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.
Author Summary The type 2 secretion system (T2SS) is a sophisticated, multi-component molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. In Vibrio cholerae, for example, the T2SS mediates the secretion of cholera toxin. We find that there are three distinct forms of T2SS, based on the sequence characteristics of the secretin. A targeting paradigm, developed for the Klebsiella-type secretin PulD, could not previously be applied to the T2SS in Vibrio cholerae and many other bacterial species whose genomes encode no homolog of the crucial targeting factor PulS (also called OutS, EtpO or GspS). Using bioinformatics we find, remarkably, that these bacteria have instead evolved a structurally distinct protein to serve in place of PulS. We crystallized and solved the structure of this distinct factor, AspS, measured its activity in novel assays for T2SS assembly, and show that the protein is essential for the function of the Vibrio-type T2SS. A structural homolog of AspS found here in Pseudomonas suggests widespread use of the pilotin-secretin targeting paradigm for T2SS assembly.