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Abstract

Enterohemorrhagic E. coli (EHEC) are highly infectious food-borne pathogens that cause severe diarrhoea in both, industrialised and developing countries all over the world. Their pathogenicity factors involve shiga-like toxins and a type III secretion system along with so called effector proteins, which are translocated directly into the cytoplasm of their host cells, usually enterocytes. Most of these proteins are encoded in pathogenicity islands within the bacterial genome that are framed by sequences of lambdoid phages. Some of these phages are still able to produce infectious particles after UV induction. In this study I generated two protein-protein interactomes, namely EHEC-host and phage lambda-E. coli. For the EHEC-host interactome, 34 effector proteins that had been previously shown to be secreted into human host cells were cloned and screened against pooled human cDNA and ORF libraries via yeast two-hybrid screening. This resulted in 35 reproducible interactions of 15 EHEC effectors with 34 human proteins, of which only four had been published previously. Inclusion of secondary human protein interactors retrieved from the BioGRID database revealed that EHEC effectors are interconnected in the human cell. The translocated intimin receptor (TIR) that was found to interact with eight human proteins was compared to its homologue in enteropathogenic E. coli (EPEC). This revealed that five of the eight EHEC TIR interactors also interact with EPEC TIR. Another interaction discovered in this study involves the EHEC effector NleF, previously a protein of unknown function, and human caspase-9. LUMIER assays against other human caspases identified caspase-4 and -8 as additional binding partners of NleF. Tests with purified enzymes revealed that NleF can potentially inhibit all three caspases. The effector decreased caspase activity significantly in HeLa cell lysate and impaired apoptosis induction in HeLa and Caco-2 cells. A collaboration partner solved the crystal structure of the NleF/caspase-9 complex, which suggested a dominant role of the carboxy-terminal four amino acids in caspase-9 binding and inhibition. I was able to confirm these findings by constructing NleF versions with mutagenized carboxy-termini. NleF versions that lacked the last four amino acids or comprised an additional carboxy-terminal alanine were unable to bind any of the three caspases or impair apoptosis. Apoptosis inhibition is a strategy often applied by viral and bacterial pathogens. Even though NleF is not the only effector protein capable of inhibiting apoptosis in human cells, direct inhibition of caspases by bacterial effectors has not been reported to date. The phage lambda-E. coli.interactome was generated during my research stay at the J. Craig Venter Institute in Rockville (USA). I screened 68 phage lambda proteins against the E. coli W3110 ORF library via yeast two-hybrid screening using two different vector systems. This resulted in 144 reproducible interacting pairs. The phage lambda and E. coli proteins involved in interactions were categorized in functional groups and analysed for interactions between phage and host groups.