Structure and Membrane Topography of the Vibrio-Type Secretin Complex from the Type 2 Secretion System of Enteropathogenic Escherichia coli

Abstract

The ␤-barrel assembly machinery (BAM) complex is the core machinery for the assembly of ␤-barrel membrane proteins, and inhibition of BAM complex activity is lethal to bacteria. Discovery of integral membrane proteins that are key to pathogenesis and yet do not require assistance from the BAM complex raises the question of how these proteins assemble into bacterial outer membranes. Here, we address this question through a structural analysis of the type 2 secretion system (T2SS) secretin from enteropathogenic Escherichia coli O127:H6 strain E2348/69. Long ␤-strands assemble into a barrel extending 17 Å through and beyond the outer membrane, adding insight to how these extensive ␤-strands are assembled into the E. coli outer membrane. The substrate docking chamber of this secretin is shown to be sufficient to accommodate the substrate mucinase SteC.IMPORTANCE In order to cause disease, bacterial pathogens inhibit immune responses and induce pathology that will favor their replication and dissemination. In Gram-negative bacteria, these key attributes of pathogenesis depend on structures assembled into or onto the outer membrane. One of these is the T2SS. The Vibriotype T2SS mediates cholera toxin secretion in Vibrio cholerae, and in Escherichia coli O127:H6 strain E2348/69, the same machinery mediates secretion of the mucinases that enable the pathogen to penetrate intestinal mucus and thereby establish deadly infections.KEYWORDS protein secretion, bacterial pathogenesis, BAM complex, transertion, outer membrane, lipoproteins, secretin E nteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are pathogens causing diseases that range from diarrhea to hemorrhagic colitis and hemolytic-uremic syndrome in humans. In order to access the epithelial layer of the human gut, these pathogens secrete mucinases that cause a thinning of the mucus layer to accelerate pathogenesis (1-3). These large mucinases are folded into functionally active forms in the bacterial periplasm before being secreted across the outer membrane (1,(4)(5)(6)(7)(8). Secretion of these mucinases is mediated by the type 2 secretion system (T2SS), with a defining component of the T2SS being the secretin: an approximately 70-kDa protein that homo-oligomerizes to form an ϳ1-MDa secretin complex (9). The secretin complex is embedded in the outer membrane and spans the periplasm (10). Diversity in the sequence features of secretins led to the classification of two major forms of the T2SS: (i) the Klebsiella type, represented in a broad and diverse set of bacterial species and which has been functionally characterized best in Klebsiella oxytoca (11), and (ii) the Vibrio type, which is found only in a few species of Vibrio and pathovars of Escherichia coli, including EPEC and EHEC (7). Other secretins, including those from the T2SS expressed by species of Pseudomonas, Legionella, and Acinetobacter, are so diverse in sequence that their relationships have remained complex and

Multiple Structures Disclose the Secretins' Secrets

“…Based on sequence analysis, the authors previously proposed that the EPEC secretin belongs to the Vibrio-type subfamily (14). In agreement with sequence similarities, a similar structural architecture can be seen between the two Vibrio-type secretins, i.e., the EPEC structure from the work of Hay et al (6) and the Vibrio structure from the work of Yan et al (15), with loops extending from the extracellular side to form the typical cap gate (Fig. 1).…”

“…Overall, with their structure-based BLAST experiment, Hay et al (6) revealed that the previous T2SS secretin classification, i.e., Vibrio and Klebsiella types (14), is definitely structurally relevant. Moreover, in providing an additional high-resolution 3D structure of a secretin, these authors are giving us a reminder of the structural diversity existing among T2SS secretins and are lending support to the existence of subtle functional differences.…”

“…In the paper by Hay et al (6), it seems obvious that secretin structures are likely to adopt slight variations that may accommodate specific functions even with secretins from the same subfamily. The study presents the cryo-EM high-resolution 3D structure of the T2SS secretin from enteropathogenic E. coli (EPEC) O127:H6 strain E2348/69 (PDB accession number 5W68) at 3.3 Å (Fig.…”

“…They are found in the T2SS to allow the release of large folded virulence factors or in type 4 pilus assembly systems (T4PS) and T3SS for the assembly and movement of cell surface appendages. A paper by Hay et al (6) describes the three-dimensional (3D) high-resolution structure of a T2SS secretin from enteropathogenic Escherichia coli (EPEC), which together with previously published structures is illuminating our understanding of these astounding objects.…”

Filamentous phages: masters of a microbial sharing economy

“…Although no clear symmetry could be observed, nanogold labelling suggested that there are 14 subunits per secretin complex . From recent near‐atomic resolution cryo‐electron microscopy models of secretins from the type 2 secretion system and previous mutagenesis experiments on the pIV gate regions , we can extrapolate some information about the structure and function of pIV. Secretins form homo‐oligomeric complexes comprised of 15 copies of the secretin subunit.…”