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

细菌被一种叫做肽聚糖层的坚固的支架状分子所包围。肽聚糖层为每种细菌提供保护，并定义它们独特的形状。在大肠杆菌和许多其他细菌中，肽聚糖层也作为第二保护层（称为外膜）的附着点，使其对抗生素具有抵抗力。外膜提供的额外保护可以使许多细菌感染难以治疗。我们对细菌蛋白质系统感兴趣，该系统在细菌繁殖时负责分解肽聚糖层。通过了解大肠杆菌中的这个系统，我们希望对细菌如何分裂以及它们如何维持其保护性外膜屏障获得实质性的新见解。涉及的关键酶是一对肽聚糖水解酶（AmiA和AmiB），它们由“激活剂”（EnvC）和内膜蛋白复合物（FtsEX）调节。在正常情况下，肽聚糖水解酶被仔细控制，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，这样肽聚糖层就可以被打破，新形成的子细胞就可以分离出来。在这个项目中，我们将结合结构生物学、生物化学和微生物学技术来精确地研究FtsEX和EnvC是如何“打开”和“关闭”肽聚糖水解酶的。首先，使用x射线晶体学，我们打算获得三维图像，以准确显示活化剂的结合如何导致肽聚糖水解酶被“打开”。其次，我们将分离所有相关的蛋白质，并将它们放在试管中，这样我们就可以研究它们的生物化学。第三，我们将通过在活细胞中制备修饰版蛋白并观察其对肽聚糖水解酶激活的影响，来验证我们目前对肽聚糖水解酶激活机制的理解。最后，我们将探讨阻断肽聚糖水解酶激活的可能性。在水解酶激活途径的任何一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。在项目结束时，我们希望建立一个近乎完整的分子理解肽聚糖水解酶是如何在分裂过程中被激活的。

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.