In vitro assembly of bacterial peptidoglycan in tethered lipid bilayer membranes

耐抗生素细菌的传播，如耐甲氧西林金黄色葡萄球菌（MRSA）和耐青霉素肺炎链球菌（肺炎的病因），对英国卫生服务的病人护理构成了真正的威胁，并已成为2005年大选中的一个政治问题。在过去的40年里，我们理所当然地认为青霉素是一种拯救生命的药物，但现在迫切需要找到抗菌作用的新目标。青霉素的作用方式是抑制肽聚糖组装的最后一步，肽聚糖是一种在所有细菌表面发现的多糖层。这一转肽化步骤，交联肽聚糖层并赋予其结构刚性，是由青霉素结合蛋白（PBPs）催化的。尽管对青霉素和头孢菌素家族的抗生素进行了大量的研究，但研究PBPs催化反应的方法仍然非常有限，因为这些酶的底物是在细胞膜中发现的复杂脂联中间体。我们已经开发了新的技术来研究这些与脂质相关的步骤。利用镀金芯片，我们可以在芯片表面沉积系留脂质双层膜（TLBMs），我们可以使用这些设备沉积脂质II底物用于PBP反应。我们可以在实验室中制备这些脂质II底物，并可以附加荧光标记，这使我们能够监测反应。在这个项目中，我们希望使用一系列表面科学方法来研究PBPs在tlbm上催化的反应，这将使我们能够a)开发新的方法来寻找新的抗菌剂；b)研究来自青霉素耐药细菌的PBPs催化的反应，并找出它们对青霉素耐药的原因。

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.