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.

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.

Venom Ex Machina? Exploring the Potential of Cell-Free Protein Production for Venom Biodiscovery.

Venoms are a complex cocktail of potent biomolecules and are present in many animal lineages. Owed to their translational potential in biomedicine, agriculture and industrial applications, they have been targeted by several biodiscovery programs in the past. That said, many venomous animals are relatively small and deliver minuscule venom yields. Thus, the most commonly employed activity-guided biodiscovery pipeline cannot be applied effectively. Cell-free protein production may represent an attractive tool to produce selected venom components at high speed and without the creation of genetically modified organisms, promising rapid and highly efficient access to biomolecules for bioactivity studies. However, these methods have only sporadically been used in venom research and their potential remains to be established. Here, we explore the ability of a prokaryote-based cell-free system to produce a range of venom toxins of different types and from various source organisms. We show that only a very limited number of toxins could be expressed in small amounts. Paired with known problems to facilitate correct folding, our preliminary investigation underpins that venom-tailored cell-free systems probably need to be developed before this technology can be employed effectively in venom biodiscovery.

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.

Helicobacters and cancer, not only gastric cancer?

The Helicobacter genus actually comprises 46 validly published species divided into two main clades: gastric and enterohepatic Helicobacters. These bacteria colonize alternative sites of the digestive system in animals and humans, and contribute to inflammation and cancers. In humans, Helicobacter infection is mainly related to H. pylori, a gastric pathogen infecting more than half of the world's population, leading to chronic inflammation of the gastric mucosa that can evolve into two types of gastric cancers: gastric adenocarcinomas and gastric MALT lymphoma. In addition, H. pylori but also non-H. pylori Helicobacter infection has been associated with many extra-gastric malignancies. This review focuses on H. pylori and its role in gastric cancers and extra-gastric diseases, as well as malignancies induced by non-H. pylori Helicobacters. Their different virulence factors and their involvement in carcinogenesis is discussed. This review highlights the importance of both gastric and enterohepatic Helicobacters in gastrointestinal and liver cancers.

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.

Therapeutic properties of Helicobacter pylori-derived vacuolating cytotoxin A in an animal model of chronic allergic airway disease.

BACKGROUND: It has previously been shown that the Helicobacter pylori (H. pylori)-derived molecule vacuolating cytotoxin A (VacA) could be suitable for the treatment of allergic airway disease. The therapeutic activity of the protein, which acts through modulation of dendritic cells (DC) and regulatory T cells (Tregs), was demonstrated in murine short-term acute models. The aim of this study is to further evaluate the therapeutic potential of VacA by determining the effectiveness of different application routes and the suitability of the protein for treating the chronic phase of allergic airway disease. METHODS: VacA was administered by the intraperitoneal (i.p.), oral (p.o.) or intratracheal (i.t.) routes, and long-term therapeutic effectiveness, allergic airway disease hallmarks, and immune phenotype were analyzed in murine models of acute and chronic allergic airway disease. RESULTS: Administration of VacA via the i.p., p.o or i.t. routes was associated with a reduction in airway inflammation. The i.p. route showed the most consistent effect in reducing airway inflammation and i.p. treatment with VacA was the only treatment that significantly reduced mucus cell hyperplasia. In a murine model of chronic allergic airway disease, both short- and long-term treatment with VacA showed a therapeutic effect, with a reduction in a variety of asthma hallmarks, including bronchoalveolar lavage eosinophilia, lung inflammation and goblet cell metaplasia. Short-term treatment was associated with induction of Tregs, while repetitive long-term administration of VacA influenced immunological memory in the lung. CONCLUSIONS: In addition to showing therapeutic efficacy in short-term models, treatment with VacA also appeared to be effective in suppressing inflammation in a chronic airway disease model. The observation that treatment was effective after administration via several different routes highlights the potential of VacA as a therapeutic agent with different routes of administration in humans.

Causal associations of circulating Helicobacter pylori antibodies with stroke and the mediating role of inflammation.

BACKGROUND: Observational studies have shown that Helicobacter pylori (H. pylori) infection and H. pylori antibodies are associated with an increased risk of stroke. However, which and how H. pylori antibodies serve as the causal determinant of the development of stroke remains largely unknown. METHODS: Genome-wide association studies (GWAS) on seven different antibodies of H. pylori-specific proteins, stroke, and stroke subtypes were included in this study. Mendelian randomization (MR) and multivariable MR (MVMR) analysis were performed to assess the causal associations between H. pylori antibodies and the development of stroke and to determine the potential mechanisms underlying the associations. RESULTS: Genetically predicted serum H. pylori vacuolating cytotoxin-A (VacA) antibody level was associated with an increased risk of all-cause stroke (odds ratio [OR] = 1.04, 95% CI 1.01-1.07, P = 0.017) and cardioembolic stroke (CES, OR = 1.11, 95% CI 1.04-1.18, P = 0.001). The results of multivariable MR (MVMR) showed that C-reactive protein (CRP), but not monocyte chemoattractant protein-1 and peptic ulcer, mediated the causal effects of VacA-positive H. pylori infection on all-cause stroke and CES. No strong causal associations were found between other H. pylori antibodies and stroke and its subtypes. CONCLUSIONS: Our results demonstrate that H. pylori VacA antibody is the only causal determinant associated with the risk of stroke in the spectrum of H. pylori-related antibodies, in which CRP may mediate the association. This study suggests that inhibition of the CRP signaling pathway may reduce the risk of stroke in patients with VacA-positive H. pylori infection.

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.

Select Toxicologic Pathology Case Studies of the Hepatobiliary System.

This case study session of the hepatobiliary system was held during the 42nd Annual Society of Toxicologic Pathology Symposium in Summerlin, Nevada. The case studies highlighed potential hepatic and biliary toxicity liabilities. This article comprises several of the case studies that were presented during the session which included copper-associated hepatitis in a dog, sinusoidal obstruction syndrome in non-human primates, hepatic cytoplasmic alteration in mice and rats, and Kupffer cell hyperplasia/granulomatous inflammation in rats. Presenters, when applicable, provided case signalment, anatomic/clinical pathology data, and diagnoses and discussed potential pathogeneses.

The active form of Helicobacter pylori vacuolating cytotoxin induces decay-accelerating factor CD55 in association with intestinal metaplasia in the human gastric mucosa.

High-level expression of decay-accelerating factor, CD55, has previously been found in human gastric cancer (GC) and intestinal metaplasia (IM) tissues. Therapeutic effects of CD55 inhibition in cancer have been reported. However, the role of Helicobacter pylori infection and virulence factors in the induction of CD55 and its association with histological changes of the human gastric mucosa remain incompletely understood. We hypothesised that CD55 would be increased during infection with more virulent strains of H. pylori, and with more marked gastric mucosal pathology. RT-qPCR and immunohistochemical analyses of gastric biopsy samples from 42 H. pylori-infected and 42 uninfected patients revealed that CD55 mRNA and protein were significantly higher in the gastric antrum of H. pylori-infected patients, and this was associated with the presence of IM, but not atrophy, or inflammation. Increased gastric CD55 and IM were both linked with colonisation by vacA i1-type strains independently of cagA status, and in vitro studies using isogenic mutants of vacA confirmed the ability of VacA to induce CD55 and sCD55 in gastric epithelial cell lines. siRNA experiments to investigate the function of H. pylori-induced CD55 showed that CD55 knockdown in gastric epithelial cells partially reduced IL-8 secretion in response to H. pylori, but this was not due to modulation of bacterial adhesion or cytotoxicity. Finally, plasma samples taken from the same patients were analysed for the soluble form of CD55 (sCD55) by ELISA. sCD55 levels were not influenced by IM and did not correlate with gastric CD55 mRNA levels. These results suggest a new link between active vacA i1-type H. pylori, IM, and CD55, and identify CD55 as a molecule of potential interest in the management of IM as well as GC treatment. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Host-Bacterium Interaction Mechanisms in Staphylococcus aureus Endocarditis: A Systematic Review.

Staphylococci sp. are the most commonly associated pathogens in infective endocarditis, especially within high-income nations. This along with the increasing burden of healthcare, aging populations, and the protracted infection courses, contribute to a significant challenge for healthcare systems. A systematic review was conducted using relevant search criteria from PubMed, Ovid's version of MEDLINE, and EMBASE, and data were tabulated from randomized controlled trials (RCT), observational cohort studies, meta-analysis, and basic research articles. The review was registered with the OSF register of systematic reviews and followed the PRISMA reporting guidelines. Thirty-five studies met the inclusion criteria and were included in the final systematic review. The role of Staphylococcus aureus and its interaction with the protective shield and host protection functions was identified and highlighted in several studies. The interaction between infective endocarditis pathogens, vascular endothelium, and blood constituents was also explored, giving rise to the potential use of antiplatelets as preventative and/or curative agents. Several factors allow Staphylococcus aureus infections to proliferate within the host with numerous promoting and perpetuating agents. The complex interaction with the hosts' innate immunity also potentiates its virulence. The goal of this study is to attain a better understanding on the molecular pathways involved in infective endocarditis supported by S. aureus and whether therapeutic avenues for the prevention and treatment of IE can be obtained. The use of antibiotic-treated allogeneic tissues have marked antibacterial action, thereby becoming the ideal substitute in native and prosthetic valvular infections. However, the development of effective vaccines against S. aureus still requires in-depth studies.

Helicobacter Pylori Virulence Factor Cytotoxin-Associated Gene A (CagA) Induces Vascular Calcification in Coronary Artery Smooth Muscle Cells.

Helicobacter pylori (H. pylori) has been associated with cardiovascular diseases. The pro-inflammatory H. pylori virulence factor cytotoxin-associated gene A (CagA) has been detected in serum exosomes of H. pylori-infected subjects and may exert systemic effects throughout the cardiovascular system. The role of H. pylori and CagA in vascular calcification was hitherto unknown. The aim of this study was to determine the vascular effects of CagA through human coronary artery smooth muscle cell (CASMC) osteogenic and pro-inflammatory effector gene expression as well as interleukin 1ß secretion and cellular calcification. CagA upregulated bone morphogenic protein 2 (BMP-2) associated with an osteogenic CASMC phenotype switch and induced increased cellular calcification. Furthermore, a pro-inflammatory response was observed. These results support that H. pylori may contribute to vascular calcification through CagA rendering CASMCs osteogenic and inducing calcification.

Transcriptional Activation of a Pro-Inflammatory Response (NF-κB, AP-1, IL-1ß) by the Vibrio cholerae Cytotoxin (VCC) Monomer through the MAPK Signaling Pathway in the THP-1 Human Macrophage Cell Line.

This study describes, to some extent, the VCC contribution as an early stimulation of the macrophage lineage. Regarding the onset of the innate immune response caused by infection, the ß form of IL-1 is the most important interleukin involved in the onset of the inflammatory innate response. Activated macrophages treated in vitro with VCC induced the activation of the MAPK signaling pathway in a one-hour period, with the activation of transcriptional regulators for a surviving and pro-inflammatory response, suggesting an explanation inspired and supported by the inflammasome physiology. The mechanism of IL-1ß production induced by VCC has been gracefully outlined in murine models, using bacterial knockdown mutants and purified molecules; nevertheless, the knowledge of this mechanism in the human immune system is still under study. This work shows the soluble form of 65 kDa of the Vibrio cholerae cytotoxin (also known as hemolysin), as it is secreted by the bacteria, inducing the production of IL-1ß in the human macrophage cell line THP-1. The mechanism involves triggering the early activation of the signaling pathway MAPKs pERK and p38, with the subsequent activation of (p50) NF-κB and AP-1 (cJun and cFos), determined by real-time quantitation. The evidence shown here supports that the monomeric soluble form of the VCC in the macrophage acts as a modulator of the innate immune response, which is consistent with the assembly of the NLRP3 inflammasome actively releasing IL-1ß.

Development of a novel tetravalent peptide that absorbs subtilase cytotoxin by targeting the receptor-binding B-subunit.

Subtilase cytotoxin (SubAB) is a major virulence factor produced by eae-negative Shiga-toxigenic Escherichia coli (STEC) that can cause fatal systemic complications. SubAB binds to target cells through multivalent interactions between its B-subunit pentamer and receptor molecules such as glycoproteins with a terminal N-glycolylneuraminic acid (Neu5Gc). We screened randomized multivalent peptide libraries synthesized on a cellulose membrane and identified a series of tetravalent peptides that efficiently bind to the receptor-binding region of the SubAB B-subunit pentamer. These peptides competitively inhibited the binding of the B-subunit to a receptor-mimic molecule containing clustered Neu5Gc (Neu5Gc-polymer). We selected the peptide with the highest inhibitory efficacy, FFP-tet, and covalently bound it to beads to synthesize FFP-tet-beads, a highly clustered SubAB absorber that displayed potency to absorb SubAB cytotoxicity through direct binding to the toxin. The efficacy of FFP-tet-beads to absorb SubAB cytotoxicity in solution was similar to that of Neu5Gc-polymer, suggesting that FFP-tet-beads might be an effective therapeutic agent against complications arising from eae-negative STEC infection.

Regulation of Enterotoxins Associated with Bacillus cereus Sensu Lato Toxicoinfection.

Bacillus cereus sensu lato (s.l.) includes foodborne pathogens, as well as beneficial microorganisms, such as bioinsecticides. Some of the beneficial and commercially used B. cereus s.l. strains have been shown to carry enterotoxin genes, the products of which can cause toxicoinfection in humans. Furthermore, recent epidemiological reports indicated that some bioinsecticidal strains have been linked with foodborne illness outbreaks. This demonstrates the need for improved surveillance of B. cereus s.l., which includes characterization of isolates' virulence capacity. However, the prediction of virulence capacity of B. cereus s.l. strains is challenging. Genetic screening for enterotoxin gene presence has proven to be insufficient for accurate discrimination between virulent and avirulent strains, given that nearly all B. cereus s.l. strains carry at least one enterotoxin gene. Furthermore, complex regulatory networks governing the expression of enterotoxins, and potential synergistic interactions between enterotoxins and other virulence factors make the prediction of toxicoinfection based on isolates' genome sequences challenging. In this review, we summarize and synthesize the current understanding of the regulation of enterotoxins associated with the B. cereus s.l. toxicoinfection and identify gaps in the knowledge that need to be addressed to facilitate identification of genetic markers predictive of cytotoxicity and toxicoinfection.

Bacillus cytotoxicus-A potentially virulent food-associated microbe.

Bacillus cytotoxicus is a member of the Bacillus cereus group with the ability to grow at high temperatures (up to 52℃) and to synthesize cytotoxin K-1, a diarrhoeagenic cytotoxin, which appears to be unique to this species and more cytotoxic than the cytotoxin K-2 produced by other members of this group. Only a few isolates of this species have been characterized with regard to their cytotoxic effects, and the role of cytotoxin K-1 as a causative agent of food poisoning remains largely unclear. Bacillus cytotoxicus was initially isolated from a food-borne outbreak, which led to three deaths, and the organism has since been linked to other outbreaks all involving plant-based food matrices. Other studies, as well as food-borne incidents reported to the UK Food Standards Agency, detected B. cytotoxicus in insect-related products and in dried food products. With insect-related food becoming increasingly popular, the association with this pathogen is concerning, requiring further investigation and evidence to protect public health. This review summarizes the current knowledge around B. cytotoxicus and highlights gaps in the literature from a food safety perspective.

Cryo-EM structures of Helicobacter pylori vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution.

Human gastric pathogen Helicobacter pylori (H. pylori) is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor secreted by H. pylori and induces multiple cellular responses. Although structural and functional studies of VacA have been extensively performed, the high-resolution structure of a full-length VacA protomer and the molecular basis of its oligomerization are still unknown. Here, we use cryoelectron microscopy to resolve 10 structures of VacA assemblies, including monolayer (hexamer and heptamer) and bilayer (dodecamer, tridecamer, and tetradecamer) oligomers. The models of the 88-kDa full-length VacA protomer derived from the near-atomic resolution maps are highly conserved among different oligomers and show a continuous right-handed ß-helix made up of two domains with extensive domain-domain interactions. The specific interactions between adjacent protomers in the same layer stabilizing the oligomers are well resolved. For double-layer oligomers, we found short- and/or long-range hydrophobic interactions between protomers across the two layers. Our structures and other previous observations lead to a mechanistic model wherein VacA hexamer would correspond to the prepore-forming state, and the N-terminal region of VacA responsible for the membrane insertion would undergo a large conformational change to bring the hydrophobic transmembrane region to the center of the oligomer for the membrane channel formation.

Exploring the potential of Cinnamomum zeylanicum oil against drug resistant Helicobacter pylori-producing cytotoxic genes.

Thirty-one of sixty dyspeptic patients tested positive for Helicobacter pylori colonization in this study, as determined by histopathology and 16S rRNA. The cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA) genes were found in 67.7 and 93.5% of H. pylori patients, respectively. The cagA gene was found to be associated with 100% of patients with duodenal erosion and ulceration identified via endoscopy examination. In addition, 86.7% of patients with cancerous and precancerous lesions, glandular atrophy, and intestinal metaplasia identified via histopathology examination. The vacA s1m1 mutation was associated with more severe forms of gastric erosion and ulceration, as well as the presence of precancerous and cancerous lesions. Eighteen (64.3%) of the twenty-eight isolates were classified as multi-drug resistant (MDR) or pan-drug resistant (PDR) H. pylori. Due to a resurgence of interest in alternative therapies derived from plants as a result of H. pylori resistance to the majority of commonly used antibiotics, the inhibitory activity of five essential oils extracted from some commonly used medicinal plants was evaluated in vitro against drug-resistant H. pylori clinical isolates. Cinnamomum zeylanicum essential oil demonstrated the highest anti-H. pylori activity when compared to the other essential oils tested. Cinnamaldehyde was the most abundant compound in C. zeylanicum (65.91%). The toxicological evaluation established the safety of C. zeylanicum oil for human use. As a result, C. zeylanicum essential oil may represent a novel antibacterial agent capable of combating drug-resistant H. pylori carrying cytotoxin genes.

Differential Expression of α-Enolase in Clinical Gastric Tissues and Cultured Normal/Cancer Cells in Response to Helicobacter pylori Infection and cagA Transfection.

Background and Objectives: The role of α-enolase (ENO1) in Helicobacter pylori-related gastric lesions might be a critical factor in the pathogenesis, but remains undefined. Materials and Methods: This study investigated the differential expression of α-enolase in clinical gastric specimens and cultured normal/cancer cells in response to H. pylori (cagA+) infection and cagA transfection using qPCR, Western blots and histochemical methods. Results: A total of 172 gastric specimens were collected from 142 patients, the former comprising chronic superficial gastritis (CSG), precancerous diseases (PCDs, including atrophic gastritis, intestinal metaplasia and dysplasia) and gastric cancer (GC) cases. Among the CSG and PCD cases, the H. pylori-infected group had significantly elevated ENO1 mRNA levels compared with the uninfected group. In the GC cases, differential ENO1 expressions were detected between the cancer tissues and the paracancerous tissues. Notably, significant difference was first detected between the GC cell (AGS) and the normal cell (GES-1) as a response of ENO1 to H. pylori infection and cagA transfection. Conclusions: This report reveals that ENO1 expression is associated with H. pylori infection, cagA transfection, co-culture duration, multiplicity of infection, gastric normal/cancerous cell lines and cellular differentiation. The findings may be crucial bases for further ascertaining H. pylori pathogenic mechanism and formulating novel therapeutic and diagnostic strategies.