Characterisation of the enterococcal sensor kinase VanSA and its ligand binding properties

Abstract

The enterococci are a leading cause of Hospital-acquired infections (HAI) in the UK, with increasing numbers identified as resistant towards “last line” glycopeptide antibiotics (GPAs) vancomycin and teicoplanin. Of the two types of vancomycin resistance in enterococci, type A (vancomycin and teicoplanin resistant) rather than type B (vancomycin resistant, teicoplanin susceptible) is the most prominent in the UK and is the system that will described in this Thesis.
All vancomycin resistances in Gram-positive bacteria including the enterococci are controlled by a two-component signal transduction system (TCS). TCSs allow the organism to appropriately respond to external stimuli, and are composed of a membrane-bound sensor kinase and a partnered cytosolic regulator which upon stimulation by an activating ligand initiate appropriated adaptive responses by the bacteria. Type A vancomycin resistance in the enterococci is regulated by the VanRASA TCS composed of VanSA (the sensor) and VanRA (the partner regulator). The activating stimulus of VanSA is still unknown.
Following successful production of intact and active membrane sensor protein VanSA, the structure and function effects of a range of candidate compounds (GPAs vancomycin and teicoplanin, peptidoglycan compounds, and sugars) were screened using biophysical approaches available at Beamline 23, Diamond Light Source Ltd, coupled with phosphorylation activity assays. Results identified GPAs as the only materials binding VanSA suggesting a change in the local tertiary structure of VanSA and roles for aromatic amino acids tryptophan and tyrosine in ligand recognition confirmed using circular dichroism, magnetic synchrotron radiation circular dichroism and fluorescence spectroscopy. Characterisation of the interaction revealed differences in the affinity of the two GPAs; 70 μM for vancomycin , and 30 μM for teicoplanin. Furthermore, only in the presence of vancomycin and teicoplanin were VanSA phosphorylation rates increased and longer-lived relative to VanSA alone, overall suggesting the roles of GPAs as the activating ligands for the onset of type A vancomycin resistance in the enterococci.