scholarly journals 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.

2021 ◽  
Author(s):  
Shiheng Zhang ◽  
Shuang Gu ◽  
Piers Rycroft ◽  
Florence Ruaudel ◽  
Frederic Delolme ◽  
...  
Keyword(s):  
Cell Wall ◽  
Outer Membrane ◽  
Secretion System ◽  
Scaffolding Protein ◽  
Inner Membrane ◽  
Dickeya Dadantii ◽  
Membrane Pore ◽  
Peptidoglycan Cell Wall ◽  
Type 2 Secretion

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.

scholarly journals Multiple approaches towards understanding the type II secretion system

2014 ◽  
Vol 70 (a1) ◽  
pp. C577-C577
Author(s):  
Connie Lu ◽  
Young-un Park ◽  
Konstantin Korotkov ◽  
Wei Mi ◽  
Stewart Turley ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Secretion System ◽  
Crystal Formation ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Inner Membrane ◽  
Membrane Pore ◽  
Membrane Complex ◽  
Type Ii Secretion System ◽  
Folded Proteins

Transport of folded proteins across membranes is a feat accomplished by few biomacromolecular machines. One of the machineries able to do so is the sophisticated type II secretion system (T2SS). It can translocate key virulence factors from the bacterial periplasm into the lumen of the gut of the human host. A prime example is the secretion of cholera toxin by Vibrio cholerae. The T2SS consists of ~12 different proteins, most of these present in multiple copies, organized into three subassemblies: (i) the Inner Membrane Platform; (ii) the Pseudopilus in the periplasm, which acts most likely as a piston pushing exoproteins through the outer membrane pore; (iii) the Outer Membrane Complex, allowing passage of ~100 kDa folded proteins. We have determined crystal structures from more than a dozen T2SS domains, yet, a full understanding of the architecture and mechanism of action of the T2SS remains a formidable challenge. Our approaches include the use of "assistant-multimers" to promote recalcitrant multimer formation and of nanobodies to overcome reluctant crystal formation. The Inner Membrane Platform is interacting with the secretion ATPase GspE which most likely needs to be hexameric for full activity. Full-length GspE co-crystallized with its major partner, the cytoplasmic domain of GspL, revealed a tremendous flexibility of this ATPase, and, most unexpectedly, also the organization of the same linear arrangement of cyto-GspL domains throughout three entirely different crystal forms. Two very different hexamers of GspE were elucidated by linking the GspE subunit to the subunit of Hcp1, which successfully acted as an "assistant hexamer", inducing hexamer formation by GspE. The dodecameric nature of the ~ 850 kDa GspD, the major component of the Outer Membrane Complex, evident in earlier electron microscopy studies, was observed in the dodecameric ring-like helix in crystals of its N-terminal domain. The contacts between GspD and the inner-membrane protein GspC will be discussed as well as the remarkably frequent occurrence of dimers of Inner Membrane Platform domains. How dimers are co-assembled with an ATPase hexamer with C6 symmetry and the Outer Membrane Complex dodecamer with C12 symmetry remains one of the many fascinating outstanding questions of the T2SS.

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

2017 ◽  
Vol 200 (5) ◽  
Author(s):  
Iain D. Hay ◽  
Matthew J. Belousoff ◽  
Rhys A. Dunstan ◽  
Rebecca S. Bamert ◽  
Trevor Lithgow
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Secretion System ◽  
Complex Activity ◽  
Content Type ◽  
Intestinal Mucus ◽  
Bam Complex ◽  
Type 2 Secretion

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.

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

2021 ◽  
Author(s):  
Guy Condemine ◽  
Bastien Le Redout
Keyword(s):  
Pathogenic Bacteria ◽  
Signal Sequence ◽  
Variable Number ◽  
Secretion System ◽  
Dickeya Dadantii ◽  
Secretion Signal ◽  
Plant Infection ◽  
Wide Range ◽  
Type 2 Secretion

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.

1H, 15N and 13C resonance assignments and secondary structure of PulG, the major pseudopilin from Klebsiella oxytoca type 2 secretion system

2017 ◽  
Vol 11 (2) ◽  
pp. 155-158 ◽  
Author(s):  
Aracelys López-Castilla ◽  
Bruno Vitorge ◽  
Léa Khoury ◽  
Nelly Morellet ◽  
Olivera Francetic ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Klebsiella Oxytoca ◽  
Resonance Assignments ◽  
Secretion System ◽  
Type 2 Secretion

Structural and Functional Studies of EpsC, a Crucial Component of the Type 2 Secretion System from Vibrio cholerae

2006 ◽  
Vol 363 (2) ◽  
pp. 311-321 ◽  
Author(s):  
Konstantin V. Korotkov ◽  
Brian Krumm ◽  
Michael Bagdasarian ◽  
Wim G.J. Hol
Keyword(s):  
Vibrio Cholerae ◽  
Secretion System ◽  
Functional Studies ◽  
Crucial Component ◽  
Type 2 Secretion

scholarly journals Agrobacterium tumefaciens VirB9, an Outer-Membrane-Associated Component of a Type IV Secretion System, Regulates Substrate Selection and T-Pilus Biogenesis

2005 ◽  
Vol 187 (10) ◽  
pp. 3486-3495 ◽  
Author(s):  
Simon J. Jakubowski ◽  
Eric Cascales ◽  
Vidhya Krishnamoorthy ◽  
Peter J. Christie
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Outer Membrane ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Substrate Selection ◽  
Inner Membrane ◽  
Protein Substrates ◽  
Type Iv ◽  
Pilus Biogenesis

ABSTRACT Agrobacterium tumefaciens translocates DNA and protein substrates between cells via a type IV secretion system (T4SS) whose channel subunits include the VirD4 coupling protein, VirB11 ATPase, VirB6, VirB8, VirB2, and VirB9. In this study, we used linker insertion mutagenesis to characterize the contribution of the outer-membrane-associated VirB9 to assembly and function of the VirB/D4 T4SS. Twenty-five dipeptide insertion mutations were classified as permissive for intercellular substrate transfer (Tra+), completely transfer defective (Tra−), or substrate discriminating, e.g., selectively permissive for transfer only of the oncogenic transfer DNA and the VirE2 protein substrates or of a mobilizable IncQ plasmid substrate. Mutations inhibiting transfer of DNA substrates did not affect formation of close contacts of the substrate with inner membrane channel subunits but blocked formation of contacts with the VirB2 and VirB9 channel subunits, which is indicative of a defect in assembly or function of the distal portion of the secretion channel. Several mutations in the N- and C-terminal regions disrupted VirB9 complex formation with the outer-membrane-associated lipoprotein VirB7 or the inner membrane energy sensor VirB10. Several VirB9.i2-producing Tra+ strains failed to elaborate T pilus at detectable levels (Pil−), and three such Tra+ Pil− mutant strains were rendered Tra− upon deletion of virB2, indicating that the cellular form of pilin protein is essential for substrate translocation. Our findings, together with computer-based analyses, support a model in which distinct domains of VirB9 contribute to substrate selection and translocation, establishment of channel subunit contacts, and T-pilus biogenesis.

scholarly journals The trans-envelope architecture and function of the type 2 secretion system: new insights raising new questions

2017 ◽  
Vol 105 (2) ◽  
pp. 211-226 ◽  
Author(s):  
Jenny-Lee Thomassin ◽  
Javier Santos Moreno ◽  
Ingrid Guilvout ◽  
Guy Tran Van Nhieu ◽  
Olivera Francetic
Keyword(s):  
Secretion System ◽  
And Function ◽  
Type 2 Secretion

scholarly journals Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform

2016 ◽  
Vol 101 (6) ◽  
pp. 924-941 ◽  
Author(s):  
Mangayarkarasi Nivaskumar ◽  
Javier Santos-Moreno ◽  
Christian Malosse ◽  
Nathalie Nadeau ◽  
Julia Chamot-Rooke ◽  
...  
Keyword(s):  
Secretion System ◽  
Type 2 Secretion
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