Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.     

Innate Immunity Crosstalk with Helicobacter pylori: Pattern Recognition Receptors and Cellular Responses

Helicobacter pylori is one of the most successful gastric pathogens that has co-existed with human for centuries. H. pylori is recognized by the host immune system through human pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), C-type lectin like receptors (CLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs), which activate downstream signaling pathways. Following bacterial recognition, the first responders of the innate immune system, including neutrophils, macrophages, and dendritic cells, eradicate the bacteria through phagocytic and inflammatory reaction. This review provides current understanding of the interaction between the innate arm of host immunity and H. pylori, by summarizing H. pylori recognition by PRRs, and the subsequent signaling pathway activation in host innate immune cells.     

The Role of microRNA in Gastric Malignancy

Helicobacter pylori (H. pylori) infection is the main cause of gastritis, gastro-duodenal ulcer, and gastric cancer. MicroRNAs (miRNAs) are small noncoding RNAs that function as endogenous silencers of numerous target genes. Many miRNA genes are expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation. Recent discoveries have shed new light on the involvement of miRNAs in gastric malignancy. However, at the same time, several miRNAs have been associated with opposing events, leading to reduced inflammation, inhibition of malignancy, and increased apoptosis of transformed cells. The regulation of miRNA expression could be a novel strategy in the chemoprevention of human gastric malignancy. In this article, the biological importance of miRNAs in gastric malignancy is summarized.     

ABO Blood Group System and Gastric Cancer: A Case-Control Study and Meta-Analysis

This study focuses on the association between the ABO blood group system and the risk of gastric cancer or Helicobacter pylori infection. The data for the ABO blood group was collected from 1045 cases of gastric cancer, whereby the patient underwent a gastrectomy in Ruijin Hospital, Shanghai. The information on the ABO blood group from 53,026 healthy blood donors was enrolled as control. We searched the Pubmed database on the relationship between ABO blood groups and gastric cancer risk for meta-analysis. In our case-control study, the risk of gastric cancer in blood group A was significantly higher than that in non-A groups (O, B and AB) (odd ratio, OR1.34; 95% confidential interval, CI 1.25–1.44). Compared with non-O groups (A, B and AB), individuals with blood group O demonstrated a reduced risk of gastric cancer (OR = 0.80; 95% CI 0.72–0.88). The proportion of H. pylori infection in blood group A individuals was significantly higher than that in non-A blood groups (OR = 1.42; 95% CI 1.05–1.93). We further combined our data with the published data of others, and crossreferenced the risk of gastric cancer with the blood type, finding consistent evidence that gastric cancer risk in the blood A group was higher than that in the non-A groups (OR = 1.11; 95% CI 1.07–1.15), and that blood type O individuals were consistently shown gastric cancer risk reduction (OR = 0.91; 95% CI 0.89–0.94). Our study concluded that there was a slightly increased risk of gastric cancer in blood group A individuals, and people with blood type A are more prone to be infected by H. pylori than other ABO blood type individuals, whereas, a slightly decreased risk of gastric cancer was identified in blood type O individuals.     

Mouse Gastric Epithelial Cells Resist CagA Delivery by the Helicobacter pylori Type IV Secretion System

The initial step in bacterial infection is adherence of the bacterium to the target cell surface. Helicobacter pylori exploits the interaction of bacterial adhesin protein HopQ with human epithelial CEACAMs (CEACAM1, 5, and 6) to stably adhere to gastric epithelial cells, which is necessary for delivery of the H. pylori CagA oncoprotein into the epithelial cells via a type IV secretion system. In contrast to human CEACAMs, however, HopQ does not interact with Ceacam1 (mouse CEACAM1) in vitro or in CHO cells ectopically expressing Ceacam1. Since the mouse genome lacks Ceacam5 and Ceacam6, no significant HopQ–Ceacam interaction may occur in mouse gastric epithelial cells. Here, we found that the mouse stomach has a much lower expression level of Ceacam1 than the expression level of CEACAM1 in the human stomach. Consistently, mouse gastric epithelial cells resist CagA delivery by cagA-positive H. pylori, and the delivery is restored by ectopic expression of human CEACAM1 or CEACAM5 in mouse gastric epithelial cells. Thus, despite the fact that mice are routinely used for H. pylori infection studies, a low expression level of Ceacam1 in the mouse stomach together with the loss or greatly reduced interaction of HopQ with Ceacams make the mouse an inappropriate model for studying the role of H. pylori-delivered CagA in gastric pathogenesis, including the development of gastric cancer.     

Outer Membrane Vesicle Production by Helicobacter pylori Represents an Approach for the Delivery of Virulence Factors CagA,VacA and UreA into Human Gastric Adenocarcinoma (AGS) Cells

Helicobacter pylori infection is the etiology of several gastric-related diseases including gastric cancer. Cytotoxin associated gene A (CagA), vacuolating cytotoxin A (VacA) and α-subunit of urease (UreA) are three major virulence factors of H. pylori, and each of them has a distinct entry pathway and pathogenic mechanism during bacterial infection. H. pylori can shed outer membrane vesicles (OMVs). Therefore, it would be interesting to explore the production kinetics of H. pylori OMVs and its connection with the entry of key virulence factors into host cells. Here, we isolated OMVs from H. pylori 26,695 strain and characterized their properties and interaction kinetics with human gastric adenocarcinoma (AGS) cells. We found that the generation of OMVs and the presence of CagA, VacA and UreA in OMVs were a lasting event throughout different phases of bacterial growth. H. pylori OMVs entered AGS cells mainly through macropinocytosis/phagocytosis. Furthermore, CagA, VacA and UreA could enter AGS cells via OMVs and the treatment with H. pylori OMVs would cause cell death. Comparison of H. pylori 26,695 and clinical strains suggested that the production and characteristics of OMVs are not only limited to laboratory strains commonly in use, but a general phenomenon to most H. pylori strains.     

Mucin 1 Gene (MUC1) and Gastric-Cancer Susceptibility

Gastric cancer (GC) is one of the major malignant diseases worldwide, especially in Asia. It is classified into intestinal and diffuse types. While the intestinal-type GC (IGC) is almost certainly caused by Helicobacter pylori (HP) infection, its role in the diffuse-type GC (DGC) appears limited. Recently, genome-wide association studies (GWAS) on Japanese and Chinese populations identified chromosome 1q22 as a GC susceptibility locus which harbors mucin 1 gene (MUC1) encoding a cell membrane-bound mucin protein. MUC1 has been known as an oncogene with an anti-apoptotic function in cancer cells; however, in normal gastric mucosa, it is anticipated that the mucin 1 protein has a role in protecting gastric epithelial cells from a variety of external insults which cause inflammation and carcinogenesis. HP infection is the most definite insult leading to GC, and a protective function of mucin 1 protein has been suggested by studies on Muc1 knocked-out mice.     

The Helicobacter pylori CagY Protein Drives Gastric Th1 and Th17 Inflammation and B Cell Proliferation in Gastric MALT Lymphoma

Background: the neoplastic B cells of the Helicobacter pylori-related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma proliferate in response to H. pylori, however, the nature of the H. pylori antigen responsible for proliferation is still unknown. The purpose of the study was to dissect whether CagY might be the H. pylori antigen able to drive B cell proliferation. Methods: the B cells and the clonal progeny of T cells from the gastric mucosa of five patients with MALT lymphoma were compared with those of T cell clones obtained from five H. pylori–infected patients with chronic gastritis. The T cell clones were assessed for their specificity to H. pylori CagY, cytokine profile and helper function for B cell proliferation. Results: 22 of 158 CD4⁺ (13.9%) gastric clones from MALT lymphoma and three of 179 CD4⁺ (1.7%) clones from chronic gastritis recognized CagY. CagY predominantly drives Interferon-gamma (IFN-γ) and Interleukin-17 (IL-17) secretion by gastric CD4⁺ T cells from H. pylori-infected patients with low-grade gastric MALT lymphoma. All MALT lymphoma-derived clones dose dependently increased their B cell help, whereas clones from chronic gastritis lost helper activity at T-to-B-cell ratios greater than 1. Conclusion: the results obtained indicate that CagY drives both B cell proliferation and T cell activation in gastric MALT lymphomas.     

Use of Fourier-Transform Infrared Spectroscopy (FT-IR) for Monitoring Experimental Helicobacter pylori Infection and Related Inflammatory Response in Guinea Pig Model

Infections due to Gram-negative bacteria Helicobacter pylori may result in humans having gastritis, gastric or duodenal ulcer, and even gastric cancer. Investigation of quantitative changes of soluble biomarkers, correlating with H. pylori infection, is a promising tool for monitoring the course of infection and inflammatory response. The aim of this study was to determine, using an experimental model of H. pylori infection in guinea pigs, the specific characteristics of infrared spectra (IR) of sera from H. pylori infected (40) vs. uninfected (20) guinea pigs. The H. pylori status was confirmed by histological, molecular, and serological examination. The IR spectra were measured using a Fourier-transform (FT)-IR spectrometer Spectrum 400 (PerkinElmer) within the range of wavenumbers 3000–750 cm⁻¹ and converted to first derivative spectra. Ten wavenumbers correlated with H. pylori infection, based on the chi-square test, were selected for a K-nearest neighbors (k-NN) algorithm. The wavenumbers correlating with infection were identified in the W2 and W3 windows associated mainly with proteins and in the W4 window related to nucleic acids and hydrocarbons. The k-NN for detection of H. pylori infection has been developed based on chemometric data. Using this model, animals were classified as infected with H. pylori with 100% specificity and 97% sensitivity. To summarize, the IR spectroscopy and k-NN algorithm are useful for monitoring experimental H. pylori infection and related inflammatory response in guinea pig model and may be considered for application in humans.     

Matrix Metalloproteinases in Helicobacter pylori–Associated Gastritis and Gastric Cancer

Gastric cancer is one of the leading causes of the cancer-related mortality worldwide. The etiology of this disease is complex and involves genetic predisposition and environmental factors, including Helicobacter pylori. Infection of the stomach with H. pylori leads to gastritis and gastric atrophy, which can progress stepwise to gastric cancer. Matrix metalloproteinases (MMPs) actively participate in the pathology development. The further progression of gastric cancer seems to be less dependent on bacteria but of intra-tumor cell dynamics. Bioinformatics data confirmed an important role of the extracellular matrix constituents and specific MMPs in stomach carcinoma invasion and metastasis, and revised their potential as predictors of the disease outcome. In this review, we describe, in detail, the impact of MMPs in H. pylori–associated gastritis and gastric cancer.     

Current Status on Stem Cells and Cancers of the Gastric Epithelium

Gastric cancer is still a leading cause of cancer-related mortality worldwide in spite of declining incidence. Gastric cancers are, essentially, adenocarcinomas and one of the strongest risk factors is still infection with Helicobacter pylori. Within the last years, it became clear that gastric self-renewal and carcinogenesis are intimately linked, particularly during chronic inflammatory conditions. Generally, gastric cancer is now regarded as a disease resulting from dysregulated differentiation of stem and progenitor cells, mainly due to an inflammatory environment. However, the situation in the stomach is rather complex, consisting of two types of gastric units which show bidirectional self-renewal from an unexpectedly large variety of progenitor/stem cell populations. As in many other tumors, cancer stem cells have also been characterized for gastric cancer. This review focuses on the various gastric epithelial stem cells, how they contribute to self-renewal and which routes are known to gastric adenocarcinomas, including their stem cells.     

The Role of Formyl Peptide Receptors in Permanent and Low-Grade Inflammation: Helicobacter pylori Infection as a Model

Formyl peptide receptors (FPRs) are cell surface pattern recognition receptors (PRRs), belonging to the chemoattractant G protein-coupled receptors (GPCRs) family. They play a key role in the innate immune system, regulating both the initiation and the resolution of the inflammatory response. FPRs were originally identified as receptors with high binding affinity for bacteria or mitochondria N-formylated peptides. However, they can also bind a variety of structurally different ligands. Among FPRs, formyl peptide receptor-like 1 (FPRL1) is the most versatile, recognizing N-formyl peptides, non-formylated peptides, and synthetic molecules. In addition, according to the ligand nature, FPRL1 can mediate either pro- or anti-inflammatory responses. Hp(2-20), a Helicobacter pylori-derived, non-formylated peptide, is a potent FPRL1 agonist, participating in Helicobacter pylori-induced gastric inflammation, thus contributing to the related site or not-site specific diseases. The aim of this review is to provide insights into the role of FPRs in H. pylori-associated chronic inflammation, which suggests this receptor as potential target to mitigate both microbial and sterile inflammatory diseases.     

In Vitro Framework to Assess the Anti-Helicobacter pylori Potential of Lactic Acid Bacteria Secretions as Alternatives to Antibiotics

Helicobacter pylori is a prevalent bacterium that can cause gastric ulcers and cancers. Lactic acid bacteria (LAB) ameliorate treatment outcomes against H. pylori, suggesting that they could be a source of bioactive molecules usable as alternatives to current antibiotics for which resistance is mounting. We developed an in vitro framework to compare the anti-H. pylori properties of 25 LAB and their secretions against H. pylori. All studies were done at acidic and neutralized pH, with or without urea to mimic various gastric compartments. Eighteen LAB strains secreted molecules that curtailed the growth of H. pylori and the activity was urea-resistant in five LAB. Several LAB supernatants also reduced the urease activity of H. pylori. Pre-treatment of H. pylori with acidic LAB supernatants abrogated its flagella-mediated motility and decreased its ability to elicit pro-inflammatory IL-8 cytokine from human gastric cells, without reverting the H. pylori-induced repression of other pro-inflammatory cytokines. This study identified the LAB that have the most anti-H. pylori effects, decreasing its viability, its production of virulence factors, its motility and/or its ability to elicit pro-inflammatory IL-8 from gastric cells. Once identified, these molecules can be used as alternatives or complements to current antibiotics to fight H. pylori infections.     

Helicobacter pylori,Protected from Antibiotics and Stresses Inside Candida albicans Vacuoles,Cause Gastritis in Mice

Due to (i) the simultaneous presence of Helicobacter pylori (ulcer-induced bacteria) and Candida albicans in the stomach and (ii) the possibility of prokaryotic–eukaryotic endosymbiosis (intravacuolar H. pylori in the yeast cells) under stresses, we tested this symbiosis in vitro and in vivo. To that end, intravacuolar H. pylori were induced by the co-incubation of C. albicans with H. pylori under several stresses (acidic pH, non-H. pylori-enrichment media, and aerobic environments); the results were detectable by direct microscopy (wet mount) and real-time polymerase chain reaction (PCR). Indeed, intravacuolar H. pylori were predominant under all stresses, especially the lower pH level (pH 2–3). Interestingly, the H. pylori (an amoxicillin-sensitive strain) inside C. albicans were protected from the antibiotic (amoxicillin), while extracellular H. pylori were neutralizable, as indicated by the culture. In parallel, the oral administration of intravacuolar H. pylori in mice caused H. pylori colonization in the stomach resulting in gastritis, as indicated by gastric histopathology and tissue cytokines, similar to the administration of free H. pylori (extra-Candida bacteria). In conclusion, Candida protected H. pylori from stresses and antibiotics, and the intravacuolar H. pylori were able to be released from the yeast cells, causing gastric inflammation with neutrophil accumulations.     

Characterization and Genomic Analysis of a New Phage Infecting Helicobacter pylori

Helicobacter pylori, a significant human gastric pathogen, has been demonstrating increased antibiotic resistance, causing difficulties in infection treatment. It is therefore important to develop alternatives or complementary approaches to antibiotics to tackle H. pylori infections, and (bacterio)phages have proven to be effective antibacterial agents. In this work, prophage isolation was attempted using H. pylori strains and UV radiation. One phage was isolated and further characterized to assess potential phage-inspired therapeutic alternatives to H. pylori infections. HPy1R is a new podovirus prophage with a genome length of 31,162 bp, 37.1% GC, encoding 36 predicted proteins, of which 17 were identified as structural. Phage particles remained stable at 37 °C, from pH 3 to 11, for 24 h in standard assays. Moreover, when submitted to an in vitro gastric digestion model, only a small decrease was observed in the gastric phase, suggesting that it is adapted to the gastric tract environment. Together with its other characteristics, its capability to suppress H. pylori population levels for up to 24 h post-infection at multiplicities of infection of 0.01, 0.1, and 1 suggests that this newly isolated phage is a potential candidate for phage therapy in the absence of strictly lytic phages.     

Cortactin Is Required for Efficient FAK,Src and Abl Tyrosine Kinase Activation and Phosphorylation of Helicobacter pylori CagA

Cortactin is a well-known regulatory protein of the host actin cytoskeleton and represents an attractive target of microbial pathogens like Helicobacter pylori. H. pylori manipulates cortactin’s phosphorylation status by type-IV secretion-dependent injection of its virulence protein CagA. Multiple host tyrosine kinases, like FAK, Src, and Abl, are activated during infection, but the pathway(s) involved is (are) not yet fully established. Among them, Src and Abl target CagA and stimulate tyrosine phosphorylation of the latter at its EPIYA-motifs. To investigate the role of cortactin in more detail, we generated a CRISPR/Cas9 knockout of cortactin in AGS gastric epithelial cells. Surprisingly, we found that FAK, Src, and Abl kinase activities were dramatically downregulated associated with widely diminished CagA phosphorylation in cortactin knockout cells compared to the parental control. Together, we report here a yet unrecognized cortactin-dependent signaling pathway involving FAK, Src, and Abl activation, and controlling efficient phosphorylation of injected CagA during infection. Thus, the cortactin status could serve as a potential new biomarker of gastric cancer development.