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Envelope Biogenesis in Gram-negative Bacteria

革兰氏阴性细菌的包膜生物发生

基本信息

批准号：
10683987
负责人：
Natividad Ruiz
金额：
$ 32.84万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10683987
关键词：
ATP phosphohydrolase ATP-Binding Cassette Transporters ATPase Domain Address Affect 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 Friends 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 model organism novel pathogen periplasm protein transport weapons

项目摘要

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.

项目总结革兰氏阴性细菌的细胞膜具有两层脂双层，即内膜 (IM)和外膜(OM)。OM不是典型的生物膜，因为虽然它的内叶含有磷脂，其外部小叶被糖脂内毒素(或脂多糖)覆盖。脂多糖分子在细胞表面密集堆积，形成了对疏水小分子的渗透性屏障否则扩散到磷脂双层。因此，革兰氏阴性细菌对很多抗生素。内毒素造成的屏障确实是很少有新的抗生素有效的主要原因。近年来，针对革兰氏阴性病原菌的研究已经取得了进展。因此，研究有机质的生物发生是不仅对理解细菌生理学很重要，而且设计出能够克服OM的障碍功能。我们的长期目标是在分子水平上了解Gram- 负性细菌会形成它们的细胞膜。在这里，我们将利用我们在遗传和生化方面的专业知识对细胞膜的研究，以研究OM生物发生所必需的两个高度保守的系统和革兰氏阴性菌大肠杆菌的生长。我们将调查LPT系统如何提取从IM中新合成的内毒素分子，使它们可以通过在细胞表面组装的蛋白质桥。我们的研究将集中在内毒素的提取和运输由LptB2FGC ATP结合盒(ABC)运输器提供动力。ABC转运蛋白由ATP驱动所有细胞都用来在细胞间转移底物的机器。它们由一台将结合和水解三磷酸腺苷所产生的能量转换到其跨膜区的三磷酸腺苷酶合作伙伴，转移底物。然而，目前尚不清楚ATPase和ATPase的动作是如何同源跨膜结构域被偶联，从而使转运蛋白能够发挥作用。LptB2FGC在功能上在结构上也不同寻常：它从IM中提取糖脂内毒素，将其置于蛋白质桥上，并且它的跨膜结构域LptF/G与另一种蛋白质LptC的跨膜(TM)螺旋相关。我们建议研究这一前所未有的结构特征在体内的作用，以及LptB2如何发挥功能在内毒素转运过程中，ATPase与跨膜区LptF/G的作用相偶联。去做因此，我们将研究LptC的TM螺旋是如何下调ATPase活性的，以及未知的功能是如何 LptF/G结构域参与内毒素转运。此外，我们还将研究大肠杆菌中的ASMA样蛋白。这一蛋白质家族大部分仍未确定特征，但我们发现它们执行的功能是对大肠杆菌的生长是必不可少的。在这一资助期间，我们将通过以下方式促进我们对这个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，并确定其本质在OM生物发生中的作用。拟议的研究将继续揭示新的机制，这些机制对于革兰氏阴性细菌的生长和与急需的抗生素的发展相关。

项目成果

期刊论文数量（22）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Correction: The O-Antigen Flippase Wzk Can Substitute for MurJ in Peptidoglycan Synthesis in Helicobacter pylori and Escherichia coli.

更正：O-抗原 Flippase Wzk 可以替代幽门螺杆菌和大肠杆菌肽聚糖合成中的 MurJ。

DOI：
10.1371/journal.pone.0170518
发表时间：
2017
期刊：
PloS one
影响因子：
3.7
作者：
Elhenawy,Wael;Davis,RebeccaM;Fero,Jutta;Salama,NinaR;Feldman,MarioF;Ruiz,Natividad
通讯作者：
Ruiz,Natividad

Function and Biogenesis of Lipopolysaccharides.