Team:Warsaw/Project/detailed

Detailed research project

Introduction

Fig 1. The overview of the system. Gene regulatory network is composed of three modules: invasion operon, endosome detection operon and cytoplasmic operon. Invasion and cytoplasmic operon are bistable switches regulated by endosome detection operon.

We are going to create E. coli strain able to enter the eukaryotic cells using the controlled expression of invasin from Yersinia sp. It leads to internalization of the bacteria into the phagosome of the host cell. To escape the endosome we will use controlled expression of bacterial hemolysin (listeriolysin) from Listeria monocytogenes. The coordination of protein expression is based on switching the E. coli cells between four physiological states: growth in the growth medium, ability to enter mammalian cells, presence in the endosome, and escape from the endosome into the cytoplasm. (Fig1.). Two component system phoP/phoQ from Salmonella typhimurium will be used to detect states. It activates PhoP dependent promoters in the endosomal conditions (low pH, low metal ions concentration). Each of the states is going to be stabilised by two feedback loops based on natural regulatory systems. System is composed of three synthetic operons [18].

Invasion operon

Fig 2. The overview of the invasion operon. It is 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. Proteins are fused with LVA sequence that provides quick proteolytic degradation by bacteria.

The invasion operon includes lacI – lactose operon repressor, llo – hemolysin from Listeria monocytogenes, inv – inwasin from Yersinia sp., phoQ/phoP 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 CL protein. The second part of the operon is gene clts that codes for thermosensitive version of the CI repressor protein from phage . It is under control of lactose promoter (Plac). This λ system of promoters and regulatory genes creates two mutually exclusive negative feedback loops and is called bistable switch[19]. It has two states depending on conditions within the cell. Cl protein binds the PR promoter inhibiting its activity and expression of the invasion operon genes. Cl 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 Cl 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 CL 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.

Endosome detection operon

Fig 3. Overview of endosomal detection operon. It is composed of cro – antirepressor from phage λ, tetR – repressor of tetracycline resistance operon, cI – repressor from phage λ and cfp – Cyan Fluorescent Protein.

The low endosomal pH limits the concentration of metal ions. In these conditions transmembrane receptor kinase phoQ is less likely to bind its ligands – divalent cations. It allows the phosphorylation of phoP by phoQ. Phosphorylated phoP activates the gene expression from phoQ promoter expression of proteins encoded by endosome detection operon. Cl protein inactivates the invasion operon. TetR protein activates cytoplasmic operon. But not all genes encoded by cytoplasmic operon are expressed in endosome because operon contains cro box that binds cro protein which stops transcription. Cro protein is encoded by endosome detection operon. After bacterium escapes into cytoplasm the endosome detection operon genes are no longer expressed because phoP is not phosphorylated in cytoplasmic conditions. Process can be monitored using CFP fluorescence protein.

The cytoplasmic operon.

Fig 4. Overview of endosomal detection operon. It is composed of YFP – Yellow Fluorescent Protein, tetR – repressor of a tetracycline resistance operon, cro box regulatory sequence from phage and λ desired genes and genes specific for desired function of bacterial strain. In this case p53 – apoptosis inducer.

Cytoplasmic operon like the invasion operon is a bistable switch. The regulatory elements are: synthetic promoter inhibited by TetR, promoter inhibited by araC, araC protein (inactivated by L-arabinose) and TetR protein (repressor of tetracycline resistance gene inactivated by tetracycline). [18] This switch controls genes expressed in the cytoplasm. Cro box confines the gene expression to the cytoplasm only. Cro protein bound to it stops the transcription while the endosome detection operon is expressed. The yellow florescence protein is expressed to allow detection of the cytoplasmic operon activity. The set of genes in the cytoplasmic operon depends on the desired functionality of bacterial strain. In case of anticancer therapy it would be p53 protein with mitochondrial leader sequence. In the strain able to conjugate with mitochondria cytoplasmic operon will control genes responsible for conjugation process.

The mechanism of proposed regulatory system.

In the medium the expression of all genetic circuits is inactive. After temperature increase to 42°C the system is activated by thermal deactivation of cI. Genes from invasion operon are transcribed. They maintain their own expression, cause internalisation of E. coli by mammalian cells and enable escape from endosome. When bacterial cell is inside the endosome the endosome detection module is activated by phoP/phoQ system. In trans expression of regulatory genes cI and tetR deactivates invasion operon and activates cytoplasmic operon. However its genes are not expressed as long as bacterial cell is inside endosome due to cro protein activity. When bacterium escapes from the endosome, the transcription from endosome detection operon is repressed and cytoplasmic operon genes are expressed.

Conjugation with mitochondria.

To place the copy of the mitochondrial genome into bacterial cell the pBACrNESd plasmid will be used. It is built basing the Bacterial Artificial Chromosome. Replication origin (ori R6K), kanamycin resistance gen (kanR) and replication complex genes (repE, parA, parC) from pBACrNESd plasmid will be fused with origin of transfer (oriTF) from natural conjugative F plasmid. Unique restriction site will be introduced to the mitochondrial genome by PCR reaction and the described gene construct will be inserted into mitochondrion. To enable conjugation proteins encoded on RP4 plasmid (Tc, MuKm, Tn7) will be placed in the cytoplasmic operon. These genes are responsible for pillus formation and transport of single stranded DNA. The cell with this system will conjugate with any structure surround by lipid bilayer. We will prepare mitochondrial DNA deficient cells using procedure described in [2]. The occurrence of conjugation will be confirmed by PCR reaction specific for sequences added to mitochondrial DNA. The quality of the DNA will be determined by analysis of restriction patterns. The expression of mitochondrial genes will be confirmed by semiquantitative time PCR [2].

Secretion of p53 into the cytoplasm

To efficiently transport p53 to the eukaryotic cells the endogenic E. coli secretion system responsible for secretion of hemolysin by uropathogenic strains will be used. It is composed of three transport proteins HlyB, HlyD and TolC [10]. TolC is present in the genome of the lab strain derived from K12 strain. To ensure efficient secretion hlyB and hlyD have to be introduced into the genome and proapoptotic protein has to be fused with 60 amino acid long C-terminal sequence of hemolysin. [5].

Directing bacterial proteins to mammalian mitochondria.

We need to be able to direct to the mammalian mitochondria some of the proteins secrete by bacteria into the cytoplasm of the host cell. It is going to be archived by fusion of mitochondrial leader sequence from human protein Suv3 to the N-terminus of proteins. It has been shown that 40 amino acids long N-terminal sequence is enough for effective transport of Suv3 through the mitochondrial membrane. [21]. First the efficiency of the process will be assessed by detection of Red Fluorescent Protein with confocal microscopy. The red fluorescence should be visible in the mitochondria of the cells invaded by bacteria expressing RFP fused with mitochondrial leader sequence. Then the ability to induce apoptosis by mitochondrially directed p53 will be investigated. Apoptosis induction will be confirmed by flow cytometry [8]. In addition we will use confocal microscopy to monitor the process. In both cases cells will be stained with propidium iodide and annexin V.