Scaffolding protein GspB/OutB facilitates assembly of the Dickeya dadantii type 2 secretion system by anchoring the outer membrane secretin pore to the inner membrane and to the peptidoglycan cell wall.

The phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall degrading enzymes and other virulence factors via the type 2 secretion system (T2SS). T2SSs are widespread among important plant, animal and human bacterial pathogens. This multi-protein complex spans the double membrane cell envelope and secretes fully folded proteins through a large outer membrane pore formed by 15 subunits of the secretin GspD. Secretins are also found in the type 3 secretion system and the type 4 pili. Usually, specialized lipoproteins termed as pilotins assist the targeting and assembly of secretins into the outer membrane. Here, we show that in Dickeya, the pilotin acts in concert with the scaffolding protein GspB. Deletion of gspB profoundly impacts secretin assembly, pectinase secretion, and virulence. Structural studies reveal that GspB possesses a conserved periplasmic Homology Region domain that interacts directly with the N-terminal secretin domain. Site-specific photo cross-linking unravels molecular details of the GspB-GspD complex in vivo. We show that GspB facilitates outer membrane targeting and assembly of the secretin pores and anchors them to the inner membrane while the C-terminal extension of GspB scaffolds the secretin channel in the peptidoglycan cell wall. Phylogenetic analysis shows that in other bacteria, GspB homologs vary in length and domain composition and act in concert with either a cognate ATPase GspA or a pilotin GspS.

Identification of new Dickeya dadantii virulence factors secreted by the type 2 secretion system

Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system named Out is necessary for bacterial virulence. Its study in D. dadantii showed that it secretes a wide range of pectinolytic enzymes. However, the full repertoire of exoproteins it can secrete has not been identified. No secretion signal present on the protein allows the identification of substrates of a type 2 secretion system. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced encoding a protein with a signal sequence. We identified four new Out-secreted proteins: the expansin YoaJ, VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that SvfA and SvfB are required for full virulence of D. dadantii and showed that Svf proteins are present with a variable number of copies, up to three in D. fanghzongdai, in other Pectobacteriaceae. This work opens the way to the study of the role in virulence of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae.

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

ABSTRACTThe β-barrel assembly machinery (BAM) complex is the core machinery for the assembly of β-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 enteropathogenicEscherichia coliO127:H6 strain E2348/69. Long β-strands assemble into a barrel extending 17 Å through and beyond the outer membrane, adding insight to how these extensive β-strands are assembled into theE. coliouter membrane. The substrate docking chamber of this secretin is shown to be sufficient to accommodate the substrate mucinase SteC.IMPORTANCEIn 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. TheVibrio-type T2SS mediates cholera toxin secretion inVibrio cholerae, and inEscherichia coliO127: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.