Biogenesis of Peptidoglycan in Escherichia coli

描述（由申请人提供）： 大多数细菌将肽聚糖（PG）包裹成网状小囊，小囊包围细胞质膜并保护其免受渗透溶解。PG球囊还提供细胞形状，并作为毒力因子锚定的支架。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。该建议的重点是跨细胞质膜的PG中间体的运输，一个鲜为人知的PG生物合成的步骤。 细菌通过将二糖五肽聚合成随后交联的聚糖链，在胞质外空间中构建PG球囊。二糖五肽在细胞质中作为脂质中间体被称为脂质II。因此，脂质II必须通过转运蛋白（翻转酶）通过未知的机制翻转穿过膜。膜蛋白MurJ已被认为是大肠杆菌中的脂质II翻转酶，因为它对于PG降解是必需的，并且它属于MOP（多药/寡糖基-脂质/多糖）输出蛋白超家族。该家族在不同的生物体中是保守的，并且它包括脂质连接蛋白的翻转酶。 低聚糖类似于脂质II。FtsW和RodA也被认为是E.大肠杆菌，但没有体内证据支持这一假设。 本建议的目的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种分析测定，以评估这些因素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。 本提案的目标2是确定MurJ如何发挥作用。将通过确定MurJ的膜拓扑结构来获得结构信息。MurJ的功能伙伴将被确定为生物化学和遗传学。将进行突变和抑制分析以鉴定稳定性、分子内和分子间相互作用和活性所需的MurJ结构域。总之，所获得的数据将揭示MurJ在PG生物发生中的基本功能。 许多最有效的抗生素靶向PG生物合成。MurJ和PG生物合成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，而在人类中不存在。 这项工作将有助于MurJ抑制剂的开发，这可能是急需的新型抗生素。此外，了解MurJ功能将促进对MOP超家族的了解，该超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。

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.