Molecular mechanisms of microbial complex carbohydrate secretion

微生物复合碳水化合物分泌的分子机制

基本信息

批准号：
9769067
负责人：
Jochen Zimmer
金额：
$ 28.28万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9769067
关键词：
ATP phosphohydrolase ATP-Binding Cassette Transporters Affect Anabolism Animals Antibiotic Resistance Antibiotics Architecture Bacillus anthracis Binding Biopolymers Burkholderia pseudomallei Carbohydrates Cell Wall Cell surface Cells Centers for Disease Control and Prevention (U.S.)Complement Complementarity Determining Regions Complex Cryoelectron Microscopy Crystallization Cytolysis Data Detergents Diffusion Ebola virus Escherichia coli Francisella tularensis Frankfurt-Marburg Syndrome Virus Gram-Negative Bacteria Health Human Hydrolysis Immune Impairment Individual Innate Immune Response Length Ligand Binding Lipid A Lipids Lipopolysaccharide Biosynthesis Pathway Lipopolysaccharides Mediating Membrane Modeling Molecular Molecular Conformation Molecular Weight Nucleotides O Antigens Oligosaccharides Pathogenicity Pathway interactions Phagocytosis Polymers Polysaccharides Positioning Attribute Predisposition Process Property Protein Engineering Salmonella Side Signal Transduction Site Structure Teichoic Acids Temperature Time Transmembrane Domain Transport Process United States National Institutes of Health Vertebral column analog antimicrobial base cell envelope crosslink drug resistant pathogen extracellular fascinate foodborne illness human pathogen in vivo inorganic phosphate insight microbial multi-drug resistant pathogen mutant novel pathogen periplasm polypeptide prevent reconstitution solute structural biology sugar

项目摘要

Antibiotic-resistant pathogens pose a significant threat to human health. The NIH identified several Gram- negative species of particular concern due to increasing antibiotic resistances. The cell envelope of Gram- negative bacteria consists of two membranes and lipopolysaccharides (LPS) form an important component of the extracellular leaflet of the outer membrane. LPS are important cell wall components that control diffusion across the outer membrane, stabilize the cell envelope, and assist in escaping host immune defenses, among other functions. Impaired LPS biosynthesis correlates with increased susceptibility to antimicrobial treatments. LPS contain a conserved core, consisting of lipid-A attached to an oligosaccharide backbone, and a hypervariable region, called the O antigen. O antigens are primarily linear complex carbohydrates that reduce the efficacy of complement-mediated cell lysis and phagocytosis as part of the innate immune response. O antigens are synthesized via two fundamentally different mechanisms. One pathway relies on assembling the polymers from short oligosaccharides in the periplasm, the other involves moving fully-assembled O antigens from the cytosolic to the periplasmic side of the inner membrane with the help of an ABC transporter. Not only is ABC transporter-mediated secretion of O antigens an important process for microbial pathogenicity, the transport of a substrate several times the size of the ABC transporter itself is fascinating from a molecular level. Taking advantage of an already determined O antigen-translocating ABC transporter structure, we propose a structural biology approach to unravel the mechanism of O antigen translocation and to identify unique features of the O antigen that regulate transporter activity. ABC transporters use ATP binding and hydrolysis to cycle between conformations that mediate substrate translocation. Our O antigen ABC transporter structure represents a nucleotide-free conformation, in which the transporter forms a continuous channel across the membrane that could accommodate a translocating O antigen. We speculate that conformational changes associated with nucleotide binding induce O antigen translocation by about 1-2 sugar units per ATP hydrolyzed. To reveal the molecular mechanism of O antigen translocation, we seek to determine the structure of the ABC transporter in a nucleotide-bound closed conformation (Aim 1). Further, many bacterial species signal completion of O antigen biosynthesis by modifying the polymer’s growing end with specific groups, such as carbohydrate, phosphate, or methyl moieties. These ‘capped O antigens’ can only be exported by transporters containing a carbohydrate-binding domain (CBD) attached to their nucleotide-binding domain. This domain was removed from our O antigen transporter to facilitate crystallization. To reveal how the CBD binds its substrate and modulates transporter functions, we propose to determine the structure and substrate binding properties of the isolated CBD (Aim 2A) and to determine the architecture of the CBD-containing full-length O antigen transporter (Aim 2B).

抗生素的病原体对人类健康构成了重大威胁。 NIH确定了几克 - 由于抗生素耐药性增加而引起的特殊关注的负面物种。革兰氏的细胞包络阴性细菌由两个膜和脂多糖（LPS）组成，构成了重要组成部分外膜的细胞外小叶。 LPS是控制扩散的重要细胞壁成分穿过外膜，稳定细胞信封，并协助逃脱宿主免疫防御措施其他功能。 LPS生物合成受损与对抗菌治疗的易感性增加相关。 LPS包含一个保守的核心，由附着在寡糖主链上的脂质A组成，A 高变量区域，称为O抗原。 o抗原是原发性线性复合碳氢化物，可减少完成介导的细胞裂解和吞噬作用的有效性是先天免疫反应的一部分。 o 抗原是通过两种根本不同的机制合成的。一条途径依赖于组装来自白质寡糖的聚合物，另一种涉及移动完全组装的O抗原从ABC转运蛋白的帮助下，从胞质到内膜的周质侧。不仅 ABC转运蛋白介导的O抗原是微生物致病性的重要过程，底物的运输几倍以ABC转运蛋白本身的大小从分子着迷等级。利用已经确定的O抗原转移ABC转运蛋白结构的优势，我们提出一种结构生物学方法，以阐明O抗原易位的机制并确定 O抗原调节转运蛋白活性的独特特征。 ABC转运蛋白使用ATP结合和水解来循环介导底物的构象运输。我们的O抗原ABC转运蛋白结构代表了无核苷酸的构象，其中转运蛋白在整个膜上形成连续的通道，可以容纳易位O 抗原。我们推测与核苷酸结合相关的构象变化会影响o抗原每个ATP水解约1-2个糖单位。揭示O抗原的分子机制易位，我们试图确定核苷酸结合的ABC转运蛋白的结构构象（目标1）。此外，许多细菌物种信号完成O抗原生物合成的完成通过特定组（例如碳氢化物，磷酸盐或甲基）修改聚合物的生长端部分。这些“上限” O抗原只能通过包含碳水化合物结合的转运蛋白出口域（CBD）附着在其核苷酸结合域上。该域从我们的O抗原中删除转运蛋白以促进结晶。揭示CBD如何结合其底物并调节转运蛋白函数，我们建议确定分离的CBD的结构和底物结合特性（AIM 2A）并确定含CBD的全长O抗原转运蛋白的结构（AIM 2B）。