Helicobacter pylori: immunoproteomics related to different pathologies

Helicobacter pylori is a Gram-negative bacterium that causes ulcer, atrophic gastritis, adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Moreover, an ongoing controversial role of this bacterium infection has been suggested in the etiopathogenesis of some extradigestive diseases. The humoral response to H. pylori during a natural infection can be used for diagnostic purposes and as a basis for vaccine development. Host-pathogen interactions may be investigated by means of immunoproteomics, which provides global information about relevant specific and nonspecific antigens, and thus might be suitable to identify novel vaccine candidates or serological markers of H. pylori infection as well as of different related diseases. In this review, we describe how several research groups used H. pylori proteomics combined with western blotting analysis, using sera from patients affected with different H. pylori-related pathologies, to investigate potential associations between host immune response and clinical outcomes of H. pylori infection, resulting in the rapid identification of novel, highly immunoreactive antigens.     

Potential antigen candidates for subunit vaccine development against Helicobacter pylori infection

Helicobacter pylori (H. pylori) is a resident bacterium in the stomach that accounts for 75% cases of gastric cancer. In this review, we comprehensively studied published papers on H. pylori vaccines using Google Scholar and NCBI databases to gather information about vaccines against H. pylori. Considering the pivotal roles of the enzyme urease (in production of NH₃ and neutralization of the acidic medium of the stomach), cytotoxin-associated gene A, and vacuolating cytotoxin A proteins in H. pylori infection, they could be the best candidates for the construction of recombinant vaccines. The outer membrane porins (Hop), blood group antigen-binding adhesin (BabA), sialic acid-binding adhesin (SabA), and outer inflammatory protein A, play significant roles in binding of bacterium to human gastric tissues, and because binding is the first step in bacterial fixation and colonization, these antigens also can be considered as suitable candidates for designing vaccines. Likely, other significant bacterial antigens, such as NapA (chemotactic factor for recruitment of human neutrophils and monocytes to the site of infection), duodenal ulcer promoting protein A (to promote duodenal ulcer), and Hsp60 (as a molecular chaperon for activation of urease enzyme), can be used in the construction of subunit vaccines. New vaccines in use currently, such as DNA vaccines and subunit vaccines, can efficiently replace the dead and attenuated vaccines. Nonetheless, the results show that urease enzyme is most used compared with bacterial components in the designing and construction of recombinant vaccines. The BabA and SabA antigens belong to the outer membrane porins family in H. pylori and are required for binding and fixation of the bacterium to the human gastric tissues.     

The Importance of Toll‐like Receptors in NF‐κB Signaling Pathway Activation by Helicobacter pylori Infection and the Regulators of this Response

Helicobacter pylori (H. pylori) is a common pathogenic bacterium in the stomach that infects almost half of the population worldwide and is closely related to gastric diseases and some extragastric diseases, including iron‐deficiency anemia and idiopathic thrombocytopenic purpura. Both the Maastricht IV/Florence consensus report and the Kyoto global consensus report have proposed the eradication of H. pylori to prevent gastric cancer as H.pylori has been shown to be a major cause of gastric carcinogenesis. The interactions between H. pylori and host receptors induce the release of the proinflammatory cytokines by activating proinflammatory signaling pathways such as nuclear factor kappa B (NF‐κB), which plays a central role in inflammation, immune response, and carcinogenesis. Among these receptors, Toll‐like receptors (TLRs) are classical pattern recognition receptors in the recognition of H. pylori and the mediation of the host inflammatory and immune responses to H. pylori. TLR polymorphisms also contribute to the clinical consequences of H. pylori infection. In this review, we focus on the functions of TLRs in the NF‐κB signaling pathway activated by H. pylori, the regulators modulating this response, and the functions of TLR polymorphisms in H.pylori‐related diseases.     

Imbalance of Gastrointestinal Microbiota in the Pathogenesis of Helicobacter pylori‐Associated Diseases

The development of new nucleotide sequencing techniques and advanced bioinformatics tools has opened the field for studying the diversity and complexity of the gastrointestinal microbiome independent of traditional cultural methods. Owing largely to the gastric acid barrier, the human stomach was long thought to be sterile. The discovery of Helicobacter pylori, the gram‐negative bacterium that infects upwards of 50% of the global population, has started a major paradigm shift in our understanding of the stomach as an ecologic niche for bacteria. Recent sequencing analysis of gastric microbiota showed that H. pylori was not alone and the interaction of H. pylori with those microorganisms might play a part in H. pylori‐associated diseases such as gastric cancer. In this review, we summarize the available literature about the changes of gastrointestinal microbiota after H. pylori infection in humans and animal models, and discuss the possible underlying mechanisms including the alterations of the gastric environment, the secretion of hormones and the degree of inflammatory response. In general, information regarding the composition and function of gastrointestinal microbiome is still in its infancy, future studies are needed to elucidate whether and to what extent H. pylori infection perturbs the established microbiota. It is assumed that clarifying the role of gastrointestinal communities in H. pylori‐associated diseases will provide an opportunity for translational application as a biomarker for the risk of serious H. pylori diseases and perhaps identify specific organisms for therapeutic eradication.     

When Helicobacter pylori invades and replicates in the cells

Autophagosome formation is induced by Helicobacter pylori infection and these autophagic vesicles are adopted for replication of H. pylori and subsequent eradication of the invading H. pylori in macrophages. Some Taiwanese clinical isolates of H. pylori can replicate in certain macrophage cell lines. After entry, there was a 5-10 fold increment of re-cultivable H. pylori from the infected permissible cells at 12 h post infection. The dividing H. pylori are observed to reside in double-layered autophagosomes. Therefore, H. pylori can be considered as a kind of intracellular microorganism. The autophagy induction by H. pylori is not only found in macrophages, but also in dendritic cells and gastric epithelial cells. This new finding has several implications for the life cycle of H. pylori in the host. The bacterium’s residence inside infected cells will increase its resistance to antimicrobial treatment, avoid neutralization by anti-H. pylori antibodies, impair antigen presentation, and alter the cellular immune response. The replication of H. pylori in autophagic vesicles, and the consequences of this provide an important hint as to why this microorganism causes so such a broad spectrum of diseases.     

Reactive oxygen species and gastric carcinogenesis: The complex interaction between Helicobacter pylori and host

Helicobacter pylori (H. pylori) is a highly successful human pathogen that colonizes stomach in around 50% of the global population. The colonization of bacterium induces an inflammatory response and a substantial rise in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), mostly derived from host neutrophils and gastric epithelial cells, which play a crucial role in combating bacterial infections. However, H. pylori has developed various strategies to quench the deleterious effects of ROS, including the production of antioxidant enzymes, antioxidant proteins as well as blocking the generation of oxidants. The host's inability to eliminate H. pylori infection results in persistent ROS production. Notably, excessive ROS can disrupt the intracellular signal transduction and biological processes of the host, incurring chronic inflammation and cellular damage, such as DNA damage, lipid peroxidation, and protein oxidation. Markedly, the sustained inflammatory response and oxidative stress during H. pylori infection are major risk factor for gastric carcinogenesis. In this context, we summarize the literature on H. pylori infection-induced ROS production, the strategies used by H. pylori to counteract the host response, and subsequent host damage and gastric carcinogenesis.     

Cloning and Characterization of a 22 kDa Outer-Membrane Protein (Omp22) from Helicobacter pylori

Helicobacter pylori is a causative agent of gastritis and peptic ulceration in humans. As the first step towards development of a vaccine against H. pylori infection, we have attempted to identify protective antigens. A potential target of vaccine development would be a H. pylori specific protein, which is surface-exposed and highly antigenic. We identified a 22 kDa outer-membrane protein (Omp22) from H. pylori, which was highly immunoreactive. By screening a H. pylori genomic DNA library with rabbit anti-H. pylori outer-membrane protein antibodies, the omp22 gene was cloned and 1.4 kb of the nucleotide sequence was determined. One open reading frame, encoding a 179-residue polypeptide, was identified and the amino acid sequence deduced showed homology with peptidoglycan-associated lipoproteins. The sequence was conserved among other H. pylori strains. Omp22 protein is expressed as a precursor polypeptide of 179 residues and undergoes lipid modification and cleavage of an 18 amino acid signal peptide to yield a mature protein. Omp22 protein in H. pylori as well as recombinant Omp22 protein expressed in E. coli was localized into the outer membrane and exposed on the cell surface. Omp22 may have the potential as a target antigen for the development of a H. pylori vaccine.     

Statistical model of the interactions between Helicobacter pylori infection and gastric cancer development

Background. The bacterium Helicobacter pylori is associated with a number of gastrointestinal diseases, such as gastric ulcer, duodenal ulcer and gastric cancer. Several histological changes may be observed during the course of infection; some may influence the progression towards cancer. The aim of this study was to build a statistical model to discover direct interactions between H. pylori and different precancerous changes of the gastric mucosa, and in what order and to what degree those may influence the development of the intestinal type of gastric cancer. Methods. To find direct and indirect interactions between H. pylori and different histological variables, log‐linear analyses were used on a case–control study. To generate mathematically and biologically relevant statistical models, a designed algorithm and observed frequency tables were used. Results. The results show that patients with H. pylori infection need to present with proliferation and intestinal metaplasia to develop gastric cancer of the intestinal type. Proliferation and intestinal metaplasia interacted with the variables atrophy and foveolar hyperplasia. Intestinal metaplasia was the only variable with direct interaction with gastric cancer. Gender had no effect on the variables examined. Conclusion. The direct interactions observed in the final statistical model between H. pylori, changes of the mucosa and gastric cancer strengthens and supports previous theories about the progression towards gastric cancer. The results suggest that gastric cancer of the intestinal type may develop from H. pylori infection, proliferation and intestinal metaplasia, while atrophy and foveolar hyperplasia interplay with the other histological variables in the disease process.     

The role of toll-like receptors in immune tolerance induced by Helicobacter pylori infection

Helicobacter pylori (H. pylori) is a gram-negative, microaerobic bacterium that colonizes the gastric mucosa in about half of the world's population. H. pylori infection can lead to various diseases. Chronic infection by H. pylori exposes the gastric mucosa to bacterial components such as lipopolysaccharide (LPS), outer membrane vesicles (OMVs), and several toxic proteins. Infected with H. pylori activates the release of pro-inflammatory factors and triggers inflammatory responses that damage the gastric mucosa. As the only microorganism that permanently colonizes the human stomach, H. pylori can suppress host immunity to achieve long-term colonization. Toll-like receptors (TLRs) play a crucial role in T-cell activation, promoting innate immune responses and immune tolerance during H. pylori infection. Among the 10 TLRs found in humans, TLR2, TLR4, TLR5, and TLR9 have been thoroughly investigated in relation to H. pylori-linked immune regulation. In the present review, we provide a comprehensive analysis of the various mechanisms employed by different TLRs in the induction of immune tolerance upon H. pylori infection, which will contribute to the research of pathogenic mechanism of H. pylori.     

Methods to monitor autophagy in H. pylori vacuolating cytotoxin A (VacA)-treated cells

Helicobacter pylori is a gram negative pathogen that infects at least half of the world’s population and is associated not only with gastric cancer but also with other diseases such as gastritis and peptic ulcers. Indeed, H. pylori is considered the single most important risk factor for the development of gastric cancer. The vacuolating cytotoxin, VacA, secreted by H. pylori promotes intracellular survival of the bacterium and modulates host immune responses. In a recent study, we reported that VacA induces autophagy. Multilamellar autophagosomes are detected in gastric epithelial cells that are distinct from the large vacuoles formed by VacA. Furthermore, inhibition of autophagy stabilizes VacA and reduces vacuolation in the cells indicating that the toxin is being degraded by autophagy, thus limiting toxin-induced host cell damage. Many of the methods that were used for this study are commonly employed techniques that were adapted for H. pylori infection and VacA intoxication. In this paper, we describe the various methods and specific protocols used for the assessment and monitoring of autophagy during H. pylori infection.     

Immunological features and the ability of inhibitory effects on enzymatic activity of an epitope vaccine composed of cholera toxin B subunit and B cell epitope from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> urease A subunit

Epitope vaccine based on urease of Helicobacter pylori is a promising option for prophylactic and therapeutic vaccination against H. pylori infection. In this study, we constructed an epitope vaccine with mucosal adjuvant cholera toxin B subunit (CTB) and an epitope (UreA_183-203) of H. pylori urease A subunit named CTB-UA. The CTB-UA fusion protein was expressed in Escherichia coli, and the purified protein was used for intraperitoneal immunization experiments in BALB/c mice. The experimental results indicated that anti-CTB-UA antibody could recognize both H. pylori urease A subunit (UreA) and urease B subunit (UreB). Besides, the CTB-UA epitope vaccine had good immunogenicity and immunoreactivity and could induce specific neutralizing antibodies which showed effectively inhibitory effect on the enzymatic activity of H. pylori urease. CTB-UA is a promising molecule to be investigated as H. pylori vaccine antigen candidate.     

Identification of a correlation between Helicobacter pylori infection and Graves' disease

Background: viral and bacterial antigens have been suspected to be able to mimic the antigenic profile of the thyroid cell membrane and to play an important role in the onset of the autoimmune diseases, such as Graves’ disease and Hashimoto thyroiditis. The Helicobacter pylori infection is worldwide diffused and is present in the developed countries up to 50% of the population. The presence of the cytotoxin‐associated gene A antigens identifies the most virulent strains of the bacterium. Previous studies have demonstrated the possible correlation between the Helicobacter pylori and Hashimoto’s thyroiditis but these results are controversial. Aims: We studied the prevalence rate of this bacterium in the Graves’ disease and two selected subgroups such as the hyperthyroid patients, at the first time of diagnosis, and the euthyroid methimazole‐treated patients. Materials and Methods: We analyzed Helicobacter pylori in fresh stool samples with an enzyme immunoassay method and the presence of cytotoxin‐associated gene A antigens with a serological test. Results: Our results show that a significative increased rate of prevalence is present in Graves' patients, when the disease is ongoing, with an overall prevalence of the strains expressing the cytotoxin‐associated gene A antigens compared to the control group. Conclusions: The association between the Helicobacter pylori and Graves’ disease suggests a possible role of this bacterium in the onset and/or the maintenance of the disease.     

Interleukin-8 is the single most up-regulated gene in whole genome profiling of <Emphasis Type="Italic">H. pylori</Emphasis> exposed gastric epithelial cells

Background  

The association between Helicobacter pylori infection and upper gastrointestinal disease is well established. However, only a small fraction of H. pylori carriers develop disease, and there are great geographical differences in disease penetrance. The explanation to this enigma lies in the interaction between the bacterium and the host. H. pylori Outer Membrane Phospholipase A (OMPLA) has been suggested to play a role in the virulence of this bacterium. The aim of this study was to profile the most significant cellular pathways and biological processes affected in gastric epithelial cells during 24 h of H. pylori exposure, and to study the inflammatory response to OMPLA⁺ and OMPLA^- H. pylori variants.     

Role of Probiotics in Pediatric Patients with Helicobacter pylori Infection: A Comprehensive Review of the Literature

Background: The current guidelines suggest the use of triple therapy as first choice treatment of Helicobacter pylori infection, although the eradication failure rate is more than 30%. Current interest in probiotics as therapeutic agents against H. pylori is stimulated not only by the clinical data showing efficacy of some probiotics in different gastrointestinal diseases but also by the increasing resistance of pathogenic bacteria to antibiotics, thus the interest for alternative therapies is a real actual topic. Aim: To review in vitro and in vivo studies on the role of probiotics in H. pylori infection focusing on the paediatric literature. Materials and methods: Pre‐clinical and clinical paediatric studies in English assessing the role of probiotics in H. pylori infection identified by MEDLINE search (1950–2009) were reviewed. Results: In vitro studies demonstrated an inhibitory activity of probiotics on H. pylori growth and that this effect is extremely strain specific. Available data in children indicate that probiotics seems to be efficacious for the prevention of antibiotic associated side‐effects, and might be of help for the prevention of H. pylori complications by decreasing H. pylori density and gastritis, and for the prevention of H. pylori colonization or re‐infection by inhibiting adhesion to gastric epithelial cells. There is no clear evidence that probiotics may increase the H. pylori eradication rate. Conclusion: Both in vitro and in vivo studies provide evidence that probiotics may represent a novel approach to the management of H. pylori infection.     

Helicobacter pylori-pulsed dendritic cells induce H. pylori-specific immunity in mice

Background: The growing concern over the emergence of antibiotic‐resistant Helicobacter pylori infection is propelling the development of an efficacious vaccine to control this highly adaptive organism. Aim: We studied the use of a dendritic cell (DC)‐based vaccine against H. pylori infection in mice. Methods: The cellular immune responses to murine bone marrow‐derived DCs pulsed with phosphate‐buffered saline (PBS‐DC) or live H. pylori SS1 (HP‐DC) were assessed in vitro and in vivo. The protective immunity against H. pylori SS1 oral challenge was compared between HP‐DC or PBS‐DC immunized mice. The effect of regulatory T‐cell (Treg) depletion by anti‐CD25 antibody on HP‐DC vaccine efficacy was also evaluated. Results: HP‐DC induced a Th1‐dominant response in vitro. In vivo, HP‐DC immunized mice were characterized by a mixed Th1/Th2 peripheral immune response. However, in the stomach, HP‐DC immunized mice expressed a higher level of IFN‐γ compared to PBS‐DC immunized mice; no difference was found for interleukin‐5 expressions in the stomach. A lower bacterial colonization post‐H. pylori challenge was observed in HP‐DC immunized mice compared to PBS‐DC immunized mice with no significant difference in gastritis severity. H. pylori‐specific Th1 response and protective immunity were further enhanced in vivo by depletion of Treg with anti‐CD25 antibody. Conclusion: DC‐based anti‐H. pylori vaccine induced H. pylori‐specific helper T‐cell responses capable of limiting bacterial colonization. Our data support the critical role of effector cellular immune response in the development of H. pylori vaccine.     

Helicobacter pylori Infection – A Boon or a Bane: Lessons from Studies in a Low‐Prevalence Population

Helicobacter pylori (H. pylori) infection is etiologically associated with gastric cancer and peptic ulcer diseases which are both important public health burdens which could be largely eliminated by H. pylori eradication. However, some investigators urge caution based on the hypothesis that eradication of H. pylori may result in an increase in the incidence of gastroesophageal reflux disease, esophageal adenocarcinoma, and childhood asthma. The ethnic Malays of northeastern Peninsular Malaysia have long had a low prevalence of H. pylori infection and, as expected, the incidence of gastric cancer and its precursor lesions is exceptionally low. The availability of a population with a low H. pylori prevalence and generally poor sanitation allows separation of H. pylori from the hygiene hypothesis and direct testing of whether absence of H. pylori is associated with untoward consequence. Contrary to predictions, in Malays, erosive esophagitis, Barrett's esophagus, distal esophageal cancers, and childhood asthma are all of low incidence. This suggests that H. pylori is not protective rather the presence of H. pylori infection is likely a surrogate for poor hygiene and not an important source of antigens involved in the hygiene hypothesis. Helicobacter pylori in Malays is related to transmission from H. pylori‐infected non‐Malay immigrants. The factors responsible for low H. pylori acquisition, transmission, and burden of H. pylori infection in Malays remain unclear and likely involves a combination of environmental, host (gene polymorphisms), and strain virulence factors. Based on evidence from this population, absence of H. pylori infection is more likely to be boon than a bane.     

Core proteome mediated therapeutic target mining and multi-epitope vaccine design for Helicobacter pylori

Helicobacter pylori is a Gram-negative spiral-shaped bacterium that infects half of the human population worldwide and causes chronic inflammation. In the present study, we used the art of computational biology for therapeutic drug targets identification and a multi-epitope vaccine against multi-strains of H. pylori. For drug target identification, we used different tools and softwares to identify human non-homologous but pathogen essential proteins, with virulent properties and involved in unique metabolic pathways of H. pylori. For this purpose, the core proteome of 84 strains of H. pylori was retrieved from EDGAR 2.3 database. There were 59,808 proteins sequences in these strains. Duplicates and paralogous protein sequence removal was followed by human non-homologous protein miningPathogen essential and virulent proteins were subjected to pathway analysis Subcellular localization of the virulent proteins was predicted and druggability was also checked, leading to 30 druggable targets based on their similarity with the approved drug targets in Drugbank. For immunoinformatics analysis, we selected two outer membrane proteins (HPAKL86_RS06305 and HPSNT_RS00950) and subjected to determined immunogenic B and T-Cell epitopes. The B and T-Cell overlapped epitopes were selected to design 9 different vaccine constructs by using linkers and adjuvants. Least allergenic and most antigenic construct (C-8) was selected as a promiscuous vaccine to elicit host immune response. Cloning and in silico expression of the constructed vaccine (C-8) was done to produce a clone having the desired (gene) vaccine construct. In conclusion, the prioritized therapeutic targets for 84 strains of H.pylori will be useful for future therapy design. Vaccine design may also prove useful in the quest for targeting multi-strains of H. pylori in patients.     

Lipopolysaccharide Structure and Biosynthesis in Helicobacter pylori

This review covers the current knowledge and gaps in Helicobacter pylori lipopolysaccharide (LPS) structure and biosynthesis. H. pylori is a Gram‐negative bacterium which colonizes the luminal surface of the human gastric epithelium. Both a constitutive alteration of the lipid A preventing TLR4 elicitation and host mimicry of the Lewis antigen decorated O‐antigen of H. pylori LPS promote immune escape and chronic infection. To date, the complete structure of H. pylori LPS is not available, and the proposed model is a linear arrangement composed of the inner core defined as the hexa‐saccharide (Kdo‐LD‐Hep‐LD‐Hep‐DD‐Hep‐Gal‐Glc), the outer core composed of a conserved trisaccharide (‐GlcNAc‐Fuc‐DD‐Hep‐) linked to the third heptose of the inner core, the glucan, the heptan and a variable O‐antigen, generally consisting of a poly‐LacNAc decorated with Lewis antigens. Although the glycosyltransferases (GTs) responsible for the biosynthesis of the H. pylori O‐antigen chains have been identified and characterized, there are many gaps in regard to the biosynthesis of the core LPS. These limitations warrant additional mutagenesis and structural studies to obtain the complete LPS structure and corresponding biosynthetic pathway of this important gastric bacterium.     

Protection against Helicobacter pylori infection by a trivalent fusion vaccine based on a fragment of urease B-UreB414

A multivalent fusion vaccine is a promising option for protection against Helicobacter pylori infection. In this study, UreB414 was identified as an antigenic fragment of urease B subunit (UreB) and it induced an antibody inhibiting urease activity. Immunization with UreB414 partially protected mice from H. pylori infection. Furthermore, a trivalent fusion vaccine was constructed by genetically linking heat shock protein A (HspA), H. pylori adhesin A (HpaA), and UreB414, resulting in recombinant HspA-HpaA-UreB414 (rHHU). Its protective effect against H. pylori infection was tested in BALB/c mice. Oral administration of rHHU significantly protected mice from H. pylori infection, which was associated with H. pylori-specific antibody production and Th1/Th2-type immune responses. The results show that a trivalent fusion vaccine efficiently combats H. pylori infection, and that an antigenic fragment of the protein can be used instead of the whole protein to construct a multivalent vaccine.