Overexpression, purification and ligand interaction studies of intact membrane sensory kinases from enterococci: towards agonist and antagonist screening

Pogson (Kelsall), Claire (2013) Overexpression, purification and ligand interaction studies of intact membrane sensory kinases from enterococci: towards agonist and antagonist screening. Masters thesis, University of Central Lancashire.

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Two component signal transduction systems are of great importance in the regulation of expression of many virulence traits in bacteria and are a vital component of the way bacteria sense and respond to their environment. The sensor kinase component is generally membrane bound and is involved in detecting any changes to its environment through binding of a specific ligand signal, resulting in activation of its partner response regulator component and thereby bringing about a response which is usually a change in expression levels of specific gene sets.
These regulatory systems are particularly important in controlling antibiotic resistances in enterococci. Enterococci are amongst the most significant agents of nosocomial infections in the UK. The present study involved an investigation of two membrane sensor kinases known to be involved in antibiotic resistance in enterococci: (1) VanSA, involved in type A resistance to vancomycin in Enterococcus faecium which involves high level of inducible resistance to both vancomycin and teicoplanin antibiotics. VanSAsenses an unknown signalling ligand, which may either be the glycopeptide antibiotics them selves or more indirect ligands such as cell wall intermediates that accumulate in response to antibiotic inhibition of its cell wall target sites. Upon activation, VanSa becomes phosphorylated and phosphorylates its response regulator (VanR), thereby activating downstream genes which produce the vancomycin resistance phenotype. The nature of the ligand is therefore unknown, and one of the aims of this study is to address this and to determine whether it is vancomycin itself or cell wall intermediates; and (2) EF3290 (HK05), which is part of the CroRS system in Enterococcus faecalis and which has been shown by knock out studies to be involved in intrinsic beta-lactam resistance in enterococci.
The present study involved an in vitro approach using purified intact VanSa and EF3290 membrane proteins to investigate the nature of the activating ligands to which they bind (and are presumably activated by). The two proteins were expressed as intact membrane proteins in Escherichia coli using the expression plasmid pTTQ18His, solubilised by detergent from purified E. coli mixed membranes (isolated by explosive decompression of E. coli cells followed by high-speed centrifugation methods), and purified using the hexa-histidine tags engineered at the C-termini of both proteins by nickel affinity chromatography. Both proteins were successfully overexpressed and purified by these methods. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was used to detect the presence and purity of the two sensor kinases. Due to the limited purity of HK05 (45%) and potential degradation no ligand association studies were done with this protein.
VanSa in detergent micelles was successfully purified and synchrotron radiation circular dichroism (SRCD) spectroscopy was used to determine whether or not vancomycin binds to the protein. Using the stabilised purified protein, far-ultraviolet (180-260 nm) SRCD revealed that the purified protein maintained structural integrity following the relatively harsh purification methods employed for this membrane protein. Measurements in this region also revealed that addition of 5-fold vancomycin caused a significant change in the secondary structure of VanSA ,demonstrating that the molecule binds directly to VanSa. Measurements in the near-UV region (250-340 nm) permit investigations of the effects of vancomycin on the tertiary structure of the protein, and revealed a significant change in the spectrum upon vancomycin addition, confirming an interaction. The region that was significantly affected was the aromatic region in particular tyrosine and tryptophan residues.
Based on the findings above, the present study provides the first evidence that vancomycin itself serves as the interacting ligand for VanSa of clinical enterococci (and thereby activating the sensor). It has also been discovered that SRCDspectroscopy is a valuable technique to study the ligand interactions between vancomycin and VanSA and therefore further experiments should be done to quantify this relationship. save this for discussionDue to the importance of vancomycin-resistant enterococci in clinical strains these results provide a starting point for agonist and antagonist screening with the hope of developing novel anti-enterococcal and anti-GRE drugs which disrupt their activity.

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