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.     

A review of the postulated mechanisms concerning the association of Helicobacter pylori with ischemic heart disease

Since its discovery, Helicobacter pylori has been implicated in the pathogenesis of several diseases, both digestive and extradigestive. Interestingly, the majority of the extradigestive-related literature is focused on two vascular manifestations: stroke and ischemic heart disease. Potential mechanisms for the establishment of a H. pylori-induced ischemic heart disease have been proposed with regard to chronic inflammation, molecular mimicry, oxidative modifications, endothelial dysfunction, direct effect of the microorganism on atherosclerotic plaques as well as changes regarding traditional or novel risk factors for ischemic heart disease or even platelet-H. pylori interactions. A positive link between H. pylori infection and ischemic heart disease has been suggested by a series of studies focusing on epidemiologic evidence, dyslipidemic alterations, upregulation of inflammatory markers or homocysteine levels, induction of hypercoagulability, oxidation of low-density lipoprotein, causation of impaired endothelial function, detection of H. pylori DNA in atherosclerotic plaques, and participation of certain antigens and antibodies in a cross-reactivity model. There are studies, however, which investigated the relationship between H. pylori and ischemic heart disease with regard to the same parameters and failed to confirm the suggested positive association. Further studies in the direction of interaction between H. pylori and the host's genotype as well as a quest for evidence towards novel risk factors for ischemic heart disease such as oxidative stress, vascular remodeling, vascular calcification, or vasomotor activity, may reveal a field of great interest, thus contributing to the determination of new potential mechanisms.     

应用生物信息学方法筛选幽门螺杆菌疫苗候选抗原

目的：应用生物信息学分析方法筛选幽门螺杆菌新的疫苗候选抗原。方法：从TIGRCMR下载幽门螺杆菌26695和J99株全基因组序列,应用生物信息学SignalP、PredTMBB、LipoP、TMHMM、Phobius、PSORT-B和SubLoc等分析软件,筛选幽门螺杆菌新的外膜蛋白和分泌蛋白疫苗候选抗原。结果：从幽门螺杆菌26695株筛选得到54个编码β-桶型跨膜蛋白、脂蛋白或分泌表达蛋白的疫苗候选蛋白抗原,从幽门螺杆菌J99株得到61个呈现上述表达方式的疫苗候选蛋白抗原;且这2株细菌的疫苗候选蛋白呈现良好的交集状况,即有43个候选疫苗蛋白是相同的。结论：用生物信息学分析方法可以从全基因组范围内快速筛选到保守的分泌或表面暴露的疫苗候选抗原,为疫苗抗原的快速筛选与鉴定奠定了基础。     

Genomics of Helicobacter pylori

During this review period, we have definitely entered into the genomic era. The Helicobacter pylori studies reported here illustrate the use of most of the technologies currently available to globally interrogate the genome of a pathogen. Global analysis of the gene content of H. pylori strains gives insight into the extent of its genetic diversity and its in vivo evolution. Our understanding of the particularities of H. pylori as a gastric pathogen colonizing a unique niche has been improved by studies aimed at: (i) the identification of H. pylori-specific genes; (ii) the establishment of correlations between the presence of one or a group of genes (or proteins) with clinical outcome; and (iii) the analysis of global regulatory circuits or responses to the extracellular signals. The response of host cells to H. pylori infection will be developed in the chapter 'H. pylori and gastric malignancies' by Sepulveda and Coehlo. Despite our knowledge of the H. pylori genome, the function of about one third of its total proteins is still unknown. Functional genomics are straightforward approaches for the identification of new gene functions or metabolic pathways as well as for the understanding of cellular processes and the detection of new virulence factors. In silico studies combined with experimental work will undoubtedly continue to develop. To date, the expansion of proteomics with refinements in mass spectrometry technology has illustrated that through immunoproteomics and comparative studies, relevant novel antigens can be identified. Genomics not only provides invaluable information on H. pylori but also opens new perspectives for diagnostic or therapeutic applications.     

Immunoproteomics of Helicobacter pylori infection and relation to gastric disease

The Gram negative bacterium Helicobacter pylori is a human pathogen which infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer. A systematic, proteome based approach was chosen to detect candidate antigens of H. pylori for diagnosis, therapy and vaccine development and to investigate potential associations between specific immune responses and manifestations of disease. Sera from patients with active H. pylori infection (n = 24), a control group with unrelated gastric disorders (n = 12) and from patients with gastric cancer (n = 6) were collected and analyzed for the reactivity against proteins of the strain HP 26695 separated by two-dimensional electrophoresis. Overall, 310 antigenic protein species were recognized by H. pylori positive sera representing about 17% of all spots separated. Out of the 32 antigens most frequently recognized by H. pylori positive sera, nine were newly identified and 23 were confirmed from other studies. Three newly identified antigens which belong to the 150 most abundant protein species of H. pylori, were specifically recognized by H. pylori positive sera: the predicted coding region HP0231, serine protease HtrA (HP1019) and Cag3 (HP0522). Other antigens were recognized differently by sera from gastritis and ulcer patients, which may identify them as candidate indicators for clinical manifestations. The data from these immunoproteomic analyses are added to our public database (http://www.mpiib-berlin.mpg.de/2D-PAGE). This platform enables one to compile many protein profiles and to integrate data from other studies, an approach which will greatly assist the search for more immunogenic proteins for diagnostic assays and vaccine design.     

Antigenic Mimicry Between Helicobacter pylori and Gastric Mucosa: Failure to Implicate Heat-Shock Protein Hsp60 Using Immunoelectron Microscopy

Background. Helicobacter pylori infection induces autoantibodies that cross-react with human gastric mucosa from infected individuals. Candidates for the antigens responsible for molecular mimicry causing autoreactivity include the heat-shock protein HspB (Hsp60, sometimes called Hsp54) or Lewis x and Lewis y carbohydrate antigens.
Objective. Our goal was to investigate the involvement of HspB (Hsp60) in autoreactivity between H. pylori and gastric biopsy tissue.
Materials and Methods. Immunoelectron microscopy was used to study cross-reactivity among biopsy tissues from a patient with gastritis, gastric ulcer, and duodenal ulcer and his own serum as well as reactivity with serum raised against HspB from H. pylori and monoclonal antibodies against Lewis antigens.
Results. The patient serum reacted with gastric mucosa, and the antibodies involved were predominantly IgG. Antibody raised to H. pylori HspB (Hsp60) reacted only with H. pylori cells but not with gastric mucosal tissue. In contrast, monoclonal antibodies specific for Lewis x and Lewis y antigens reacted with both H. pylori and human gastric epithelial tissue.
Conclusions. Hsp60 (Hsp54) is unlikely to be involved in autoreactivity seen in individuals infected with H. pylori. In contrast, we could not rule out the role of Lewis x and Lewis y carbohydrate antigens, expressed as a component of H. pylori lipopolysaccharides, in molecular mimicry and autoantibody production.     

Expression of histo-blood group antigens by lipopolysaccharides of Helicobacter pylori strains from asian hosts: the propensity to express type 1 blood-group antigens

Past studies have shown that the cell surface lipopolysaccharides (LPSs) of the ubiquitous human gastric pathogen Helicobacter pylori (a type 1 carcinogen) isolated from people residing in Europe and North America express predominantly type 2 Lewis x (Le(x)) and Le(y) epitopes and, infrequently, type 1 Le(a), Le(b), and Le(d) antigens. This production of Lewis blood-group structures by H. pylori LPSs, similar to those found in the surfaces of human gastric cells, allows the bacterium to mimic its human niche. In this study, LPSs of H.pylori strains extracted from patients living in China, Japan, and Singapore were chemically and serologically analyzed. When compared with Western H.pylori LPSs, these Asian strains showed a stronger tendency to produce type 1 blood groups. Of particular interest, and novel observations in H.pylori, the O-chain regions of strains F-58C and R-58A carried type 1 Le(a) without the presence of type 2 Le(x), strains R-7A and H607 were shown to have the capability of producing the type 1 blood group A antigen, and strains CA2, H507, and H428 expressed simultaneously the difucosyl isomeric antigens, type 1 Le(b) and type 2 Le(y). The apparent proclivity for the production of type 1 histo-blood group antigens in Asian H.pylori LPSs, as compared with Western strains, may be an adaptive evolutionary effect in that differences in the gastric cell surfaces of the respective hosts might be significantly dissimilar to select for the formation of different LPS structures on the resident H.pylori strain.     

Helicobacter pylori attaches to NeuAc alpha 2,3Gal beta 1,4 glycoconjugates produced in the stomach of transgenic mice lacking parietal cells

Helicobacter pylori infection of the human stomach is associated with altered acid secretion, loss of acid-producing parietal cells, and, in some hosts, adenocarcinoma. We have used a transgenic mouse model to study the effects of parietal cell ablation on H. pylori pathogenesis. Ablation results in amplification of the presumptive gastric epithelial stem cell and its immediate committed daughters. The amplified cells produce sialylated oncofetal carbohydrate antigens that function as receptors for H. pylori adhesins. Attachment results in enhanced cellular and humoral immune responses. NeuAc alpha 2,3Gal beta 1,4 glycoconjugates may not only facilitate persistent H. pylori infection in a changing gastric ecosystem, but by promoting interactions with lineage progenitors and/or initiated cells contribute to tumorigenesis in patients with chronic atrophic gastritis.     

Evaluation of ribotyping for characterization and identification of Enterococcus haemoperoxidus and Enterococcus moraviensis strains

Periodontopathic Campylobacter rectus strains possess 41- and 68-kDa proteinaceous antigens which share antigenicity with antigens of Helicobacter pylori strains. H. pylori strains have a 54-kDa antigen which reacts with C. rectus strains. We found that the salivary IgA levels against H. pylori were correlated with those against C. rectus. These cross-reactive antigens of C. rectus may affect the serological diagnosis of H. pylori infections, especially when saliva is used. It is possible that these cross-reacting antigens may relate to the induction of immunopathological responses against both microorganisms.     

Helicobacter pylori, T cells and cytokines: the "dangerous liaisons"

Helicobacter pylori infection is the major cause of gastroduodenal pathologies, but only a minority of infected patients develop chronic and life threatening diseases, as peptic ulcer, gastric cancer, B-cell lymphoma, or autoimmune gastritis. The type of host immune response against H. pylori is crucial for the outcome of the infection. A predominant H. pylori-specific Th1 response, characterized by high IFN-gamma, TNF-alpha, and IL-12 production associates with peptic ulcer, whereas combined secretion of both Th1 and Th2 cytokines are present in uncomplicated gastritis. Gastric T cells from MALT lymphoma exhibit abnormal help for autologous B-cell proliferation and reduced perforin- and Fas-Fas ligand-mediated killing of B cells. In H. pylori-infected patients with autoimmune gastritis cytolytic T cells infiltrating the gastric mucosa cross-recognize different epitopes of H. pylori proteins and H+K+ ATPase autoantigen. These data suggest that peptic ulcer can be regarded as a Th1-driven immunopathological response to some H. pylori antigens, whereas deregulated and exhaustive H. pylori-induced T cell-dependent B-cell activation can support the onset of low-grade B-cell lymphoma. Alternatively, H. pylori infection may lead in some individuals to gastric autoimmunity via molecular mimicry.     

Pathogenesis of Helicobacter pylori Infection

Helicobacter pylori induces chronic inflammation of the gastric mucosa, but only a proportion of infected individuals develop peptic ulcer disease or gastric carcinoma. Reasons underlying these observations include differences in bacterial pathogenicity as well as in host susceptibility. Numerous studies published in the last year provided new insight into H. pylori virulence factors, their interaction with the host and consequences in pathogenesis. These include the role of bacterial genetic diversity in host colonization and persistence, outer membrane proteins and modulation of adhesin expression, new aspects of VacA functions, and CagA and its phosphorylation-dependent and -independent cellular effects. This article will also review the recent novel findings on the interactions of H. pylori with diverse host epithelial signaling pathways and events involved in the initiation of carcinogenesis, including genetic instability and dysregulation of DNA repair.     

Lewis antigens in Helicobacter pylori: biosynthesis and phase variation

The lipopolysaccharides (LPS) of most Helicobacter pylori strains contain complex carbohydrates known as Lewis antigens that are structurally related to the human blood group antigens. Investigations on the genetic determinants involved in the biosynthesis of Lewis antigens have led to the identification of the fucosyltransferases of H. pylori, which have substrate specificities distinct from the mammalian fucosyltransferases. Compared with its human host, H. pylori utilizes a different pathway to synthesize the difucosylated Lewis antigens, Lewis y. and Lewis b. Unique features in the H. pylori fucosyltransferase genes, including homopolymeric tracts mediating slipped-strand mispairing and the elements regulating translational frameshifting, enable H. pylori to produce variable LPS epitopes on its surface. These new findings have provided us with a basis to further examine the roles of molecular mimicry and phase variation of H. pylori Lewis antigen expression in both persistent infection and pathogenesis of this important human gastric pathogen.     

Flow cytometric analysis of the localization of Helicobacter pylori antigens during different growth phases

Previous studies on the localization of several different Helicobacter pylori antigens have been contradictory. We have therefore examined by using both one- and two-color flow cytometry (FCM), immunofluorescence (IF), and immunoelectron microscopy (IEM), the possible surface localization of some H. pylori antigens that may be important virulence factors. All four methods detected the lipopolysaccharide and the N-acetyl-neuroaminyllactose-binding hemagglutinin protein (HpaA) as surface-exposed, while the urease enzyme was not detected at all and the neutrophil activating protein only in low concentration on the surface of the H. pylori bacteria during culture of H. pylori in liquid broth for 11 days. The FCM analysis was found to be quite sensitive and specific and also extremely fast compared with IF and IEM, and therefore the preferred method for detection of surface-localized antigens of H. pylori.     

Detection of specific Helicobacter pylori DNA and antigens in stool samples in dyspeptic patients and healthy subjects

In this study stool samples from dyspeptic patients and healthy subjects were used for detection of specific Helicobacter pylori antigens and DNA by immunoenzymatic test (PPHpSA) and semi-nested PCR (ureA-PCR), respectively. The H. pylori status was estimated by invasive endoscopy-based rapid urease test and histology or noninvasive urea breath test (UBT), and by serology (ELISA, Western blot). The coincidence of H. pylori-negative invasive tests or UBT and negative antigen or DNA stool tests was very high (mean 95%). The PPHpSA results were found positive for 56% and ureA-PCR for 26% of individuals with H. pylori infection confirmed by invasive tests or UBT. The detection of specific H. pylori antigens and especially DNA in feces is not sufficient as a one-step diagnosis of H. pylori infection.     

Synthesis of mono- and di-fucosylated type I Lewis blood group antigens by Helicobacter pylori.

The identification of Helicobacter pylori isolates that expresses exclusively type I Lewis antigens is necessary to determine the biosynthetic pathway of these antigens. Fast-atom bombardment MS provides evidence that the H. pylori isolate UA1111 expresses predominantly Leb, with H type I and Lea in lesser amounts. Cloning and expression of the H. pylori fucosyltransferases (FucTs) allow comparisons with previously identified H. pylori enzymes and determination of the enzyme specificities. Although all FucTs, one alpha(1,2) FucT and two alpha(1,3/4) FucTs, appear to be functional in this isolate, their activities are lower and enzyme specificities are different to other H. pylori FucTs previously characterized. Studies of the cloned enzyme activities and mutational analysis indicate that Lea acts as the substrate for the synthesis of Leb. This is different from the human Leb biosynthetic pathway, but analogous to the biosynthetic pathway utilized by H. pylori for the production of Ley.     

Helicobacter pylori from asymptomatic hosts expressing heptoglycan but lacking Lewis O-chains: Lewis blood-group O-chains may play a role in Helicobacter pylori induced pathology.

Helicobacter pylori is a widespread Gram-negative bacterium responsible for the onset of various gastric pathologies and cancers in humans. A familiar trait of H. pylori is the production of cell-surface lipopolysaccharides (LPSs; O-chain --> core --> lipid A) with O-chain structures analogous to some mammalian histo-blood-group antigens, those being the Lewis determinants (Lea, Leb, Lex, sialyl Lex, Ley) and blood groups A and linear B. Some of these LPS antigens have been implicated as autoimmune, adhesion, and colonization components of H. pylori pathogenic mechanisms. This article describes the chemical structures of LPSs from H. pylori isolated from subjects with no overt signs of disease. Experimental data from chemical- and spectroscopic-based studies unanimously showed that these H. pylori manufactured extended heptoglycans composed of 2- and 3-linked D-glycero-alpha-D-manno-heptopyranose units and did not express any blood-group O-antigen chains. The fact that another H. pylori isolate with a similar LPS structure was shown to be capable of colonizing mice indicates that H. pylori histo-blood-group structures are not an absolute prerequisite for colonization in the murine model also. The absence of O-chains with histo-blood groups may cause H. pylori to become inept in exciting an immune response. Additionally, the presence of elongated heptoglycans may impede exposure of disease-causing outer-membrane antigens. These factors may render such H. pylori incapable of creating exogenous contacts essential for pathogenesis of severe gastroduodenal diseases and suggest that histo-blood groups in the LPS may indeed play a role in inducing a more severe H. pylori pathology.     

Role of Helicobacter pylori Infection in the Treatment and Outcome of Chronic Urticaria

Introduction:  Chronic urticaria is thought to have numerous causative factors including a large variety of infectious conditions, food intake, and drugs. The impact of Helicobacter pylori infection has been studied with ambiguous results. The aim of this study was to investigate the course of chronic urticaria in H. pylori -positive patients undergoing eradication compared to H. pylori -negative urticaria patients.
Patients and Methods:  We included 74 urticaria patients with positive H. pylori breath test and 74 age- and sex-matched H. pylori -negative controls. All urticaria patients underwent an extensive diagnostic work-up to search for trigger foci. H. pylori -infected patients were submitted to eradication therapy. Mean follow-up time was 58 months.
Results:  Neither the prevalence of H. pylori nor the eradication therapy had an influence on the clinical course of chronic urticaria. In 81.1% of H. pylori -infected patients at least one additional infectious focus was found. Nevertheless, it could be shown that individuals that described any kind of symptom relief presented with higher serum IgE levels at diagnosis (198.1 vs 115.7 kU/L, p = .027) but this effect was independent of H. pylori infection.
Conclusions:  In conclusion there is no evidence that eradication of H. pylori improves the outcome in patients with chronic urticaria. The high rate of spontaneous remission and the coexisistance of multiple foci will always obscure the evaluation of any specific antimicrobial therapy.     

Pathophysiology and clinical relevance of Helicobacter pylori.

Considerable knowledge has recently accumulated on the mechanism by which Helicobacter pylori (H. pylori) induces chronic gastritis. Although H. pylori is not an invasive bacterium, soluble surface constituents can provoke pepsinogen release from gastric chief cells or trigger local inflammation in the underlying tissue. Urease appears to be one of the prime chemoattractants for recruitment and activation of inflammatory cells. Release of cytokines, such as tumor necrosis factor alpha, interleukin 1 and 6, and oxygen radicals, leads to a further tissue inflammation accompanied by a potent systemic IgA and IgG type of immune response. Chronic inflammation and antigens on glandular epithelial cells lead to a progressive destruction with loss of the epithelial barrier function. Within the gastric mucosa, patches of intestinal metaplasia develop, which may be a risk factor for subsequent development of gastric carcinoma. Hyperacidity in duodenal ulcer patients induces gastric metaplasia in the duodenal bulb, which represents a target for H. pylori colonization and ulcer formation. H. pylori can be detected in the majority of patients with peptic ulcers and, compared to age-matched healthy people, it is also found more often in patients with dyspepsia and gastric carcinoma. Although H. pylori can be detected in healthy people, the marked reduction of the ulcer recurrence rate by eradication of H. pylori (80 percent versus 20 percent relapse within one year) suggests that H. pylori is a major risk factor for duodenal ulcer formation. The potential role of H. pylori in non-ulcer dyspepsia and carcinogenesis is under investigation. Current regimens aimed at eradicating H. pylori use a combination of several drugs that are potentially toxic. Since the risk of complications may exceed the potential benefit in most patients, eradication treatment should be limited to clinical trials and to patients with aggressive ulcer disease. New drug regimens, e.g., the combination of proton pump inhibitors with one antibiotic, may provide less toxic alternatives. Beyond ulcer treatment, effective and well-tolerated eradication regimens may have a place in prophylaxis of gastric carcinoma.     

Lewis epitopes on outer membrane vesicles of relevance to Helicobacter pylori pathogenesis

BACKGROUND: Helicobacter pylori extrudes protein- and lipopolysaccharide-enriched outer membrane vesicles from its cell surface which have been postulated to act to deliver virulence factors to the host. Lewis antigen expression by lipopolysaccharide of H. pylori cells has been implicated in a number of pathogenic roles. The aim of this study was to further characterize the expression of lipopolysaccharide on the surface of these outer membrane vesicles and, in particular, expression of Lewis antigens and their association with antibody production in the host. MATERIALS AND METHODS: H. pylori strains were examined for outer membrane vesicle production using transmission electron microscopy and Lewis antigen expression probed using immunoelectron microscopy. Sera from patients were analyzed for cross-reacting anti-Lewis antibodies and, subsequently, absorbed using outer membrane vesicle preparations to remove the cross-reacting antibodies. RESULTS: The formation of outer membrane vesicles by H. pylori was observed in both in vitro and in vivo samples. Furthermore, vesicles were produced following culture in either liquid or solid medium by all strains examined. Moreover, we observed the presence of Lewis epitopes on outer membrane vesicles using immunoelectron microscopy and immunoblotting. Circulating anti-Lewis antibodies were found in the sera of gastric cancer patients but not in the sera of H. pylori-negative control subjects. Absorption of patient sera with outer membrane vesicles decreased the levels of anti-Lewis autoantibodies. CONCLUSIONS: Our results demonstrate the ability of H. pylori to generate outer membrane vesicles bearing serologically recognizable Lewis antigens on lipopolysaccharide molecules which may contribute to the chronic immune stimulation of the host. The ability of these vesicles to absorb anti-Lewis autoantibodies indicates that they may, in part, play a role in putative autoimmune aspects of H. pylori pathogenesis.