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Protein subassemblies of the Helicobacter pylori Cag type IV secretion system revealed by localization and interaction studies.

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.

SUBMITTER: Kutter S

PROVIDER: S-EPMC2258873 | biostudies-other | 2008 Mar

REPOSITORIES: biostudies-other

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Protein subassemblies of the Helicobacter pylori Cag type IV secretion system revealed by localization and interaction studies.

Kutter Stefan S Buhrdorf Renate R Haas Jürgen J Schneider-Brachert Wulf W Haas Rainer R Fischer Wolfgang W

Journal of bacteriology 20080104 6

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 ...[more]

PMID: 18178731

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Project description:Helicobacter pylori colonizes about half of humans worldwide, and its presence in the gastric mucosa is associated with an increased risk of gastric adenocarcinoma, gastric lymphoma, and peptic ulcer disease. H. pylori strains carrying the cag pathogenicity island (cagPAI) are associated with increased risk of disease progression. The cagPAI encodes the Cag type IV secretion system (CagT4SS), which delivers the CagA oncoprotein and other effector molecules into human gastric epithelial cells. We visualized structures of native and mutant CagT4SS machines on the H. pylori cell envelope by cryoelectron tomography. Individual H. pylori cells contain multiple CagT4SS nanomachines, each composed of a wheel-shaped outer membrane complex (OMC) with 14-fold symmetry and an inner membrane complex (IMC) with 6-fold symmetry. CagX, CagY, and CagM are required for assembly of the OMC, whereas strains lacking Cag3 and CagT produce outer membrane complexes lacking peripheral components. The IMC, which has never been visualized in detail, is configured as six tiers in cross-section view and three concentric rings surrounding a central channel in end-on view. The IMC contains three T4SS ATPases: (i) VirB4-like CagE, arranged as a hexamer of dimers at the channel entrance; (ii) a hexamer of VirB11-like Cagα, docked at the base of the CagE hexamer; and (iii) VirD4-like Cagβ and other unspecified Cag subunits, associated with the stacked CagE/Cagα complex and forming the outermost rings. The CagT4SS and recently solved Legionella pneumophila Dot/Icm system comprise new structural prototypes for the T4SS superfamily.IMPORTANCE Bacterial type IV secretion systems (T4SSs) have been phylogenetically grouped into two subfamilies. The T4ASSs, represented by the Agrobacterium tumefaciens VirB/VirD4T4SS, include "minimized" machines assembled from 12 VirB- and VirD4-like subunits and compositionally larger systems such as the Helicobacter pylori CagT4SS T4BSSs encompass systems closely related in subunit composition to the Legionella pneumophila Dot/IcmT4SS Here, we present structures of native and mutant H. pylori Cag machines determined by in situ cryoelectron tomography. We identify distinct outer and inner membrane complexes and, for the first time, visualize structural contributions of all three "signature" ATPases of T4SSs at the cytoplasmic entrance of the translocation channel. Despite their evolutionary divergence, the CagT4SS aligns structurally much more closely to the Dot/IcmT4SS than an available VirB/VirD4 subcomplex. Our findings highlight the diversity of T4SSs and suggest a structural classification scheme in which T4SSs are grouped as minimized VirB/VirD4-like or larger Cag-like and Dot/Icm-like systems.

Project description:UnlabelledBacterial type IV secretion systems (T4SSs) can function to export or import DNA, and can deliver effector proteins into a wide range of target cells. Relatively little is known about the structural organization of T4SSs that secrete effector proteins. In this report, we describe the isolation and analysis of a membrane-spanning core complex from the Helicobacter pylori cag T4SS, which has an important role in the pathogenesis of gastric cancer. We show that this complex contains five H. pylori proteins, CagM, CagT, Cag3, CagX, and CagY, each of which is required for cag T4SS activity. CagX and CagY are orthologous to the VirB9 and VirB10 components of T4SSs in other bacterial species, and the other three Cag proteins are unique to H. pylori. Negative stain single-particle electron microscopy revealed complexes 41 nm in diameter, characterized by a 19-nm-diameter central ring linked to an outer ring by spoke-like linkers. Incomplete complexes formed by Δcag3 or ΔcagT mutants retain the 19-nm-diameter ring but lack an organized outer ring. Immunogold labeling studies confirm that Cag3 is a peripheral component of the complex. The cag T4SS core complex has an overall diameter and structural organization that differ considerably from the corresponding features of conjugative T4SSs. These results highlight specialized features of the H. pylori cag T4SS that are optimized for function in the human gastric mucosal environment.ImportanceType IV secretion systems (T4SSs) are versatile macromolecular machines that are present in many bacterial species. In this study, we investigated a T4SS found in the bacterium Helicobacter pylori. H. pylori is an important cause of stomach cancer, and the H. pylori T4SS contributes to cancer pathogenesis by mediating entry of CagA (an effector protein regarded as a "bacterial oncoprotein") into gastric epithelial cells. We isolated and analyzed the membrane-spanning core complex of the H. pylori T4SS and showed that it contains unique proteins unrelated to components of T4SSs in other bacterial species. These results constitute the first structural analysis of the core complex from this important secretion system.

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Protein subassemblies of the Helicobacter pylori Cag type IV secretion system revealed by localization and interaction studies.

Publications

Protein subassemblies of the Helicobacter pylori Cag type IV secretion system revealed by localization and interaction studies.

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