Dissecting Gram-negative envelope biogenesis

世界卫生组织(WHO)将抗药性病原体确定为对全球健康、粮食安全和发展的最大威胁之一。任何人都可能受到影响，无论年龄或国籍。越来越多的病原体对目前的抗生素治疗产生抗药性，这清楚地表明需要对这些快速适应的病原体采取行动。尽管可以获得最现代的药物和医院，但不可治愈的感染对患者有几种影响，从更长的住院时间到最终死亡。因此，迫切需要在病原体的研究上投入更多的资源，以便能够治疗它们引起的感染。因此，世界卫生组织发出警告，要采取行动，防止我们前往抗生素后区域，在那里，常见的感染和轻伤将再次致命。世界卫生组织已经将最令人担忧的病原体列为优先事项，以鼓励像BBSRC这样的资助者和科学家解决即将变得无法治疗的病原体。在这份被归类为关键的名单的顶部，完全是革兰氏阴性细菌。这项研究计划的重点是了解革兰氏阴性菌是如何建立它们最重要的结构之一--细胞膜的。进一步了解这一过程将有助于我们设计策略，克服病原体对我们使用的抗生素的耐药性。细菌细胞膜是一种多层结构，可以保护细胞免受不可预测的和往往是有害的环境的影响，包括接触抗生素。特别是，革兰氏阴性细菌的细胞膜具有特殊的吸引力，因为它既是结构元件，又是渗透性屏障。这种低通透性是由不对称的脂双层(称为外膜)造成的，它阻止包括许多抗生素在内的有毒化合物进入细胞。确定哪些基因在维持包膜的结构和不透性方面发挥作用，是了解细菌如何保护自己的基础。它还帮助我们找到新的方法来克服这种渗透性障碍，并提供抗生素来治疗感染。尽管需要这种研究，但用于检测革兰氏阴性细菌包膜完整性的全基因组筛查仍然缺乏。本提案中概述的工作将填补这一知识空白。我将开发一种全基因组、高通量的分析方法，以强有力地量化参与革兰氏阴性包膜生物合成的潜在基因网络。可以通过从基因组中删除任何基因并分析其缺失的后果(例如，对抗生素的反应差异)来研究任何基因的功能。这可以使用数千个病原体的突变株系统地完成，每个突变株都缺乏一个单一的基因。我建议使用一组单缺失突变体来分析革兰氏阴性杆菌、铜绿假单胞菌和肺炎克雷伯菌的包膜生物发生。这项工作将揭示每个缺失基因对许多不同抗生素和环境压力反应的影响。由此产生的应激反应图将提供有关药物未知作用模式的知识，以及这些细菌如何在受到挑战时保持其包膜完整性。通过分析这些网络，我可以确定在这些过程中发挥基础性作用的基因。一旦我确定了重要的基因或途径，我将进一步研究它们的细胞功能。为此，我将利用我在分子生物学方面的专业知识来了解其他基因是否相互依赖于已确定的关键角色(遗传相互作用)，以及如果我们能够确定这些蛋白质是(蛋白质相互作用)的一部分的蛋白质机制。这些观察将有助于确定潜在的药物靶点，并有助于克服细胞膜构成的分子屏障，最终导致更好地治疗革兰氏阴性细菌感染。

The World Health Organization (WHO) identified antibiotic resistant pathogens as one of the biggest threats to global health, food security and development. Anyone can be affected, regardless of age or nationality. The growing number of pathogens that are resistant to current antibiotic treatments clearly signal a need to act against these rapidly adapting pathogens. Despite access to the most modern medicines and hospitals, non-treatable infections impact patients in several ways, ranging from longer hospital stays, to ultimately death. Thus, there is an urgent need to invest more resources in research on pathogens to be able to treat infections they cause. Therefore, the WHO issued a warning to act to prevent us from heading for a post-antibiotic area, where common infections and minor injuries would once again be deadly. The WHO have prioritised a list of the most concerning pathogens to encourage funders like the BBSRC and scientists to tackle the pathogens that are close to becoming untreatable. At the top of this list, classed as critical, are solely Gram-negative bacteria. This research proposal focusses on understanding how Gram-negative bacteria build one of their most important structures - their cell envelope. Furthering our knowledge about this process will help us to design strategies to overcome pathogen resistance to the antibiotics we use. The bacterial cell envelope is a multi-layered structure that protects the cell from its unpredictable and often hostile environment, including exposure to antibiotics. In particular, Gram-negative bacterial cell envelopes hold special interest because of the combined property of being both a structural element and a permeability barrier. The low permeability is conferred by the asymmetric lipid bilayer, referred to as the outer membrane, which prevents toxic compounds, including many antibiotics, from entering the cell. Defining which genes play a role in maintaining the structure and impermeability of the envelope is fundamental to understanding how bacteria protect themselves. It also helps us to find new ways to overcome this permeability barrier and to deliver antibiotics to treat infections. Despite the need for this kind of research, genome-wide screens to assay envelope integrity in Gram-negative bacteria are still missing. The work outlined in this proposal will fill this knowledge gap. I will develop a genome-wide, high-throughput assay to robustly quantify the underlying network of genes involved in Gram-negative envelope biosynthesis. The function of any gene can be studied by deleting it from the genome and analysing the consequences of its deletion (e.g. differences in responses to antibiotics). This can be done systematically using thousands of mutants of a pathogen, each mutant deficient in a single gene. I propose to use a collection of single deletion mutants to profile envelope biogenesis for the Gram-negative pathogens Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. This work will uncover the effect of each deleted gene on responses to many different antibiotics and environmental stresses. The resulting stress-response maps will provide knowledge about the uncharted mode of action of drugs and how those bacteria maintain their envelope integrity when challenged. By analysing these networks, I can identify genes that play fundamental roles in these processes. Once I have identified important genes or pathways, I will further investigate their cellular function. For this, I will use my expertise in molecular biology to understand if other genes are co-dependent on identified key players (genetic interactions) and if we can identify the protein machineries these proteins are part of (protein interactions). These observations will aid in the identification of potential drug targets and help to overcome the molecular barrier posed by the cell envelope, ultimately leading to better treatment of Gram-negative bacterial infections.