• AGS
• CagA
• H. pylori
• IL-8
• infection
• minimum inhibitory concentration
• silver nanoparticles

• Helicobacter pylori/drug effects
• Bacterial Proteins/metabolism
• Antigens, Bacterial/metabolism
• Silver/pharmacology
• Silver/metabolism
• Humans
• Metal Nanoparticles
• Helicobacter Infections/microbiology
• Helicobacter Infections/drug therapy
• Signal Transduction/drug effects
• Host-Pathogen Interactions
• Epithelial Cells/microbiology
• Virulence Factors/metabolism
• Cell Line
• Anti-Bacterial Agents/pharmacology
• Cell Line, Tumor

• I 4360 Austrian Science Fund FWF
• P 31507 Austrian Science Fund FWF

• CYP1A1
• Campylobacter
• Campylobacter concisus
• gastritis
• oral Campylobacter

• Extracellular vesicles
• Gastric fibroblast
• Helicobacter pylori
• SNAI2
• miRNA-124-3p

• Humans
• Helicobacter pylori
• Stomach Neoplasms
• Cell Line, Tumor
• MicroRNAs/genetics
• Cell Proliferation
• Aniline Compounds
• Benzylidene Compounds

• Helicobacter pylori
• exosomes
• extracellular vesicles
• gastric cancer
• malignancy

• Agencia Nacional de Investigación y Desarrollo
• National Institutes of Health
• Fondo Nacional de Desarrollo Científico y Tecnológico
• Fondo de Financiamiento de Centros de Investigación en Áreas Prioritarias

• Epstein-Barr virus
• Helicobacter pylori
• coinfection
• gankyrin
• gastric cancer

• CagL
• Helicobacter pylori
• polymorphism
• type IV secretion system

• CagY
• Helicobacter
• PAMP
• PRR
• T4SS
• TLR5
• flagellin
• innate and adaptive immunity
• signal transduction
• type IV secretion

Helicobacter pylori (H. pylori) infection promotes the metastasis of gastric carcinoma cells by modulating signal transduction pathways that regulate cell proliferation, motility, and invasion. Astaxanthin (ASTX), a xanthophyll carotenoid, is known to inhibit cancer cell migration and invasion, however the mechanism of action of ASTX in H. pylori-infected gastric epithelial cells is not well understood. To gain insight into this process, we carried out a comparative RNA sequencing (RNA-Seq) analysis of human gastric cancer AGS (adenocarcinoma gastric) cells as a function of H. pylori infection and ASTX administration. The results were used to identify genes that are differently expressed in response to H. pylori and ASTX. Gene ontology (GO) analysis identified differentially expressed genes (DEGs) to be associated with cell cytoskeleton remodeling, motility, and/or migration. Among the 20 genes identified, those encoding c-MET, PI3KC2, PLCγ1, Cdc42, and ROCK1 were selected for verification by real-time PCR analysis. The verified genes were mapped, using signaling networks contained in the KEGG database, to create a signaling pathway through which ASTX might mitigate the effects of H. pylori-infection. We propose that H. pylori-induced upregulation of the upstream regulator c-MET, and hence, its downstream targets Cdc42 and ROCK1, is suppressed by ASTX. ASTX is also suggested to counteract H. pylori-induced activation of PI3K and PLCγ. In conclusion, ASTX can suppress H. pylori-induced gastric cancer progression by inhibiting cytoskeleton reorganization and reducing cell motility through downregulation of c-MET, EGFR, PI3KC2, PLCγ1, Cdc42, and ROCK1.

• Helicobacter pylori
• astaxanthin
• cell migration
• cell motility
• gastric carcinoma

• Adenocarcinoma/drug therapy
• Adenocarcinoma/genetics
• Adenocarcinoma/microbiology
• Adenocarcinoma/pathology
• Antineoplastic Agents/pharmacology
• Cell Line, Tumor
• Cell Movement/drug effects
• Gene Expression Profiling
• Gene Expression Regulation, Neoplastic
• Gene Regulatory Networks
• Helicobacter pylori/pathogenicity
• Host-Pathogen Interactions
• Humans
• Neoplasm Invasiveness
• Protein Interaction Maps
• Signal Transduction
• Stomach Neoplasms/drug therapy
• Stomach Neoplasms/genetics
• Stomach Neoplasms/microbiology
• Stomach Neoplasms/pathology
• Transcriptome
• Xanthophylls/pharmacology

Helicobacter pylori (H. pylori) infection is associated with some gastric diseases, such as gastritis, peptic ulcer, and gastric cancer. CagA and VacA are known virulence factors of H. pylori, which play a vital role in severe clinical outcomes. Additionally, the expression of outer membrane proteins (OMPs) helps H. pylori attach to gastric epithelial cells at the primary stage and increases the virulence of H. pylori. In this review, we have summarized the paralogs of H. pylori OMPs, their genomic loci, and the different receptors of OMPs identified so far. We focused on five OMPs, BabA (HopS), SabA (HopP), OipA (HopH), HopQ, and HopZ, and one family of OMPs: Hom. We highlight the coexpression of OMPs with other virulence factors and their relationship with clinical outcomes. In conclusion, OMPs are closely related to the pathogenic processes of adhesion, colonization, persistent infection, and severe clinical consequences. They are potential targets for the prevention and treatment of H. pylori–related diseases.

• Helicobacter pylori
• Outer membrane proteins
• Virulence factors

Helicobacter pylori typically colonizes the human stomach, but it can occasionally be detected in the oral cavity of infected persons. Clinical outcome as a result of gastric colonization depends on presence of the pathogenicity island cagPAI that encodes a type-IV secretion system (T4SS) for translocation of the effector protein CagA and ADP-heptose. Upon injection into target cells, CagA is phosphorylated, which can be demonstrated by in vitro infection of the gastric epithelial cell line AGS, resulting in cell elongation. Here we investigated whether H. pylori can exert these responses during interaction with cells from the oral epithelium. To this purpose, three oral epithelial cell lines, HN, CAL-27 and BHY, were infected with various virulent wild-type H. pylori strains, and CagA delivery and ADP-heptose-mediated pro-inflammatory responses were monitored.

All three oral cell lines were resistant to elongation upon infection, despite similar bacterial binding capabilities. Moreover, T4SS-dependent CagA injection was absent. Resistance to CagA delivery was shown to be due to absence of CEACAM expression in these cell lines, while these surface molecules have recently been recognized as H. pylori T4SS receptors. Lack of CEACAM expression in HN, CAL-27 and BHY cells was overcome by genetic introduction of either CEACAM1, CEACAM5, or CEACAM6, which in each of the cell lines was proven sufficient to facilitate CagA delivery and phosphorylation upon H. pylori infection to levels similar to those observed with the gastric AGS cells. Pro-inflammatory responses, as measured by interleukin-8 ELISA, were induced to high levels in each cell line and CEACAM-independent.

These results show that lack of CEACAM receptors on the surface of the oral epithelial cells was responsible for resistance to H. pylori CagA-dependent pathogenic activities, and confirms the important role for the T4SS-dependent interaction of these receptors with H. pylori in the gastric epithelium.

• AGS
• BHY
• CAL-27
• CEACAM
• CagA
• HN
• Signaling
• T4SS
• Type IV secretion
• Tyrosine kinases

The gastric pathogen and carcinogen Helicobacter pylori(H. pylori) encodes a type IV secretion system for translocation of the effector protein CagA into host cells. Injected CagA becomes tyrosine-phosphorylated at the five amino acid residue Glutamate-Proline- Isoleucine-Tyrosine-Alanine (EPIYA)-sequence motifs. These phosphorylated EPIYA-sites represent recognition motifs for binding of multiple host factors, which then manipulate signaling pathways to trigger gastric disease. Thus, efficient detection of single phosphorylated EPIYA-motifs in CagA is required. Detection of phospho-CagA is primarily performed using commercial pan-phosphotyrosine antibodies. However, those antibodies were originally generated to recognize many phosphotyrosines in various mammalian proteins and are not optimized for use in bacteria. To address this important limitation, we synthesized 11-mer phospho- and non-phospho-peptides from EPIYA-motifs A, B, and C, and produced three phospho-specific and three non-phospho-specific rabbit polyclonal CagA antibodies. These antibodies specifically recognized the corresponding phosphorylated and non-phosphorylated EPIYA-motifs, while the EPIYA-C antibodies also recognized the related East-Asian EPIYA-D motif. Otherwise, no cross-reactivity of the antibodies among EPIYAs was observed. Western blotting demonstrated that each EPIYA-motif can be predominantly phosphorylated during H. pylori infection. This represents the first complete set of phospho-specific antibodies for an effector protein in bacteria, providing useful tools to gather information for the categorization of CagA phosphorylation, cancer signaling, and gastric disease progression.

• Abl and Src tyrosine kinases
• CagA
• EPIYA motifs
• Helicobacter pylori
• cagPAI
• dotblot
• signaling
• type IV secretion

• C57BL/6 mice
• CagA-positive Helicobacter pylori
• Immortalized human gastric epithelial cells (GES-1)
• N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)
• Wnt/β-catenin signaling pathway
• cancer stem cell-like properties
• epithelial-mesenchymal transition (EMT)

Afadin is a cytoplasmic protein of the adherens junctions, which regulates the formation and stabilization of both the adherens and the tight junctions. Aberrant expression of Afadin has been shown in cancer and its loss has been associated with epithelial-to-mesenchymal transition (EMT). EMT is characterized by the change from an epithelial to a mesenchymal phenotype, with modifications on the expression of adhesion molecules and acquisition of a migratory and invasive cell behavior. While it is known that Helicobacter pylori disrupts the tight and the adherens junctions and induces EMT, the effect of the bacteria on Afadin is still unknown. The aim of this study was to disclose the effect of H. pylori on Afadin and its impact in the induction of an EMT phenotype in gastric cells. Using two different cell lines, we observed that H. pylori infection decreased Afadin protein levels, independently of CagA, T4SS, and VacA virulence factors. H. pylori infection of cell lines recapitulated several EMT features, displacing and downregulating multiple proteins from cell–cell junctions, and increasing the expression of ZEB1, Vimentin, Slug, N-cadherin, and Snail. Silencing of Afadin by RNAi promoted delocalization of junctional proteins from the cell–cell contacts, increased paracellular permeability, and decreased transepithelial electrical resistance, all compatible with impaired junctional integrity. Afadin silencing also led to increased expression of the EMT marker Snail, and to the formation of actin stress fibers, together with increased cell motility and invasion. Finally, and in line with our in vitro data, the gastric mucosa of individuals infected with H. pylori showed decrease/loss of Afadin membrane staining at cell–cell contacts significantly more frequently than uninfected individuals. In conclusion, Afadin is downregulated by H. pylori infection in vitro and in vivo, and its downregulation leads to the emergence of EMT and to the acquisition of an aggressive phenotype in gastric cells, which can contribute to gastric carcinogenesis.

• Afadin
• Helicobacter pylori
• cell–cell junction disruption
• epithelial to mesenchymal transition
• gastric cancer

• Carvacrol
• gastric cells
• genotoxicity
• pro-oxidant activity
• thymol

• Adherens junction
• CagA
• E-cadherin
• Epithelial barrier
• HtrA serine protease
• Protease
• Tight junction
• Type IV secretion T4SS
• UreA
• VacA

• Adherens Junctions/genetics
• Adherens Junctions/metabolism
• Adherens Junctions/microbiology
• Animals
• Bacterial Proteins/genetics
• Bacterial Proteins/metabolism
• Epithelial Cells/metabolism
• Epithelial Cells/microbiology
• Gastric Mucosa/metabolism
• Helicobacter Infections/genetics
• Helicobacter Infections/metabolism
• Helicobacter Infections/microbiology
• Helicobacter pylori/genetics
• Helicobacter pylori/physiology
• Humans
• Signal Transduction
• Stomach/microbiology
• Tight Junctions/genetics
• Tight Junctions/metabolism
• Tight Junctions/microbiology
• Virulence Factors/genetics
• Virulence Factors/metabolism

• P 24074 Austrian Science Fund FWF

H. pylori induce ETS2 and Twist1 expression in the infected GCC.

Helicobacter pylori infection is one of the most potent factors leading to gastric carcinogenesis. The seven in absentia homologue (Siah2) is an E3 ubiquitin ligase which has been implicated in various cancers but its role in H. pylori-mediated gastric carcinogenesis has not been established. We investigated the involvement of Siah2 in gastric cancer metastasis which was assessed by invasiveness and migration of H. pylori-infected gastric epithelial cancer cells. Cultured gastric cancer cells (GCCs) MKN45, AGS and Kato III showed significantly induced expression of Siah2, increased invasiveness and migration after being challenged with the pathogen. Siah2-expressing stable cells showed increased invasiveness and migration after H. pylori infection. Siah2 was transcriptionally activated by E26 transformation-specific sequence 2 (ETS2)- and Twist-related protein 1 (Twist1) induced in H. pylori-infected gastric epithelial cells. These transcription factors dose-dependently enhanced the aggressiveness of infected GCCs. Our data suggested that H. pylori-infected GCCs gained cell motility and invasiveness through Siah2 induction. As gastric cancer biopsy samples also showed highly induced expression of ETS2, Twist1 and Siah2 compared with noncancerous gastric tissue, we surmise that ETS2- and Twist1-mediated Siah2 up-regulation has potential diagnostic and prognostic significance and could be targeted for therapeutic purpose.

• ETS2
• H. pylori
• Siah2
• Twist1
• gastric cancer
• metastasis

Helicobacter pylori (H. pylori) infection is highly prevalent in human, affecting nearly half of the world’s population; however, infection remains asymptomatic in majority of population. During its co-existence with humans, H. pylori has evolved various strategies to maintain a mild gastritis and limit the immune response of host. On the other side, presence of H. pylori is also associated with increased risk for the development of various gastric pathologies including gastric cancer (GC). A complex combination of host genetics, environmental agents, and bacterial virulence factors are considered to determine the susceptibility as well as the severity of outcome in a subset of individuals. GC is one of the most common cancers and considered as the third most common cause of cancer related death worldwide. Many studies had proved H. pylori as an important risk factor in the development of non-cardia GC. Although both H. pylori infection and GC are showing decreasing trends in the developed world, they still remain a major threat to human population in the developing countries. The current review attempts to highlight recent progress in the field of research on H. pylori induced GC and aims to provide brief insight into H. pylori pathogenesis, the role of major virulence factors of H. pylori that modulates the host environment and transform the normal gastric epithelium to neoplastic one. This review also emphasizes on the mechanistic understanding of how colonization and various virulence attributes of H. pylori as well as the host innate and adaptive immune responses modulate the diverse signaling pathways that leads to different disease outcomes including GC.

• Cag pathogenicity island
• Gastric cancer
• Gastric mucosa
• Helicobacter pylori
• Type IV secretion system

• Helicobacter pylori
• casein kinase 2
• gastric epithelial cells
• invasion
• migration
• α-/β-catenin

• Amino Acid Motifs
• Amino Acid Sequence
• Antigens, Bacterial/metabolism
• Bacterial Proteins/metabolism
• Cell Line, Tumor
• Gastric Mucosa/metabolism
• Helicobacter Infections/metabolism
• Helicobacter pylori/metabolism
• Humans
• Molecular Sequence Data
• Phosphorylation/physiology
• Phosphotyrosine/metabolism
• Sequence Alignment

• R01DK62813 NIDDK NIH HHS
• R21 AI078237 NIAID NIH HHS
• R21 AI088337 NIAID NIH HHS
• P30 DK056338 NIDDK NIH HHS
• R01 DK062813 NIDDK NIH HHS

The trefoil peptides (TFF1, TFF2 and TFF3) are a family of small highly conserved proteins that play an essential role in epithelial regeneration within the gastrointestinal tract, where they are mainly expressed. TFF1 expression is strongly induced after mucosal injury and it has been proposed that tff1 functions as a gastric tumor suppressor gene. Several studies confirm that tff1 expression is frequently lost in gastric cancer because of deletions, mutations or methylation of the tff1 promoter. Infection by Helicobacter pylori (H. pylori) results in chronic gastritis and it can lead to the development of gastric or duodenal ulcers. Moreover, it is known that there is a strong link to the development of gastric cancer. It has been shown that H. pylori interacts with the dimeric form of TFF1 and that the rough form of lipopolysaccharide mediates this interaction. We have previously reported that the carboxy-terminus of TFF1 is able to specifically bind copper ions (Cu) and that Cu binding favours the homodimerization of the peptide, thus enhancing its motogenic activity. Here, we report that the Cu-TFF1 cuprocomplex promotes adherence of H. pylori to epithelial cells. Adherence of H. pylori to gastric adenocarcinoma cells, AGS AC1 cells, induced to hyper-express TFF1 was enhanced compared to noninduced cells. Copper further promoted this interaction. A H. pylori mutant unable to bind TFF1 did not show enhanced infection of induced cells. Cu treatment induced a thickening of the mucus layer produced by the colorectal adenocarcinoma mucus secreting, goblet cells, HT29-E12 and promoted H. pylori colonisation. Finally, SPR analysis shows that the C-terminus of TFF1, involved in the binding of copper, is also able to selectively bind H. pylori RF-LPS.

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25⁺ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies (“humming bird”) compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells.

Infection with the bacterium Helicobacter pylori (H. pylori) mainly affects children in the developing countries who are at risk to progress to gastric cancer (GC) as adults after many years of persistent infection, especially with strains which are positive for the oncogenic virulence factor CagA. Eradication of H. pylori by antibiotics is a treatment of choice but may also alter the susceptibility to allergies and other tumor types. Thus, novel diagnostic or prognostic markers are needed which detect early molecular changes in the stomach mucosa during the transition of chronic inflammation to cancer. In our study, we found that the tumor suppressor caveolin-1 (Cav1) is reduced upon infection with H. pylori, and CagA was sufficient but not necessary for this down-regulation. Loss of Cav1 was caused by H. pylori-dependent activation of sterol-responsive element-binding protein-1 (SREBP1), and this event abolished the interaction of Cav1 with p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1), a second bona fide tumor suppressor in gastric tissue. Conclusively, Cav1 and DLC1 may constitute novel molecular markers in the H. pylori-infected gastric mucosa before neoplastic transformation of the epithelium.

• Animals
• Antigens, Bacterial/genetics
• B-Lymphocytes/immunology
• Bacterial Proteins/genetics
• Caveolin 1/deficiency
• Caveolin 1/genetics
• Caveolin 1/metabolism
• Cell Line
• Dogs
• Enzyme Activation
• GTPase-Activating Proteins/metabolism
• Gastric Mucosa/immunology
• Gastric Mucosa/metabolism
• Gastric Mucosa/microbiology
• Gastritis/immunology
• Gastritis/microbiology
• Gastritis/prevention & control
• HEK293 Cells
• Helicobacter Infections/immunology
• Helicobacter Infections/microbiology
• Helicobacter Infections/prevention & control
• Helicobacter pylori/genetics
• Helicobacter pylori/immunology
• Helicobacter pylori/metabolism
• Helicobacter pylori/pathogenicity
• Humans
• Macrophages/immunology
• Madin Darby Canine Kidney Cells
• Mice
• Mice, Inbred BALB C
• Mice, Inbred C57BL
• Mice, Knockout
• Parietal Cells, Gastric
• Sterol Regulatory Element Binding Protein 1/metabolism
• T-Lymphocytes, Regulatory/immunology
• Tumor Suppressor Proteins/metabolism
• p120 GTPase Activating Protein/metabolism

• CagA
• CagL
• Helicobacter pylori
• T4SS

• CagL
• ERK kinase
• binding motifs
• cortactin
• integrin interaction
• α5β1

Helicobacter pylori is a specific gastric pathogen that colonizes the stomach in more than 50% of the world’s human population. Infection with this bacterium can induce several types of gastric pathology, ranging from chronic gastritis to peptic ulcers and even adenocarcinoma. Virulent H. pylori isolates encode components of a type IV secretion system (T4SS), which form a pilus for the injection of virulence proteins such as CagA into host target cells. This is accomplished by a specialized adhesin on the pilus surface, the protein CagL, a putative VirB5 ortholog, which binds to host cell β₁ integrin, triggering subsequent delivery of CagA across the host cell membrane. Like the human extracellular matrix protein fibronectin, CagL contains an RGD (Arg-Gly-Asp) motif and is able to trigger intracellular signaling pathways by RGD-dependent binding to integrins. While CagL binding to host cells is mediated primarily by the RGD motif, we identified an auxiliary binding motif for CagL–integrin interaction. Here, we report on a surface exposed FEANE (Phe-Glu-Ala-Asn-Glu) interaction motif in spatial proximity to the RGD sequence, which enhances the interactions of CagL with integrins. It will be referred to as RGD helper sequence (RHS). Competitive cell adhesion assays with recombinant wild type CagL and point mutants, competition experiments with synthetic cyclic and linear peptides, and peptide array experiments revealed amino acids essential for the interaction of the RHS motif with integrins. Infection experiments indicate that the RHS motif plays a role in the early interaction of H. pylori T4SS with integrin, to trigger signaling and to inject CagA into host cells. We thus postulate that CagL is a versatile T4SS surface protein equipped with at least two motifs to promote binding to integrins, thereby causing aberrant signaling within host cells and facilitating translocation of CagA into host cells, thus contributing directly to H. pylori pathogenesis.

• CagL
• ERK kinase
• binding motifs
• cortactin
• integrin interaction
• α5β1

• Animals
• Antibodies, Viral/pharmacology
• Cell Line/virology
• Cell Shape/drug effects
• Helicobacter pylori/physiology
• Humans
• Inclusion Bodies, Viral/metabolism
• Microscopy, Electron, Scanning
• Microscopy, Electron, Transmission
• Microscopy, Immunoelectron
• Parainfluenza Virus 5/immunology
• Parainfluenza Virus 5/pathogenicity
• Parainfluenza Virus 5/ultrastructure
• Rabbits
• Rubulavirus Infections/transmission
• Rubulavirus Infections/virology
• Virion/immunology
• Virion/pathogenicity
• Virion/ultrastructure

• virulence factors
• cag pathogenicity island
• outer inflammatory protein

Helicobacter pylori is the causative agent for developing gastritis, gastric ulcer, and even gastric cancer. Virulent strains carry the cag pathogenicity island (cagPAI) encoding a type-IV secretion system (T4SS) for injecting the CagA protein. However, mechanisms of sensing this pathogen through Toll-like receptors (TLRs) and downstream signalling pathways in the development of different pathologies are widely unclear. Here, we explored the involvement of TLR-2 and TLR-5 in THP-1 cells and HEK293 cell lines (stably transfected with TLR-2 or TLR-5) during infection with wild-type H. pylori and isogenic cagPAI mutants. H. pylori triggered enhanced TLR-2 and TLR-5 expression in THP-1, HEK293-TLR2 and HEK293-TLR5 cells, but not in the HEK293 control. In addition, IL-8 and TNF-α cytokine secretion in THP-1 cells was induced in a cagPAI-dependent manner. Furthermore, we show that HEK293 cells are not competent for the uptake of T4SS-delivered CagA, and are therefore ideally suited for studying TLR signalling in the absence of T4SS functions. HEK293 control cells, which do not induce TLR-2 and TLR-5 expression during infection, only secreted cytokines in small amounts, in agreement with T4SS functions being absent. In contrast, HEK293-TLR2 and HEK293-TLR5 cells were highly competent for inducing the secretion of IL-8 and TNF-α cytokines in a cagPAI-independent manner, suggesting that the expression of TLR-2 or TLR-5 has profoundly changed the capability to trigger pro-inflammatory signalling upon infection. Using phospho-specific antibodies and luciferase reporter assays, we further demonstrate that H. pylori induces IRAK-1 and IκB phosphorylation in a TLR-dependent manner, and this was required for activation of transcription factor NF-κB. Finally, NF-κB activation in HEK293-TLR2 and HEK293-TLR5 cells was confirmed by expressing p65-GFP which was translocated from the cytoplasm into the nucleus. These data indicate that H. pylori-induced expression of TLR-2 and TLR-5 can qualitatively shift cagPAI-dependent to cagPAI-independent pro-inflammatory signalling pathways with possible impact on the outcome of H. pylori-associated diseases.

• Antigens, Bacterial/metabolism
• Bacterial Proteins/metabolism
• Cell Line
• Genomic Islands/genetics
• HEK293 Cells
• Helicobacter Infections
• Helicobacter pylori/physiology
• Humans
• I-kappa B Proteins/metabolism
• Inflammation Mediators/metabolism
• Interleukin-1 Receptor-Associated Kinases/metabolism
• Interleukin-8/metabolism
• NF-kappa B/metabolism
• Phosphorylation
• Phosphoserine/metabolism
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• Signal Transduction
• Toll-Like Receptor 2/genetics
• Toll-Like Receptor 2/metabolism
• Toll-Like Receptor 5/genetics
• Toll-Like Receptor 5/metabolism
• Transcription Factor AP-1/metabolism
• Transcriptional Activation
• Tumor Necrosis Factor-alpha/metabolism
• Up-Regulation/genetics

• R56 DK064371 NIDDK NIH HHS
• R56DK064371 NIDDK NIH HHS

Helicobacter pylori is a pathogenic bacterium that colonizes more than 50% of the world's population, which leads to a tremendous medical burden. H. pylori infection is associated with such varied diseases as gastritis, peptic ulcers, and two forms of gastric cancer: gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. This association represents a novel paradigm for cancer development; H. pylori is currently the only bacterium to be recognized as a carcinogen. Therefore, a significant amount of research has been conducted to identify the bacterial factors and the deregulated host cell pathways that are responsible for the progression to more severe disease states. Two of the virulence factors that have been implicated in this process are cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA), which are cytotoxins that are injected and secreted by H. pylori, respectively. Both of these virulence factors are polymorphic and affect a multitude of host cellular pathways. These combined facts could easily contribute to differences in disease severity across the population as various CagA and VacA alleles differentially target some pathways. Herein we highlight the diverse types of cellular pathways and processes targeted by these important toxins.

• CagA
• Helicobacter
• VacA
• cell-signaling
• gastric cancer
• toxin

Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA⁺/VacA⁺H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.

The gram-negative bacterium Helicobacter pylori is the main causative agent of peptic ulcer and gastric cancer in humans. Our work sheds light on a new molecular mechanism by which H. pylori would exert its highly efficient colonization strategy of the human host. In this paper, we show that the H. pylori CagA protein counteracts, by two distinct non-overlapping mechanisms, the apoptotic activity of the H. pylori VacA toxin on human gastric epithelial cells so as to allow a protection of the bacterium niche against VacA, giving a rationale for the association of these two virulence factors in the most pathogenic H. pylori strains. This is a new, highly ingenious mechanism by which a bacterium locally protects its ecological niche against the action of one of its own virulence factors. However, while exerting a beneficial role for survival and growth of the bacterium by counteracting VacA toxin, CagA injection in the gastric epithelial cells triggers proinflammatory and anti-apoptotic responses which are detrimental for the human host in the long-term and favor the development of ulcer and cancer.

Highly dynamic integrin-based focal adhesions provide an important structural basis for anchoring the cellular actin cytoskeleton to the surrounding extracellular matrix. The human pathogen Helicobacter pylori (H. pylori) directly targets integrins with drastic consequences on the epithelial cell morphology and migration, which might contribute to the disruption of the gastric epithelium in vivo. In this review, we summarize the recent findings concerning the complex mechanism through which H. pylori interferes with host integrin signaling thereby deregulating focal adhesions and the actin cytoskeleton of motile epithelial cells.

• Cell-Matrix Junctions/microbiology
• Cell-Matrix Junctions/pathology
• Epithelium/microbiology
• Epithelium/pathology
• Helicobacter pylori/growth & development
• Helicobacter pylori/pathogenicity
• Humans
• Intercellular Junctions/microbiology
• Intercellular Junctions/pathology
• Models, Biological

Host–pathogen interactions involve protein expression changes within both the host and the pathogen. An understanding of the nature of these interactions provides insight into metabolic processes and critical regulatory events of the host cell as well as into the mechanisms of pathogenesis by infectious microorganisms. Pathogen exposure induces changes in host proteins at many functional levels including cell signaling pathways, protein degradation, cytokines and growth factor production, phagocytosis, apoptosis, and cytoskeletal rearrangement. Since proteins are responsible for the cell biological functions, pathogens have evolved to manipulate the host cell proteome to achieve optimal replication. Intracellular pathogens can also change their proteome to adapt to the host cell and escape from immune surveillance, or can incorporate cellular proteins to invade other cells. Given that the interactions of intracellular infectious agents with host cells are mainly at the protein level, proteomics is the most suitable tool for investigating these interactions. Proteomics is the systematic analysis of proteins, particularly their interactions, modifications, localization and functions, that permits the study of the association between pathogens with their host cells as well as complex interactions such as the host–vector–pathogen interplay. A review on the most relevant proteomic applications used in the study of host–pathogen interactions is presented.

• Actins/metabolism
• Bacterial Outer Membrane Proteins/genetics
• Bacterial Outer Membrane Proteins/metabolism
• Bacterial Outer Membrane Proteins/physiology
• Cell Line
• Cytoskeleton/metabolism
• Epithelial Cells/cytology
• Epithelial Cells/metabolism
• Epithelial Cells/microbiology
• Extracellular Signal-Regulated MAP Kinases/metabolism
• Focal Adhesion Protein-Tyrosine Kinases/genetics
• Focal Adhesion Protein-Tyrosine Kinases/metabolism
• Helicobacter pylori/genetics
• Helicobacter pylori/metabolism
• Helicobacter pylori/physiology
• Humans
• Immunoblotting
• Immunoprecipitation
• Microscopy, Fluorescence
• Mutation
• Phosphorylation
• RNA, Small Interfering/genetics
• src-Family Kinases/metabolism

• P30 DK056338 NIDDK NIH HHS
• R01 DK062813 NIDDK NIH HHS
• DK62813 NIDDK NIH HHS
• DK56338 NIDDK NIH HHS

• Adherens Junctions/metabolism
• Animals
• Antigens, Bacterial/metabolism
• Bacterial Proteins/metabolism
• Cadherins/metabolism
• Cell Adhesion
• Cell Line, Tumor
• Cell Movement
• Cell Polarity
• Dogs
• Epithelial Cells/metabolism
• Epithelial Cells/microbiology
• Helicobacter Infections/microbiology
• Helicobacter pylori/pathogenicity
• Humans
• Phosphorylation
• Protein Transport
• Transcriptional Activation/genetics
• beta Catenin/genetics