Structural basis for bacterial peptidoglycan hydrolase activation in cell division and intrinsic resistance

细菌被称为肽聚糖层的强支架样分子包围。肽聚糖层为每种细菌提供保护，并定义了它们独特的形状。在大肠大肠杆菌和许多其他细菌的肽聚糖层也作为第二层保护层（称为外膜）的附着点，该保护层赋予对抗生素的抗性。由外膜赋予的额外保护可以使许多细菌感染难以治疗。我们感兴趣的是一个系统的细菌蛋白质，负责打破肽聚糖层时，细菌繁殖。我们希望获得有关细菌如何分裂以及它们如何维持其保护性外膜屏障的实质性新见解。所涉及的关键酶是一对肽聚糖水解酶（AmiA和AmiB），其由“激活剂”（EnvC）和内膜蛋白复合物（FtsEX）调节。在正常情况下，小心控制肽聚糖水解酶以防止它们咀嚼肽聚糖层。然而，在分裂过程中，FtsEX和EnvC提供的信号被用来打开这些蛋白质，从而打破肽聚糖层并分离新形成的子细胞。在这个项目中，我们将使用结构生物学，生物化学和微生物学技术的组合来精确地研究FtsEX和EnvC是如何打开和关闭肽聚糖水解酶的。首先，利用X射线晶体学，我们打算获得三维图像，以准确地显示激活剂的结合是如何导致肽聚糖水解酶被“打开”的。2其次，我们将分离所有相关的蛋白质，并将它们放在试管中，以便我们可以研究它们的生物化学。第三，我们将测试我们目前的理解肽聚糖水解酶的激活机制，通过在活细胞中的蛋白质的修改版本，并观察对肽聚糖氢化酶激活的影响。最后，我们将探讨阻断肽聚糖水解酶激活的可能性。在水解酶活化途径的任何沿着点成功抑制将验证这些蛋白质作为未来药物开发的潜在靶点。在该项目结束时，我们将期望建立一个接近完整的分子理解，在分裂过程中如何激活肽聚糖水解酶。

Bacteria are surrounded by a strong scaffolding-like molecule called the peptidoglycan layer. The peptidoglycan layer provides protection to each bacterium and defines their distinctive shapes. In E. coli, and many other bacteria, the peptidoglycan layer also serves as a point of attachment for a second protective layer (termed the outer membrane) which confers resistance to antibiotics. The additional protection conferred by the outer membrane can make many bacterial infections difficult to treat.We are interested in a system of bacterial proteins that are responsible for breaking down the peptidoglycan layer when bacteria reproduce.By understanding this system in E. coli, we hope to gain substantial new insights into how bacteria divide, and also, how they maintain their protective outer membrane barrier.The key enzymes involved are a pair of peptidoglycan hydrolases (AmiA and AmiB) that are regulated by an 'activator' (EnvC) and an inner membrane protein complex (FtsEX). Under normal circumstances, the peptidoglycan hydrolases are carefully controlled to prevent them from chewing up the peptidoglycan layer. However, during division, a signal provided by FtsEX and EnvC is used to switch these proteins 'on' so that the peptidoglycan layer can be broken and newly-formed daughter cells can be separated.In this project we will use a combination of structural biology, biochemistry and microbiological techniques to investigate precisely how the peptidoglycan hydrolases are turned 'on' and 'off' by FtsEX and EnvC.Firstly, using X-ray crystallography, we intend to obtain 3-dimensional images that will show exactly how binding of the activator causes the peptidoglycan hydrolase to be switched 'on'.Secondly, we will isolate all the proteins involved and bring them together in a test tube so that we can study their biochemistry. Thirdly, we will test our current understanding of the peptidoglycan hydrolase activation mechanism by making modified versions of the proteins in live cells and observing the effect on peptidoglycan hydsrolase activation. Finally, we will explore the possibility of blocking the peptidoglycan hydrolase activation. Successful inhibition at any point along the hydrolase activation pathway would validate these proteins as potential targets for future drug development.At the end of the project we would expect to establish a near-complete molecular understanding of how petidoglycan hydrolases are activated during division.