scholarly journals The Type IV Secretion System Component VirB5 Binds to the trans-Zeatin Biosynthetic Enzyme Tzs and Enables Its Translocation to the Cell Surface of Agrobacterium tumefaciens

2007 ◽  
Vol 190 (5) ◽  
pp. 1595-1604 ◽  
Author(s):  
Khaled Ahmed Aly ◽  
Lilian Krall ◽  
Friedrich Lottspeich ◽  
Christian Baron
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Cell Surface ◽  
Minor Component ◽  
Secretion System ◽  
Type Iv Secretion ◽  
System Component ◽  
Type Iv Secretion System ◽  
Biosynthetic Enzyme ◽  
Interacting Protein ◽  
Type Iv

ABSTRACT VirB5 is a minor component of the extracellular T pilus determined by the Agrobacterium tumefaciens type IV secretion system. To identify proteins that interact with VirB5 during the pilus assembly process, we purified VirB5 as a recombinant fusion protein and, by using a gel overlay assay, we detected a 26-kDa interacting protein in Agrobacterium cell lysates. The VirB5-binding protein was purified from A. tumefaciens and identified as the cytokinin biosynthetic enzyme Tzs. The VirB5-Tzs interaction was confirmed using pulldown assays with purified proteins and the yeast two-hybrid system. An analysis of the subcellular localization in A. tumefaciens showed that Tzs was present in the soluble as well as the membrane fraction. Tzs was extracted from the membranes with the mild detergent dodecyl-β-d-maltoside in complexes of different molecular masses, and this association was strongly reduced in the absence of VirB5. Using immunoelectron microscopy, we also detected Tzs on the Agrobacterium cell surface. A functional type IV secretion system was required for efficient translocation to the surface, but Tzs was not secreted into the cell supernatant. The fact that Tzs localizes on the cell surface suggests that it may contribute to the interaction of Agrobacterium with plants.

scholarly journals Membrane and Core Periplasmic Agrobacterium tumefaciens Virulence Type IV Secretion System Components Localize to Multiple Sites around the Bacterial Perimeter during Lateral Attachment to Plant Cells

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Julieta Aguilar ◽  
Todd A. Cameron ◽  
John Zupan ◽  
Patricia Zambryski
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Cells ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Content Type ◽  
Protein Substrates ◽  
Type Iv

ABSTRACTType IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain ofAgrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in theA. tumefaciensoctopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression followingvirinduction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles.vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiplevir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates.IMPORTANCETransfer of DNA and/or proteins to host cells through multiprotein type IV secretion system (T4SS) complexes that span the bacterial cell envelope is critical to bacterial pathogenesis. Early reports suggested that T4SS components localized at the cell poles. Now, higher-resolution deconvolution fluorescence microscopy reveals that all structural components of theAgrobacterium tumefaciens vir-T4SS, as well as its transported protein substrates, localize to multiple foci around the cell perimeter. These results lead to a new model ofA. tumefaciensattachment to a plant cell, whereA. tumefacienstakes advantage of the multiplevir-T4SS along its length to make intimate lateral contact with plant cells and thereby effectively transfer DNA and/or proteins through thevir-T4SS. The T4SS ofA. tumefaciensis among the best-studied T4SS, and the majority of its components are highly conserved in different pathogenic bacterial species. Thus, the results presented can be applied to a broad range of pathogens that utilize T4SS.

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 Association and Evidence for Linked Recognition of Type IV Secretion System Proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale

2011 ◽  
Vol 80 (1) ◽  
pp. 215-227 ◽  
Author(s):  
Kaitlyn Morse ◽  
Junzo Norimine ◽  
Guy H. Palmer ◽  
Eric L. Sutten ◽  
Timothy V. Baszler ◽  
...  
Keyword(s):  
T Cell ◽  
Outer Membrane ◽  
Anaplasma Marginale ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Interacting Protein ◽  
Content Type ◽  
Cell Responses ◽  
Type Iv

ABSTRACTLike several other bacterial pathogens,Anaplasma marginalehas an outer membrane that induces complete protection from infection and disease. However, the proteins that confer protective immunity and whether protection requires interacting proteins and/or linked T-cell and immunoglobulin G epitopes are not known. Our goal is to target the conserved type IV secretion system (T4SS) to identify conserved, immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which a B cell is optimally activated by a helper T cell that responds to the same, or physically associated, antigen.A. marginaleT4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11, and VirD4 were screened for their ability to induce IgG and to stimulate CD4+T cells from outer membrane-vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T-cell responses in the majority of cattle, although three animals with major histocompatibility complex class II DRB3 restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine againstA. marginale.

scholarly journals AnIn VivoHigh-Throughput Screening Approach Targeting the Type IV Secretion System Component VirB8 Identified Inhibitors ofBrucella abortus2308 Proliferation

2010 ◽  
Vol 79 (3) ◽  
pp. 1033-1043 ◽  
Author(s):  
Athanasios Paschos ◽  
Andreas den Hartigh ◽  
Mark A. Smith ◽  
Vidya L. Atluri ◽  
Durga Sivanesan ◽  
...  
Keyword(s):  
Secretion System ◽  
Bacterial Virulence ◽  
Type Iv Secretion ◽  
Host Cells ◽  
System Component ◽  
Type Iv Secretion System ◽  
Promising Alternative ◽  
Type Iv ◽  
Two Hybrid

ABSTRACTAs bacterial pathogens develop resistance against most currently used antibiotics, novel alternatives for treatment of microbial infectious diseases are urgently needed. Targeting bacterial virulence functions in order to disarm pathogens represents a promising alternative to classical antibiotic therapy. Type IV secretion systems, which are multiprotein complexes in the cell envelope that translocate effectors into host cells, are critical bacterial virulence factors in many pathogens and excellent targets for such “antivirulence” drugs. The VirB8 protein from the mammalian pathogenBrucellawas chosen as a specific target, since it is an essential type IV secretion system component, it participates in multiple protein-protein interactions, and it is essential for the assembly of this translocation machinery. The bacterial two-hybrid system was adapted to assay VirB8 interactions, and a high-throughput screen identified specific small-molecule inhibitors. VirB8 interaction inhibitors also reduced the levels of VirB8 and of other VirB proteins, and many of them inhibitedvirBgene transcription inBrucella abortus2308, suggesting that targeting of the secretion system has complex regulatory effectsin vivo. One compound strongly inhibited the intracellular proliferation ofB. abortus2308 in a J774 macrophage infection model. The results presented here show thatin vivoscreens with the bacterial two-hybrid assay are suited to the identification of inhibitors ofBrucellatype IV secretion system function.

scholarly journals The Brucella suis Type IV Secretion System Assembles in the Cell Envelope of the Heterologous Host Agrobacterium tumefaciens and Increases IncQ Plasmid pLS1 Recipient Competence

2006 ◽  
Vol 74 (1) ◽  
pp. 108-117 ◽  
Author(s):  
Anna Carle ◽  
Christoph Höppner ◽  
Khaled Ahmed Aly ◽  
Qing Yuan ◽  
Amke den Dulk-Ras ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Mammalian Cells ◽  
Cell Envelope ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Heterologous Host ◽  
Type Iv ◽  
Brucella Suis

ABSTRACT Pathogenic Brucella species replicate within mammalian cells, and their type IV secretion system is essential for intracellular survival and replication. The options for biochemical studies on the Brucella secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. To overcome these problems, we heterologously expressed the Brucella suis virB operon in the closely related α2-proteobacterium Agrobacterium tumefaciens and showed that the VirB proteins assembled into a complex. Eight of the twelve VirB proteins were detected in the membranes of the heterologous host with specific antisera. Cross-linking indicated protein-protein interactions similar to those in other type IV secretion systems, and the results of immunofluorescence analysis supported the formation of VirB protein complexes in the cell envelope. Production of a subset of the B. suis VirB proteins (VirB3-VirB12) in A. tumefaciens strongly increased its ability to receive IncQ plasmid pLS1 in conjugation experiments, and production of VirB1 further enhanced the conjugation efficiency. Plasmid recipient competence correlated with periplasmic leakage and the detergent sensitivity of A. tumefaciens, suggesting a weakening of the cell envelope. Heterologous expression thus permits biochemical characterization of B. suis type IV secretion system assembly.

scholarly journals Agrobacterium tumefaciens VirB6 Domains Direct the Ordered Export of a DNA Substrate Through a Type IV Secretion System

2004 ◽  
Vol 341 (4) ◽  
pp. 961-977 ◽  
Author(s):  
Simon J. Jakubowski ◽  
Vidhya Krishnamoorthy ◽  
Eric Cascales ◽  
Peter J. Christie
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Iv ◽  
Dna Substrate
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