The type III secretion system 'translocation-stop' activity of EspZ

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.

To orchestrate host cell pathways, the bacteria must deliver Type III secreted effectors in the required order and amounts. Moreover, it is necessary to control the timing of delivery, and the duration of action and location of effectors inside host cells. Our recent research has indicated that the effector protein EspZ plays an important role in controlling the flow of effector proteins into host cells. It appears to do this only once injected, and host cells that have been engineered to express EspZ are resistant to injection of effectors. Bacteria that lack EspZ cause excessive damage to host cells, likely because they inject effectors at elevated levels. These data suggest that EspZ is a novel natural inhibitor of Type III secretion that may arrest the injection process once it has been delivered into host cells. Our pilot data suggest that EspZ may interact with other LEE-encoded proteins to close the pore created by the syringe, and that it may be modified once it enters host cells. The nature and consequences of interactions between EspZ and other proteins, and of modification of EspZ, are not known. We therefore propose to:

1. Define when EspZ is injected into host cells, and whether targeting of the protein to the host cell membrane coincides with arrest of Type III secretion.
2. Identify proteins that interact with EspZ and determine how such interactions arrest Type III secretion.
3. Define the nature and consequences of modification of EspZ inside host cells.
4. Determine the role of EspZ in bacterial persistence and disease in animal models.

An understanding of how EspZ modulates Type III secretion will aid the rational design of strategies to control EHEC and EPEC infections and carriage by farm animals. Inhibitors of the process may be used to treat infections in humans or reservoir hosts. Moreover, we will explore the possibility of creating transgenic animals that are refractory to infection as a consequence of expression of EspZ in intestinal cells.

'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. Our data indicate that the LEE-encoded effector EspZ is modified upon entry into host cells and is integrated into the host cell plasma membrane where it functions as an inhibitor of further effector translocation. Ectopic expression of EspZ in eukaryotic cells renders them refractory to infection, and cells infected with AE pathogens are resistant to super-infection in an EspZ-dependent manner. Bacteria lacking EspZ cause cell damage, likely owing to accumulation of effectors to toxic levels. Our yeast-2-hybrid analysis has indicated that EspZ may interact with a constituent of the translocation pore (EspD), however the molecular mechanism by which it arrests Type III secretion is not known. We propose to define the kinetics of EspZ translocation, its insertion into the host cell plasma membrane and the onset of 'translocation-stop' activity. We will map the protein-protein interaction sites of EspZ and its partner proteins, and determine the role of the partner proteins and interacting residues in its 'translocation-stop' activity. The electrophoretic mobility of EspZ shifts on entry into cells and we propose to define the nature and consequences of post-translational modification. We will also evaluate the impact of mutation and over-espression of espZ on intestinal persistence of enterohaemorrhagic E. coli in calves and Citrobacter rodentium in mice. Unravelling the mode of action of EspZ will have major implications for the design of novel strategies to control infections by pathogenic E. coli.

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. Subunit vaccines and inhibitors targeting the Type III secretion apparatus have been described; however it remains a challenge to ensure that antibodies or drugs are present in adequate quantities at key sites of infection to prevent deployment of the system. We have discovered a novel natural inhibitor of Type III secretion (EspZ) that acts within host cells to block further injection of effectors. The finding that cells expressing EspZ become refractory to infection by EHEC and EPEC raises the possibility of creating transgenic livestock resistant to colonisation. The feasibility of this approach was recently demonstrated by researchers at the Roslin Institute, who created transgenic chickens unable to transmit avian influenza virus owing to constitutive expression of a stem-loop RNA that acts as a decoy for the viral polymerase [Lyall et al. 2011. Science 331:223-6]. Prof. Frankel will use his honorary position at the Sanger Institute to explore this approach in mice based on data expected to arise from the project. Attenuated bacteria engineered to deliver EspZ may also be useful to prevent super-infection in reservoir hosts.

Type III secretion is vital for the ability of a plethora of bacterial pathogens of animals and plants to cause disease. Data on how EspZ arrests translocation of effectors, for example via its interactions with components of the translocation pore, can be exploited for the rational design of novel drugs. The data will therefore to be of interest to industry, and the applicants have proven records of interaction with several industry partners and of protecting intellectual property. An understanding of the basis of control of Type III secretion may also be relevant to policy makers, for example as the epidemic and zoonotic potential of AE pathogens may be dictated by levels of EspZ-controlled T3SS activity. The applicants have 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 applicants have described their BBSRC-funded research on E. coli and its applications to varied audiences, for example via recent television and radio interviews (Frankel) and a BBC film on E. coli O157 for the Health Explained series (Stevens). The applicants have also produced lay articles on their research, and a workshop on a simulated E. coli outbreak for schools (see Pathways to Impact). 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