Outer Membrane Targeting of Passenger Proteins by the Vacuolating Cytotoxin Autotransporter of
            <i>Helicobacter pylori</i>

Fischer, Wolfgang; Buhrdorf, Renate; Gerland, Elke; Haas, Rainer

doi:10.1128/iai.69.11.6769-6775.2001

Cited by 62 publications

(57 citation statements)

References 37 publications

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“…The correct deletions were verified by PCR (data not shown). To test the individual P12⌬hp0015 (P224) and P12⌬hp0016 (P225) mutant strains for their natural transformation competence, they were transformed with (i) plasmid pDH29 carrying an H. pylori recA gene interrupted by an erythromycin resistance gene cassette (erm) or (ii) chromosomal DNA carrying a streptomycin resistance mutation (str) (21). Each of the deletions resulted in complete abrogation of natural transformation competence for plasmid as well as chromosomal DNA in the corresponding mutant strains (transformation frequency, Ͻ10 Ϫ9 ) (Table 3; Fig.…”

Section: Resultsmentioning

confidence: 99%

Functional and Topological Characterization of Novel Components of the comB DNA Transformation Competence System in Helicobacter pylori

Karnholz¹,

Hoefler²,

Odenbreit³

et al. 2006

J Bacteriol

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Helicobacter pylori is one of the most diverse bacterial species known. A rational basis for this genetic variation may be provided by its natural competence for genetic transformation and high-frequency recombination. Many bacterial competence systems have homology with proteins that are involved in the assembly of type IV pili and type II secretion systems. In H. pylori, DNA uptake relies on a transport system related to type IV secretion systems (T4SS) designated the comB system. The prototype of a T4SS in Agrobacterium tumefaciens consists of 11 VirB proteins and VirD4, which form the core unit necessary for the delivery of single proteins or large nucleoprotein complexes into target cells. In the past we identified proteins ComB4 and ComB7 through ComB10 as being involved in the process of DNA uptake in H. pylori. In this study we identified and functionally characterized further (T4SS-homologous) components of the comB transformation competence system. By combining computer prediction modeling, experimental topology determination, generation of knockout strains, and genetic complementation studies we identified ComB2, ComB3, and ComB6 as essential components of the transformation apparatus, structurally and functionally homologous to VirB2, VirB3, and VirB6, respectively. comB2, comB3, and comB4 are organized as a separate operon. Thus, for the H. pylori comB system, all T4SS core components have been identified except for homologues to VirB1, VirD4, VirB5, and VirB11.Helicobacter pylori, a highly motile gram-negative bacterial pathogen, is the principal cause of chronic active gastritis and peptic ulcer disease in humans and has been implicated in the development of gastric mucosa-associated lymphoid tissue lymphoma and adenocarcinoma (38,49). H. pylori colonizes a very special habitat at the surface of gastric epithelial cells or in the mucus layer covering the epithelium, which suggests that this bacterium has evolved specialized features for gastric adaptation. A comparison of the two published genome sequences shows considerable diversity in gene content, with about 7% of all putative genes being strain specific (2). The panmictic population structure of H. pylori is believed to result from frequent recombination during mixed colonization by unrelated strains (20,50).H. pylori is naturally competent for genetic transformation (36). Natural transformation competence in bacteria is a complex process, involving DNA binding, uptake/translocation, and recombination. In general, bacterial competence systems have homology with proteins that are involved in the assembly of type IV pili and type II secretion systems and form a structure that partially spans the cell envelope (see reference 13 for a recent review). In H. pylori, DNA uptake is not mediated by a type IV pilus-based apparatus but relies on a transport system related to type IV secretion systems (T4SS) designated the comB system (26).The Vir system of Agrobacterium tumefaciens, necessary for the transfer of its transferred DNA (T-DNA) into plant ce...

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Section: Resultsmentioning

confidence: 99%

Functional and Topological Characterization of Novel Components of the comB DNA Transformation Competence System in Helicobacter pylori

Karnholz¹,

Hoefler²,

Odenbreit³

et al. 2006

J Bacteriol

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“…The seminal work on secretion has also demonstrated the potential biotechnological exploitation of the autotransporter secretion pathway, namely, by displaying heterologous proteins on the bacterial surface (453). The possible applications of this autodisplay system are numerous and include (i) exposure of antigenic determinants for vaccine development (142,267,316), (ii) expression of peptide libraries for epitope mapping or antibody specificity test (258), (iii) display of receptor or ligand for binding assays or purification (499,500), (iv) functional domain analyses of a heterologous protein (59,499,500), and (v) bioconversion by expressing enzymatic activity on the bacterial surface (229,230,276). In conclusion, further understanding of the autotransporter secretion pathway and the functions of the passenger domains will facilitate a richer view of the mechanisms and evolution of bacterial pathogenesis and provide important practical applications for the medical and biotechnological communities.…”

Section: Future Directionsmentioning

confidence: 99%

Type V Protein Secretion Pathway: the Autotransporter Story

Henderson

Navarro-Garcı́a

Desvaux

et al. 2004

Microbiol Mol Biol Rev

713

795

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Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have developed several systems for protein secretion. The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins. Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria. Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis. However, most of the autotransporter proteins remain to be characterized. In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function

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“…Secretion of VacA is believed to proceed via a type V or auto-transporter mechanism (6,(34)(35)(36)(37)(38). The secreted 88-kDa VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55, which remain together after proteolysis (27,36,(39)(40)(41)(42).…”

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confidence: 99%

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

et al. 2016

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Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly ␤-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the ␤-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the ␤-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly. Helicobacter pylori is a Gram-negative bacterium that persistently colonizes the stomach in about half of the human population worldwide (1-3). Most people tolerate the presence of this organism for long periods without any adverse consequences, but a small subset of H. pylori-infected individuals develop gastric adenocarcinoma or peptic ulcer disease.H. pylori is a relatively noninvasive organism that lives in the gastric mucus layer overlying gastric epithelial cells, sometimes adhering to the gastric epithelium. Many H. pylori-induced alterations in the gastric mucosa are attributable to the actions of secreted bacterial proteins on host cells (4-6). One such protein is the secreted vacuolating toxin, VacA (4, 7-10). VacA causes multiple alterations in gastric epithelial cells, including swelling of endosomes (vacuolation) (11, 12), permeabilization of mitochondrial membranes, and activation of mitogen-activated protein kinases (4, 7-9). In addition to its effects on gastric epithelial cells, VacA can disrupt the functions of many types of immune cells (T cells, B cells, neutrophils, eosinophils, and monocytes) (4, 7-9, 13-16). The cellular activities of VacA are attributed mainly to its ability to form anion-selective channels in host cells (17-21). VacA lacks sequence similarity to any other known bacterial toxins.H. pylori strains isolated from unrelated individuals are genetically heterogeneous, and the risk of developing gastric...

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Outer Membrane Targeting of Passenger Proteins by the Vacuolating Cytotoxin Autotransporter of Helicobacter pylori

Cited by 62 publications

References 37 publications

Functional and Topological Characterization of Novel Components of the comB DNA Transformation Competence System in Helicobacter pylori

Functional and Topological Characterization of Novel Components of the comB DNA Transformation Competence System in Helicobacter pylori

Type V Protein Secretion Pathway: the Autotransporter Story

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

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