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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. strains carrying the pathogenicity island (PAI) are associated with increased risk of disease progression. The PAI encodes the Cag type IV secretion system (Cag), which delivers the CagA oncoprotein and other effector molecules into human gastric epithelial cells. We visualized structures of native and mutant Cag machines on the cell envelope by cryoelectron tomography. Individual cells contain multiple Cag 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 Cag and recently solved Dot/Icm system comprise new structural prototypes for the T4SS superfamily. Bacterial type IV secretion systems (T4SSs) have been phylogenetically grouped into two subfamilies. The T4ASSs, represented by the VirB/VirD4, include "minimized" machines assembled from 12 VirB- and VirD4-like subunits and compositionally larger systems such as the Cag T4BSSs encompass systems closely related in subunit composition to the Dot/Icm Here, we present structures of native and mutant Cag machines determined by 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 Cag aligns structurally much more closely to the Dot/Icm 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.
Copyright © 2019 Hu et al.
Gastric cancer is a leading cause of cancer-related death worldwide. Helicobacter pylori infection is the strongest known risk factor for this malignancy. An important goal is to identify H. pylori-infected persons at high risk for gastric cancer, so that these individuals can be targeted for therapeutic intervention. H. pylori exhibits a high level of intraspecies genetic diversity, and over the past two decades, many studies have endeavored to identify strain-specific features of H. pylori that are linked to development of gastric cancer. One of the most prominent differences among H. pylori strains is the presence or absence of a 40-kb chromosomal region known as the cag pathogenicity island (PAI). Current evidence suggests that the risk of gastric cancer is very low among persons harboring H. pylori strains that lack the cag PAI. Among persons harboring strains that contain the cag PAI, the risk of gastric cancer is shaped by a complex interplay among multiple strain-specific bacterial factors as well as host factors. This review discusses the strain-specific properties of H. pylori that correlate with increased gastric cancer risk, focusing in particular on secreted proteins and surface-exposed proteins, and describes evidence from cell culture and animal models linking these factors to gastric cancer pathogenesis. Strain-specific features of H. pylori that may account for geographic variation in gastric cancer incidence are also discussed.
Copyright © 2016 Cover.
Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the "pilus". Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface(. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.
Helicobacter pylori causes numerous alterations in gastric epithelial cells through processes that are dependent on activity of the cag type IV secretion system (T4SS). Filamentous structures termed "pili" have been visualized at the interface between H. pylori and gastric epithelial cells, and previous studies suggested that pilus formation is dependent on the presence of the cag pathogenicity island (PAI). Thus far, there has been relatively little effort to identify specific genes that are required for pilus formation, and the role of pili in T4SS function is unclear. In this study, we selected 7 genes in the cag PAI that are known to be required for T4SS function and investigated whether these genes were required for pilus formation. cagT, cagX, cagV, cagM, and cag3 mutants were defective in both T4SS function and pilus formation; complemented mutants regained T4SS function and the capacity for pilus formation. cagY and cagC mutants were defective in T4SS function but retained the capacity for pilus formation. These results define a set of cag PAI genes that are required for both pilus biogenesis and T4SS function and reveal that these processes can be uncoupled in specific mutant strains.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. H. pylori expresses a repertoire of virulence factors that increase gastric cancer risk, including the cag pathogenicity island and the vacuolating cytotoxin (VacA). One host element that promotes carcinogenesis within the gastrointestinal tract is Krüppel-like factor 5 (KLF5), a transcription factor that mediates key cellular functions. To define the role of KLF5 within the context of H. pylori-induced inflammation and injury, human gastric epithelial cells were co-cultured with the wild-type cag(+) H. pylori strain 60190. KLF5 expression was significantly upregulated following co-culture with H. pylori, but increased expression was independent of the cag island or VacA. To translate these findings into an in vivo model, C57BL/6 mice were challenged with the wild-type rodent-adapted cag(+) H. pylori strain PMSS1 or a PMSS1 cagE(-) isogenic mutant. Similar to findings in vitro, KLF5 staining was significantly enhanced in gastric epithelium of H. pylori-infected compared to uninfected mice and this was independent of the cag island. Flow cytometry revealed that the majority of KLF5(+) cells also stained positively for the stem cell marker, Lrig1, and KLF5(+)/Lrig1(+) cells were significantly increased in H. pylori-infected versus uninfected tissue. To extend these results into the natural niche of this pathogen, levels of KLF5 expression were assessed in human gastric biopsies isolated from patients with or without premalignant lesions. Levels of KLF5 expression increased in parallel with advancing stages of neoplastic progression, being significantly elevated in gastritis, intestinal metaplasia, and dysplasia compared to normal gastric tissue. These results indicate that H. pylori induces expression of KLF5 in gastric epithelial cells in vitro and in vivo, and that the degree of KLF5 expression parallels the severity of premalignant lesions in human gastric carcinogenesis.
Gastric adenocarcinoma is strongly associated with Helicobacter pylori infection; however, most infected persons never develop this malignancy. H. pylori strains harboring the cag pathogenicity island (cag+), which encodes CagA and a type IV secretion system (T4SS), induce more severe disease outcomes. H. pylori infection is also associated with iron deficiency, which similarly augments gastric cancer risk. To define the influence of iron deficiency on microbial virulence in gastric carcinogenesis, Mongolian gerbils were maintained on iron-depleted diets and infected with an oncogenic H. pylori cag+ strain. Iron depletion accelerated the development of H. pylori-induced premalignant and malignant lesions in a cagA-dependent manner. H. pylori strains harvested from iron-depleted gerbils or grown under iron-limiting conditions exhibited enhanced virulence and induction of inflammatory factors. Further, in a human population at high risk for gastric cancer, H. pylori strains isolated from patients with the lowest ferritin levels induced more robust proinflammatory responses compared with strains isolated from patients with the highest ferritin levels, irrespective of histologic status. These data demonstrate that iron deficiency enhances H. pylori virulence and represents a measurable biomarker to identify populations of infected persons at high risk for gastric cancer.
The cag pathogenicity island is a well-characterized virulence determinant. It is composed of 32 genes that encode a type IV bacterial secretion system and is linked with a more severe clinical outcome. The following chapters will explore the manipulation of bacterial factors in order to understand their role in gastric mucosal disease.
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 (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. ΔcagI and ΔcagL mutant strains fail to form pili, whereas a ΔcagH 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.
Chronic gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulceration and distal gastric cancer, and adherence of H. pylori to gastric epithelial cells is critical for induction of inflammation. One H. pylori constituent that increases disease risk is the cag pathogenicity island, which encodes a secretion system that translocates bacterial effector molecules into host cells. Decay-accelerating factor (DAF) is a cellular receptor for H. pylori and a mediator of the inflammatory response to this pathogen. H. pylori induces DAF expression in human gastric epithelial cells; therefore, we sought to define the mechanism by which H. pylori up-regulates DAF and to extend these findings into a murine model of H. pylori-induced injury. Co-culture of MKN28 gastric epithelial cells with the wild-type H. pylori cag(+) strain J166 induced transcriptional expression of DAF, which was attenuated by disruption of a structural component of the cag secretion system (cagE). H. pylori-induced expression of DAF was dependent upon activation of the p38 mitogen-activated protein kinase pathway but not NF-kappaB. Hypergastrinemic INS-GAS mice infected with wild-type H. pylori demonstrated significantly increased DAF expression in gastric epithelium versus uninfected controls or mice infected with an H. pylori cagE(-) isogenic mutant strain. These results indicate that H. pylori cag(+) strains induce up-regulation of a cognate cellular receptor in vitro and in vivo in a cag-dependent manner, representing the first evidence of regulation of an H. pylori host receptor by the cag pathogenicity island.
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.