Old Herborn University Seminar Monograph 18: From friends to foes. Editors: Peter J. Heidt, Tore Midtvedt, Volker Rusch, and Dirk van der Waaij. Herborn Litterae, Herborn-Dill, Germany: 1-10 (2005).

HELICOBACTER: CHRONIC EFFECTS AND ROLE IN HOST MICROECOLOGY TORKEL WADSTRÖM and ÅSA LJUNGH Department of Medical Microbiology, Dermatology and Infection, Lund University, Lund, Sweden

SUMMARY pylori is the first named species of the Helicobac- ter/Wolinella family, now including more than 20 species and about 10 candidate species. The organisms are all micro-aerophilic “mucino- philes” with a few exceptions. H. pylori is the prototype for a number of bile-sensitive species colonising the stomach of most mammals, in- cluding dolphins and whales. The low toxicity of the lipopolysaccharide (LPS) and a number of properties unique for these species determine how they may cause life-long infections. H. pylori carries the vacA toxin as well as a set of other virulence traits permitting optimal early coloni- sation of the host, e.g. in childhood. The cagA pathogenicity island (PAI) makes cagA+ strains of H. pylori more virulent than cagA- strains to develop chronic active gastritis, gastric atrophy and pre-cancerous le- sions in the host as well as in mouse and mongolian gerbil models. H. pylori as well as a number of entero-hepatic bile-tolerant species are camouflaged from the innate immune system of the GI epithelial cell surfaces, yet cagA+ H. pylori transcribe NF-κB to the nucleus of these cells and of macrophages and other cells. At least H. pylori evades the host immune system by a number of responses such as molecular mimicry of the H–K adenosine triphosphatase and of gastric cell surface fucosylated antigens. The degree of inflammation is modulated by the IL-1β cytokine polymorphism and probably by a number of other host factors. The co-evolution of H. pylori and man back to the origin of mankind is clearly defined with a sophisticated haemostasis between the H. pylori as a pathogen. Alternative scenarios in the 21st century in several parts of the world with a “clean” Helicobacter-free human stomach are ad- dressed as well as recent reports of a newly discovered gastro-oeso- phageal microflora and the rapid increase in GERD, Barrett´s oesopha- gus, oesophageal cancer and obesity as well as changes in living condi- tions in Western societies.

INTRODUCTION lives in the mu- the area of colonisation is invariably in- cus layer overlying the gastric epithe- flamed (chronic superficial gastritis; lium and does not appear to invade tis- Northfield et al., 1994). Most infected sues. However, the mucosa underneath persons do not show clinical manifesta-

1 Table 1: Entero-hepatic bile-tolerant Helicobacter species ————————————————————————————— Species Comment ————————————————————————————— Helicobacter pylori some strains are bile-tolerant Helicobacter pullorum common in chicken Helicobacter bilis common in rodents Helicobacter hepaticus common in rodents Helicobacter cholecystus common in hamster Helicobacter canis common in dogs Helicobacter rappinii certain subtype common in sheep Helicobacter ganmani anaerobic ————————————————————————————— tions of the inflammation. Studies that manii as a second gastric species. This include human volunteers, experimental species and some others are highly fas- animal infections and treatment of pa- tidious and difficult or impossible to tients with antimicrobial agents show culture in vitro under micro-aerophilic that H. pylori plays a critical role in this or anaerobic conditions with H. gan- inflammation and in these diseases. mani as the prototype of the second Much evidence suggest that H. pylori is group (Robertson et al., 2001). All spe- an indigenous microbe of the human cies are highly motile and possess non- stomach and that most, if not all, mam- sheathed or sheathed flagellae enabling malian species harbour related Helico- them to swim in the mucin layer (An- bacter species with a long co-evolution dersen and Wadström, 2001). Suer- of microbe and host (Blaser, 1998; baum and colleagues (Schreiber et al., Richter, 2001). H. pylori probably 2004) recently showed that H. pylori evolved from bile-tolerant enteric Heli- prefers a specific part of the gastric cobacter species colonising rodents and mucin layer, probably regulated by acid other mammals, including primates and secretion, H. pylori urease and ammonia man (Fox et al., 2001; Tables 1 and 2). formation, a metabolite most toxic for The phylogenetic tree of the gastric mucosa. Urease-negative as includes Sulphurospirillum, Arcobacter, well as catalase-negative mutants are un- Campylobacter, Helicobacter and able to colonise and infect the mouse Wolinellae (On, 2001; Figure 1). stomach, suggesting that ammonia pro- More than 20 species of Helicobac- duction and the redox potential are cru- ter are recognised today, with H. heil- cial to initiate the infection. Several ge-

Table 2: Evidence that Helicobacter pylori infection of humans is of ancient origin. ——————————————————————————————————————— • Extensive genetic heterogeneity • Acid-secreting stomachs arose early (300 million years ago!) in vertebrates • Helicobacter genus is highly prevalent in the stomach and gut of all vertebrates? • H. pylori-like organisms are widely present in the stomach of primates • High incidence among human populations in Asia and Africa of H. pylori (>80-90%) • H. pylori is adapted to persist for lifetime in the human stomach ———————————————————————————————————————

2 Figure 1: Representation of the phylogenetic tree of proteobacteria (modified from On, 2001). netic studies of H. pylori isolates from a not a post mortem “by-stander” was single human stomach show that these not addressed properly until Marshall microbes are highly adaptive organisms, and Warren (1984) in 1982 grew the which partly explains that this pathogen first Campylobacter pyloridis (later C. can persist for decades in a single stom- pylori and renamed to Helicobacter py- ach inducing a low grade tissue inflam- lori in 1989). By drinking viable in vitro mation (Blaser and Atherton, 2004). cultured H. pylori cells, Marshall and This adaptation involves mutations and colleagues showed that it induced acute recombination, and many strains may be achlorhydria and dyspepsia, which was classified as hypermutation phenotypes. suppressed or cured by a bismuth-anti- H. pylori is able to maximise diversity biotic therapy. of genetic sequences under strong se- Later, in vitro co-culture studies of lective pressure while maintaining alleles H. pylori and gastric cancer epithelial critical for its lifestyle (Björkholm et al., (AGS) cells showed that strains con- 2004). taining the 35 to 40 kilobase cag patho- Helicobacter-like organisms, resem- genicity island (PAI) flanked by specific bling the syphilis spirochete, were re- 39 basepair direct DNA repeats induced ported by several pathologists in human a higher cytokine response (IL-8), and and animal stomachs already in the pe- promoted an anti-apoptotic pathway riod from1880 to 1890, including beau- aiding persistence of the organism in the tiful studies in dogs by Bizzozeroni in gastric mucosa (Crabtree, 2001). Italy, describing a species today named Another reason for its persistence is H. bizzozeron (On, 2001). However, its the molecular mimicry, in part due to the possible role as a gastric pathogen and low biological activity of its lipopoly-

3 saccharide (LPS)(Moran et al. 2000; vates CD4+ T cells in the stomach. This Blaser and Atherton, 2004). Molecular leads to gastric autoimmunity in geneti- mimicry between H. pylori antigens and cally susceptible individuals via mo- H+,K+-adenosine triphosphatase acti- lecular mimicry (Amedei et al., 2003).

H. PYLORI PATHOGENESIS – A MULTIPLE STEP INFECTION TO CHRONIC GASTRITIS AND GASTRIC ATROPHY Early development of mouse models Toll-like receptor (TLR4) and not gas- has clearly given good opportunities to tric TLR4 (Bäckhed et al, 2003). CagA- elucidate the H. pylori pathogenesis, and positive strains induce transcription of to develop alternative prophylactic and NF-κB in the epithelium through rec- treatment schedules to standard proton ognition of Nod1, an innate intracellular pump inhibitor (PPI) and antibiotics pathogen-recognition molecule, recog- (Hamilton-Miller, 2003). Mice given the nising soluble bacterial peptidoglycan vacuolating (vac) toxin orally developed fragments (Kim et al., 2004). How such ulcers. However, strains producing a vac molecules as well as other cell surface, toxin with an S1/m2 mid-region seem to extra-cellular and cell lysis molecules, bind poorly to specific cell lines and in- including nucleic acids, are delivered to duce less tissue damage and cell mem- the gastric mucosa is poorly understood. brane pores (Blaser and Atherton, Further studies are needed to define new 2004). Moreover, the S2 genotype is possible interventions, such as probiotic- associated with a lack of the cag PAI based strategies including anti- and may induce a less severe gastric in- Helicobacter peptides and bacteriocins flammation. Transient oral and gastric (Hamilton-Miller, 2003; Lorca et al., H. pylori colonisation occurs in chil- 2001). dren, as shown in a study from Dhaka, The H. pylori infection down-regu- Bangladesh (Casswall et al., 1999). It is lates the immune response, suppresses likely that H. pylori is a paediatric in- T-cell proliferation and induces selective fection, “achieved” soon after weaning T-cell apoptosis (Shirin and Moss, in all primitive societies (Blaser, 1988). 1998; Lundgren et al., 2003). The early Weaning habits such as maternal gastric colonisation involves a Lewis B chewing of food and early rotavirus and binding cell surface (HOP) protein as other viral infections changing the gas- well as a number of other adhesins, such tric physiology influence the time of ac- as sialic acid lectins (SAL´s) recognis- quisition. Ongoing infection can be de- ing cell surface mucin and glycolipid tected by faecal immunomagnetic bead molecules (Gerhard et al., 2001; Falk et based PCR or antigen detection methods al., 2000). Inhibition studies with milk (Weingart et al., 2004). A humoral as glycoconjugates (Hirmo et al., 1998; well as local immune response is rapidly Wang et al., 2000a; Wang et al., 2001) induced. Antibody titres remain for and a probiotic strain of Lactic Acid several decades but interestingly, cagA+ (LAB) could prevent, suppress strains disappear more rapidly (Perez- or cure H. pylori infection in a mouse Perez et al., 2002). model (Cruchet et al., 2003). The H. pylori LPS is an anergic low H. pylori further induces a rapid, toxicity endotoxin with a unique lipid A early neutrophilic activation by a specific core structure (Hynes and Wadström, molecule (HPNAP) (Teneberg et al., 2004). It stimulates only macrophage 1997). This induces a rapid cell uptake

4 Figure 2: Divergent responses to Helicobacter pylori infection. through lectino-phagocytosis by SAL´s Figure 2). Polymorphism of the TNF-α and glycosaminoglycan (GAG) surface and IL-10 genes may have a similar lectins. Other chronic infections, such as modulating effect on the outcome of a a specific helminth or parasite infection chronic inflammation after one or two may modulate the Th1/Th2 immune re- decades. A sophisticated somatostatin sponse to a predominant Th2 response regulation of gastrin release, a growth in black Africans, “the African factor for H. pylori, creates a feedback enigma”. This may reflect a genetic loop reversal after curing of an H. pylori predisposition selected by malaria (Fox infection (Zhao et al., 2003). A persis- et al., 2000; Bennedsen et al., 1999). tent increased tissue gastrin level in- A specific IL-1β polymorphism in- crease the parietal cell mass and en- duced by H. pylori increases the risk of hances the process of gastric metaplasia severe gastritis proceeding to gastric in the duodenum associated with H. atrophy, hypochlorhydria and adenocar- pylori inflammation and duodenal ulcer cinoma (Blaser and Atherton, 2004; disease (Wang et al., 2000b).

H. PYLORI IN THE 21ST CENTURY H. pylori still infects the majority of other parts of Eastern Europe (Forman children in the non-industrialised world, and Graham, 2004). However, in West- leading to pangastritis and stomach ern societies with a low incidence of H. atrophy. Depending on food intake, i.e. pylori infection in children (< 2% today a high or low level of fruit, antioxidants in Scania, Southern Sweden), the human and possibly food carcinogens, the risk stomach homeostasis and health should of gastric malignancies varies between be studied since pangastritis leads to a 2.7 and more than 12-fold in various reduction of gastric acid production studies with high prevalences in Japan, (Sande et al., 2001; Figure 2). It is likely Northern-China, the Baltic countries and that an increased acid production is

5 Figure 3: Disruption of tight junctions by microbes and microbial products. *: ZO1= zona occludens 1. associated with GERD-reflux oesoph- inhibit transcription of NF-κB and agitis and related conditions, such as DNA mutations in the epithelium Barrett´s oesophagus and pre-malignant (Wang et al., 2000b). The relative role of epithelial changes (Fitzgerald, 2001). food carcinogens, such as nitrosamines Moreover, H. pylori infection is as- and water rich in nitrates, in gastric sociated with elevated serum leptin levels carcinoma development should be (Breidert et al., 1999; Matarese and studied in various geographical regions Lechler, 2004). A weight gain is com- of the world. Likewise, in patients on a mon after H. pylori eradication (Azuma long-standing PPI regime to suppress et al., 2001), possibly predisposing to acid reflux (GERD) disease, gastric adult as well as adolescent obesity. A overgrowth by enteric microbes with high intake of antioxidant-rich food and potential carcinogen production (c.f. food supplements can inhibit free reac- enterococci) should be investigated. tive oxygen metabolites (ROM´s) and

THE IL-10 -/- MOUSE AND HELICOBACTER-INDUCED GASTRITIS AND COLITIS LAB of the upper mouse stomach cobacter and other bacterial gas- form a barrier towards Salmonella, Heli- trointestinal pathogens. An early germ-

6 free (GF) mouse model to study anti- and enteric Helicobacter infections. IL- Helicobacter effects of L. gasserii was 10 is associated with several traits such developed in Japan (Kabir et al., 1997). as gut permeability regulation, which More recent studies indicate that GF seems important for LAB as well as an- mice are not readily colonised by H. tioxidant anti-Helicobacter effects. pylori and enteric Helicobacter sp. (E. Similar mechanisms were proposed for Norin, H.-O. Nilsson, and T. Wad- Salmonella and other enteropathogens, ström, unpublished observations). including (Figure 3). However, an IL-10 -/- mouse derived The complete genome of H. hepaticus from C57-black mouse responded to H. has been published (Suerbaum et al., pylori with a more severe gastric in- 2003). This will provide a valuable tool flammation than Balb-c, and this mouse to identify virulence genes. strain seems promising to optimise a However, in the near future well de- mouse H. pylori gastric cancer model fined conditions to create and keep Heli- (Kullberg et al., 2003). cobacter-free mouse colonies should be The IL-10 -/- mouse is susceptible to addressed, including a modified Schaed- H. pylori as well as to natural and ex- ler flora to stimulate studies on chronic perimental H. hepaticus and other en- experimental models of inflammatory teric Helicobacter species (H. bilis, H. conditions and to avoid interference of ganmani, etc., see Table 1). murine Helicobacter induced inflamma- A first H. hepaticus colitis study in tions in various experimental models. IL-10 -/- mice by Pena and co-workers These include inflammatory bowel dis- (Pena et al., 2004) suggests that this ease (IBD) in dextran sulphate and other model may become the model of choice chemically as well as microbe induced to study effects of probiotic microbes as IBD-like syndromes. well as other therapies towards gastric

CONCLUSIONS The gastric as well as the intestinal bilise TJ:s, e.g. by probiotic and anti- epithelium is an interactive barrier that oxidant treatment. This may also be an directs neutrophil movement. Specific important step in inflammatory bowel peptides act via Toll-like receptors and disease (IBD) research in IL-10 knock- induce NF-κB transcription with pro- out mice as well as in human patients. duction of pro-inflammatory cytokines. Further comparative studies on the H. pylori and several enteric Helico- pathogenesis of H. pylori and enteric bacter species may disrupt tight junc- IBD-inducing species (Sturegård et al., tions (TJ:s) (Figure 4) in a similar way 2004) may reveal new preventive and as discussed for enteric pathogens such curative methods for chronic gastric and as . Ongoing studies in enteric inflammations. several laboratories aim at means to sta-

ACKNOWLEDGEMENTS Our studies were supported by the Swedish Research Council (16x-04723), the Medical Faculty, Lund University and the University Hospital of Lund by ALF- grant.

7 LITERATURE

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