Award details

Membrane sensory proteins and environmental cues of Enterococcus faecalis

Reference	BB/D001641/1
Principal Investigator / Supervisor	Dr Mary Phillips-Jones
Co-Investigators / Co-Supervisors	Professor Peter Henderson
Institution	University of Leeds
Department	Inst of Molecular & Cellular Biology
Funding type	Research
Value (£)	211,042
Status	Completed
Type	Research Grant
Start date	04/01/2006
End date	03/04/2009
Duration	39 months

Abstract

Using Enterococcus faecalis as a model infection agent, the three specific objectives of this proposal are as follows. (1) Build on our specialist expertises and successes with the SK group of membrane proteins by overexpressing and purifying the full complement of 16 membrane (and 1 soluble) SKs in this bacterium in their intact form (prioritising the 9 known or predicted virulence-associated sensors). (2) Identify or confirm the signals/ligands that interact with these proteins. We will use a range of candidate and other signal molecules in SK phosphorylation assays to identify the external signals sensed by these SKs; some already have known or strong candidates. (3) Identify the structural changes that occur in these proteins upon ligand (signal) binding using FTIR, fluorescence and CD spectroscopy, with stopped-flow measurements as appropriate. Finally, we will undertake crystallisation trials to initiate structural characterisation. Ultimately, knowledge of the structure and the environmental triggers (ligands) identified by these proteins will be used to devise strategies that inactivate ligand-protein binding, affecting signal sensing and therefore virulence activation. This project exploits a structural genomics approach for elucidating key environmental responses of a pathogenic microorganism.

Summary

Disease-causing bacteria depend on being able to sense (and respond to) changes in their environment in order to survive and multiply in their host and surroundings. They have evolved specific sensory proteins located in their outer layers (the membrane), and they use the information from these proteins to switch on/off production of virulence and other disease-enhancing factors (e.g. toxins). Almost every pathogenic bacterium possesses these sensory proteins, and the most common group are known as two-component signal transduction systems (TCSs). They consist of a sensor kinase protein (SK, usually located in the membrane) that senses the outside environmental signal, and a partner response regulator protein (RR) located inside the cell that receives the SK information and effects a response /usually a change in the expression of various genes, including those encoding virulence factors. Most bacteria possess multiple TCS pairs, each one tuned in to a different specific environmental cue. For example, the infective bacterium Enterococcus faecalis possesses 17 TCS pairs. This bacterium is an important pathogen of humans -causing endocarditis, urinary tract and bloodstream infections, wound infections and infections of indwelling foreign devices, and is of additional concern because of its readily acquired resistance to a wide range of antibiotics including last-line glycopeptide antibiotics such as vancomycin. Enterococci have now become the second most important agent of hospital-infections. The aim of this proposal is to study the 17 SKs of E. faecalis, in order to understand what environmental signals are being sensed, how the proteins respond to their signal, how signal information is passed across the membrane, and ultimately the atomic structures of the proteins. We will prioritise the 9 SKs that are implicated in virulence, but structural knowledge of any will be highly informative. To achieve these aims, we must study the SKs in their intact forms, completewith membrane spanning portions, and we have the relevant specialist expertise for this. Ultimately, such studies will lead to new strategies to combat infections by E. faecalis that are based on inhibitors of these signal sensing/transduction systems, including those involved in activation of virulence factors and/or host defence systems.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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