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）聚合成网状球囊，包围细胞质膜并保护其免受渗透裂解。 PG 球囊还提供细胞形状并充当锚定毒力因子的支架。 PG 球囊的生物发生对于生存能力至关重要，我们的长期目标是在分子水平上了解它。该提案的重点是 PG 中间体跨细胞质膜的运输，这是 PG 生物发生中一个鲜为人知的步骤。 细菌通过将二糖五肽聚合成随后交联的聚糖链，在胞质外空间构建其 PG 球囊。二糖五肽在细胞质中作为脂质中间体产生，称为脂质 II。因此，脂质 II 必须通过转运蛋白（翻转酶）通过未知机制翻转穿过膜。膜蛋白 MurJ 被认为是大肠杆菌中的脂质 II 翻转酶，因为它对于 PG 合成至关重要，并且属于 MOP（多药/寡糖基脂质/多糖）输出蛋白超家族。该家族在多种生物体中是保守的，它包括脂质连接的翻转酶 与脂质II相似的寡糖。 FtsW 和 RodA 也被认为是大肠杆菌中的脂质 II 翻转酶，但没有体内证据支持这一假设。 该提案的目标 1 是确定 MurJ、FtsW 和 RodA 是否参与体内脂质 II 易位。将开发两种基于差异标记和质谱分析的分析方法，以评估这些因子的消耗如何影响脂质 II 的水平和膜拓扑结构。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。 该提案的目标 2 是确定 MurJ 如何运作。通过确定 MurJ 的膜拓扑结构可以获得结构信息。 MurJ 的功能伙伴将通过生化和基因鉴定。将进行突变和抑制分析，以确定稳定性、分子内和分子间相互作用以及活性所需的 MurJ 结构域。总之，获得的数据将揭示 MurJ 在 PG 生物发生中的基本功能。 许多最有效的抗生素以 PG 生物发生为目标。 MurJ 和 PG 生物发生所需的其他蛋白质是抗生素的潜在靶标，因为 PG 在大多数细菌中是必需的，而在人类中则不存在。 拟议的工作将有助于 MurJ 抑制剂的开发，这可能被证明是急需的新型抗生素。此外，了解 MurJ 功能将增进对输出蛋白 MOP 超家族的了解，该家族包括参与细菌发病机制和某些类型的人类先天性糖基化疾病的成员。