Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS.

II型分泌系统（Type II Secretion System，T2SS）是一类分子机器，可介导完全折叠的蛋白质底物跨细菌外膜分泌。该装置的关键组分之一为分泌素（secretin）：一种整合型多聚体外膜蛋白，构成分泌孔道。我们的研究表明，可依据分泌素孔道的序列特征，区分三类不同的T2SS。对其中两类——克雷伯菌型（Klebsiella-type）与弧菌型（Vibrio-type）T2SS的详细比较分析显示，二者还可通过介导其转运与外膜组装的引导蛋白（pilotin）进一步区分。我们解析了霍乱弧菌（Vibrio cholerae）来源的新型引导蛋白AspS的晶体结构，证实其存在趋同进化现象：AspS在功能上与克雷伯菌及其他细菌中的引导蛋白等效，但结构上并无关联。AspS可结合弧菌型分泌素中的特定靶向序列，加速分泌素的组装动力学；且AspS的同源蛋白不仅存在于所有弧菌物种中，也存在于少数携带弧菌型T2SS的埃希菌（Escherichia）和志贺菌（Shigella）菌株中。