Subversion of actin cytoskeleton dynamics by the WxxxE effector proteins Map, EspM2 and EspS

Escherichia coli bacteria live in the human gut and are normally harmless. However, several strains of E. coli have acquired additional genes that enable them to inflict damage and cause disease. One example of such pathogenic E. coli, which is among the most important bacterial causes of diarrhoeal disease worldwide and responsible for deaths of hundreds of thousands of babies and young infants in developing countries each year, is enteropathogenic E. coli (EPEC). Like EPEC, other diarrhoeal pathogens, including enterohaemorrhagic E. coli (EHEC), Salmonella and Shigellea are also important enteric pathogens responsible for child morbidity and mortality in developing countries. These pathogens are also common causes of travellers? diarrhoea. Moreover, EHEC infection can lead to haemorrhagic colitis and haemolytic uraemic syndrome (HUS); EHEC-induced HUS is now the leading cause of acute paediatric renal failure in the UK and US. Salmonella typhi is the causative agent of typhoid fever. In both developing and developed countries the economical burden of these diseases is vast.
We now know that disease is caused when these bacteria directly inject virulence proteins, known as effectors, into gut cells. Therefore, understanding what these different effectors do inside the cell is crucial to understanding how they cause disease. Based on a common and a critical motif, a number of effector proteins expressed by these pathogens were recently grouped into one category (refer to as the WxxxE effectors). In this study we will focus on three example WxxxE proteins and study their function and role in disease using in vitro (biochemistry and cell culture methodologies) and in vivo models. The study will undoubtedly reveal new insights into the function of WxxxE proteins and mechanisms of bacterial colonisation. Better understanding of these effectors is essential for development of effective treatments and will enhance our general understanding of how pathogenic bacteria hijack cell functions for their own advantage.

Infections with diarrhoeal pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively), Salmonella and Shigella account for a large proportion of child morbidity and mortality. These pathogens are also common causes of travellers? diarrhoea. Although the infection strategy and disease manifestation are different, these enteric pathogens all use a type III secretion system (T3SS) to inject effector proteins into the mammalian cell which subvert normal cellular functions for the benefit of the bacterium. Prominent targets of the T3SS effectors are the Rho GTPases and the actin cytoskeleton.

In January 2006 Alto et al (Cell 124, 133-145, 2006) assembled several known T3SS effectors into a single family that share the common motif Trp-xxx-Glu (WxxxE). Membrane localisation seems to be critical for the function of these WxxxE effectors, which include the Salmonella (SifA and SifB), the Shigella (IpgB1 and IPgB2) and the EPEC, EHEC and C. rodentium (a mouse pathogen that shares many virulence factors and mechanisms with the human pathogens EPEC and EHEC) effector proteins Map, EspM2 and EspS. We present preliminary data showing that Map triggers filopodia formation by activating the Rho GTPase Cdc42, while EspM2 triggers formation of stress fibers by activating RhoA. In contrast, EspS induces assembly of unique ?star-shaped? actin-rich structures although the GTPase involved is not yet identified.

The aim of this investigation is to use Map, EspM2 and EspS as model WxxxE effector proteins that induce distinct actin structures by activation of different Rho GTPases. We will identify host cell binding partners, including membrane targeting proteins and the guanine nucleotide exchange factors (GEFs) involved in activation of the Rho GTPases. We will use biochemical methods for protein interaction and cell culture approaches to dissect cell signalling. Although studying infection using cultured cell lines provides useful indicators for possible roles of bacterial effectors in disease, it is essential that conclusions drawn from such in vitro studies are confirmed in vivo. Accordingly we will use the C. rodentium model to assess the role of Map, EspM2, EspS and their interacting host cell proteins in colonisation and infection.

This project will lead to better understanding of the function of the WxxxE effectors in bacterial enteric pathogens. Such knowledge is essential for development of new and effective treatments which will benefit millions of patients suffering from diarrhoeal disease in both developing and developed countries.