Team:Warsaw/Project/theory

Theoretical basis

Entrance of bacteria into eukaryotic cells

Many bacterial species are able to invade an eukaryotic cell. One of the crucial proteins for this process is invasin. It is capable of selective interaction with integrins, which are present on eukaryotic membrane. It triggers signalling cascade indispensable to start endocytosis [9]. This interaction occurs via the invasin N-terminal domain, which is able to promote effective endocytosis of bacteria that synthesize invasin by cells normally unable to undertake phagocytosis [3]. Majority of the enteropathogenic bacteria are incapable to divide within the phagosome, but after leaving the phagosome they can proliferate rapidly in the cytoplasm. Escape from the phagosome is possible because of hemolysin which causes membrane permeabilisation and phagosome disruption [16]. It was presented previously that transfection with gene encoding hemolysin is sufficient to enable Bacillus subtilis and Escherichia coli to escape phagosomal vesicle [1].

Mitochondrial transformation

Electron transport chain function can be impaired by a variety of defects acting on the DNA level: not only point mutation and local deletions in genes encoding proteins of the chain, but also deletions of huge fragments or even whole mitochondrial genome (mtDNA). Correct mitochondrion contains 2 to 10 copies of mtDNA. Defects in mitochondrial DNA result in severe medical disorders especially in tissues which consume large amount of energy like nervous and muscle tissue. Effects of this impairments are neuroand myopathies [20]. The standard therapy based on bypassing interrupted pathways does not apply under mentioned circumstances. Administration of enzyme cofactors or other metabolically active substances is not effective and may be applied only as supplementary therapy. We are forced to seek novel treatment strategies to cure patients suffering from mitochondrial disorders [7]. Currently the most commonly used method of mitochondrial transformation is the "gene gun" approach which is based on delivery of metal beads coated with DNA into the cell. This method is mostly limited to plants and yeast and has low therapeutic potential [7]. Current mammalian mitochondrial transformation techniques like electroporation are also limited. Whole process takes place ex vivo and causes irreversible mitochondrion destructions which make its efficient reintroduction to the cell almost impossible. The conjugation between bacteria and mitochondria free from these side effects. Moreover, this phenomenon has been proven to act in vitro. Conjugative plasmid had been placed in mitochondrion, where the expression of encoding genes occurred [22]. More recently a conjugation between two strains of bacteria within the cytoplasm of a mammalian cell has been observed . These findings suggest that conjugation between a bacterium and a mitochondrion is feasible [10].

Bacterial systems of protein secretion

Bacteria in natural environment use diverse, effective transport and secretion systems. In gram negative bacteria like E. coli common are type I secretion systems e.g. well characterized hemolysin secretion system. Hemolysin is responsible for pathogenicity of some E. coli strains. Systems using fusion of protein of interest to hemolysin C-terminus result in secretion of the protein into the growth medium[6] The same method can be use to secret bacterial proteins into the eukaryotic cell. It is currently used by designing new generation vaccines[5].

Induction of apoptosis by p53

Mitochondrial outer membrane permabilization MOMP is one of the main programmed cell death mechanisms. The process is regulated by PTPc complex and Bcl2 family proteins [17]. Many cancer types involve disruption of this process therefore inducing MOMP in the tumour cells is regarded as potential therapeutic solution. Factors inducing MOMP include p53 protein. It is mutated in almost half of the cancer cases. Protein p53 by acting as a transcription regulator in the nucleus arrests the cell cycle and induce apoptosis [18]. In the cytoplasm p53 interacts with mitochondrial membrane and can enter the mitochondrium. It has been proven that these processes induce apoptosis and do not influence the cell cycle. In the mitochondrium p53 is an effector protein that activates soluble (cytochrom c, Smac) and insoluble (AIF, EndoG) apoptototic factors. Moreover interaction of p53 and Bax protein in vitro leads to permabilisation of lipid bilayer analogous to mitochondrial membrane. [12] Because p53 is inactive in tumour cells many research groups focused on designing gene therapies based on p53 gene delivery. Most frequently adenoviruses and eukaryotic plasmids were used. The research results confirmed increased effectiveness of chemotherapeutic drugs after introduction of p53 gene copy into the cell [4]. It has been shown that p53 with the mitochondrial leader sequence is enough to induce apoptosis in mammalian cells. The fusion protein Lp53 with mitochondrial leader sequence from ornithine transcarbamylase was used. In terms of apoptosis induction fusion protein was almost as effective as standard p53. Previous research did not include delivery of p53 into tumour cells by bacterial vectors. Secretion of p53 into the cytoplasm by bacteria has an advantage over gene therapy because it does not disrupt genome integrity. Results of p53 integration with host genome are not fully characterized yet, but too high level of p53 is shown to accelerate ageing. Our project proposes a method to induce apoptosis without arresting the cell cycle. Cell cycle arrest decreases the sensitivity of cancer cells to chemotherapeutic agents, because they can affect only proliferating cells [14].