Dissecting Gram-negative envelope biogenesis

剖析革兰氏阴性包膜生物发生

• 批准号: MR/V027204/1
• 负责人: Manuel Banzhaf
• 金额: $ 135.99万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FV027204%2F1
• 关键词: Dissecting; Gram; negative; envelope; biogenesis

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.

世界卫生组织（WHO）将抗生素耐药性病原体确定为全球健康、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。对当前抗生素治疗产生耐药性的病原体数量不断增加，这清楚地表明需要对这些快速适应的病原体采取行动。尽管可以使用最现代的药物和医院，但不可治疗的感染仍会以多种方式影响患者，从延长住院时间到最终死亡。因此，迫切需要投入更多资源进行病原体研究，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，要求我们采取行动，防止我们走向后抗生素时代，普通感染和轻伤将再次致命。世界卫生组织优先列出了最受关注的病原体清单，以鼓励 BBSRC 等资助者和科学家解决几乎无法治疗的病原体。在此列表的顶部，被归类为关键细菌，仅是革兰氏阴性细菌。该研究计划的重点是了解革兰氏阴性细菌如何构建其最重要的结构之一 - 细胞膜。进一步了解这一过程将有助于我们设计策略来克服病原体对我们使用的抗生素的耐药性。细菌细胞包膜是一种多层结构，可以保护细胞免受不可预测且通常充满敌意的环境的影响，包括接触抗生素。特别是，革兰氏阴性细菌细胞包膜因其作为结构元件和渗透性屏障的综合特性而受到特别关注。低渗透性是由不对称脂质双层（称为外膜）赋予的，它可以防止有毒化合物（包括许多抗生素）进入细胞。定义哪些基因在维持包膜的结构和不渗透性方面发挥作用对于理解细菌如何保护自身至关重要。它还帮助我们找到克服这种渗透性屏障并提供抗生素来治疗感染的新方法。尽管需要此类研究，但用于测定革兰氏阴性细菌包膜完整性的全基因组筛选仍然缺失。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、高通量的检测方法，以稳健地量化参与革兰氏阴性包膜生物合成的潜在基因网络。任何基因的功能都可以通过从基因组中删除它并分析其删除的后果（例如对抗生素反应的差异）来研究。这可以通过使用病原体的数千个突变体来系统地完成，每个突变体都缺乏一个基因。我建议使用一系列单缺失突变体来分析革兰氏阴性病原体大肠杆菌、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删除的基因对许多不同抗生素和环境压力的反应的影响。由此产生的应激反应图将提供有关药物未知作用模式以及这些细菌在受到挑战时如何保持包膜完整性的知识。通过分析这些网络，我可以识别在这些过程中发挥基本作用的基因。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共同依赖于已确定的关键参与者（遗传相互作用），以及我们是否可以识别这些蛋白质所属的蛋白质机器（蛋白质相互作用）。这些观察结果将有助于识别潜在的药物靶点，并有助于克服细胞被膜构成的分子屏障，最终更好地治疗革兰氏阴性细菌感染。

项目成果

Plasmid permissiveness of wastewater microbiomes can be predicted from 16S rRNA sequences by machine learning.

废水微生物组的质粒容许度可以通过机器学习从 16S rRNA 序列预测。

• DOI: http://dx.10.1093/bioinformatics/btad400
• 发表时间: 2023
• 期刊: Bioinformatics (Oxford, England)
• 作者: Moradigaravand D

Membrane staining and phospholipid tracking in Pseudomonas aeruginosa PAO1 using the phosphatidylcholine mimic propargyl-choline

使用模拟磷脂酰胆碱对铜绿假单胞菌 PAO1 进行膜染色和磷脂追踪

• DOI: http://dx.10.1099/acmi.0.000690.v1
• 发表时间: 2023
• 作者: Graham C

ChemGAPP: a tool for chemical genomics analysis and phenotypic profiling.

ChemGAPP：化学基因组学分析和表型分析的工具。

• DOI: http://dx.10.1093/bioinformatics/btad171
• 发表时间: 2023
• 期刊: Bioinformatics (Oxford, England)
• 作者: Doherty HM

The lipoprotein DolP affects cell separation in Escherichia coli, but not as an upstream regulator of NlpD.

脂蛋白 DolP 影响大肠杆菌中的细胞分离，但不是 NlpD 的上游调节因子。

• DOI: http://dx.10.1099/mic.0.001197
• 发表时间: 2022
• 期刊: Microbiology (Reading, England)
• 作者: Boelter G

LI-Detector: a Method for Curating Ordered Gene-Replacement Libraries.

LI-Detector：一种整理有序基因替换文库的方法。

• 发表时间: 2022
• 作者: []