Trapped translocation intermediates establish the route for export of capsular polysaccharides across Escherichia coli outer membranes
Trapped translocation intermediates establish the route for export of capsular polysaccharides across Escherichia coli outer membranes
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DOI：
10.1073/pnas.1400341111
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发表时间：
2014-06-03
影响因子：
11.1
通讯作者：
Whitfield, Chris
中科院分区：
文献类型：
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作者：
Nickerson, Nicholas N.;Mainprize, Iain L.;Whitfield, Chris
革兰氏阴性菌的外膜（OM）旨在排除潜在的有害分子。这种特性对细菌提出了挑战，因为细菌必须分泌蛋白质和大的糖复合物才能生长、分裂和存活。参与运输这些分子的蛋白质已经被鉴定，但由于在输出途径期间难以捕获“快照”，它们的确切作用通常未得到解决。Wza是OM多糖输出蛋白大家族的原型。在大肠杆菌中，Wza是组装胶囊所必需的，胶囊是由长链多糖组成的保护性表面涂层。Wza产生跨越OM的八聚体α-螺旋通道，但大部分蛋白质作为包围广泛管腔的大周质结构存在。靶向Wza内腔内的残基，用于UV光交联非天然氨基酸对苯甲酰基-L-苯丙氨酸的位点特异性掺入。使用这种体内光交联策略，我们能够捕获Wza内腔内的多糖易位中间体，为我们的知识提供了第一个明确的证据，即新生荚膜多糖链通过Wza门户离开细胞。
The outer membrane (OM) of Gram-negative bacteria is designed to exclude potentially harmful molecules. This property presents a challenge for bacteria that must secrete proteins and large glycoconjugates to grow, divide, and persist. Proteins involved in trafficking such molecules have been identified, but their precise roles are often unresolved due to the difficulty in capturing "snapshots" during the export pathway. Wza is the prototype for the large family of OM polysaccharide export proteins. In Escherichia coli, Wza is essential for the assembly of a capsule, a protective surface coat composed of long-chain polysaccharides. Wza creates an octameric a-helical channel spanning the OM, but the bulk of the protein exists as a large periplasmic structure enclosing an extensive lumen. Residues within the lumen of Wza were targeted for site-specific incorporation of the UV photo-cross-linkable unnatural amino acid p-benzoyl-L-phenylalanine. Using this in vivo photo-cross-linking strategy, we were able to trap polysaccharide translocation intermediates within the lumen of Wza, providing the first unequivocal evidence to our knowledge that nascent capsular polysaccharide chains exit the cell through the Wza portal.