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

Lead Research Organisation: University of Glasgow
Department Name: School of Life Sciences

Abstract

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

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.

Publications

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Beckham KS (2012) The structure of an orthorhombic crystal form of a 'forced reduced' thiol peroxidase reveals lattice formation aided by the presence of the affinity tag. in Acta crystallographica. Section F, Structural biology and crystallization communications

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Gabrielsen M (2012) Useable diffraction data from a multiple microdomain-containing crystal of Ascaris suum As-p18 fatty-acid-binding protein using a microfocus beamline. in Acta crystallographica. Section F, Structural biology and crystallization communications

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Gabrielsen M (2010) Expression, purification, crystallization and initial X-ray diffraction analysis of thiol peroxidase from Yersinia pseudotuberculosis. in Acta crystallographica. Section F, Structural biology and crystallization communications

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Gabrielsen M (2012) Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus. in Acta crystallographica. Section F, Structural biology and crystallization communications

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Holmes A (2010) The EspF effector, a bacterial pathogen's Swiss army knife. in Infection and immunity

 
Description The most significant achievement was understanding the substrate that YhaO transported (ie D serine) and correlating this with the actual biology of the organism. We showed that YhaO "senses" D serine in the environment, that D serine can have deleterious effects on the bacteria and that the bacteria respond by expressing their flagella.



We successfully crystallized several proteins associated with regulation of the Type Three Secretion System. This is in line with the overall aim of my group - to develop intervention strategies to combat this organism.
Exploitation Route The type three secretion system is a fascinating example of how bacteria interact with host cells. We have found it is of wide interest to a general audience and will continue to develop animations and interactive tools to showcase our research. As our understanding of the regulation is developed, we will modify the animation to reflect our current research and disseminate this using widely used web sources such as u tube and itunes university. The knowledge that D serine can affect expression of the flagella and T3SS is important for the production of current O157 vaccines. Modifying the D serine concentration in the media would dramatically affect their relative expression which could be exploited to improve vaccine yields.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.gla.ac.uk/researchinstitutes/iii/research/infection/infresgroups/andrewroe/mainpage/#d.en.127468