A biochemical and molecular analysis of the YhaO membrane protein in Escherichia coli O157:H7

Summary E. coli O157 is a dangerous bacterium that can cause serious illness in humans. The disease caused by O157 can lead to kidney failure or even death, especially if it infects the very young or eldery people. There is no effective treatment at the moment. Normally, O157 lives in cattle where no obvious disease is caused but if milk or meat products get contaminated with O157 then it can enter the food chain and affect humans. In both humans and cattle O157 manages to bind to the gut wall using an apparatus that pokes through its cell wall, this is known as its type three secretion system. When and how O157 decides to make its type three secretion system is not fully understood. If we know exactly how type three secretion is controlled then we can take advantage of this knowledge by using new agents that block its function. In addition we will be able to force the bacteria to produce the type three secretion system on a large scale which could be used in vaccine production. In this work we want to understand how O157 senses key signals from the environment and uses these signals to affect when it makes the type three secretion system. We have found a protein that sits on the edge of O157 and want to know what it senses, what shape it is and how best to stop it from working.

Technical summary E. coli O157causes serious and life threatening infections which can affect some 1000 people every year in the UK. One key virulence factor for E. coli O157 is the locus for enterocyte effacement which encodes its type three secretion system (T3SS) used to deliver effector proteins into eukaryotic cells. Exactly how and when the T3SS is controlled is not fully understood. Our ongoing work has identified a membrane protein that appears to sense environmental signals and affect expression of the T3SS. However, how this signal is transduced from the membrane to affect gene expression is not clear- the membrane protein is not a typical 2 component sensor and topology models are likely to be unreliable as no membrane protein with a periplasmic C terminus has been crystallised. We aim to identify the target substrate for the membrane protein, understand how gene expression is affected and gain important information as to the structre of the sensory protein.