Abstract
The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.
Highlights
Pathogenic bacteria commonly employ the Type II Secretion System (T2SS) for the release of protein toxins
Vibrio vulnificus and Aeromonas hydrophila are two bacterial species that are emerging as significant human pathogens
The major virulence mechanism utilized by these species is the Type II secretion system (T2SS) that functions in exporting toxic proteins produced by the bacterium into the infected individual
Summary
Pathogenic bacteria commonly employ the Type II Secretion System (T2SS) for the release of protein toxins. The opportunistic pathogen Aeromonas hydrophila, which is a causative agent of skin and soft tissue infections as well as gastrointestinal tract infections, utilizes the T2SS for the secretion of a wide variety of degradative and toxic proteins, including proteases, lipases, amylases, enterotoxins and the wellstudied pore-forming toxin aerolysin [3]. For these reasons, the assembly, structure and function of the T2SS is an important focus of research on the pathogenesis of these and many other bacterial infections. The secretin and the assembly platform are thought to assemble independently and transiently, and there is only a single, recent example where an intact T2SS has been purified and characterized in a stable form [12]
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