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Helicobacter pylori exploits a unique repertoire of type IV secretion system components for pilus assembly at the bacteria-host cell interface.

ABSTRACT: Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor ?5?1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. ?cagI and ?cagL mutant strains fail to form pili, whereas a ?cagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS.

SUBMITTER: Shaffer CL

PROVIDER: S-EPMC3164655 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Helicobacter pylori exploits a unique repertoire of type IV secretion system components for pilus assembly at the bacteria-host cell interface.

Shaffer Carrie L CL Gaddy Jennifer A JA Loh John T JT Johnson Elizabeth M EM Hill Salisha S Hennig Ewa E EE McClain Mark S MS McDonald W Hayes WH Cover Timothy L TL

PLoS pathogens 20110901 9

Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (anoth ...[more]

PMID: 21909278

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Project description:BackgroundHelicobacter pylori infection is associated with risk for chronic gastritis (CG), gastric ulcer (GU), duodenal ulcer (DU), and gastric cancer (GC). The H. pylori Cag type IV secretion system (TFSS) translocates the virulence factor cytotoxin-associated gene A protein into host cells and plays an important role in initiating gastric carcinogenesis. The CagL and CagI proteins are components of the TFSS. The Arg-Gly-Asp (RGD) motif of CagL, and the six most distal C-terminal amino acids (Ser-Lys-Ile-Ile-Val-Lys, and Ser-Lys-Val-Ile-Val-Lys) of CagL and CagI are essential for TFSS adhesion to host cells. Additionally, the CagL variant Tyr58Glu59 was previously shown to be associated with GC patients.ResultsWe isolated 43 H. pylori isolates from 17 CG, 8 GU, 8 DU, and 10 GC patients in Southeast Asia. Total DNAs were extracted and sequenced with MiSeq. H. pylori strain ATCC 26695, which was isolated from CG patients, was used as a reference. We examined the full sequences of H. pylori cagL and cagI using whole-genome sequencing (WGS), and analyzed whether single nucleotide variants and amino acid changes (AACs) correlated with adverse clinical outcomes. Three isolates were excluded from the analysis due to cagPAI rearrangements. CagL RGD motifs were conserved in 39 isolates (97.5%). CagL-Glu59 and Ile234 in the C-terminal motif were more common in 10 H. pylori isolates from GC patients (p < 0.001 and p < 0.05, respectively). When 5 Vietnamese isolates from GC patients were excluded, CagL-Glu59 still remains significant (p < 0.05), but not Ile234. CagL-Tyr58 was seen in only one isolate. The CagI C-terminal motif was completely conserved across all 40 isolates, and there were no significant AACs in CagI.ConclusionsUsing WGS, we analyzed genetic variants in clinical H. pylori isolates and identified putative novel and candidate variants in uncharacterized CagL and CagI sequences that are related to gastric carcinogenesis. In particular, CagL-Glu59 has the possible association with GC.

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:Background & aimsPeptic ulcer disease and gastric cancer are caused most often by Helicobacter pylori strains that harbor the cag pathogenicity island, which encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into host cells. cagY is an essential gene in the T4SS and has an unusual DNA repeat structure that predicts in-frame insertions and deletions. These cagY recombination events typically lead to a reduction in T4SS function in mouse and primate models. We examined the role of the immune response in cagY-dependent modulation of T4SS function.MethodsH pylori T4SS function was assessed by measuring CagA translocation and the capacity to induce interleukin (IL)8 in gastric epithelial cells. cagY recombination was determined by changes in polymerase chain reaction restriction fragment-length polymorphisms. T4SS function and cagY in H pylori from C57BL/6 mice were compared with strains recovered from Rag1-/- mice, T- and B-cell-deficient mice, mice with deletion of the interferon gamma receptor (IFNGR) or IL10, and Rag1-/- mice that received adoptive transfer of control or Ifng-/- CD4+ T cells. To assess relevance to human beings, T4SS function and cagY recombination were assessed in strains obtained sequentially from a patient after 7.4 years of infection.ResultsH pylori infection of T-cell-deficient and Ifngr1-/- mice, and transfer of CD4+ T cells to Rag1-/- mice, showed that cagY-mediated loss of T4SS function requires a T-helper 1-mediated immune response. Loss of T4SS function and cagY recombination were more pronounced in Il10-/- mice, and in control mice infected with H pylori that expressed a more inflammatory form of cagY. Complementation analysis of H pylori strains isolated from a patient over time showed changes in T4SS function that were dependent on recombination in cagY.ConclusionsAnalysis of H pylori strains from mice and from a chronically infected patient showed that CagY functions as an immune-sensitive regulator of T4SS function. We propose that this is a bacterial adaptation to maximize persistent infection and transmission to a new host under conditions of a robust inflammatory response.

Dataset Information

Helicobacter pylori exploits a unique repertoire of type IV secretion system components for pilus assembly at the bacteria-host cell interface.

Publications

Helicobacter pylori exploits a unique repertoire of type IV secretion system components for pilus assembly at the bacteria-host cell interface.

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