Subversion of actin signaling pathways by enterohaemorrhagic and enteropathogenic E. coli

Escherichia coli constitute part of the normal gut flora, while several strains acquired additional genes that enable them to cause disease. Enteropathogenic (EPEC) and enterohaemorrhagic Escherichai coli (EHEC, known E. coli O157) cause disease when they inject specific virulence proteins, known as effectors, into the mammalian cell.
EPEC is the leading cause of infantile diarrhoea, morbidity and mortality in developing countries. EHEC (particularly E. coli O157:H7), which is predominant in developed countries, cause diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome (HUS); the young and the elderly are most at risk. It is estimated that ca. 73,000 and 1,000 human EHEC infections occur in the US and the UK each year, respectively. EHEC-induced HUS is the leading cause of acute paediatric renal failure in the UK and US.

Elucidating the infection strategy of EPEC and EHEC is totally dependent on the identification of proteins and signal transduction pathways that are targeted by the effectors. The aim of this project is to investigate the physiological role of several key EPEC and EHEC effectors during infection.

We will first investigate the mechanism by which the EPEC and EHEC effectors Tir and EspT subvert signalling within human cells in culture. We will then determine translate the data gathered in the laboratory to gut infection using an EPEC and EHEC mouse model. Studying pathogenesis in vivo is essential for understanding host pathogen interaction. While conducting animal experiments we take particular care of the 3Rs principals. Indeed, our infection model won the 2006 NC3Rs prizes.

Rehydration and nutritional supplementation are the only effective treatments for EPEC and EHEC infection; treatment with antibiotics is countered productive. HUS patients may require dialysis and in sever cases kidney transplantation. No vaccines or specific treatment are currently available against EPEC and EHEC infections. Better understanding of colonisation processes and the role of virulence factors is essential for the development of new specific and effective treatments.

Infection of mucosal surfaces by a pathogenic bacterium is a process involving coordinated activity of diverse virulence factors, which provide the bacterium with a high level of adaptation and a competitive edge leading to efficient colonisation and dissemination. The diarrhoeal pathogens enteropathogenic (EPEC) and enterohaemorrhagic (EHEC), Escherichia coli, and the mouse pathogen Citrobacter rodentium are striking examples of such specialised pathogens that use attaching and effacing (A/E) lesion formation as a major mechanism of tissue targeting and infection. The ability to form A/E lesions and disease is dependent on a type III secretion system and translocation of effector proteins.
Tir is one of the major EPEC and EHEC effectors, which is involved in A/E lesion formation and actin polymerisation. Tir EPEC binds the adaptor protein Nck leading to activation of N-WASP. In contrast, EHEC translocate the effector TccP (aka EspFU), which mimics Nck in terms of activation of N-WASP and the actin-signalling pathway. Importantly, Tir does not bind TccP directly. Recently it was reported that IRTKS and/or IRSp53 link Tir and TccP.

EPEC and EHEC, which are considered extracellular pathogens, also encode effector proteins Map, EspM and EspT (belonging to the WxxxE effector family), which that Rho GTPases, leading to formation of filopodia, stress fibres and lamelleopodia, respectively. Importantly, we recently found that expression of EspT leads to EPEC cell invasion, defining a new category of EPEC (invasive EPEC).

In this study we will employ contemporary cell biology and state of the art microscopical techniques to dissect the function of Tir and TccP and the WxxxE effectors. In particular, we will employ dual wavelength live cell imaging and fluorescence resonance energy transfer (FRET) to determine the dynamics and protein-protein interactions involved in actin pedestal formation.

We will then investigate the relevance of the in vitro finding to host pathogen interaction in vivo using the EPEC and EHEC mouse pathogen Citrobacter rodentium. C. rodentium, which shares many of the virulence factors with EPEC and EHEC, colonise the mouse colonic mucosa via A/E lesions. We will use site directed C. rodentium mutants, C. rodentium over expressing effector proteins (from the chromosome) and microscopy to localise the effectors and host cell partner proteins on infected mucosal surfaces and determine a role in gut infection.