Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system

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.

Bacterial secretins: Mechanisms of assembly and membrane targeting

“…Studies of the PulS‐PulD interaction showed that the role of the pilotin is to protect the secretin from degradation in the periplasm; in its absence, secretin assembly can occur within the IM, leading to the initiation of the phage shock response . More recent secretin assembly studies have confirmed the role of pilotins in T2SS secretin stability in the outer membrane . It is of interest that T2SS secretins have been classified as Vibrio ‐type, Klebsiella ‐type or Pseudomonas ‐type based not only on sequence homologies, but also on the identity of their cognate pilotins …”

“…In the case of interaction studies performed with PulS and the S‐domain of PulD, all four helices were shown to interact with a disordered segment of the S‐domain that undergoes a disorder‐to‐order transition and folds into a helix upon binding . However, pilotins from the AspS/GspS β family display a completely different fold, consisting of a 5‐stranded β‐sheet flanked by 4 α‐helices . This fold is reminiscent of a cupped hand, where the central region of the β‐sheet, which is highly hydrophobic, could represent the palm (EpsS in Figure ).…”

“…This analysis indicated that upon binding to α12, EpsS could undergo a minor conformational modification, as if “closing” its hand on the C‐terminal helix of EpsD (Figures and ). Mutational studies performed on E. coli and V. cholerae highlighted the importance of hydrophobic regions of both the S‐domain and the pilotins for secretin stability in the outer membrane and bacterial toxicity …”

“…33 However, pilotins from the AspS/GspS β family display a completely different fold, consisting of a 5-stranded β-sheet flanked by 4 α-helices. 15,36,38 This fold is reminiscent of a cupped hand, where the central region of the β-sheet, which is highly hydrophobic, could represent the palm (EpsS in Figure 3).…”

“…Cryo‐EM structures of secretins (T2SS, T4PS) and injectisome needle complex (T3SS). The resolution of the maps for the T2SS (EpsD from V. vulnificus , 3.4 Å) and T3SS (InvG, PrgH, PrgK, PrgI from S. typhimurium ; ~3.6 Å) allowed for the positioning of the monomer (orange) including the S‐domain (red). The T3SS needle is indicated in cyan.…”