Biogenesis of Peptidoglycan in Escherichia coli

大肠杆菌中肽聚糖的生物发生

• 批准号: 8505507
• 负责人: Natividad Ruiz
• 金额: $ 24.44万
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8505507
• 关键词: Affect; Alleles; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Proteins; Binding; Biochemical Genetics; Biogenesis; Bioinformatics; Biological Assay; Biotin; Cell Shape; Cell Wall; Cell membrane; Cells; Congenital Disorders; Cytolysis; Cytoplasm; Data; Development; Disaccharides; Environment; Escherichia coli; Family; Future; Goals; Human; Knowledge; Label; Life; Link; Lipids; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Metabolic Diseases; Molecular; Mutation; Names; Oligosaccharides; Organism; Pathogenesis; Peptidoglycan; Polysaccharides; Process; Protein Family; Proteins; Reaction; Research; Role; Shelter facility; Signal Pathway; Spectrometry, Mass, Electrospray Ionization; Structure; System; Testing; Virulence Factors; Work; Yeasts; base; capsule; cell type; combat; crosslink; exoskeleton; glycosylation; in vivo; inhibitor/antagonist; lipid transport; lipooligosaccharide; member; mutant; novel; protein complex; scaffold; small molecule

DESCRIPTION (provided by applicant): Most bacteria polymerize peptidoglycan (PG) into a mesh-like sacculus that surrounds the cytoplasmic membrane and protects it against osmotic lysis. The PG sacculus also provides cell shape and serves as a scaffold to which virulence factors are anchored. Biogenesis of the PG sacculus is essential for viability, and our long-term goal is to understand it at the molecula level. This proposal focuses on the transport of PG intermediates across the cytoplasmic membrane, a poorly understood step in PG biogenesis. Bacteria build their PG sacculus in the extracytoplasmic space by polymerizing a disaccharide pentapeptide into glycan strands that are later crosslinked. The disaccharide pentapeptide is made in the cytoplasm as a lipid intermediate known as lipid II. Therefore, lipid II must be flipped across the membrane by a transporter (a flippase) through an unknown mechanism. The membrane protein MurJ has been proposed to be the lipid II flippase in Escherichia coli since it is essential for PG synthess and it belongs to the MOP (multidrug/oligosaccharidyl- lipid/polysaccharide) superfamily of exporters. This family is conserved in diverse organisms and it includes flippases of lipid-linked oligosaccharides that are similar to lipid II. FtsW and RodA have also been proposed to be lipid II flippases in E. coli, but there is no in vivo evidence supporting this hypothesis. Aim 1 of thi proposal is to determine whether MurJ, FtsW, and RodA are involved in lipid II translocation in vivo. Two analytical assays based on differential labeling and mass spectrometry will be developed to assess how depletion of these factors affects the levels and membrane topology of lipid II. The data obtained will be crucial for understanding the roles of MurJ, FtsW, and RodA in PG biogenesis. Aim 2 of this proposal is to determine how MurJ functions. Structural information will be obtained by determining the membrane topology of MurJ. Functional partners of MurJ will be identified biochemically and genetically. Mutation and suppression analyses will be conducted to identify domains of MurJ required for stability, intra- and inter- molecular interactions, and activity. Together, the data obtained will uncover the essential function of MurJ in PG biogenesis. Many of the most effective antibiotics target PG biogenesis. MurJ and other proteins required for PG biogenesis are potential targets for antibiotics since PG is essential in most bacteria and is absent in humans. The proposed work will aid in the development of MurJ inhibitors, which may prove to be much-needed novel antibiotics. In addition, understanding MurJ function will advance knowledge of the MOP superfamily of exporters, which includes members that are involved in bacterial pathogenesis and certain types of human congenital disorders of glycosylation.

描述（由申请人提供）： 大多数细菌将肽聚糖（PG）聚合成围绕细胞质膜的网状囊中，并保护其免受渗透裂解。 PG Sacculus还提供细胞形状，并用作固定毒力因子的支架。 PG囊的生物发生对于生存力至关重要，我们的长期目标是在分子水平上理解它。该提案的重点是PG中间体在整个细胞质膜上的运输，这是PG生物发生的一步知识。 细菌通过将二糖五肽聚合到聚糖链中，在外质质空间中建立PG囊，随后被交联。二糖五肽是在细胞质中作为脂质中间体制成的，称为脂质II。因此，必须通过未知机制将脂质II通过转运蛋白（Flippase）翻转膜。膜蛋白murj已被认为是大肠杆菌中的脂质II Flippase，因为它对于PG合成是必不可少的，并且属于Exporters的MoP（多剂/寡糖基脂糖/多糖）超级家族。这个家庭在多种生物中保守，其中包括脂质连接的flippase 类似于脂质II的寡糖。 FTSW和RODA还被认为是大肠杆菌中的脂质II flippase，但没有体内证据支持这一假设。 提案的目标1是确定MURJ，FTSW和RODA是否参与体内脂质II易位。将开发基于差异标记和质谱法的两个分析测定，以评估这些因素的耗竭如何影响脂质II的水平和膜拓扑。获得的数据对于理解MURJ，FTSW和RODA的作用至关重要 在PG生物发生中。 该建议的目标2是确定MURJ的功能。结构信息将通过确定MURJ的膜拓扑来获得。 MURJ的功能伙伴将在生化和遗传上识别。将进行突变和抑制分析，以确定稳定性，内和分子间相互作用和活性所需的MURJ域。共同获得的数据将发现MURJ在PG生物发生中的基本功能。 许多最有效的抗生素靶向PG生物发生。 PG生物发生所需的MURJ和其他蛋白质是抗生素的潜在靶标，因为PG在大多数细菌中都是必不可少的，并且在人类中不存在。 拟议的工作将有助于开发MURJ抑制剂，这可能被证明是急需的新型抗生素。此外，了解MURJ功能将提高出口商的MOP超家族知识，其中包括涉及细菌发病机理和某些类型的人类先天性糖基化的成员。