Protein–protein interactions and protein self-assembly involved in enteropathogenic Escherichia coli infection

Abstract

Enteropathogenic Escherichia coli (EPEC) is one the most prevalent pathogens infecting children worldwide, with one of the major symptoms including watery diarrhoea. During EPEC infection, bacterial effector proteins are injected into epithelial cells by a type three secretion system (T3SS). Bacterial effectors have evolved to manipulate the host defence system and hijack crucial innate immunity signalling pathways. This thesis aims to provide a deeper insight into the most important protein interactions during EPEC infection. It studies and elaborates on different stages of the infection: colonisation, signalling and cell hijacking. Using cell-free protein expression, single molecule fluorescence techniques and a protein interaction proximity assay we studied the assembly process of the T3SS and its docking to the host plasma membrane. We also screened the main effector proteins causing disease against host proteins involved in inflammation, apoptosis and cell survival pathways. Our laboratory has observed that many innate immune proteins signal through the formation of higherorder assembly structures. We investigated the ability of EPEC bacterial effectors to affect the formation of these polymer-like assemblies. Our experiments uncovered a novel interaction between EspF and the tumour necrosis factor receptor-associated factor 2 (TRAF2). We characterized the behaviour of TRAF2 and TRAF2-EspF interaction in vitro and in cells. We discovered that bacterial proteins can not only bind to some host proteins but also alter the formation of the higher-order assemblies (oligomers or aggregates) that some immune proteins carry out to serve as scaffold or to amplify signal transduction. Finally, using proteomics and cell culture techniques, we validated the data obtained in the protein screen and obtained information about the most affected pathways during EPEC infection and the cell location of the effectors. Overall, this research contributes to understanding how EPEC causes disease onset and would provide useful information for future drug development against EPEC infection.