Ler a versatile global regulator from E. coli O157 and related strains

Already E. coli is generally a 'friendly bacterium', some kinds of E. coli, particularly E. coli O157, can cause severe gut infections in humans, posing a special threat to the very young and the very old. Infection is commonly associated with consumption of beef and related products (especially hamburgers), but can also result from consumption of contaminated vegetables or from farm visits. To gain a foothold in the human bowel, E. coli O157 uses a sophisticated molecular syringe (called a type III secretion system) to inject its own proteins into human cells. Deployment of the syringe is in turn controlled by regulation of the genes that encode its components. We are interested in a bacterial protein called Ler that regulates these type III secretion genes. Although interest in the past has focused exclusively on how Ler regulates type III secretion, we have preliminary evidence to suggest that Ler regulates many other facets of the bacterial cell. We now wish to confirm and expand on these findings to gain a global all-round view of Ler's function in gene regulation. We also wish to investigate Ler's interactions with other proteins and test our hypothesis that Ler is involved in a system for sensing when proteins are secreted and changing the expression of genes in response to such secretion. A better understanding of how Ler regulates virulence in E. coli will pave the way for the development and introduction of novel vaccine strains providing new approaches to the prevention and control not only of this fearsome pathogen, but also potentially of other infectious micro-organisms. In this work, we benefit from a (1) world-class team of applicants (all three have published in top journals such as Proceedings of the National Academy of Science in the last year), (2) an exceptional research environment with unique access to key technologies and information and (3) input from superb collaborators, who have broad experience of the kind of questions we are trying to address in other settings.

This proposal draws on the expertise of three applicants with an international reputation (all three with PNAS papers in the last year) to address important questions within the AF theme 'Control of Food-borne Pathogens'. Enterohaemorrhagic strains of E. coli (EHEC), especially E. coli O157:H7, present a formidable challenge to the Agri-Food industry. Virulence in EHEC relies on a type III secretion system, encoded by the locus for enterocyte effacement (the LEE) to mediate translocation of effector proteins from the bacterial cytoplasm into the cytoplasm of eukaryotic cells. The LEE-encoded regulator, Ler, plays a pivotal role in the regulation of gene expression within the LEE. Ler is homologous to the intensively studied global regulator, H-NS. Building on this homology and on our own preliminary work, we now wish to address a number of important research questions concerning Ler: (1) Is Ler, like H-NS, a global regulator binding to multiple sites throughout the genome? We have preliminary transcriptomics data to suggest that Ler influences many genes outside the LEE, plus we have unique local expertise in transcriptomics and chromatin immunoprecipitation to perform timely studies that define the global effects of Ler in different settings and distinguish direct from indirect influences. (2) Does Ler engage in dimerisation and homo- and hetero-oligomerisation, and how might these activities relate to its function? With local expertise in appropriate techniques and input from a leading expert on H-NS (Ladbury), we are well placed to address this question. (3) Do protein-protein interaction networks converging on Ler provide a route for coupling protein secretion to transcriptional regulation? We will explore the protein-protein interaction networks that converge on Ler and explore the hypothesis that these pathways provide a route for coupling protein secretion to transcriptional regulation.