Molecular Characterization of Antimicrobial Resistance and Virulence Genotyping among Helicobacter pylori-Positive Dyspeptic Patients in North Iran.

BACKGROUND: Iran has a relatively high prevalence of H. pylori, which correlates with high-risk areas for gastric cancer worldwide. METHODS: Our study aimed to investigate the underlying genetic mechanisms associated with resistance to metronidazole (frxA, rdxA), clarithromycin (23S rRNA), tetracycline (16S rRNA), and fluoroquinolone (gyrA) in H. pylori-positive dyspeptic patients using PCR and sequencing. We further examined the potential correlation between resistance profiles and various virulence genotypes. RESULTS: The rates of genetic mutations associated with resistance to metronidazole, fluoroquinolone, clarithromycin, and tetracycline were found to be 68%, 32.1%, 28.4%, and 11.1%, respectively. Well-documented multiple antibiotic resistance mutations were detected, such as rdxA and frxA (with missense and frameshift alterations), gyrA (Asp91, Asn87), 23S rRNA (A2142G, A2143G), and 16S rRNA (triple-base-pair substitutions AGA926-928→TTC). The cagA+ and vacA s1/m1 types were the predominant genotypes in our study. With the exception of metronidazole and tetracycline, no significant correlation was observed between the cagA+ and cagL+ genotypes and resistance-associated mutations. CONCLUSION: The prevalence of antibiotic resistance-associated mutations in H. pylori was remarkably high in this region, particularly to metronidazole, ciprofloxacin, and clarithromycin. By conducting a simultaneous screening of virulence and resistance genotypes, clinicians can make informed decisions regarding the appropriate therapeutic regimen to prevent the escalation of antibiotic resistance against H. pylori infection in this specific geographical location.

Design and Application of pH-Responsive Liposomes for Site-Specific Delivery of Cytotoxin from Cobra Venom.

Background: Current immunotherapies with unexpected severe side effects and treatment resistance have not resulted in the desired outcomes for patients with melanoma, and there is a need to discover more effective medications. Cytotoxin (CTX) from Cobra Venom has been established to have favorable cytolytic activity and antitumor efficacy and is regarded as a promising novel anticancer agent. However, amphiphilic CTX with excellent anionic phosphatidylserine lipid-binding ability may also damage normal cells. Methods: We developed pH-responsive liposomes with a high CTX load (CTX@PSL) for targeted acidic-stimuli release of drugs in the tumor microenvironment. The morphology, size, zeta potential, drug-release kinetics, and preservation stability were characterized. Cell uptake, apoptosis-promoting effects, and cytotoxicity were assessed using MTT assay and flow cytometry. Finally, the tissue distribution and antitumor effects of CTX@PSL were systematically assessed using an in vivo imaging system. Results: CTX@PSL exhibited high drug entrapment efficiency, drug loading, stability, and a rapid release profile under acidic conditions. These nanoparticles, irregularly spherical in shape and small in size, can effectively accumulate at tumor sites (six times higher than free CTX) and are rapidly internalized into cancer cells (2.5-fold higher cell uptake efficiency). CTX@PSL displayed significantly stronger cytotoxicity (IC50 0.25 µg/mL) and increased apoptosis in than the other formulations (apoptosis rate 71.78±1.70%). CTX@PSL showed considerably better tumor inhibition efficacy than free CTX or conventional liposomes (tumor inhibition rate 79.78±5.93%). Conclusion: Our results suggest that CTX@PSL improves tumor-site accumulation and intracellular uptake for sustained and targeted CTX release. By combining the advantages of CTX and stimuli-responsive nanotechnology, the novel CTX@PSL nanoformulation is a promising therapeutic candidate for cancer treatment.

Immunoinformatics-Based Designing of Novel and Potent Multi-Epitope PSA D15 and Cag11 Immunogens for Helicobacter pylori Immunodiagnostic Assay Development.

Helicobacter pylori (H. pylori) strain is the most genetically diverse pathogenic bacterium and now alarming serious human health concern ranging from chronic gastritis to gastric cancer and human death all over the world. Currently, the majority of commercially available diagnostic assays for H. pylori is a challenging task due to the heterogeneity of virulence factors in various geographical regions. In this concern, designing of universal multi-epitope immunogenic biomarker targeted for all H. pylori strains would be crucial to successfully immunodiagnosis assay and vaccine development for H. pylori infection. Hence, the present study aimed to explore the potential immunogenic epitopes of PSA D15 and Cag11 proteins of H. pylori, using immunoinformatics web tools in order to design novel immune-reactive multi-epitope antigens for enhanced immunodiagnosis in humans. Through an in silico immunoinformatics approach, high-ranked B-cell, MHC-I, and MHC-II epitopes of PSA D15 and Cag11 proteins were predicted, screened, and selected. Subsequently, a novel multi-epitope PSA D15 and Cag11 antigens were designed by fused the high-ranked B-cell, MHC-I, and MHC-II epitopes and 50S ribosomal protein L7/L12 adjuvant using linkers. The antigenicity, solubility, physicochemical properties, secondary and tertiary structures, 3D model refinement, and validations were carried. Furthermore, the designed multi-epitope antigens were subjected to codon adaptation and in silico cloning, immune response simulation, and molecular docking with receptor molecules. A novel, stable multi-epitope PSA D15 and Cag11 H. pylori antigens were developed and immune simulation of the designed antigens showed desirable levels of immunological response. Molecular docking of designed antigens with immune receptors (B-cell, MHC-I, MHC-II, and TLR-2/4) revealed robust interactions and stable binding affinity to the receptors. The codon optimized and in silico cloned showed that the designed antigens were successfully expressed (CAI value of 0.95 for PSA D15 and 1.0 for Cag11) after inserted into pET-32ba (+) plasmid of the E. coli K12 strain. In conclusion, this study revealed that the designed multi-epitope antigens have a huge immunological potential candidate biomarker and useful in developing immunodiagnostic assays and vaccines for H. pylori infection.

A High-Homology Region Provides the Possibility of Detecting ß-Barrel Pore-Forming Toxins from Various Bacterial Species.

The pathogenicity of many bacteria, including Bacillus cereus and Staphylococcus aureus, depends on pore-forming toxins (PFTs), which cause the lysis of host cells by forming pores in the membranes of eukaryotic cells. Bioinformatic analysis revealed a region homologous to the Lys171-Gly250 sequence in hemolysin II (HlyII) from B. cereus in over 600 PFTs, which we designated as a "homologous peptide". Three ß-barrel PFTs were used for a detailed comparative analysis. Two of them-HlyII and cytotoxin K2 (CytK2)-are synthesized in Bacillus cereus sensu lato; the third, S. aureus α-toxin (Hla), is the most investigated representative of the family. Protein modeling showed certain amino acids of the homologous peptide to be located on the surface of the monomeric forms of these ß-barrel PFTs. We obtained monoclonal antibodies against both a cloned homologous peptide and a 14-membered synthetic peptide, DSFNTFYGNQLFMK, as part of the homologous peptide. The HlyII, CytK2, and Hla regions recognized by the obtained antibodies, as well as an antibody capable of suppressing the hemolytic activity of CytK2, were identified in the course of this work. Antibodies capable of recognizing PFTs of various origins can be useful tools for both identification and suppression of the cytolytic activity of PFTs.

Combined Use of Helicobacter pylori Genotyping and CDX2 Expression as a Predictor of Malignant Potential in Gastric Intestinal Metaplasia.

OBJECTIVE: Gastrointestinal metaplasia (GIM) has a close relationship with gastric cancer (GC), but it is unclear how to judge which GIM could develop into GC. This study aimed to assess the role of CDX2 and its association with Helicobacter pylori (H.pylori) genotypes in GIM. METHODS: CagA and vacA genes were identified via PCR in 466 H. pylori-positive gastric tissues, including gastritis (n=104), GIM diagnosed endoscopically (GIM-1; n=82), gastric cancer (GC; n=173), and paired adjacent GIM tumors resected surgically (GIM-2; n=107). GIM was subclassified per the HID- AB pH2.5-PAS as follows: type I (n=23), type II (n=43), and type III (n=16) in GIM-1; type I (n=8), type II (n=40), and type III (n=59) in GIM-2. CDX2 expression was evaluated immunohistochemically. RESULTS: In GIM-1, the infection rate of vacAm2 (55.8%) and vacAs1m2 (53.5%) was higher in subtype II than in others (P<0.05), while that of vacAm1 (49.2%) and vacAs1m1 (33.9%) was higher in subtype III than in others. The cagA+ rate was higher in subtypes I (75.0%) and III (64.4%) than in subtype II (40.0%; P<0.05) respectively. CDX2 was upregulated in subtype I than in subtypes II and III in GIM-1 and GIM-2. In GIM-2 and GC, CDX2 was downregulated in vacAm1, vacAs1m1, and cagA+ (P<0.05). The predominant genotype was vacAs1m2 in subtype II of GIM-1, CDX2 expression remaining unaltered; however, the predominant genotype was cagA+ vacAs1m1 in subtypes II and III of GIM-2, negatively correlated with CDX2 expression. CONCLUSION: These GIM subtypes (cagA+ vacAs1m1 H. pylori-positive GIM with negative CDX2 expression) resemble GC and should be evaluated similar to cancerous GIM.

Dual RNA sequencing of Helicobacter pylori and host cell transcriptomes reveals ontologically distinct host-pathogen interaction.

Helicobacter pylori is a highly successful pathogen that poses a substantial threat to human health. However, the dynamic interaction between H. pylori and the human gastric epithelium has not been fully investigated. In this study, using dual RNA sequencing technology, we characterized a cytotoxin-associated gene A (cagA)-modulated bacterial adaption strategy by enhancing the expression of ATP-binding cassette transporter-related genes, metQ and HP_0888, upon coculturing with human gastric epithelial cells. We observed a general repression of electron transport-associated genes by cagA, leading to the activation of oxidative phosphorylation. Temporal profiling of host mRNA signatures revealed the downregulation of multiple splicing regulators due to bacterial infection, resulting in aberrant pre-mRNA splicing of functional genes involved in the cell cycle process in response to H. pylori infection. Moreover, we demonstrated a protective effect of gastric H. pylori colonization against chronic dextran sulfate sodium (DSS)-induced colitis. Mechanistically, we identified a cluster of propionic and butyric acid-producing bacteria, Muribaculaceae, selectively enriched in the colons of H. pylori-pre-colonized mice, which may contribute to the restoration of intestinal barrier function damaged by DSS treatment. Collectively, this study presents the first dual-transcriptome analysis of H. pylori during its dynamic interaction with gastric epithelial cells and provides new insights into strategies through which H. pylori promotes infection and pathogenesis in the human gastric epithelium. IMPORTANCE: Simultaneous profiling of the dynamic interaction between Helicobacter pylori and the human gastric epithelium represents a novel strategy for identifying regulatory responses that drive pathogenesis. This study presents the first dual-transcriptome analysis of H. pylori when cocultured with gastric epithelial cells, revealing a bacterial adaptation strategy and a general repression of electron transportation-associated genes, both of which were modulated by cytotoxin-associated gene A (cagA). Temporal profiling of host mRNA signatures dissected the aberrant pre-mRNA splicing of functional genes involved in the cell cycle process in response to H. pylori infection. We demonstrated a protective effect of gastric H. pylori colonization against chronic DSS-induced colitis through both in vitro and in vivo experiments. These findings significantly enhance our understanding of how H. pylori promotes infection and pathogenesis in the human gastric epithelium and provide evidence to identify targets for antimicrobial therapies.

Helicobacter pylori infection and Parkinson's Disease: etiology, pathogenesis and levodopa bioavailability.

Parkinson's disease (PD), a neurodegenerative disorder with an unknown etiology, is primarily characterized by the degeneration of dopamine (DA) neurons. The prevalence of PD has experienced a significant surge in recent years. The unidentified etiology poses limitations to the development of effective therapeutic interventions for this condition. Helicobacter pylori (H. pylori) infection has affected approximately half of the global population. Mounting evidences suggest that H. pylori infection plays an important role in PD through various mechanisms. The autotoxin produced by H. pylori induces pro-inflammatory cytokines release, thereby facilitating the occurrence of central inflammation that leads to neuronal damage. Simultaneously, H. pylori disrupts the equilibrium of gastrointestinal microbiota with an overgrowth of bacteria in the small intestinal known as small intestinal bacterial overgrowth (SIBO). This dysbiosis of the gut flora influences the central nervous system (CNS) through microbiome-gut-brain axis. Moreover, SIBO hampers levodopa absorption and affects its therapeutic efficacy in the treatment of PD. Also, H. pylori promotes the production of defensins to regulate the permeability of the blood-brain barrier, facilitating the entry of harmful factors into the CNS. In addition, H. pylori has been found to induce gastroparesis, resulting in a prolonged transit time for levodopa to reach the small intestine. H. pylori may exploit levodopa to facilitate its own growth and proliferation, or it can inflict damage to the gastrointestinal mucosa, leading to gastrointestinal ulcers and impeding levodopa absorption. Here, this review focused on the role of H. pylori infection in PD from etiology, pathogenesis to levodopa bioavailability.

[Redox Biology Involved in the Toxicity of Enterohemorrhagic Escherichia coli Toxin SubAB].

AB5 toxins of pathogenic bacteria enter host cells and utilize the retrograde trafficking pathway to translocate to the cytoplasm and exert its pathogenesis. Cholera toxin and Shiga toxin reach the endoplasmic reticulum (ER), and the A subunit undergoes redox regulation by ER proteins to become active fragments, which pass through the ER membrane and translocate to the cytoplasm. By acting on molecular targets in the cytoplasm, the normal function of host cells are disrupted, causing diseases. ER chaperone proteins such as protein disulfide isomerase (PDI) and binding immunoglobulin protein (BiP) induce conformational changes triggered by the reduction of disulfide bonds in the A subunit. This is thought to be dependent on cysteine thiol-mediated redox regulation, but the detailed mechanism remains unclear. On the other hand, subtilase cytotoxin (SubAB), produced by enterohemorrhagic Escherichia coli (EHEC), localizes to the ER without translocating to the cytoplasm and cleaves BiP as a substrate. Therefore, it is thought that ER stress-based cytotoxicity and intestinal bleeding occur without translocating to the cytoplasm. We reported that PDI is involved in BiP cleavage through SubAB localization to the ER. Like other AB5 toxins, this indicates the involvement of redox regulation via chaperone proteins in the ER, but also suggests that SubAB does not translocate to the cytoplasm because it cleaves BiP. Although there are few reports on the redox state of ER protein thiols, it is suggested that polysulfidation, which is discussed in this symposium, may be involved.

High-throughput virtual screening, pharmacophore modelling and antagonist effects of small molecule inhibitors against cytotoxin-induced cytotoxicity.

Cobra venom cytotoxins (CTX) cause dermonecrosis in envenomed patients who suffered from limb amputations due to the limitation of serotherapy-based antivenoms. This study aimed to identify small molecule inhibitors against CTX. A structure-based high-throughput virtual screening (HTVS) was conducted based on a conserved CTX, using the Natural Product Activity and Species Source (NPASS) screening library. The hits were valerenic acid, 1-oxo-2H-isoquinoline-4-carboxylic acid, acenaphthene, and 5-bromopyrrole-2-carboxamide, which interacted with contemporary antivenom binding site A and functional loops I-III of CTX, respectively, in molecular docking studies. Furthermore, molecular dynamic simulations were performed along with analysis of ligand fitness through their pharmacophore and pharmacokinetics properties. The antagonist effects of these hits on CTX-induced cytotoxicity were examined in human keratinocytes (HaCaT). Despite having a low binding affinity (KD = 14.45 × 10-4 M), acenaphthene demonstrated a significant increase of cell viability at 6 h and 24 h in experimental envenomed HaCaT. It also demonstrated the highest neutralization potency against CTX with a median effective concentration (EC50) of 0.05 mL/mg. Acenaphthene interacted with the functional loop II, which is the crucial cytotoxic site of CTX. It has an aromatic ring as its primary pharmacophoric feature, commonly used for rational drug design. In conclusion, acenaphthene could be a promising lead compound as a small molecule inhibitor.Communicated by Ramaswamy H. Sarma.

G-protein-coupled estrogen receptor prevents nuclear factor-kappa B promoter activation by Helicobacter pylori cytotoxin-associated gene A in gastric cancer cells.

Helicobacter pylori is a well-known pathogen that causes chronic gastritis, leading to the development of gastric cancer. This bacterium has also been detected in dogs, and symptoms similar to those in humans have been reported. The cytotoxin-associated gene A (CagA) is involved in pathogenesis through aberrant activation of host signal transduction, including the nuclear factor-kappa B (NF-κB) pathway. We have previously shown the anti-inflammatory effect of the G-protein-coupled estrogen receptor (GPER) via inhibiting of NF-κB activation in several cells. Therefore, here, we investigated the effect of GPER on CagA-mediated NF-κB promoter activity and showed that CagA overexpression in gastric cancer cells activated the NF-κB reporter and induced interleukin 8 (il-8) expression, both of which were inhibited by the GPER agonist.

Computational insights into VacA toxin inhibition: harnessing FDA-approved antibiotics against Helicobacter pylori.

Cancer is a condition in which a few of the body's cells grow beyond its control and spread to other outward regions. Globally, gastric cancer (GC) is third most common cause of cancer-related mortality and the fourth most common kind of cancer. Persistent infection of VacA-positive Helicobacter pylori (H. pylori) modulates cellular physiology and leads to GC. About ∼70% of H. pylori are positive for vacuolating cytotoxin-A (VacA), and it infects ∼80-90% of world populations. Herein, for first time, we repurposed FDA-approved gram-negative antibiotics, which are feasible alternatives to existing regimens and may be used in combinatorial treatment against VacA-positive H. pylori. Out of 110 FDA-approved antibiotics, we retrieved 92 structures, which were screened against the VacA protein. Moreover, we determined that the top eight hit antibiotics viz; cefpiramide, cefiderocol, eravacycline, doxycycline, ceftriaxone, enoxacin, tedizolid, and cefamandole show binding free energies of -9.1, -8.9, -8.1, -8.0, -7.9, -7.8, -7.8 and -7.8 Kcal/mol, respectively, with VacA protein. Finally, we performed 100 ns duplicate MD simulations on the top eight selected antibiotics showing strong VacA binding. Subsequently, five antibiotics, including cefiderocol, cefpiramide, doxycycline, enoxacin, and tedizolid show stable ligand protein distance and good binding affinity revealed by the MM-PBSA scheme. Among the five antibiotics cefiderocol act as the most potent inhibitor (-28.33 kcal/mol). Furthermore, we also identified the hotspot residue like Asn-506, Tyr-529, and Phe-483 which control the interaction. Concisely, we identified antibiotics that can be repurposed against VacA of H. pylori and explored their molecular mechanism of interaction with VacA.Communicated by Ramaswamy H. Sarma.

Molecular pathogenesis of Chlamydia trachomatis.

Chlamydia trachomatis is a strict intracellular human pathogen. It is the main bacterial cause of sexually transmitted infections and the etiologic agent of trachoma, which is the leading cause of preventable blindness. Despite over 100 years since C. trachomatis was first identified, there is still no vaccine. However in recent years, the advancement of genetic manipulation approaches for C. trachomatis has increased our understanding of the molecular pathogenesis of C. trachomatis and progress towards a vaccine. In this mini-review, we aimed to outline the factors related to the developmental cycle phase and specific pathogenesis activity of C. trachomatis in order to focus priorities for future genetic approaches. We highlight the factors known to be critical for developmental cycle stages, gene expression regulatory factors, type III secretion system and their effectors, and individual virulence factors with known impacts.

Venomous Noodles: The Evolution of Toxins in Nemertea through Positive Selection and Gene Duplication.

Some, probably most and perhaps all, members of the phylum Nemertea are poisonous, documented so far from marine and benthic specimens. Although the toxicity of these animals has been long known, systematic studies on the characterization of toxins, mechanisms of toxicity, and toxin evolution for this group are scarce. Here, we present the first investigation of the molecular evolution of toxins in Nemertea. Using a proteo-transcriptomic approach, we described toxins in the body and poisonous mucus of the pilidiophoran Lineus sanguineus and the hoplonemertean Nemertopsis pamelaroeae. Using these new and publicly available transcriptomes, we investigated the molecular evolution of six selected toxin gene families. In addition, we also characterized in silico the toxin genes found in the interstitial hoplonemertean, Ototyphlonemertes erneba, a meiofaunal taxa. We successfully identified over 200 toxin transcripts in each of these species. Evidence of positive selection and gene duplication was observed in all investigated toxin genes. We hypothesized that the increased rates of gene duplications observed for Pilidiophora could be involved with the expansion of toxin genes. Studies concerning the natural history of Nemertea are still needed to understand the evolution of their toxins. Nevertheless, our results show evolutionary mechanisms similar to other venomous groups.

Host cell sensing and restoration of mitochondrial function and metabolism within Helicobacter pylori VacA intoxicated cells.

IMPORTANCE: Persistent human gastric infection with Helicobacter pylori is the single most important risk factor for development of gastric malignancy, which is one of the leading causes of cancer-related deaths worldwide. An important virulence factor for Hp colonization and severity of gastric disease is the protein exotoxin VacA, which is secreted by the bacterium and modulates functional properties of gastric cells. VacA acts by damaging mitochondria, which impairs host cell metabolism through impairment of energy production. Here, we demonstrate that intoxicated cells have the capacity to detect VacA-mediated damage, and orchestrate the repair of mitochondrial function, thereby restoring cellular health and vitality. This study provides new insights into cellular recognition and responses to intracellular-acting toxin modulation of host cell function, which could be relevant for the growing list of pathogenic microbes and viruses identified that target mitochondria as part of their virulence strategies.

Expression and purification of epitope vaccine against four virulence proteins from Helicobacter pylori and construction of label-free electrochemical immunosensor.

The epitope vaccine against four virulence proteins (FVpE) from Helicobacter pylori (H. pylori) was expressed and purified. Western blot and Enzyme-linked Immunosorbent Assays (ELISA) were used to identify and investigate the immunoreactivity of FVpE protein. The immune-sensing platform based on titanium carbide/colloidal gold nanoparticles@carbon nanofiber/ionic liquid composites electrode was constructed for immobilizing FVpE. Electrochemical impedance spectroscopy (EIS) was used to study the electrochemical properties of the modified electrodes. The relevant influenced factors were optimized including pH value, antigen concentration, and incubating time. The prepared H. pylori label-free electrochemical immunosensor was used for antibody detection using differential pulse voltammetry (DPV). Under the optimal experimental conditions, the linear ranges of H. pylori antibodies, including anti-H. pylori, cytotoxin-associated gene A (CagA), vacuolating cytotoxin-associated gene A (VacA), and urease A (UreA), were all 0.1-5 ng mL-1, except urease B (UreB, 0.1-4.5 ng mL-1). The selectivity study showed that other antibodies had little influence on the detection of H. pylori antibodies. The immunosensor could be used to detect serum samples, and the recoveries were in the range of 68.5%-100.5%.

Chloroquine inhibited Helicobacter pylori-related gastric carcinogenesis by YAP-ß-catenin-autophagy axis.

YAP participates in autophagy associated with many diseases. In this study, we demonstrate that YAP promotes autophagy by interacting with beclin 1, upregulating beclin 1 and LC3B-II protein expression, and promoting autophagosome formation after H. pylori infection in a vacuolating cytotoxin A-dependent manner. The protein levels of ß-catenin in the cytoplasm and nuclei of GES-1 cells and the mRNA levels of Axin2, Myc, Lgr5, and Ccnd1 were increased in H. pylori-infected cells or YAP-overexpressed cells, but were decreased in YAP-silenced cells. The ß-catenin inhibitor XAV939 significantly downregulated autophagy, whereas the activator LiCl showed opposite effects. An H. pylori-infected mouse model of gastric carcinoma was successfully established. The mouse model showed that H. pylori infection, when combined with NMU, promoted the tumorigenesis of gastric tissues; increased IL-1ß, IL-6, and TNF-α levels; promoted NO release; and increased the expression of beclin 1, LC3B-II more than NMU alone. Chloroquine inhibited these phenomena, but did not completely attenuate the effects of H. pylori. These results demonstrate that chloroquine can be used as a drug for the treatment of H. pylori-related gastric cancer, but the treatment should simultaneously remove H. pylori.

Restoration of mitochondrial structure and function within Helicobacter pylori VacA intoxicated cells.

The Helicobacter pylori vacuolating cytotoxin (VacA) is an intracellular, mitochondrial-targeting exotoxin that rapidly causes mitochondrial dysfunction and fragmentation. Although VacA targeting of mitochondria has been reported to alter overall cellular metabolism, there is little known about the consequences of extended exposure to the toxin. Here, we describe studies to address this gap in knowledge, which have revealed that mitochondrial dysfunction and fragmentation are followed by a time-dependent recovery of mitochondrial structure, mitochondrial transmembrane potential, and cellular ATP levels. Cells exposed to VacA also initially demonstrated a reduction in oxidative phosphorylation, as well as increase in compensatory aerobic glycolysis. These metabolic alterations were reversed in cells with limited toxin exposure, congruent with the recovery of mitochondrial transmembrane potential and the absence of cytochrome c release from the mitochondria. Taken together, these results are consistent with a model that mitochondrial structure and function are restored in VacA-intoxicated cells.

A novel role for Helicobacter pylori cytotoxin-associated gene A in negative regulation of autophagy in human gastric cells.

BACKGROUND: Autophagy plays an important role in carcinogenesis and tumor progression in many cancers, including gastric cancer. Cytotoxin-associated gene A (CagA) is a well-known virulent factor in Helicobacter pylori (H. pylori) infection that plays a critical role in gastric inflammation and gastric cancer development. However, its role in autophagy during these processes remains unclear. Therefore, we aimed to clarify the role of CagA in autophagy in CagA-related inflammation. METHODS: We evaluated the autophagic index of AGS cells infected with wild-type cagA-positive H. pylori (Hp-WT) and cagA-knockout H. pylori (Hp-ΔcagA) and rat gastric mucosal (RGM1) cells transfected with CagA genes. To identify the mechanisms underlying the down regulation of autophagy in AGS cells infected with H. pylori, we evaluated protein and mRNA expression levels of autophagy core proteins using western blotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR). To determine whether autophagy induced the expression of the pro-inflammatory mediator, cyclooxygenase-2 (COX-2), we evaluated COX-2 expression in AGS cells treated with an autophagy inducer and inhibitor and infected with H. pylori. In addition, we evaluated whether COX-2 protein expression in AGS cells influenced beclin-1 (BECN1) expression with si-RNA transfection when infected with H. pylori. RESULTS: Autophagic flux assay using chloroquine showed that autophagy in AGS cells was significantly suppressed after H. pylori infection. The autophagic index of AGS cells infected with Hp-WT was decreased significantly when compared with that in AGS cells infected with Hp-ΔcagA. The autophagic index of RGM1 cells transfected with CagA was lower, suggesting that CagA inhibits autophagy. In addition, BECN1 expression levels in AGS cells infected with Hp-WT were reduced compared to those in AGS cells infected with Hp-ΔcagA. Furthermore, COX-2 expression in AGS cells infected with H. pylori was controlled in an autophagy-dependent manner. When AGS cells were transfected with small interfering RNA specific for BECN1 and infected with Hp-WT and Hp-ΔcagA, COX-2 was upregulated significantly in cells infected with Hp-ΔcagA. CONCLUSIONS: In conclusion, the H. pylori CagA protein negatively regulated autophagy by downregulating BECN1. CagA-induced autophagy inhibition may be a causative factor in promoting pro-inflammatory mediator production in human gastric epithelial cells.

Transcriptional analysis of Helicobacter pylori cytotoxic-associated gene-pathogenicity island in response to different pH levels and proton pump inhibitor exposure.

BACKGROUND: Long-term use of proton pump inhibitors (PPIs) can increase the risk of gastric cancer in Helicobacter pylori-infected patients; nevertheless, there is no data about their impact on the pathogenicity of H. pylori. This study aimed at investigating the transcriptional alteration of key gene mediators of cytotoxin-associated gene-pathogenicity island (cag-PAI) among clinical H. pylori isolates in response to omeprazole at different pH levels. METHODS: Accordingly, H. pylori isolates with the same virulence genotypes selected from the gastric biopsies of patients and transcriptional alteration in the cag-PAI genes studied in the presence or absence of omeprazole (2 mg/mL) at pH 2.0, 4.0 and 7.0 after 30 and 90 minutes of the treatment. Relative changes in the transcriptional levels were recorded in each assay, separately. RESULTS: Of 18 H. pylori isolates, the cag-PAI empty site was detected in four strains, while the presence of cagA, cagL and cagY was characterized in 77.7%, 83.3% and 83.3% of the cag-PAI-positive strains, respectively. Transcriptional analysis of the selected strains showed up-regulation of cagA and cagL, mainly at pH 2.0 and 4.0 after 30 and 90-minute exposure. A diversity in the expression levels of cag-PAI genes was seen among the strains at the extent and time of induction. CONCLUSION: Our results showed that omeprazole could increase the expression of H. pylori cagA and cagL at acidic pH. Heterogeneity among the strains probably has an impact on the extent of their interplay with PPIs. Further studies are needed to establish this correlation.

Immune response modulation in inflammatory bowel diseases by Helicobacter pylori infection.

Many studies point to an association between Helicobacter pylori (H. pylori) infection and inflammatory bowel diseases (IBD). Although controversial, this association indicates that the presence of the bacterium somehow affects the course of IBD. It appears that H. pylori infection influences IBD through changes in the diversity of the gut microbiota, and hence in local chemical characteristics, and alteration in the pattern of gut immune response. The gut immune response appears to be modulated by H. pylori infection towards a less aggressive inflammatory response and the establishment of a targeted response to tissue repair. Therefore, a T helper 2 (Th2)/macrophage M2 response is stimulated, while the Th1/macrophage M1 response is suppressed. The immunomodulation appears to be associated with intrinsic factors of the bacteria, such as virulence factors - such oncogenic protein cytotoxin-associated antigen A, proteins such H. pylori neutrophil-activating protein, but also with microenvironmental changes that favor permanence of H. pylori in the stomach. These changes include the increase of gastric mucosal pH by urease activity, and suppression of the stomach immune response promoted by evasion mechanisms of the bacterium. Furthermore, there is a causal relationship between H. pylori infection and components of the innate immunity such as the NLR family pyrin domain containing 3 inflammasome that directs IBD toward a better prognosis.