In vitro assembly of bacterial peptidoglycan in tethered lipid bilayer membranes

The spread of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and penicillin-resistant Streptococcus pneumoniae (the cause of pneumonia) poses a real threat to patient care in the UK health service, and has become a political issue in the 2005 general election. For the last 40 years we have taken penicillin for granted as a life-saving medicine, but there is now an urgent need to find new targets for antibacterial action. The mode of action of penicillin is to inhibit the final step of the assembly of peptidoglycan, a polysaccharide layer found on the surface of all bacteria. This transpeptidation step, which cross-links the peptidoglycan layer and gives it structural rigidity, is catalysed by the penicillin-binding proteins (PBPs). In spite of the large amount of work carried out on the penicillin and cephalosporin families of antibiotics, there are still very limited methods to study the reaction catalysed by the PBPs, because the substrates for these enzymes are complex lipid-linked intermediates found in the cell membrane. We have developed new techniques to study these lipid-linked steps. Using gold-coated chips, we can deposit tethered lipid bilayer membranes (TLBMs) on the surface of the chip, and we can use these devices to deposit the lipid II substrate for the PBP reaction. We can prepare these lipid II substrates in the lab, and can attach fluorescent labels, which allow us to monitor the reaction. In this project we want to use a range of surface science methods to study the reactions catalysed by the PBPs on TLBMs, which will allow us to a) develop new ways to find new antibacterial agents; b) study the reactions catalysed by PBPs from penicillin-resistant bacteria, and find out why they have become resistant to penicillin.

The project involves the application of tethered lipid bilayer membranes (TLBMs) and surface science methods to the study of the lipid-linked step of bacterial peptidoglycan biosynthesis.We have already shown that we can deposit lipid- linked intermediates in TLBMs, and that we can use surface plasmon resonance (SPR) to detect the polymerisation of peptidoglycan on the surface of TLBMs. We have also developed a range of fluorescently-labelled peptidoglycan precursors, containing fluorescent labels at positions 3, 4, and 5 of the pentapeptide chain. We will use TLBMs to study in vitro peptidoglycan assembly, monitored in situ using fluorescence spectroscopy and surface plasmon resonance. The peptidoglycan layer formed in situ will be characterised by mass spectrometry and atomic force microscopy. The processing of different fluorescent substrates will be monitored by fluorescence microscopy. We will apply new surface science techniques to in vitro peptidoglycan formation. Quartz crystal microbalance with dissipation (QCM-D) will be used to probe transglycosylation and transpeptidation of the peptidoglycan layer. ATR-IR will be used, with a beta-cyanoalanine-labelled substrate, to monitor transpeptidation. Neutron reflectivity and tip-modified atomic force microscopy will also be used to study in vitro peptidoglycan formation. New fluorescence- or SPR-based methods for monitoring peptidoglycan synthesis will be applied to array formats, with the aim of developing new screening methods for peptidoglycan synthesis inhibitors. These arrays will be tested using known natural product inhibitors, and small libraries of synthetic enzyme inhibitors. TLBMs will be used to follow the processing of lipid II by individual penicillin-binding proteins (PBPs), both bifunctional class A PBPs (PBP-1b from S. pneumoniae or E. coli) and monofunctional class B PBPs (PBP-2x or PBP-2a from S. pneumoniae). These studies will allow new insight into the specific roles of individual PBPs, and will allow a detailed study of PBPs from penicillin-resistant S. pneumoniae.