Biogenesis of Peptidoglycan in Escherichia coli

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，包裹在细胞膜周围，保护细胞膜免受渗透溶解。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是必不可少的，我们的长期目标是在分子水平上了解它。这项建议侧重于PG中间体通过细胞膜的运输，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后来被交联的糖链，在胞质外空间建立它们的PG球囊。二糖五肽是以脂类II的形式在细胞质中产生的，因此，脂类II必须通过一种未知的机制被转运蛋白(一种翻转酶)翻转到细胞膜上。膜蛋白Murj被认为是大肠杆菌中的脂类II翻转酶，因为它是PG合成所必需的，并且属于MOP(多药/寡糖-脂/多糖)超家族出口蛋白。这个家族在不同的生物体中都是保守的，它包括脂类相关的翻转酶。 低聚糖类似于脂类II。FtsW和RodA也被认为是大肠杆菌中的脂类II翻转酶，但没有体内证据支持这一假说。这项建议的目的1是确定Murj、FtsW和RodA是否参与体内脂质II的转位。基于差示标记和质谱学的两种分析方法将被用来评估这些因子的缺乏如何影响脂质II的水平和膜拓扑结构。所获得的数据将对理解Murj、FtsW和RodA的作用至关重要 在PG的生物发生中。这项提案的目标2是确定MUJ是如何运作的。结构信息将通过确定Murj的膜拓扑来获得。Murj的功能性合作伙伴将通过生物化学和遗传学进行确定。将进行突变和抑制分析，以确定稳定所需的MurJ结构域、分子内和分子间相互作用以及活性。总之，所获得的数据将揭示Murj在PG生物发生中的重要功能。许多最有效的抗生素都是针对PG生物发生的。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不可少的，而在人类中则不存在。这项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功能将促进对MOP出口蛋白超家族的了解，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障碍的成员。 公共卫生相关性： 抗生素耐药性的增加是一个世界性的问题，可以通过开发新的抗生素来解决。这个项目试图了解一个未知的抗生素靶标--基本细菌蛋白Murj的功能。我们的研究将(1)促进新型MURJ抑制剂的未来开发和表征，以及(2)促进我们对与MURJ相关的蛋白质家族的了解，该家族包括参与细菌发病机制的因素和统称为CDG的某些形式的人类先天性代谢性疾病。

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. PUBLIC HEALTH RELEVANCE: The increase in antibiotic resistance is a world-wide problem that can be combated by developing new antibiotics. This project seeks to understand the function of an unexplored antibiotic target, the essential bacterial protein MurJ. Our research will (1) facilitate the futur development and characterization of novel MurJ inhibitors, and (2) advance our knowledge of a family of proteins related to MurJ that includes factors involved in bacterial pathogenesis and certain forms of the human congenital metabolic diseases collectively known as CDG.