scholarly journals VirB1* Promotes T-Pilus Formation in the vir-Type IV Secretion System of Agrobacterium tumefaciens

2007 ◽  
Vol 189 (18) ◽  
pp. 6551-6563 ◽  
Author(s):  
John Zupan ◽  
Cheryl A. Hackworth ◽  
Julieta Aguilar ◽  
Doyle Ward ◽  
Patricia Zambryski
Keyword(s):  
Protein Interactions ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Protein Protein Interactions ◽  
Additional Function ◽  
Type Iv ◽  
Lytic Transglycosylase ◽  
Peptidoglycan Cell Wall ◽  
Two Hybrid

ABSTRACT The vir-type IV secretion system of Agrobacterium is assembled from 12 proteins encoded by the virB operon and virD4. VirB1 is one of the least-studied proteins encoded by the virB operon. Its N terminus is a lytic transglycosylase. The C-terminal third of the protein, VirB1*, is cleaved from VirB1 and secreted to the outside of the bacterial cell, suggesting an additional function. We show that both nopaline and octopine strains produce abundant amounts of VirB1* and perform detailed studies on nopaline VirB1*. Both domains are required for wild-type virulence. We show here that the nopaline type VirB1* is essential for the formation of the T pilus, a subassembly of the vir-T4SS composed of processed and cyclized VirB2 (major subunit) and VirB5 (minor subunit). A nopaline virB1 deletion strain does not produce T pili. Complementation with full-length VirB1 or C-terminal VirB1*, but not the N-terminal lytic transglycosylase domain, restores T pili containing VirB2 and VirB5. T-pilus preparations also contain extracellular VirB1*. Protein-protein interactions between VirB1* and VirB2 and VirB5 were detected in the yeast two-hybrid assay. We propose that VirB1 is a bifunctional protein required for virT4SS assembly. The N-terminal lytic transglycosylase domain provides localized lysis of the peptidoglycan cell wall to allow insertion of the T4SS. The C-terminal VirB1* promotes T-pilus assembly through protein-protein interactions with T-pilus subunits.

scholarly journals Protein-Protein Interactions among Helicobacter pylori Cag Proteins

2006 ◽  
Vol 188 (13) ◽  
pp. 4787-4800 ◽  
Author(s):  
Valerie J. Busler ◽  
Victor J. Torres ◽  
Mark S. McClain ◽  
Oscar Tirado ◽  
David B. Friedman ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Protein Interactions ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Protein Protein Interactions ◽  
Chromosomal Dna ◽  
2D Dige ◽  
Type Iv ◽  
H Pylori

ABSTRACT Many Helicobacter pylori isolates contain a 40-kb region of chromosomal DNA known as the cag pathogenicity island (PAI). The risk for development of gastric cancer or peptic ulcer disease is higher among humans infected with cag PAI-positive H. pylori strains than among those infected with cag PAI-negative strains. The cag PAI encodes a type IV secretion system that translocates CagA into gastric epithelial cells. To identify Cag proteins that are expressed by H. pylori during growth in vitro, we compared the proteomes of a wild-type H. pylori strain and an isogenic cag PAI deletion mutant using two-dimensional difference gel electrophoresis (2D-DIGE) in multiple pH ranges. Seven Cag proteins were identified by this approach. We then used a yeast two-hybrid system to detect potential protein-protein interactions among 14 Cag proteins. One heterotypic interaction (CagY/7 with CagX/8) and two homotypic interactions (involving H. pylori VirB11/ATPase and Cag5) were similar to interactions previously reported to occur among homologous components of the Agrobacterium tumefaciens type IV secretion system. Other interactions involved Cag proteins that do not have known homologues in other bacterial species. Biochemical analysis confirmed selected interactions involving five of the proteins that were identified by 2D-DIGE. Protein-protein interactions among Cag proteins are likely to have an important role in the assembly of the H. pylori type IV secretion apparatus.

scholarly journals Molecular dissection of protein-protein interactions between integrin α5β1 and the Helicobacter pylori Cag type IV secretion system

FEBS Journal ◽  
2017 ◽  
Vol 284 (23) ◽  
pp. 4143-4157 ◽  
Author(s):  
Thomas Koelblen ◽  
Célia Bergé ◽  
Mickaël V. Cherrier ◽  
Karl Brillet ◽  
Luisa Jimenez-Soto ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Protein Interactions ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Molecular Dissection ◽  
Protein Protein Interactions ◽  
Integrin Α5β1 ◽  
Type Iv

scholarly journals AtlA Functions as a Peptidoglycan Lytic Transglycosylase in the Neisseria gonorrhoeae Type IV Secretion System

2007 ◽  
Vol 189 (15) ◽  
pp. 5421-5428 ◽  
Author(s):  
Petra L. Kohler ◽  
Holly L. Hamilton ◽  
Karen Cloud-Hansen ◽  
Joseph P. Dillard
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Lytic Transglycosylases ◽  
Type Iv ◽  
Lytic Transglycosylase ◽  
Enzymatic Function ◽  
Type Iv Secretion Systems

ABSTRACT Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis.

scholarly journals Protein Subassemblies of the Helicobacter pylori Cag Type IV Secretion System Revealed by Localization and Interaction Studies

2008 ◽  
Vol 190 (6) ◽  
pp. 2161-2171 ◽  
Author(s):  
Stefan Kutter ◽  
Renate Buhrdorf ◽  
Jürgen Haas ◽  
Wulf Schneider-Brachert ◽  
Rainer Haas ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Protein Interactions ◽  
Protein Transport ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Transport Systems ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Type Iv ◽  
Small Proteins

ABSTRACT Type IV secretion systems are possibly the most versatile protein transport systems in gram-negative bacteria, with substrates ranging from small proteins to large nucleoprotein complexes. In many cases, such as the cag pathogenicity island of Helicobacter pylori, genes encoding components of a type IV secretion system have been identified due to their sequence similarities to prototypical systems such as the VirB system of Agrobacterium tumefaciens. The Cag type IV secretion system contains at least 14 essential apparatus components and several substrate translocation and auxiliary factors, but the functions of most components cannot be inferred from their sequences due to the lack of similarities. In this study, we have performed a comprehensive sequence analysis of all essential or auxiliary Cag components, and we have used antisera raised against a subset of components to determine their subcellular localization. The results suggest that the Cag system contains functional analogues to all VirB components except VirB5. Moreover, we have characterized mutual stabilization effects and performed a comprehensive yeast two-hybrid screening for potential protein-protein interactions. Immunoprecipitation studies resulted in identification of a secretion apparatus subassembly at the outer membrane. Combining these data, we provide a first low-resolution model of the Cag type IV secretion apparatus.

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 Interaction between Protein Subunits of the Type IV Secretion System of Bartonella henselae

2004 ◽  
Vol 186 (14) ◽  
pp. 4796-4801 ◽  
Author(s):  
Alireza Shamaei-Tousi ◽  
Rachel Cahill ◽  
Gad Frankel
Keyword(s):  
Bartonella Henselae ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Core Complex ◽  
Protein Subunits ◽  
Type Iv ◽  
Yeast Two Hybrid System ◽  
Periplasmic Proteins ◽  
Two Hybrid

ABSTRACT In this study we used the yeast two-hybrid system to identify interactions between protein subunits of the virB type IV secretion system of Bartonella henselae. We report interactions between inner membrane and periplasmic proteins, the pilus polypeptide, and the core complex and a novel interaction between VirB3 and VirB5.

scholarly journals Peptidoglycan degradation by specialized lytic transglycosylases associated with type III and type IV secretion systems

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3455-3467 ◽  
Author(s):  
Doris Zahrl ◽  
Maria Wagner ◽  
Karin Bischof ◽  
Michaela Bayer ◽  
Barbara Zavecz ◽  
...  
Keyword(s):  
Secretion System ◽  
Type Iv Secretion ◽  
Conjugative Plasmid ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Lytic Transglycosylases ◽  
Type Iii ◽  
Type Iv ◽  
Lytic Transglycosylase ◽  
Type Iv Secretion Systems

Specialized lytic transglycosylases are muramidases capable of locally degrading the peptidoglycan meshwork of Gram-negative bacteria. Specialized lytic transglycosylase genes are present in clusters encoding diverse macromolecular transport systems. This paper reports the analysis of selected members of the specialized lytic transglycosylase family from type III and type IV secretion systems. These proteins were analysed in vivo by assaying their ability to complement the DNA transfer defect of the conjugative F-like plasmid R1-16 lacking a functional P19 protein, the specialized lytic transglycosylase of this type IV secretion system. Heterologous complementation was accomplished using IpgF from the plasmid-encoded type III secretion system of Shigella sonnei and TrbN from the type IV secretion system of the conjugative plasmid RP4. In contrast, neither VirB1 proteins (Agrobacterium tumefaciens, Brucella suis) nor IagB (Salmonella enterica) could functionally replace P19. In vitro, IpgF, IagB, both VirB1 proteins, HP0523 (Helicobacter pylori) and P19 displayed peptidoglycanase activity in zymogram analyses. Using an established test system and a newly developed assay it was shown that IpgF degraded peptidoglycan in solution. IpgF was active only after removal of the chaperonin GroEL, which co-purified with IpgF and inhibited its enzymic activity. A mutant IpgF protein in which the predicted catalytic amino acid, Glu42, was replaced by Gln, was completely inactive. IpgF-catalysed peptidoglycan degradation was optimal at pH 6 and was inhibited by the lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A.

scholarly journals The putative lytic transglycosylase VirB1 from Brucella suis interacts with the type IV secretion system core components VirB8, VirB9 and VirB11

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3469-3482 ◽  
Author(s):  
Christoph Höppner ◽  
Anna Carle ◽  
Durga Sivanesan ◽  
Sabine Hoeppner ◽  
Christian Baron
Keyword(s):  
Protein Interactions ◽  
Gel Filtration ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Expression Vectors ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Type Iv ◽  
Brucella Suis ◽  
Core Components

VirB1-like proteins are believed to act as lytic transglycosylases, which facilitate the assembly of type IV secretion systems via localized lysis of the peptidoglycan. This paper presents the biochemical analysis of interactions of purified Brucella suis VirB1 with core components of the type IV secretion system. Genes encoding VirB1, VirB8, VirB9, VirB10 and VirB11 were cloned into expression vectors; the affinity-tagged proteins were purified from Escherichia coli, and analyses by gel filtration chromatography showed that they form monomers or homo-multimers. Analysis of protein–protein interactions by affinity precipitation revealed that VirB1 bound to VirB9 and VirB11. The results of bicistron expression experiments followed by gel filtration further supported the VirB1–VirB9 interaction. Peptide array mapping identified regions of VirB1 that interact with VirB8, VirB9 and VirB11 and underscored the importance of the C-terminus, especially for the VirB1–VirB9 interaction. The binding sites were localized on a structure model of VirB1, suggesting that different portions of VirB1 may interact with other VirB proteins during assembly of the type IV secretion machinery.

High prevalence of functional type IV secretion system for CagA protein in Japanese clinical Helicobacter pylori

2001 ◽  
Vol 120 (5) ◽  
pp. A652-A653
Author(s):  
Y HIRATA ◽  
S MAEDA ◽  
Y MITUNO ◽  
M AKANUMA ◽  
T KAWABE ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Functional Type ◽  
Caga Protein ◽  
Type Iv ◽  
High Prevalence
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