Envelope Biogenesis in Gram-negative Bacteria

革兰氏阴性细菌的包膜生物发生

• 批准号: 10251349
• 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/10251349
• 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 革兰氏阴性菌的细胞被膜的特征在于具有两个脂质双层，即内膜 (IM)和外膜（OM）。OM不是典型的生物膜，因为虽然其内部小叶 含有磷脂，其外部小叶被糖脂LPS（或脂多糖）覆盖。LPS分子 密集地堆积在细胞表面，形成针对小疏水分子的渗透屏障， 否则扩散穿过磷脂双层。因此，革兰氏阴性菌自然对 许多抗生素。LPS所造成的屏障确实是很少有新抗生素有效的主要原因。 针对革兰氏阴性病原体的抗生素近年来已经开发出来。因此，研究有机质的生物成因， 不仅对了解细菌生理学很重要，而且对设计抗菌策略也很重要， 克服了OM的屏障作用。我们的长期目标是在分子水平上了解革兰氏- 阴性细菌构建它们的细胞包膜。在这里，我们将利用我们在遗传和生物化学方面的专业知识， 细胞被膜的研究，以调查两个高度保守的系统，是必不可少的OM生物合成 和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 从IM新合成的LPS分子，使得它们可以通过细胞外膜转运， 在细胞表面组装的蛋白质桥。我们的研究将集中在如何LPS提取和运输 由LptB 2FGC ATP结合盒（ABC）转运蛋白提供动力。ABC转运蛋白由ATP驱动 所有细胞都使用这种机器来将底物跨细胞区室转移。它们由一个 将结合和水解ATP产生的能量转换到其跨膜结构域的ATP酶 伴侣，转移底物。然而，仍然不清楚ATP酶和 关联的跨膜结构域被偶联，使得转运蛋白可以发挥功能。LptB 2FGC在功能上 结构上也不寻常：它从IM中提取糖脂LPS，将其置于蛋白质桥上， 跨膜结构域LptF/G与另一种蛋白质LptC的跨膜（TM）螺旋缔合。我们 我建议研究这种前所未有的结构特征在体内的作用，以及LptB 2的功能如何改变。 在LPS转运循环期间，ATP酶与跨膜结构域LptF/G的作用偶联。做 因此，我们将研究LptC的TM螺旋如何下调ATP酶活性， LptF/G结构域参与LPS转运。此外，我们还将对大肠杆菌中的AsmA-like蛋白进行研究。杆菌 这个蛋白质家族仍然没有被描述，但我们发现它们具有一种功能， 对E.杆菌在此资助期内，我们将通过以下方式推进我们对该蛋白质家族的理解： 进行结构-功能分析，确定其潜在合作伙伴，并确定其基本 在OM生物发生中的作用。拟议的研究将继续揭示新的机制，这是至关重要的 革兰氏阴性菌的生长和相关的急需的抗生素的开发。