EspO orthologs and NleF: type III secretion system effectors of enteric pathogens that modulate apoptosis and inflammation

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. Enteropathogenic E. coli (EPEC) strains are a related subset of bacteria that cause acute watery diarrhoea in infants in the developing world. Shigella strains, which are closely related to E. coli, cause bacillary dysentery (aka Shigellosis). Shigellosis, which is usually acquired by ingestion of contaminated food or water, remains a global endemic disease responsible for ca. 165 million cases of severe dysentery and 1 million deaths annually, particularly in children less than five years of age. Non-typhoidal Salmonella enterica (NTS) strains cause an estimated 1 billion cases of self-limiting foodborne gastroenteritis annually. More recently, multiple antibiotic-resistant strains have emerged as important causes of fatal invasive bacteraemia, particularly in sub-Saharan regions. Importantly, although considered high priority, no vaccines are yet available for either of these pathogens, the development of which relies on better understanding of their biology and infection strategies.

EPEC, EHEC, Shigella and Salmonella rely on a 'molecular syringe' to colonise the intestines and produce disease. This syringe serves to inject a set of bacterial proteins termed effectors into cells lining the intestines. This process, known as Type III secretion (T3S), enables the bacteria to take control of processes inside host cells for their own benefit. Our research has shown that T3S 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.

Infected hosts fight bacterial infection by mounting immune responses while infected cells might assist in clearing the pathogen by undergoing programme cell death (aka apoptosis). Importantly, low-level inflammation may assist the pathogens in establishing a foothold within the host as it modulates the gut physiology and the normal gut flora. It is therefore not surprising that the pathogenic E. coli, Shigella and Salmonella inject T3S effector proteins that subvert both inflammation and apoptosis. We recently found that the EPEC and EHEC T3SS effector NleF binds a host cell protein called caspase-4, which plays a role in both apoptosis and inflammation. Similarly, we found the effector EspO of EHEC, and it's family members in Shigella (OspE) and Salmonella (SopO) bind a host cell protein called Hax-1, which is a major inhibitor of apoptosis and has an indirect role in host immune responses.

The broad aim of this work is to characterise the mechanism of action and to understand the role during infection of the EspO family members and NleF. In particular, our aim (which is also our strength) is to translate results obtained by biochemical and cell biology assays in vitro to pathogen-host interactions in vivo. The specific aims include:

1. Determine the structure of the EspO:Hax-1 and NleF:caspase-4 complexes
2. Determine the intracellular interactome of EspO family members and NleF
3. Dissect the EspO family members and NleF cell signalling pathways using cell culture models
4. Study the role of EspO, SopO and NleF and their host cell partner proteins in pathogen-host interaction in animal models in vivo

1. The effector proteins will be over-produced in E. coli either individually or through co-expression and purified using our standard protocol. Purified proteins and their interactions will be characterised biochemically and biophysically using native-PAGE, SEC-MALS and ITC. Proteins and their stable complexes will be subject to crystallisation experiments. Crystals will be screened for their diffraction quality using our in-house X-ray setup before data collection at the diamond synchrotron radiation source. Phase information will be obtained either through molecular replacement or Se-Met derivatised crystals and MAD phasing.

2. We will use protein-protein interaction assays (e.g. co-IP, column pull-down and yeast two hybrid) together with proteome analysis of co-IP material to identify the entire signalling complexes formed by the effectors and their cellular targets during infection and transfection.

3. We will generate inducible, stable, effector cells lines and use them for localisation and functional studies during infection and trasfection by employing conventional cell biology and microscopy techniques (immunofluorescent and immungold EM). We will also use other cell types (including polarised epithelial cells and macrophages) to study the role of the effectors and their target proteins in pathogen-host interaction.

4. We will use wild type and isogenic effector mutants (S. Typhimurium and the EPEC/EHEC-like mouse pathogen Citrobacter rodentium), expressing bioluminescent or fluorescent reporters to study infection dynamics and clearance in real time and 3D using the IVIS technology. We will use histology and immunoflorescent staining to study the localisation of the effectors' targets in healthy and infected tissues. We will use immunohistochemistry and cytokine qRT-PCR to measure host immune responses and knockout mice infected with bioluminescent bacteria to determine the role of the effectors' targets on the infection.

Foodborne bacterial pathogens cause an estimated 626,000 cases of acute enteritis in humans in the UK per annum at a recurring cost of £1.5bn. 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. Shigella and non-typhoidal Salmonella enterica remain a major health concern world-wide, the former is a main cause of morbidity and mortality in young children and diarrhoeal diseases amongst travellers and army personnel. Importantly, although considered high priority, no vaccines are yet available for either of these pathogens, the development of which relies on better understanding of their biology and infection strategies.

We are collaborating with Novartis Vaccine and Diagnostics (NVD) in development of a pan E. coli vaccine using the C. rodentium model. The outcome of this project would have direct implications on the development and assessment of the vaccine. Although a vaccine is a long term objective, once produced it would have major national and global health and economical benefits. We are also collaborating with PerkinElmer (PE) in the development of new optical bacterial reporters for in vivo imaging of infections with Gram-negative and Gram-positive pathogens. The new vectors would be licenced to Imperial College and distributed by PE, thus making direct financial contribution to the PI's lab and Imperial College as well as providing novel tools to the wider scientific community.

By examining the cell biology of host responses to infection, bacterial pathogens in general, and our selected effecters in particular will provide valuable insights into the physiological and pathological functions of key host cell proteins and signalling pathways. For example, little is currently known about the functions of human caspase-4 and this study would therefor be of great interest to the scientific community working on apoptosis and inflammation.

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

The applicant was a board member of the BBSRC AgriFood committee and now serves as a member on the MRC Infection and Immunity Board. Such links will be used to inform Policy Makers on the implications of research arising from the project.