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

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 motif
2. Identify the host cell partner and substrate proteins of NleG-EDL and NleG7-EDL and study cell signaling
3. Determine the effect of NleG-EDL on the nuclear proteome, nuclear proteins' turnover and the transcriptome
5. 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) models

An 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.

'Attaching & effacing' (AE) E. coli exert a substantial burden on human and animal health and use Type III secretion to colonise intestinal epithelia and induce pathology. The locus of enterocyte effacement (LEE)-encoded Type III secretion system of such pathogens mediates injection of tens of bacterial effectors into enterocytes. These orchestrate cellular pathways in a manner requiring exquisite temporal and spatial control. Among the EHEC effectors are the NleG family members that mimic eukaryotic RING finger/U-box ubiquitin E3 ligases. Our data show that the EHEC EDL933 ubiquitin ligase effectors NleG and NleG7, which target the nucleus and cytosol respectively, contain a carboxy terminal PDZ-binding motif. Moreover, one of the NleG effectors in the EHEC-like mouse pathogen Citrobacter rodetnium, which is homologues to the NleG-EDL (we named NleG-CR) also contains a PDZ-binding motif. Following ectopic expression we found co-localisation of NleG and NleG-CR with ubiquitin in the nucleus, which coincided with diminished histone H3 straining. Moreover, both NleG and NleG-CR bind PDZ1 of the mammalian scaffold protein Na(+) /H(+) exchanger regulatory factor 1 (NHERF1). The PDZ binding partner of NleG7 is not yet known. The focus of this study is the NleG effectors with a PDZ-binding motif. We will employ biochemical, protein-protein interaction, cellular and molecular methodologies to determine the intracellular trafficking of the NleGs, the involvement of NHERF1, their host cell partner and substrate proteins, the effect of NleG on the nuclear proteome and the transcriptome, and the effect of NleG7 on the cytosolic proteome and cell signalling. We will use bovine explants ex vivo and the mouse model to study the role NleGs play in infection of mucosal surfaces, colonisation and pathology. Unravelling the mode of action of the NleGs would have major implications on our understudying of EHEC infection and potentially for designing novel control strategies.

Foodborne bacterial pathogens cause an estimated 626,000 cases of acute enteritis in humans in the UK per annum [1] at a recurring cost of £1.5bn [2]. Global demand for safe nutritious food is fast accelerating but some pathogens remain an intractable threat to food security. Shiga toxin-producing E. coli are a great concern to the public and are the leading antecedent to acute paediatric renal failure in many countries. Infections by related enteropathogenic E. coli are an important cause of infant diarrhoea and mortality in the developing world. Novel ways to control infection are needed, not least as many classes of antibiotic are contraindicated in the treatment of EHEC infections owing to induction of Shiga toxin synthesis. Currently there are no vaccines or specific treatment for EHEC infections.

We have discovered a novel infection strategy employed by EHEC as while remaining extracellular it injects effectors that appear to modulate host (and potentially bacterial effector) protein turnover and transcription. Together with the Co-I Dr. Jyoti Choudhary, Prof. Frankel will use his honorary position at the Sanger Institute to explore their proteomics and transcriptomics platforms. Type III secretion is vital for the ability of a plethora of bacterial pathogens of animals, humans and plants to cause disease. Data on how NleGs modulate protein turnover and gene expression can be exploited for the rational design of novel drugs. The data will therefore to be of interest to industry, and the applicant have proven records of interaction with several industry partners and of protecting intellectual property. The applicant has previously associated the carriage or expression of selected effectors with pathogenic potential and further work of this kind is envisaged.

In addition to extending national scientific competitiveness and instilling valuable training (see Academic Beneficiaries), we expect the project will provide further opportunities for public engagement and education. The applicant has described his BBSRC-funded research on E. coli and its applications to varied audiences, for example via recent television and radio interviews. We will continue to use such media to disseminate the findings of our work to schools and the general public.

1. http://www.defra.gov.uk/publications/files/pb13571-zoonoses2009-110125.pdf
2. http://www.food.gov.uk/multimedia/pdfs/csr0910a.pdf