Investigation of the first step of a virulence regulatory pathway in a bacterial hospital-acquired infection agent

Abstract

Enterococci are significant agents of hospital-acquired infection (HAI) in the UK, including endocarditis, urinary tract infections and infections of surgical sites and indwelling foreign devices. These bacteria account for over 20% of HAI cases in the UK, which in total cost the National Health Service an estimated £1 billion per annum. Two significant clinical features of these bacteria are: (1) their ability to adhere to host tissues such as heart valves and the urinary tract and to prosthetics such as catheters and to then establish biofilms (thick layers of bacterial cells that are difficult to treat with antibiotics); and (2) their ability to produce proteases that weaken heart valves and other tissues. One mechanism by which these bacteria activate production of these virulence features is through the Fsr signal transduction pathway, in which the first step is activation of the FsrC membrane sensory protein by a pheromone called gelatinase biosynthesis-activating pheromone (GBAP). Investigations of this important interaction and its potential inhibition have formerly been hampered by the technical challenges associated with obtaining and working with purified FsrC because it is a hydrophobic membrane protein. However, the facilities at the Circular Dichroism beamline (B23) at Diamond Light Source allowed us to successfully investigate FsrC-GBAP and FsrC-inhibitor binding using relatively small amounts of purified intact protein, and to define stabilising conditions under which quantitative ligand binding data for FsrC and presumably other membrane proteins more generally can be obtained using synchrotron radiation circular dichroism (SRCD) spectroscopy.