Envelope Biogenesis in Gram-negative Bacteria

革兰氏阴性细菌的包膜生物发生

• 批准号: 10462796
• 负责人: Natividad Ruiz
• 金额: $ 33.69万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-05 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462796
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; ATPase Domain; Address; Affect; Animal Model; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Physiology; Bacteriophages; Behavior; Binding; Biochemical; Biogenesis; Bioinformatics; Biological; Carrier Proteins; Cell Wall; Cell membrane; Cell surface; Cells; Communities; Complex; Coupled; Cytoplasm; Data; Development; Diffuse; Environment; Escherichia coli; Eukaryotic Cell; Family; Funding; Genetic; Globular Region; Glycolipids; Goals; Gram-Negative Bacteria; Growth; Hydrophobicity; Immune system; In Vitro; Individual; Infection; Knowledge; Lipid Bilayers; Lipids; Lipopolysaccharides; Membrane; Membrane Proteins; Molecular; Movement; Natural Resistance; Nucleotides; Organism; Pathway interactions; Peptidoglycan; Permeability; Phospholipids; Protein Family; Proteins; Research; Resources; Role; Signal Transduction; Structure; Surface; System; Transmembrane Domain; Travel; Virulence; Work; antimicrobial; antimicrobial drug; aqueous; bacterial community; bacterial resistance; cell community; cell envelope; cell motility; env Gene Products; environmental change; global health; in vivo; lipid transport; membrane biogenesis; novel; pathogen; periplasm; protein transport; weapons

PROJECT SUMMARY The cell envelope of Gram-negative bacteria is characterized by having two lipid bilayers, the inner membrane (IM) and the outer membrane (OM). The OM is not a typical biological membrane because while its inner leaflet contains phospholipids, its outer leaflet is covered with the glycolipid LPS (or lipopolysaccharide). LPS molecules are densely packed at the cell surface, creating a permeability barrier against small hydrophobic molecules that otherwise diffuse across phospholipid bilayers. As a result, Gram-negative bacteria are naturally resistant to many antibiotics. The barrier imposed by LPS is indeed the main reason why very few novel antibiotics effective against Gram-negative pathogens have been developed in recent years. Therefore, studying OM biogenesis is not only important to understand bacterial physiology, but also to devise antimicrobial strategies that can overcome the barrier function of the OM. Our long-term goal is to understand at the molecular level how Gram- negative bacteria build their cell envelope. Here, we will leverage our expertise in genetic and biochemical studies of the cell envelope to investigate two highly conserved systems that are essential for OM biogenesis and growth of the Gram-negative bacterium Escherichia coli. We will investigate how the Lpt system extracts newly synthesized LPS molecules from the IM so that they can be transported across the cell envelope through a protein bridge to be assembled at the cell surface. Our studies will focus on how LPS extraction and transport is powered by the LptB2FGC ATP-binding cassette (ABC) transporter. ABC transporters are ATP-driven machines that all cells use to translocate substrates across cellular compartments. They are powered by an ATPase that transduces the energy derived from binding and hydrolyzing ATP to its transmembrane-domain partners, which translocate the substrate. However, it remains unknown how the actions of the ATPase and cognate transmembrane domains are coupled so that the transporter can function. The LptB2FGC is functionally and structurally unusual: it extracts the glycolipid LPS from the IM to place it onto a protein bridge, and its transmembrane domains LptF/G associate with the transmembrane (TM) helix of another protein, LptC. We propose to investigate the in vivo role of this unprecedented structural feature, and how the function of the LptB2 ATPase is coupled to the action of the transmembrane domains LptF/G during the LPS transport cycle. To do so, we will investigate how LptC’s TM helix downregulates ATPase activity, and how uncharacterized functional domains of LptF/G participate in LPS transport. In addition, we will also study the AsmA-like proteins in E. coli. This family of proteins remain mostly uncharacterized, but we have discovered they perform a function that is essential for growth of E. coli. In this funding period, we will advance our understanding of this protein family by conducting structure-function analyses, identifying their potential partners, and determining their essential function in OM biogenesis. The proposed research will continue to reveal novel mechanisms that are crucial for the growth of Gram-negative bacteria and relevant the development of much needed antibiotics.

项目摘要 革兰氏阴性细菌的细胞包膜的特征是有两个脂质双层，即内膜 （IM）和外膜（OM）。 OM不是典型的生物膜，因为它的内部小叶 含有磷脂，其外部小叶覆盖有糖脂LPS（或脂多糖）。 LPS分子 垂直在细胞表面，形成了针对小疏水分子的渗透性屏障 否则跨磷脂双层散布。结果，革兰氏阴性细菌自然具有抗性 许多抗生素。 LPS施加的障碍确实是很少有新型抗生素有效的主要原因 近年来已经开发了针对革兰氏阴性病原体。因此，研究OM生物发生是 了解细菌生理学不仅很重要，而且还要设计可以制定抗菌策略 克服OM的屏障功能。我们的长期目标是在分子水平上了解如何掌握 阴性细菌建立其细胞包膜。在这里，我们将利用我们在遗传和生化方面的专业知识 细胞包络的研究研究了两个对OM生物发生必不可少的高度保守的系统 革兰氏阴性细菌大肠杆菌的生长。我们将研究LPT系统如何提取 来自IM的新合成的LPS分子可以通过细胞包膜运输 要在细胞表面组装的蛋白桥。我们的研究将重点介绍LP的提取和运输方式 由LPTB2FGC ATP结合盒（ABC）转运蛋白提供动力。 ABC转运蛋白是ATP驱动的 所有细胞都用来将底物转移到细胞隔间的机器。他们由 将源自结合和水解ATP得出的能量转化为跨膜域的ATPase 合作伙伴，底物易位。但是，尚不清楚ATPase和 同源式运输膜结构域是耦合的，以便转运蛋白可以起作用。 LPTB2FGC在功能上是 在结构上很不寻常：它从IM提取糖脂LPS将其放在蛋白质桥上，并将其放在蛋白质桥上 跨膜结构域LPTF/G与另一种蛋白LPTC的跨膜（TM）螺旋相关。我们 提出研究这种前所未有的结构特征的体内作用，以及LPTB2的功能如何 在LPS传输周期中，ATPase与跨膜结构域LPTF/G的作用耦合。做 因此，我们将研究LPTC的TM螺旋如何下调ATPase活性，以及​​如何未表征功能 LPTF/g参与LPS运输的域。此外，我们还将研究大肠杆菌中的ASMA样蛋白。 这个蛋白质家族大部分仍然没有表征，但我们发现它们执行了一个功能 大肠杆菌的生长至关重要。在这个资助期间，我们将通过 进行结构功能分析，确定其潜在伴侣并确定其必不可少的 在OM生物发生中的功能。拟议的研究将继续揭示新的机制，这些机制对于 革兰氏阴性细菌的生长以及相关的急需抗生素的发展。