Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway.
Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway.
复制标题
多肽通过三聚体自转运蛋白途径顺序易位穿过细菌外膜。
DOI：
10.1128/mbio.01973-19
复制
发表时间：
2019
期刊：
影响因子：
6.4
通讯作者：
Bernstein,HarrisD
中科院分区：
文献类型：
--
作者：
Sikdar,Rakesh;Bernstein,HarrisD
三聚体自转运粘附素（TAAs）是由三个相同亚基组成的细菌外膜（OM）蛋白家族。每个亚基含有N-末端胞外（“过客”）结构域和短C-末端区段，其将四条β链贡献给单个12链β桶。乘客域跨OM易位的机制和易位反应的能量学知之甚少。为了解决这些问题，我们研究了由大肠杆菌CFT 073产生的TAA UpaG乘客结构域的修饰版本的分泌。使用SpyTag-SpyCatcher系统来探测乘客结构域定位，我们发现与UpaG乘客结构域融合的固有无序多肽和人工二硫键结合的多肽都有效地分泌，但相对缓慢。令人惊讶的是，我们还发现，在某些情况下，与单个三聚体相关的三个非天然乘客结构域片段顺序分泌。光交联实验表明，不完全组装的UpaG衍生物仍然结合到桶装配机械（BAM）复合物，直到所有三个乘客域完全分泌。总之，我们的结果强烈表明，多肽通过TAA途径的分泌与β桶结构域的组装协调，并且细胞外空间中乘客结构域的折叠使分泌速率最大化。此外，我们的工作提供了一个前所未有的顺序模式的蛋白质易位的证据，至少在特定的实验条件下。重要三聚体自转运粘附素（TAAs）是专门的细菌外膜蛋白组成的三个相同的亚基。TAA含有大的胞外结构域，其三聚化并促进毒力，但它们分泌的机制知之甚少。我们发现，天然TAA的胞外结构域分泌迅速，但无序和人工折叠的多肽融合到天然乘客结构域分泌缓慢，顺序的方式。我们的研究结果强烈表明，天然胞外结构域的有效分泌是由它们的三聚体出口后，但替代能源可以利用分泌非天然多肽。此外，我们获得的证据表明，TAA细胞外结构域的连接跨膜结构域组装完成之前分泌。
Trimeric autotransporter adhesins (TAAs) are a family of bacterial outer membrane (OM) proteins that are comprised of three identical subunits. Each subunit contains an N-terminal extracellular (“passenger”) domain and a short C-terminal segment that contributes four β strands to a single 12-stranded β barrel. The mechanism by which the passenger domains are translocated across the OM and the energetics of the translocation reaction are poorly understood. To address these issues, we examined the secretion of modified versions of the passenger domain of UpaG, a TAA produced by Escherichia coli CFT073. Using the SpyTag-SpyCatcher system to probe passenger domain localization, we found that both intrinsically disordered polypeptides fused to the UpaG passenger domain and artificially disulfide-bonded polypeptides were secreted effectively but relatively slowly. Surprisingly, we also found that in some cases, the three nonnative passenger domain segments associated with a single trimer were secreted sequentially. Photo-cross-linking experiments indicated that incompletely assembled UpaG derivatives remained bound to thebarrelassemblymachinery (Bam) complex until all three passenger domains were fully secreted. Taken together, our results strongly suggest that the secretion of polypeptides through the TAA pathway is coordinated with the assembly of the β barrel domain and that the folding of passenger domains in the extracellular space maximizes the rate of secretion. Furthermore, our work provides evidence for an unprecedented sequential mode of protein translocation, at least under specific experimental conditions.IMPORTANCETrimeric autotransporter adhesins (TAAs) are specialized bacterial outer membrane proteins consisting of three identical subunits. TAAs contain large extracellular domains that trimerize and promote virulence, but the mechanism by which they are secreted is poorly understood. We found that the extracellular domains of a native TAA were secreted rapidly but that disordered and artificially folded polypeptides fused to native passenger domains were secreted in a slow, sequential fashion. Our results strongly suggest that the efficient secretion of native extracellular domains is driven by their trimerization following export but that alternative energy sources can be harnessed to secrete nonnative polypeptides. Furthermore, we obtained evidence that TAA extracellular domains are secreted before the assembly of the linked membrane spanning domain is completed.