Protein import through the E. coli cell envelope

由于抗生素耐药细菌的不可阻挡的增长，我们正在迅速耗尽用于医药和农业的抗生素。30多年来，制药行业的药物发现计划未能提供任何新型抗生素，这意味着我们的社会正迅速接近抗生素出现之前的时代，在那个时代，常规手术面临着潜在致命感染的高度风险，最近全球各地关于抗菌素耐药性的报告强调了这一观点。此外，保护我们的食物免受细菌病原体的侵害日益受到关注；例如，2011年欧洲各地的食品受到大肠杆菌O157:H7污染，导致多人死亡。显然，需要新的办法来应对这个问题。一组尚未被用于治疗细菌感染的分子是蛋白质细菌素。细菌素是细菌在争夺营养时产生的蛋白质抗生素，用于杀死近亲。这一建议的重点是一类被称为粘菌素的细菌素。大肠杆菌素是由大肠杆菌制造的，用于杀死其他大肠杆菌，最近已被证明在控制粮食作物中的大肠杆菌O157:H7感染方面非常有效。我的建议集中在了解这些对人体细胞没有活性的有毒蛋白质是如何绕过细菌的防御（称为“细胞包膜”）以杀死它们的。我们使用非致病性大肠杆菌K-12作为模型。我们最近在了解粘菌素如何易位到大肠杆菌（易位是它们从细胞外部移动到细胞内部的过程）方面取得了两项重大进展。首先，我们分离出黏菌素与细胞表面目标结合的复合物，这为它们进入细胞做了准备，并获得了初步数据，表明我们将能够确定这种易位态的三维结构。其次，我们设计了新的基于荧光的显微镜工具，用于实时可视化这些分子进入细菌的过程，使我们能够解决迄今为止无法调查的问题。该提案有三个具体目标：1 .利用晶体学方法确定大肠杆菌中与构成易位机制的蛋白质结合的粘菌素的三维结构；当细胞被紫外线照射时，使用浸透光反应性交联基团的粘连蛋白（可以通过基因工程）作为捕获粘连蛋白的手段。这种方法将使我们能够跟踪这些分子穿过细胞包膜的路径，并识别它们所接触的蛋白质；为了利用我们开发的显微镜工具来跟踪粘菌素穿过细胞包膜的过程，并利用蛋白质工程技术来解剖它们的进入机制。

We are rapidly running out of antibiotics for use in medicine and agriculture due to the inexorable rise of antibiotic resistant bacteria. The failure of drug discovery programmes in the pharmaceutical industry to furnish any new classes of antibiotics in over 30 years means our society is rapidly approaching a time akin to the pre-antibiotic era where routine operations carried a high degree of risk from potentially lethal infections, a view emphasized by recent reports on antimicrobial resistance from across the globe. Moreover, protecting our food against bacterial pathogens is an increasing concern; for example, contamination of food products by E. coli O157:H7 in 2011 across Europe resulted in multiple deaths.Clearly, new approaches are needed to respond to this problem. A group of molecules that have yet to be exploited in the treatment of bacterial infections are protein bacteriocins. Bacteriocins are protein antibiotics produced by bacteria to kill their close related neighbours during competition for nutrients. This proposal focuses on one class of bacteriocins known as colicins. Colicins are made by the bacterium Escherichia coli to kill other E. coli, and have recently been shown to be highly effective at controlling E. coli O157:H7 infections in food crops.My proposal is focused on understanding how these toxic proteins, which have no activity against human cells, circumvent the defences of bacteria (known as the 'cell envelope') in order to kill them. As our model, we use the non-pathogenic organism E. coli K-12. We have recently made two major advances in understanding how colicins translocate into E. coli (translocation is the process by which they move from the outside of the cell to the inside). First, we have isolated complexes of colicins bound to their cell surface targets, which primes them for entry into the cell, and have obtained preliminary data to suggest that we will be able to determine three dimensional structures for such translocation-competent states. Second, we have devised new fluorescence-based microscopy tools for visualizing the entry of these molecules into bacteria in real time, allowing us to address questions that have until now been impossible to investigate.The proposal has three specific objectives:1. To use crystallographic methods to determine the three dimensional structures of colicins bound to proteins that constitute the translocation machinery in E. coli;2. To use colicins impregnated with photoreactive cross-linking groups (which can be engineered genetically) as a means of trapping colicins as they pass through the cell envelope when cells are illuminated with UV light. This approach will allow us to follow the path taken by these molecules through the cell envelope and identify proteins they come into contact with;3. To exploit the microscopy tools we have developed to track colicins as they pass through the cell envelope and dissect their mechanism of entry using protein engineering.