The NleG type III secretion system effectors of E. coli O157

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Escherichia coli is a bacterium that often inhabits the intestines of warm-blooded animals. Subsets of E. coli have evolved the ability to cause disease. One such group are enterohaemorrhagic E. coli (EHEC), which can cause bloody diarrhoea in humans. Infections can involve life-threatening complications affecting the kidneys and are frequently acquired via the food chain and farm environment from ruminants. Cattle are a major reservoir of EHEC, including the O157:H7 form that has caused serious outbreaks in recent years. Enteropathogenic E. coli (EPEC) are a related subset of bacteria that cause acute watery diarrhoea in infants in the developing world. Both types of E. coli rely on a 'molecular syringe' to colonise the intestines and produce disease. This syringe, encoded by a cluster of genes called the locus of enterocyte effacement (LEE), serves to inject a set of bacterial proteins termed effectors into cells lining the intestines. This process, known as Type III secretion, enables the bacteria to take control of processes inside host cells for their own benefit. Our research has shown that Type III secretion is vital for adherence of EHEC and EPEC to the gut lining and to interfere with the induction of host responses that might otherwise resolve the infection. The type III secretion system effectors target diverse signalling pathways and the main cellular organelles. For example, the effectors Tir, Map, EspH and EspM modulate the cellular cytoskeleton, NleB, NleC and NleE block innate immune responses and NleH maintains viability of infected cells. Moreover, the effector EspG is targeted to the Golgi, Map and EspF are targeted to the mitochondria; EspF also targets the nucleolus. Importantly, no EHEC effectors were thus far found to target the nucleus. Recently, a large family of Type III EHEC effectors named NleG was shown to have a ubiquitin ligase activity. Ubiquitin and deubiquitylating enzymes are key regulators of protein turnover, cell signaling, transcription, cell cycle, and DNA damage repair. It is therefore not surprising that bacterial pathogens hijack the cellular ubiquitylation machinery as part of their infection strategies. The goals of this project are to study two NleG effectors that contain a PDZ-binding motif at the C-terminus (PDZ binding motif mediate the interaction of these NleGs with host cellular proteins), NleG-EDL and NleG7-EDL. Importantly, while NleG-EDL is targeted to the nucleus, NleG7-EDL is found diffused in the cytosol. In particular we aim to:1. Determine the intracellular trafficking of NleG-EDL and NleG7-EDL and the role of the PDZ-binding motif2. Identify the host cell partner and substrate proteins of NleG-EDL and NleG7-EDL and study cell signaling3. Determine the effect of NleG-EDL on the nuclear proteome, nuclear proteins' turnover and the transcriptome5. Determine the effect of NleG7-EDL on the cytocsolic proteome and proteins' turnover 6. Determine the role of NleGs during infection using ex vivo (bovine intestinal in vitro organ clutters) and in vivo (the EHEC-like moue pathogen Citrobacter rodetnium) modelsAn understanding of how the NleGs modulate the function of infected cells is likely to aid in rational design of strategies to control EHEC and EPEC infections and carriage by farm animals.