invasion operon genes. cI_ts protein is inactivated by high temperature 42°C it allows the expression of invasion operon that codes for lacI. lacI stops gene expression from the Plac promoter ( the expression of cI protein). IPTG that binds and inactivates lacI can be used to switch on the expression from Plac promoter. Another way to control the operon is in trans expression of cI or lacI genes. Activated invasion operon causes internalisation of bacteria by mammalian cells. It also detects endosomal localisation using PhoP/PhoQ system. Expression of green fluorescence protein allows observation of the process using confocal microscopy.

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