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Clinical Microbiology Reviews, July 2006, p. 449-490, Vol. 19, No. 3
0893-8512/06/$08.00+0     doi:10.1128/CMR.00054-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Pathogenesis of Helicobacter pylori Infection

Department of Gastroenterology and Hepatology, Erasmus MC—University Medical Center, Rotterdam, The Netherlands

SUMMARY

INTRODUCTION

HISTORY

MICROBIOLOGY

Genus Description and Phylogeny

Gastric Helicobacter species.

(i) Helicobacter felis.

(ii) Helicobacter mustelae.

(iii) Helicobacter acinonychis.

(iv) Helicobacter heilmannii.

Enterohepatic Helicobacter species.

Microbiology of H. pylori

Genome, plasmids, and strain diversity.

Morphology.

Growth requirements.

Metabolism.

(i) Respiration and oxidative stress defense.

(ii) Nitrogen metabolism.

(iii) Metal metabolism.

Cell envelope, outer membrane, and LPS.

Gene regulation.

EPIDEMIOLOGY

Prevalence and Geographical Distribution

Transmission and Sources of Infection

CLINICAL ASPECTS OF H. PYLORI-ASSOCIATED DISEASES

Disease Types

Acute and chronic gastritis.

(i) Acute gastritis.

(ii) Chronic gastritis.

Peptic ulcer disease. (i) Definitions.

(ii) Association with H. pylori.

(iii) Ulcer epidemiology.

(iv) Ulcer complications.

(v) H. pylori and NSAIDs.

Non-ulcer dyspepsia.

Atrophic gastritis, intestinal metaplasia, and gastric cancer.

Gastric MALT lymphoma.

GERD.

Extragastroduodenal disorders.

Histopathology

DIAGNOSIS AND TREATMENT

Diagnosis

Treatment

Vaccination

PATHOGENESIS OF INFECTION

H. pylori-Associated Pathogenesis

Animal Models

Mouse.

Mongolian gerbil.

Guinea pig.

Gnotobiotic piglets.

Nonhuman primates.

Role of H. pylori Virulence Factors

cag PAI.

VacA vacuolating cytotoxin.

Acid resistance.

Adhesins and outer membrane proteins.

(i) BabA (HopS).

(ii) OipA (HopH).

(iii) SabA (HopP).

LPS.

Immune Response

Role of antibodies in protective immunity.

Immune modulation.

Activation of the innate immune response.

Resistance to phagocytosis and modulation of dendritic cell activity.

Regulatory T cells.

Contribution of Host Genetics

IL-1.

Other cytokines.

CONCLUSIONS

REFERENCES

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INTRODUCTION

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It has been known for more than a century that bacteria are present in the human stomach (56). These bacteria, however, were thought to be contaminants from digested food rather than true gastric colonizers. About 20 years ago, Barry Marshall and Robin Warren described the successful isolation and culture of a spiral bacterial species, later known as Helicobacter pylori (684), from the human stomach. Self-ingestion experiments by Marshall (398) and Morris (442) and later experiments with volunteers (443) demonstrated that these bacteria can colonize the human stomach, thereby inducing inflammation of the gastric mucosa. Marshall developed a transient gastritis after ingestion of H. pylori; the case described by Morris developed into a more persistent gastritis, which resolved after sequential therapy with first doxycycline and then bismuth subsalicylate. These initial data strongly stimulated further research, which showed that gastric colonization with H. pylori can lead to variety of upper gastrointestinal disorders, such as chronic gastritis, peptic ulcer disease, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. This knowledge had a major clinical impact with regard to the management of these diseases. In addition, the persistence of a pathogen in an environment long thought to be sterile also resulted in insights into the pathogenesis of chronic diseases. This discovery resulted in the awarding of the 2005 Nobel Prize in Physiology or Medicine to Robin Warren and Barry Marshall for their "discovery of the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease."

The number of peer-reviewed publications on Helicobacter has rapidly increased, from less than 200 in 1990 to approximately 1,500 per year over the last few years (PubMed [www.pubmed.gov]). Despite this wide attention important issues, such as the transmission route of H. pylori, are still poorly understood. Although the prevalence of H. pylori in the Western world is decreasing, gastric colonization by H. pylori remains widespread in the developing world. Infection with H. pylori can be diagnosed by a variety of tests and can often be successfully treated with antibiotics. Unfortunately, the increase in antibiotic resistance is starting to affect the efficacy of treatment, and, in spite of the impact of H. pylori, preventive vaccination strategies still do not exist. A better understanding of H. pylori persistence and pathogenesis is thus mandatory to aid the development of novel intervention and prevention strategies. This review focuses on the pathogenesis of H. pylori infection, with emphasis on its microbiological aspects.

HISTORY

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By the late 19th and early 20th centuries, several investigators had reported the presence of spiral microorganisms in the stomachs of animals (56). Soon afterward, similar spiral bacteria were observed in humans (328, 379, 497), some of whom had peptic ulcer disease or gastric cancer. The etiological role of these bacteria in the development of peptic ulcer disease and gastric cancer was considered at the time, and patients were sometimes even treated with high doses of the antimicrobial compound bismuth (497). This possibility was later discarded as irrelevant, probably because of the high prevalence of these spiral bacteria in the stomachs of persons without any clinical signs. The bacteria observed in human stomachs were thus considered to be bacterial overgrowth or food contaminants until the early 1980s. At this time, Warren and Marshall performed their groundbreaking experiments, leading to the identification of a bacterium in 58 of 100 consecutive patients, with successful culture and later demonstration of eradication of the infection with bismuth and either amoxicillin or tinidazole (397-401, 684). The organism was initially named "Campylobacter-like organism," "gastric Campylobacter-like organism," "Campylobacter pyloridis," and "Campylobacter pylori" but is now named Helicobacter pylori in recognition of the fact that this organism is distinct from members of the genus Campylobacter (234). It soon became clear that this bacterium causes chronic active gastritis, which in a subset of subjects may progress to other conditions, in particular, peptic ulcer disease, distal gastric adenocarcinomas, and gastric lymphomas (168).

MICROBIOLOGY

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Helicobacter species can be subdivided into two major lineages, the gastric Helicobacter species and the enterohepatic (nongastric) Helicobacter species. Both groups demonstrate a high level of organ specificity, such that gastric helicobacters in general are unable to colonize the intestine or liver, and vice versa. An extensive review of non-pylori Helicobacter species is available (587), and here we briefly discuss those Helicobacter species that are either associated with human disease or have relevance for animal models of human Helicobacter infections (Table 1).

(i) Helicobacter felis. The spiral-shaped Helicobacter felis was first isolated from the stomach of a cat (353) and was later also found in dogs. Subsequently designated H. felis (493), it was probably also the Helicobacter species originally described by Bizzozero in 1893 (56). H. felis is one of the Helicobacter species with zoonotic potential (344, 351). It has a helical morphology with typical periplasmic fibers, which can be used for microscopic identification. H. felis requires high humidity and can only poorly, if at all, be cultured on standard growth media used for the culture of H. pylori (344). H. felis is highly motile; on agar plates it does not really form colonies but rather grows as a lawn (235).

The significance of H. felis in gastric disorders of cats and dogs is somewhat unclear, since there is no clear association between canine and feline gastritis and H. felis infection (78, 144, 566-568). It is therefore possible that H. felis comprises part of the normal gastric flora in cats and dogs. In contrast, H. felis has been used in murine models of Helicobacter infection (352), where it can induce gastritis, epithelial cell proliferation, and apoptosis (85, 98, 417). Murine infection with H. felis results in a mononuclear cell-predominant inflammatory response in the gastric corpus that may progress to atrophic gastritis (180, 432, 433).

There is at present little information available about the virulence genes, physiology, or metabolism of H. felis, since H. felis is only poorly amenable to the genetic techniques used for H. pylori. The bacterium contains a urease gene cluster resembling that of other gastric Helicobacter species (181, 182), as well as two flagellin genes (flaA and flaB) (297). The latter genes have been inactivated, and this resulted in truncated flagella and reduced motility. Mutation of flaA also resulted in the inability to colonize a murine model of infection (297).

(ii) Helicobacter mustelae. The ferret pathogen H. mustelae was isolated shortly after H. pylori and was originally classified as Campylobacter pylori subsp. mustelae (197, 202, 630). It was subsequently shown to have characteristics different from H. pylori (198) and was later classified as H. mustelae (234). H. mustelae a is relatively small rod, which has multiple polar and lateral sheathed flagella. Interestingly, H. mustelae is phylogenetically closer to the enterohepatic Helicobacter species (242), based on its 16S rRNA gene sequence, urease sequences, and fatty acid profile (242, 256), but to our knowledge H. mustelae has not been implicated in enteric colonization in ferrets.

The ferret stomach resembles the human stomach at both the anatomical and physiological levels (193), and gastritis, gastric ulcer, gastric adenocarcinoma, and MALT lymphoma in ferrets have all been described (163, 199, 200). H. mustelae infection is very common in ferret populations (295), and this suggests that H. mustelae is a member of the resident flora of the ferret stomach. H. mustelae shares many virulence factors with H. pylori, including a urease enzyme (139), motility (604), and molecular mimicry of host blood group antigens (465). Ultrastructural studies have shown that H. mustelae adheres intimately to gastric epithelial cells in a manner that closely resembles the adherence of H. pylori (199, 475). H. mustelae also induces an autoantibody response similar to that observed in H. pylori-infected humans (464).

The similarities between these two natural infections suggest that H. mustelae infection of the ferret is a suitable model to characterize the role played by Helicobacter virulence factors in vivo (199). H. mustelae is also amenable to genetic manipulation; thus, H. mustelae is an interesting candidate for investigation of the role of Helicobacter virulence factors in the natural host. This will be aided by the ongoing determination of the complete genome sequence of H. mustelae (available at http://www.sanger.ac.uk/Projects/H_mustelae).

(iii) Helicobacter acinonychis. H. acinonychis, a pathogen of cheetahs and other big cats (formerly named Helicobacter acinonyx [145]), is currently the closest known relative to H. pylori (242) and has been suggested to have diverged from its last common ancestor (H. pylori) only relatively recently (241). The presence of H. acinonychis is associated with chronic gastritis and ulceration, a frequent cause of death of cheetahs in captivity (444). Furthermore, eradication treatment of H. acinonychis led to the resolution of gastric lesions in tigers (77), similar to the effect of antibiotic treatment of H. pylori infection (228). H. acinonychis is susceptible to antibiotic therapy, as used for H. pylori infection, and utilizes similar mechanisms for antimicrobial resistance (509).

H. acinonychis is genetically amenable, by techniques similar to those developed for H. pylori, and H. acinonychis shares several virulence factors with H. pylori but contains only a degenerate copy of the vacA gene (111) and lacks the cag pathogenicity island (PAI) (241). Recently, mouse-colonizing strains of H. acinonychis have been described (111); this should allow further comparisons of the pathogenic properties of H. acinonychis, as well as comparison with the pathogenesis of H. pylori infection. Furthermore, the pending release of the complete genome sequence of H. acinonychis (241) will give further insight into the evolutionary relationship between H. acinonychis and H. pylori.

(iv) Helicobacter heilmannii. The diverse species H. heilmannii was originally designated Gastrospirillum hominis and is a Helicobacter species with a wide host range (583, 586). It has been isolated from several domestic and wild animals, including dogs, cats, and nonhuman primates, and is also observed in a small percentage of humans with gastritis (194). In the latter, colonization may reflect a zoonosis, as there is an association between colonization with this bacterium and close contact with dogs and cats carrying the same bacterium (598). Its morphology resembles that of H. felis, but H. heilmannii lacks the periplasmic fibers.

Human H. heilmannii infection may result in gastritis and dyspeptic symptoms (4), and in sporadic cases even in ulcer disease, but the inflammation is usually less marked than in H. pylori-positive subjects and may be spontaneously transient (598). In a mouse model of infection, different H. heilmannii isolates of both human and animal origin were able to induce gastric B-cell MALT lymphoma (476). Characterization of this Helicobacter species is difficult, since it has not been successfully cultured in vitro, and it may be necessary to make a further subdivision of the species H. heilmannii. Recent phylogenetic analyses have led to the proposal of the species designation "Candidatus Helicobacter heilmannii," but this is mostly based on 16S rRNA and urease sequence analyses and thus awaits further confirmation (478).

Enterohepatic Helicobacter species. Enterohepatic Helicobacter species colonize the lower gastrointestinal tract, including the ileum, colon, and biliary tree of humans and other mammals. They cause persistent infections, which are associated with chronic inflammation and epithelial cell hyperproliferation that can lead to neoplastic disease, and are associated with human hepatobiliary disease (31, 587, 664). The group of enterohepatic Helicobacter species consists of many different species, differing in morphology, ultrastructure, growth conditions, and the presence or absence of the urease virulence factor (587). Only one of these species has been more than superficially characterized, the murine pathogen H. hepaticus, and is discussed here.

The enterohepatic pathogen H. hepaticus infects rodents, in which it may cause chronic active hepatitis, hepatic tumors, and proliferative typhlocolitis (681, 683). It was initially isolated from a colony of male A/JCr mice with a high incidence of hepatitis and hepatic cancer (683). Subsequently it was shown that several inbred strains of mice were susceptible to hepatic lesions after infection with H. hepaticus. In addition, many commercially available mouse strains were shown to be naturally infected with H. hepaticus (562).

Although it was first identified in the liver, the primary site of H. hepaticus colonization is the intestinal tract; it has not been found in the stomach (587). In immunocompetent mice, infection with H. hepaticus results in mild intestinal inflammation, but in immunodeficient and SCID mice, infection with H. hepaticus leads to severe colitis, typhlitis, and proctitis, which resemble lesions found in animal models of inflammatory bowel disease (682). H. hepaticus is among several Helicobacter species identified in rodents with disease of the hepatobiliary or intestinal tracts, including H. bilis, H. muridarum, and H. trogontum (587). In a recent study, mice were fed a lithogenic diet and were coinfected with H. hepaticus and Helicobacter rodentium. These mice developed cholesterol gallstones at 80% prevalence by 8 weeks, suggesting a link between infection with enterohepatic Helicobacter species and gallstone formation (404). In comparison, this association is not found when these mice are infected with H. pylori (405). H. hepaticus infection of mice can be treated with antibiotics (189, 534), and this results in resolution of lesions associated with the infection.

H. hepaticus is morphologically similar to Campylobacter species, with bipolar sheathed flagella (201). It is urease, oxidase, and catalase positive and grows on most standard H. pylori growth media, including ß-cyclodextrin-supplemented media (45). Growth conditions are similar to those employed for H. pylori, and selective antibiotic supplements used for H. pylori can also be used for isolation and subsequent cultivation of H. hepaticus (45). Although it has been well established that infection with H. hepaticus causes diverse diseases in rodents, relatively little is known about its mechanisms of virulence. Several putative virulence factors of H. hepaticus have been identified, including the cytolethal distending toxin (CDT) and a potent urease enzyme (43), but mutational analysis demonstrating the role of these virulence factors in colonization or hepatic diseases is available only for CDT (712). Recently, the complete genome sequence of H. hepaticus was determined (605), and this revealed the presence of a potential PAI, coined HHGI1 (65, 605). Furthermore, H. hepaticus is also genetically amenable by both electroporation (415, 712) and natural transformation (45), albeit to a lower efficiency than H. pylori and other gastric Helicobacter species. Taken together, this makes H. hepaticus an attractive organism for elucidation of the molecular mechanisms involved in adaptation to the enteric and hepatic niches and of the mechanisms of enterohepatic pathogenesis.

In contrast to other bacterial pathogens that are highly clonal (such as Shigella dysenteriae and Mycobacterium tuberculosis), H. pylori is genetically heterogeneous, suggesting a lack of clonality. This results in every H. pylori-positive subject carrying a distinct strain (300), although differences within relatives may be small. The genetic heterogeneity is possibly an adaptation of H. pylori to the gastric conditions of its host, as well as to the distinct patterns of the host-mediated immune response to H. pylori infection (333). Genetic heterogeneity is thought to occur via several methods of DNA rearrangement and the introduction and deletion of foreign sequences (1, 176, 603). The latter usually have an aberrant G+C content and often carry genes involved in virulence. A striking example of this in H. pylori is the cag PAI, but other plasticity regions have also been suggested to play a role in the pathogenesis of H. pylori infection (115, 357, 462, 545).

Diversity is also seen at the nucleotide level via several mechanisms, including transcriptional and translational phase variation and mutation (1, 175, 602, 606). Phase variation often occurs via reversible slipped-strand mispairing in homopolymeric G or C tracts, which causes a shift in translation of the affected gene, thus resulting in phase variation via a single mutation. This leads to a reversible phenotypic diversity with only minor genetic variation. Several virulence genes, such as the sabA, sabB, hopZ, and oipA outer membrane protein-encoding genes, display such phenotypic variation, as do lipopolysaccharide (LPS) biosynthetic enzymes (18, 28, 117, 387).

Morphology. H. pylori is a gram-negative bacterium, measuring 2 to 4 µm in length and 0.5 to 1 µm in width. Although usually spiral-shaped, the bacterium can appear as a rod, while coccoid shapes appear after prolonged in vitro culture or antibiotic treatment (342). These coccoids cannot be cultured in vitro and are thought to represent dead cells (342), although it has been suggested that coccoid forms may represent a viable, nonculturable state (162). The organism has 2 to 6 unipolar, sheathed flagella of approximately 3 µm in length, which often carry a distinctive bulb at the end (481). The flagella confer motility and allow rapid movement in viscous solutions such as the mucus layer overlying the gastric epithelial cells (481). In contrast to many other pathogens of the gastrointestinal tract, it lacks fimbrial adhesins.

Growth requirements. A key feature of H. pylori is its microaerophilicity, with optimal growth at O₂ levels of 2 to 5% and the additional need of 5 to 10% CO₂ and high humidity. There is no need for H₂, although it is not detrimental to growth. Many laboratories utilize standard microaerobic conditions of 85% N₂, 10% CO₂, and 5% O₂ for H. pylori culture. Growth occurs at 34 to 40°C, with an optimum of 37°C. Although its natural habitat is the acidic gastric mucosa, H. pylori is considered to be a neutralophile. The bacterium will survive brief exposure to pHs of <4, but growth occurs only at the relatively narrow pH range of 5.5 to 8.0, with optimal growth at neutral pH (554, 595).

H. pylori is a fastidious microorganism and requires complex growth media. Often these media are supplemented with blood or serum. These supplements may act as additional sources of nutrients and possibly also protect against the toxic effects of long-chain fatty acids. The latter function may also be performed by more defined medium supplements, such as ß-cyclodextrins or IsoVitaleX, or by using activated charcoal (616). Commonly used solid media for routine isolation and culture of H. pylori consist of Columbia or brucella agar supplemented with either (lysed) horse or sheep blood or, alternatively, newborn or fetal calf serum. For primary isolation but also routine culture, selective antibiotic mixtures are available, although these are not required per se. The often used Dent supplement consists of vancomycin, trimethoprim, cefsoludin, and amphotericin B (121), whereas the alternatively used Skirrow supplement consists of vancomycin, trimethoprim, polymyxin B, and amphotericin B (573). Both selective supplements are commercially available. Liquid media usually consist of either brucella, Mueller-Hinton, or brain heart infusion broth supplemented with 2 to 10% calf serum or 0.2 to 1.0% ß-cyclodextrins, often together with either Dent or Skirrow supplement. Growth of H. pylori in chemically defined media has been reported (526), but these are not suitable for routine growth and isolation of H. pylori. Most of the commercially available synthetic media, such as tissue culture media, do not support the growth of H. pylori without the addition of serum, perhaps with the exception of Ham's F-12 nutrient mixture (622).

Isolation of H. pylori from gastric biopsy samples is difficult and not always successful. Cultures should be inspected from day 3 to day 14. H. pylori forms small (1-mm), translucent, smooth colonies (254). Upon successful subculturing, H. pylori isolates tend to adapt to the growth conditions used in the laboratory. Subsequently, good growth can generally be achieved following 1 to 3 days of incubation when reference strains and laboratory-adapted isolates of H. pylori are used. It should be noted that once a culture reaches the stationary phase, the growth rate rapidly declines, accompanied by the morphological change to a coccoid form (342). Prolonged culture does not lead to any significant increase in colony size but rather leads to a transition to the unculturable coccoid state. To facilitate optical detection of H. pylori, plates can be supplemented with triphenyltetrazolium chloride (TTC) to a final concentration of 0.004% (690). In the presence of TTC, H. pylori colonies appear dark red via the reduction of TTC to deep red formazan (60) and develop a golden shine. H. pylori can be stored for the long term at –80°C in brain heart infusion or brucella broth supplemented with either 15 to 20% glycerol or 10% dimethyl sulfoxide, but optimal viability requires the use of cultures less than 48 h old, with more than 90% spiral-shaped cells.

Metabolism. H. pylori exhibits a narrow host and target organ range, but infection is usually lifelong. This suggests strong adaptation to its natural habitat, the mucus layer overlying the gastric epithelial cells. As a consequence, H. pylori lacks several of the biosynthetic pathways commonly found in less specialized bacteria, such as many enteric bacteria (14, 49, 126, 392, 628). It has been inferred from genomic comparisons and metabolic studies that H. pylori has a stripped-down metabolic route with very few redundancies and lacks biosynthetic pathways for some amino acids. As a consequence, H. pylori can be grown only in chemically defined medium with the additional amino acids arginine, histidine, isoleucine, leucine, methionine, phenylalanine and valine, and some strains also require alanine and/or serine (456, 526). H. pylori is urease, catalase, and oxidase positive, characteristics which are often used in identification of H. pylori. H. pylori can catabolize glucose, and both genomic and biochemical information indicates that other sugars cannot be catabolized by H. pylori (49, 126, 392, 456).

Our understanding of the metabolism of H. pylori has been greatly facilitated by the availability of two complete genome sequences (14, 628). Many of the previously described biochemical deficiencies of H. pylori were confirmed by the apparent lack of genes encoding the corresponding enzymes for these metabolic routes (49, 126, 311, 312, 392). However, it must be stressed that many of these analyses are based on sequence homology and await further experimental validation. In this section we discuss only those systems involved in or connected to the virulence of H. pylori; for in-depth reviews of H. pylori metabolism, see references 126, 311, 312, and 392.

(i) Respiration and oxidative stress defense. H. pylori is a microaerophilic bacterium that does not tolerate high oxygen conditions, but it requires at least 2% O₂ (420). This is because H. pylori uses oxygen as a terminal electron acceptor. H. pylori cannot utilize alternative electron acceptors, such as nitrate or formate, although there is a single report on anaerobic growth of H. pylori using fumarate (578). In the human host, H. pylori is thought to be exposed to oxidative stress produced by the active immune response. To combat such forms of oxidative stress, H. pylori expresses several key components of bacterial oxidative stress resistance; these include the superoxide stress defense mediated via the iron-cofactored superoxide dismutase (SodB) (38, 166, 561) and the peroxide stress defense mediated via catalase (KatA) and alkyl hydroperoxide reductase (AhpC) (257, 474). In addition, the two thioredoxins, its respective reductases, and the thiol-peroxidase Tcp mediate resistance to both nitrosative and oxidative stresses (36, 94, 473, 474, 675). The neutrophil activating protein (HP-NAP) (see also "Pathogenesis of Infection," below) is a member of the Dps family and is thought to protect DNA from the detrimental effects of reactive oxygen species (38, 473, 474). As indicated by its name, HP-NAP is also implicated in the activation of neutrophils, leading to the formation of reactive oxygen species (172), but this function is still under debate and may well be an artifact (38, 436). The formation of reactive oxygen species is connected with iron metabolism, as oxygen radicals can be produced via the Fenton and Haber-Weiss reactions (Fe²⁺ + O₂ Fe³⁺ + O₂^– and Fe²⁺ + H₂O₂ Fe³⁺ + OH^– + OH·). Thus, it is not surprising that proteins involved in iron metabolism, such as the global iron-responsive regulator Fur, the FeoB iron transporter, the iron-storage protein ferritin, and the iron-cofactored SodB, are involved in oxidative stress resistance (166, 474, 561, 659, 670). Interestingly, mutation of the carbon storage regulator protein CsrA also affects oxidative stress resistance, although the mechanism underlying this phenomenon has not yet been elucidated (38). Many of the components of oxidative stress defense are essential for host colonization, as the absence of one or more factors often results in lower levels or absence of colonization by H. pylori in animal models (8, 38, 73, 257, 258, 473, 474, 561, 673-675).

(ii) Nitrogen metabolism. Amino acids and urea are the two major sources of nitrogen in the gastric environment. Since ammonia is a key component in nitrogen metabolism as well as acid resistance (595), it is not surprising that H. pylori can utilize several alternative sources of ammonia (71, 406, 422, 574, 575, 656). The different pathways contributing to ammonia synthesis are regulated in response to different stimuli, which probably allows H. pylori to switch different pathways on or off depending on the environmental conditions (summarized in Fig. 1).

View larger version (26K): [in this window] [in a new window]   FIG. 1. Schematic representation of the relationships between acid resistance (urease activity and urea transport), nitrogen metabolism (ammonia production), metal metabolism (iron uptake and nickel uptake), and gene regulation (Fur and NikR) in H. pylori.

In addition to facilitating survival and growth in acidic conditions, the ammonia produced via enzymatic degradation of urea is used for amino acid biosynthesis. The importance of ammonia in H. pylori metabolism and virulence is underlined by the presence of several alternative routes for ammonia production, via enzymatic degradation of diverse amides as well as amino acids (Fig. 1) (71, 406, 422, 574, 575, 656). H. pylori expresses two paralogous amidases, the wide-range aliphatic amidase AmiE as well as the formamidase AmiF. Together with four amino acid deaminases, these probably serve as sources for ammonia in environments low in urea. It is remarkable that H. pylori also expresses several key components of the eukaryotic urea cycle (418) and thus may be able to produce urea from ammonia. One of the major components of the H. pylori urea cycle is the arginase (RocF) enzyme, which converts L-arginine to L-ornithine and urea. Mutation of the rocF gene did not affect urease activity but significantly reduced the acid resistance of H. pylori (406). Since the rocF mutation also decreased the activity of one of the amino acid deaminases via an unknown mechanism, it is unclear whether the reduced acid resistance is a direct effect of the lack of arginase or an indirect effect (406). H. pylori arginase is also implicated in the control of nitric oxide production in eukaryotic cells (232), and arginine metabolism of the eukaryotic cell may also be controlled by H. pylori (231).

(iii) Metal metabolism. Metals play an important role in the metabolism of all organisms, which is reflected by the wide variety of chemical reactions in which they are involved. Metals are cofactors of enzymes, catalyzing basic functions such as electron transport, redox reactions, and energy metabolism, and are essential for maintaining the osmotic pressure of the cell. Since both metal limitation and metal overload delay growth and can cause cell death, metal homeostasis is of critical importance to all living organisms.

(a) Nickel. H. pylori requires efficient acquisition of nickel, as this is the metal cofactor of the essential colonization factors urease and hydrogenase. Nickel availability in human serum is very low (2 to 11 nM), and the nickel concentration in ingested food varies significantly depending on the diet and on food sources (91, 608). One certain and several potential nickel transporters have been identified in H. pylori. The NixA protein (HP1077) is a 37-kDa protein located in the cytoplasmic membrane and has been demonstrated to have a high affinity for nickel (Fig. 1) (206, 428). Mutation of the nixA gene results in strongly reduced nickel transport and lowered urease activity (40). Absence of nixA also affected colonization efficiency in a mouse model of H. pylori infection, presumably due to the reduced urease activity (459). A second putative nickel transport system may be encoded by the abcCD locus (266), since abcC nixA double mutants showed only residual urease activity. However, a role of the abcCD system in nickel transport has not yet been demonstrated. A third system thought to be involved in nickel transport is the Dpp dipeptide permease, but mutation of this gene did not affect overall urease activity (112). It is still unclear how nickel enters H. pylori; however, since mutation of nixA complemented the nickel sensitivity of an H. pylori nikR mutant (95, 167, 655), this strongly supports an important role of NixA in nickel transport of H. pylori.

Several urease- and hydrogenase-associated systems have adapted to the central role of nickel, as is demonstrated by the effect of hydrogenase accessory proteins on urease activity (46, 413, 414) and the presence of a nickel-binding motif on the HspA chaperone (299). Next to the nickel transporters and urease/hydrogenase accessory proteins, H. pylori also expresses one or two small, very histidine-rich proteins (Hpn), which show strong binding to nickel (225, 429). Mutation of the hpn gene rendered H. pylori sensitive to nickel (429), which is suggestive of a role of Hpn in nickel storage or nickel sequestration, but this remains to be proven experimentally.

(b) Iron. In tissues of human or animal hosts, the concentration of free iron is too low to support bacterial growth, as most iron is complexed into hemoglobin or chelated by transferrin in serum or by lactoferrin at mucosal surfaces (652). Iron sources available in the gastric mucosa are lactoferrin, heme compounds released from damaged tissues, and iron derived from pepsin-degraded food. It is thought that metals such as iron display increased solubility under the acidic conditions of the gastric mucosa and that eukaryotic iron-complexing proteins display lowered binding affinity under these conditions. The H. pylori genome encodes 11 proteins predicted to be involved in iron transport and 2 proteins thought to function as iron storage proteins (14, 49, 628, 652). In the acidic, microaerobic gastric environment, ferrous iron (Fe²⁺) is thought to constitute the main form of free iron, and this is transported by H. pylori via the FeoB protein (HP0687) (659). FeoB-mediated iron acquisition is of major importance to H. pylori, as isogenic feoB mutants were unable to colonize the gastric mucosa of mice (659). H. pylori also possesses ferric reductase activity, which converts ferric iron (Fe³⁺) to Fe²⁺, which is then subsequently transported by the FeoB system (695). However, the importance of ferric reductase activity in gastric colonization remains to be assessed. Finally, H. pylori also possesses several ferric iron transport systems (652, 657). Due to the insolubility of ferric iron, ferric iron transport requires an outer membrane receptor to transport the iron over the outer membrane, as well as an ABC transporter to transport the iron from the periplasm to the cytoplasm. H. pylori has three copies of the putative ferric citrate outer membrane receptor FecA and three copies of the FrpB outer membrane receptor, for which the substrate is unknown (14, 49, 628, 652). There are two copies of the periplasmic binding protein CeuE and finally a single inner membrane permease (FecD) and an ATP-binding protein (FecE). Currently, only mutants in the fecDE system and in one of the fecA genes have been described (659). Rather surprisingly, this did not affect iron transport. Thus, the contribution of ferric iron uptake in H. pylori remains to be clarified. Two iron storage proteins in H. pylori have been characterized, the Pfr ferritin and HP-NAP bacterioferritin. The 19-kDa Pfr ferritin serves as an intracellular iron deposit and protects H. pylori against iron toxicity and free iron-mediated oxidative stress (47, 48, 125, 205, 670). Iron stored in Pfr can be released and reused to support growth under iron-limited conditions (670). HP-NAP was originally isolated as an immunodominant protein that activates neutrophilic granulocytes in vitro (172). It was subsequently shown to also mediate adhesion of H. pylori to mucin (452). The HP-NAP protein is homologous both to bacterioferritins and to the DNA-binding proteins of the Dps family (137, 631). However, a role of HP-NAP in H. pylori iron storage, although suggested, is yet to be demonstrated (631).

(c) Copper. Copper is a cofactor for several proteins involved in electron transport, oxidases, and hydroxylases, but may also contribute to the formation of reactive oxygen species (525). H. pylori expresses several proteins which either are involved in copper transport or may act as copper chaperones. It is currently unclear whether there is a specific import system for copper or whether it is transported by other metal transporters such as FeoB or NixA. However, H. pylori expresses several proteins involved in copper export, including the CopA (HP1073) and CopA2 (HP1503) P-type ATPases (41, 44, 216, 416) and the CrdA (HP1365) copper resistance determinant (671). Furthermore, H. pylori also expresses a small protein, CopP, that may function as a copper chaperone (41, 44, 216, 416).

(d) Cobalt. The trace metal cobalt is a cofactor of the arginase enzyme, which plays an important role in nitrogen metabolism of H. pylori (406, 419) but also in modulating the immune response to H. pylori (231, 232). It has been noted that H. pylori is exquisitely sensitive to cobalt in vitro (95), and it has been suggested that cobalt may be used in nonantibiotic therapy of H. pylori infections (68, 74).

Cell envelope, outer membrane, and LPS. The overall composition of the cell envelope of H. pylori is similar to that of other gram-negative bacteria. It consists of an inner (cytoplasmic) membrane, periplasm with peptidoglycan, and an outer membrane. The outer membrane consists of phospholipids and LPS. The H. pylori outer membrane phospholipid moiety contains cholesterol glucosides (69, 256, 618, 619), which is very rare in bacteria. LPS usually consists of lipid A, core oligosaccharide, and an O side chain. The lipid A moiety of H. pylori LPS has low biological activity compared to lipid A from other bacteria (446). Clinical isolates of H. pylori produce high-molecular-weight (smooth) LPS with an O antigen, but during in vitro culturing the bacteria may convert to rough LPS variants, which lack the O side chain (440, 672). The O side chain of H. pylori can be fucosylated and mimics Lewis blood group antigens (Lewis x [Le^x] and Le^y), aiding molecular mimicry of host antigens and associated immune evasion (20). The O antigen can also mimic other blood group antigens (437). H. pylori LPS displays phase variation through length variation of poly(C) tracts in the genes encoding -1,3-fucosyltransferases (18) and a poly(C) tract and poly(TAA) repeats in the gene encoding the -1,2-fucosyltransferase (676). This LPS phase variation contributes to population heterogeneity and may allow adaptation of H. pylori to changing conditions in the gastric mucosa (440, 619, 620).

The H. pylori genome encodes a large array of outer membrane proteins, which have been grouped into five paralogous families (13, 128). The largest gene family consists of 21 Hop and 12 Hor outer membrane proteins, and this family includes the known (putative) adhesins of H. pylori (171). Other families include porins (127), iron transporters (see above), flagellum-associated proteins, and proteins of unknown function. The outer membrane of H. pylori often also contains urease and heat shock proteins, which are otherwise found only in the cytoplasm. Although specific export of these proteins cannot be completely excluded (649), this may be due to "altruistic lysis," a process wherein part of the population of bacterial cells lyses and releases its cytoplasmic proteins, which are subsequently used by surviving bacteria to coat their outer membrane with proteins from lysed cells (395, 507). The function of this process is not yet understood, but it can be envisaged that it aids in protection against environmental stresses and functions in diversion of the immune response of the host.

Gene regulation. Rapid responses to stressful changes in environmental conditions are often mediated via changes in transcription of sets of genes that encode some factor involved in dealing with these stresses. Examples of this are the expression of oxidative stress defense genes in response to oxidative stress. In many bacteria, such stress-responsive systems are often encoded by genes organized in an operon, and the transcription is regulated by one or two regulatory proteins.

The only known niche of H. pylori is the human gastric mucosa, and this niche was considered to offer relatively stable conditions and limited competition with other bacteria. This hypothesis was consistent with the relatively low number of predicted regulatory proteins in the H. pylori genome (122). H. pylori lacks several two-component regulatory systems, which are thought to function as sensors of changes in the environment. The H. pylori genome contains only 4 complete two-component regulatory systems, which strongly contrasts to the 36 complete two-component regulatory systems found in Escherichia coli K-12 (479, 701). The repertoire of regulatory proteins mediating regulation in response to cytoplasmic changes is similar to that of related bacteria, such as Campylobacter, but also lower than that of E. coli. Overall, H. pylori contains only three sigma factors (⁸⁰, ²⁸, and ⁵⁴), two metalloregulatory proteins, two heat shock regulators, four complete two-component regulatory systems, two orphan response regulators, and a few other regulatory proteins (122, 546, 658).

It is becoming apparent, however, that the gastric mucosa may not be the relatively stable environment previously envisaged. Examples of complex, overlapping regulatory responses are becoming apparent with comparison of transcriptional profiling studies using different environmental stimuli (73, 165, 422, 423, 653, 658, 688). The regulatory responses to iron restriction, nickel, and acid show considerable overlap with each other and with growth phase-regulated genes (Fig. 1) (95, 165-167, 422, 423, 653, 654, 657, 658, 688). In addition, regulatory proteins seem to have acquired extra functions to compensate for the lack of specific regulatory proteins, as seen in the Fur-responsive regulation of the paralogous amidases of H. pylori (73, 95, 653, 656).

Transcriptional profiling studies using array hybridization indicate that approximately half of the H. pylori genome is expressed under in vitro conditions (95, 165, 422, 423, 626, 688). Of these genes, 5 to 10% display regulated expression in response to diverse environmental stresses, suggesting that H. pylori may not so much respond to specific stresses as combat environmental stresses, such as decreases in pH, using general stress-responsive mechanisms. This view is supported by the fact that the response of H. pylori to different environmental stimuli appears to be multifactorial and to be mediated via several regulatory systems, which may be connected via regulatory cross talk and cascades (658). This is exemplified by the nickel-responsive regulator NikR, which controls transcription of the iron-responsive regulator Fur and thus mediates regulation of both nickel- and iron-responsive genes (55, 73, 95, 653, 658). In addition, the HP0166-HP0165 (ArsRS) two-component regulatory system is implicated in acid-responsive regulation of urease expression (505, 506), and mutation of genes involved in posttranscriptional modification of RNA may also affect the overall expression level of genes (2, 38). Thus, it may well be that there is a hierarchical structure of regulatory networks in H. pylori, which allows moderate but optimal responses to environmental stresses in the gastric environment. Alternatively, the overlap between the different regulons may allow modulation of the level of gene expression rather than switching expression of genes on or off.

EPIDEMIOLOGY

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CLINICAL ASPECTS OF H. PYLORI-ASSOCIATED DISEASES

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Colonization with H. pylori is not a disease in itself but a condition that affects the relative risk of developing various clinical disorders of the upper gastrointestinal tract and possibly the hepatobiliary tract. Testing for H. pylori therefore has no relevance by itself but should be performed to find the cause of an underlying condition, such as peptic ulcer disease, or for the purpose of disease prevention, such as in subjects with familial gastric cancer. In these cases, a positive test result justifies treatment and a negative test result may indicate the need to search for other etiologic factors or preventive measures. For these reasons, a correct understanding of the clinical course of H. pylori-associated disorders and the effect of H. pylori eradication is needed.

View larger version (14K): [in this window] [in a new window]   FIG. 2. Schematic representation of the factors contributing to gastric pathology and disease outcome in H. pylori infection.

View larger version (79K): [in this window] [in a new window]   FIG. 3. (A) Gastric glands abundantly colonized with Helicobacter pylori, shown as dark, curved bacilli closely aligning with the mucosal surface. (B) Endoscopic view of a gastric ulcer, with a clean base at the angulus.

(ii) Chronic gastritis. When colonization does become persistent, a close correlation exists between the level of acid secretion and the distribution of gastritis (Fig. 4). This correlation results from the counteractive effects of acid on bacterial growth versus those of bacterial growth and associated mucosal inflammation on acid secretion and regulation. This interaction is crucial in the determination of outcomes of H. pylori infection. In subjects with intact acid secretion, H. pylori in particular colonizes the gastric antrum, where few acid-secretory parietal cells are present. This colonization pattern is associated with an antrum-predominant gastritis. Histological evaluation of gastric corpus specimens in these cases reveals limited chronic inactive inflammation and low numbers of superficially colonizing H. pylori bacteria. Subjects in whom acid secretion is impaired, due to whatever mechanism, have a more even distribution of bacteria in antrum and corpus, and bacteria in the corpus are in closer contact with the mucosa, leading to a corpus-predominant pangastritis (339). The reduction in acid secretion can be due to a loss of parietal cells as a result of atrophic gastritis, but it can also occur when acid-secretory capacity is intact but parietal cell function is inhibited by vagotomy or acid-suppressive drugs, in particular, proton pump inhibitors (PPIs) (339). The resulting active inflammation of the corpus mucosa further augments hypochlorhydria, paralleling the acute phase of infection, as local inflammatory factors such as cytokines, including interleukin-1ß (IL-1ß), have a strong suppressive effect on parietal cell function. This is illustrated by various observations. Firstly, H. pylori corpus gastritis is often associated with hypochlorhydria, and eradication therapy leads to increased acid secretion in these subjects (158, 533). Secondly, H. pylori corpus gastritis augments the acid-suppressive effects of PPIs (663). As a result, H. pylori-positive patients with gastroesophageal reflux disease (GERD) may respond somewhat faster to PPI treatment both with respect to symptom resolution and with healing of esophagitis (275), but this effect is minimal and largely irrelevant in daily clinical practice. This means that there is no general need to take H. pylori status into account when decisions on the dose of PPI treatment for GERD must be made. A third observation in support of the acid-suppressive effects of active corpus gastritis comes from more recent, important research showing that subjects with proinflammatory genotypes have a higher risk of corpus-predominant pangastritis, predisposing them to atrophic gastritis, intestinal metaplasia, and gastric cancer (157).

View larger version (14K): [in this window] [in a new window]   FIG. 4. Acid secretion and the associated pattern of gastritis play an important role in disease outcome in H. pylori infection. The figure displays the correlations between the pattern of H. pylori colonization, inflammation, acid secretion, gastric and duodenal histology, and clinical outcome.

Peptic ulcer disease. (i) Definitions. Gastric or duodenal ulcers (commonly referred to as peptic ulcers) are defined as mucosal defects with a diameter of at least 0.5 cm penetrating through the muscularis mucosa (Fig. 3B). Gastric ulcers mostly occur along the lesser curvature of the stomach, in particular, at the transition from corpus to antrum mucosa (661). Duodenal ulcers usually occur in the duodenal bulb, which is the area most exposed to gastric acid. In Western countries, duodenal ulcers are approximately fourfold more common than gastric ulcers; elsewhere, gastric ulcers are more common. Duodenal ulcers in particular occur between 20 and 50 years of age, while gastric ulcers predominantly arise in subjects over 40 years old.

(ii) Association with H. pylori. Both gastric and duodenal ulcer diseases are strongly related to H. pylori. In initial reports from all over the world in the first decade after the discovery of H. pylori, approximately 95% of duodenal ulcers and 85% of gastric ulcers occurred in the presence of H. pylori infection (338). Several cohort studies estimated that the lifetime risk for ulcer disease in H. pylori-positive subjects is 3 to 10 times higher than in H. pylori-negative subjects (460) and that 10 to 15% of H. pylori-positive subjects developed ulcer disease during long-term follow-up (571; D. J. E. Cullen, J. Collins, K. J. Christiansen, J. Epis, J. R. Warren, and K. J. Cullen, abstract from the Digestive Diseases Week 1993, Gastroenterology 104:A60, 1993). These data came from studies in developed areas of the world. It is unknown whether H. pylori-positive subjects in developing countries have similar disease risks. Introduction of H. pylori eradication regimens completed the evidence for a causal relation between H. pylori and ulcer disease by showing that eradication of this bacterium strongly reduced the risk of recurrent ulcer disease (522). This has had a major impact on the treatment and course of peptic ulcer disease in daily clinical practice. In earlier days, this disease was a chronic, recurrent disorder with high morbidity, frequently requiring acid-suppressive maintenance therapy or surgery. Approximately 50% of patients with H. pylori-associated peptic ulcer disease suffered ulcer recurrence within 1 year (268, 522). Eradication of H. pylori dramatically changes the natural course of ulcer disease and almost completely prevents ulcer recurrence (268, 522, 634, 645). Ulcer recurrences after H. pylori eradication therapy can be due to persistent or renewed H. pylori infection, use of NSAIDs, or idiopathic ulcer disease.

Ulcer development in the presence of H. pylori is influenced by a variety of host and bacterial factors. Ulcers mostly occur at sites where mucosal inflammation is most severe (661) (Fig. 4). In subjects with decreased acid output, this usually is the gastric transitional zone between corpus and antrum, giving rise to gastric ulcer disease. If acid production is normal to high, the most severe inflammation usually is found in the distal stomach and proximal duodenum, giving rise to juxta-pyloric and duodenal ulcer disease.

(iii) Ulcer epidemiology. The incidence of peptic ulcer disease has shown large variation over the past 150 years. In the late 19th century, the risk of developing peptic ulcers rose strongly in subsequent birth cohorts and then declined in subsequent generations. The birth cohorts with th