Maintaining cell wall integrity in Gram-Negative Bacteria

BBSRC：Christopher Graham：BB/M01116X/1病原菌对抗生素的抗药性的发展已经破坏了现代和未来的医疗保健，并已被公认为具有与气候变化一样重要的影响的世界性问题。我们正处于后抗生素时代的初始阶段，就感染的危险而言，这类似于19世纪之前的世界。或者在这方面特别令人担忧的是革兰氏阴性细菌，因为它们的外膜对现有和未来的抗生素进入构成了特殊的障碍。近年来，由于复杂的经济问题，制药业减少了为抗生素发现提供资金的能力和承诺，然而，很大一部分成功的抗生素是基于一般针对细菌细胞壁的抗生素。革兰氏阴性菌和革兰氏阳性菌都有内膜和细胞壁肽聚糖聚合物，但第三层外层，即外膜，是革兰氏阴性菌所独有的。这一外膜是革兰氏阴性细菌对大多数抗生素的最大防御，因为它为它们的进入提供了显着的障碍，并在革兰氏阴性细菌生理中发挥着重要的生物学作用。我们的假设是，细胞壁肽聚糖和外膜生物发生的过程必须联系在一起，如果我们理解这个过程，那么它可能会导致组合抗生素策略的使用，从而协同影响这两个系统。这将使我们能够真正分解革兰氏阴性细菌病原体，破坏外膜，因此，允许影响药物的肽聚糖通过。我们有初步数据表明，肽聚糖合成系统的破坏实际上会导致外膜的不稳定和细菌敏感性的增加，并假设外膜和肽聚糖层是通过复杂的相互作用同时和局部创建的。我们已经与加拿大麦克马斯特大学的洛里·伯罗斯教授建立了合作关系，他拥有专业的遗传工具和设施，使我们能够在最重要的革兰氏阴性病原体之一--白僵菌中探索这一假说。华威大学目前没有这样做的设施或专业知识，但我们与加拿大的合作将使我们能够获得这方面的知识，并促进未来在大肠杆菌中进行平行研究。在这项研究中，我们的目标是通过制作细菌菌株来显示负责外膜和肽聚糖合成的蛋白质的协调，在这些菌株中，个别蛋白质对被特别荧光标记，从而使我们能够使用适当的显微镜来显示它们的共存。这将在分子水平上确定这些过程是如何联系在一起的，以及可能涉及哪些蛋白质。以前，这两个外膜肽聚糖的生物发生都是作为单独的事件进行研究的，但在这种合作中获得的知识可能具有基本的生物学意义，并为未来同时针对这两个过程的抗菌策略奠定基础。在加拿大的研究将由一位才华横溢的英国博士生承担，他将学习新的技能，暴露在一个完全不同的研究环境中，从而从这次国际培训机会中获益良多。

BBSRC: Christopher Graham: BB/M01116X/1The development of resistance to antibiotics by pathogenic bacteria already undermines modern and future healthcare and has been acknowledged as a worldwide issue with an impact as important as climate change. We are in initial stages of a post-antibiotics era, akin to the pre-19th century world in terms of the danger from infections. Or particular concern in this respect are Gram-negative bacteria since their outer membrane presents a particular barrier to entry of existing and future antibiotics. The pharmaceutical industry has reduced in its capacity and commitment to funding antibiotic discovery in recent years due to complex economic issues, however, a large proportion of successful antibiotics have been those based on targeting the cell walls of bacteria generally. Both Gram-negative bacteria and Gram-positive bacteria have an inner membrane and cell wall peptidoglycan polymer however a third outer layer known as the outer membrane is unique to Gram-negatives. This outer membrane in Gram-negative bacteria's greatest defence against most antibiotics since it provides a significant barrier to their entry as well as having an important biological role in Gram-negative bacterial physiology. Our hypothesis is that the process of cell wall peptidoglycan and outer membrane biogenesis must be linked and if we understood this process then it may lead to the use of combinatorial antibiotics strategies that affect both systems synergistically. This would allow us to truly break down Gram-negative bacterial pathogens, disrupting the outer-membrane, therefore, allowing peptidoglycan affecting drugs to pass. We have preliminary data that shows disruption of the peptidoglycan synthesis system actually causes outer membrane destabilisation and increased bacterial susceptibility, and hypothesise the outer membrane and peptidoglycan layers are created simultaneously and locally through complex interactions.We have established a collaboration with Professor Lori Burrows at McMaster University in Canada who has the specialist genetic tools and facilities to enable us to explore this hypothesis in P. aeuginosa, one of the most important Gram-Negative pathogens. The University of Warwick does not currently have the facilities or expertise to do this but our collaboration with Canada will allow this knowledge to be gained and facilitate a parallel study in E.coli in the future. We aim in this study, to show the co-ordination of the proteins responsible for outer membrane and peptidoglycan synthesis by making bacterial strains in which individual pairs of proteins are specially fluorescently tagged allowing us to use appropriate microscopy to visualise their co-localisation. This will establish at a molecular level how these processes are linked and what proteins may be involved. Previously both outer membrane peptidoglycan biogenesis has been studied as separate events but knowledge gained in this collaboration could be of fundamental biological importance as well as the basis upon which future antimicrobial strategies that target the two processes in parallel are based. The study in Canada will be undertaken by a highly talented UK PhD student who will learn new skills, be exposed to an entirely different research environment and thus gain enormously from this international training opportunity.