UMEÅ UNIVERSITY MEDICAL DISSERTATIONS Abstract No. 83, ISSN 0345-7532, ISBN 91-7305-548-4 From the Department of Odontology, Division of Oral Microbiology, Faculty of Medicine, Umeå University, Umeå, Sweden Helicobacter pylori Adhesion and Patho-adaptation The role of BabA and SabA adhesins in persistent infection and chronic inflammation by Jafar Mahdavi Umeå, 2004 Print & Media To my lovely family; Vina, Melissa and Mona Print & Media Vision without action is a daydream, Action without vision is a nightmare. Derived from The Analects of Confucius Print & Media Contents Page Preface 5 Abbreviations 6 Abstract 8 Introduction 9 1. A brief history of H. pylori infection 9 2. Microbiology of Helicobacter 9 3. Epidemiology 10 4. The Lewis and ABO blood group systems and H. pylori infection 11 5. Biodiversity 13 5.1 Habitat diversity 13 5.2 Genetic diversity 13 5.3 Tropism and patho-adaptation 16 5.4 Biofilm formation 17 6. Virulence factors and persistence of infection 17 6.1 Adhesins 19 6.1.1 Consequences of bacterial adhesion 19 6.1.1.1 Effect on the bacterial cells 20 6.1.1.2 Effect on the host cells 21 6.2 Invasion of host cells 22 6.3 H. pylori enzymes 23 6.4 VacA and CagA 23 6.5 H. pylori LPS 24 3 Print & Media 7. Diseases associated with H. pylori infection 25 7.1 Gastritis 25 7.2 Peptic ulcer disease 27 7.3 Gastric adenocarcinoma 27 7.4 Barrett’s esophagus 28 Aims 29 Results and Discussion 30 8. The role of MUC5AC in H. pylori adherence 30 9. Gastric mucosa sialylation and inflammation 30 10. Identification of inflammation associated H. pylori adhesive activities 32 11. The role of BabA and SabA in persistent infection and 32 chronic inflammation 11.1 phase variation and biofilm formation 33 11.2 SabA and BabA in relation to patho-adaptation 34 12. The role of LPS in H. pylori adherence 35 Specific conclusions 37 Future perspectives 38 Acknowledgements 40 References 41 Appendix I-IV 53 4 Print & Media Preface This thesis is based on the following papers, which are referred to in the text by their Roman numerals: I. Van de Bovenkamp J.H., Mahdavi J., Korteland-Van Male A., Büller H., Einerhand A., Borén T., and Dekker J. The MUC5AC glycoprotein is the primary receptor for Helicobacter pylori in the human stomach. Helicobacter 2003, 8, 521-532. II. Mahdavi J., Sondén B., Hurtig M., Olfat O.F., Forsberg L., Roche N., Ångström J., Larsson T., Teneberg S., Karlsson K.A., Altraja S., Wadström T., Kersulyte D., Berg E.D., Dubois A., Petersson C., Magnusson K.M., Norberg T., Lindh F., Lundskog B.B., Arnqvist A., Hammarström L., and Borén T. Helicobacter pylori SabA adhesin in persistent infection and chronic inflammation. Science 2002, 297, 573-578. III. Lindén S., Mahdavi J., Olsen C., Borén T., Carlstedt I., and Dubois A. Effects of Helicobacter pylori inoculation on host glycosylation and H. pylori adhesion sites in rhesus monkey. Submitted. IV. Mahdavi J., Borén T., Vandenbroucke-Grauls C., and Appelmelk B.J. Limited role of lipopolysaccharide Lewis antigens in adherence of Helicobacter pylori to the human gastric epithelium. Infection & Immunity 2003, 71, 2876-2880. 5 Print & Media Abbreviations ALeb: blood group A derivative of Lewis b. BLeb: blood group B derivative of Lewis b. BabA: blood group antigen binding adhesin. cagA: cytotoxin-associated gene A. cag-PAI: cag-pathogenicity island. FimH: adhesive subunit of type I fimbriae. GERD: gastro-esophageal reflux disease. GI: gastrointestinal. IARC: International Agency for Research on Cancer. ICAM: intercellular adhesion molecule. IL-8: interleukin-8. IVEC: in vitro explant culture. LPS: lipopolysaccharide. Le a, b, x and y: Lewis blood group antigen a, b, x and y. MALT: mucosa-associated lymphoid tissue. MAP: mitogen-activated protein kinase. NADH: nicotinamide adenine dinucleotide (co-enzyme). NAP: neutrophil activating protein. NF-B: nuclear factor-B. NMR: nuclear magnetic resonance. NIH: National Institutes of Health. OMP: outer membrane protein. P-(pap)-fimbriae: pyelonephritis-associated pilus. PMN: polymorphonuclear leukocyte. RBC: red blood cell. ROS: reactive oxygen species. SabA: sialic acid binding adhesin. sLex: sialyl-Lewis x antigen. sLea: sialyl-Lewis a antigen. sdiLex: sialyl-dimeric Lewis x antigen. TLR: toll-like receptor. TNF: tumor necrosis factor alpha. 6 Print & Media UTI: urinary tract infection. VacA: vacuolating cytotoxin A. 7 Print & Media Helicobacter pylori Adhesion and Patho-adaptation The role of BabA and SabA adhesins in persistent infection and chronic inflammation Helicobacter pylori (H. pylori) is a human-specific gastric pathogen which is responsible for a spectrum of diseases ranging from superficial gastritis to gastric and duodenal ulceration, and which is also highly associated with gastric cancer. The pathogenesis of severe gastric disorders caused by H. pylori is multifactorial and involves complex interactions between the microbe and the gastric mucosa. H. pylori expresses several adhesion proteins. These molecules have important roles in the establishment of persistent infection and chronic inflammation, which cause tissue damage. The aim of this thesis was to study the attachment of this bacterium to human gastric epithelium, mediated by blood group antigens in both health and disease. One of the best-characterized H. pylori adhesins is the histo-blood group antigen binding adhesin (BabA), which binds specifically to the Lewis b antigen (Leb) in the gastric mucosa. A protective mucus layer lines the stomach. The mucosal glycosylation patterns (GPs) vary between different cell lineages, different locations along the gastrointestinal (GI) tract and different developmental stages. In addition, GPs undergo changes during malignant transformation. MUC5AC is a mucin molecule produced by the surface epithelium. Three distinctly different types of human gastrointestinal tissue were studied by bacterial adherence analysis in situ. MUC5AC is the most important carrier of Leb and the new results demonstrate that it forms major receptors for H. pylori adherence. By analysing an H. pylori babA-deletion mutant, a novel adhesin-receptor binding mode was found. Surprisingly, the mutant bound efficiently to both human gastric mucosa and to gastric mucosa of Leb transgenic mice. The sialylated and fucosylated blood group antigen, sialyl-dimeric-Lewis x (sdiLex), was structurally identified as the new receptor. A positive correlation was found between adherence of H. pylori to sialyl- Lewis x (sLex) and elevated levels of inflammation response in the human gastric mucosa. These results were supported by detailed analysis of sialylated and fucosylated blood group antigen glycosylation patterns and, in addition, in situ bacterial adherence to gastric mucosa of experimentally challenged Rhesus monkey. The cognate sialic acid- binding adhesin (SabA) was purified by the retagging technique, and the corresponding sabA-gene was identified. H. pylori lipopolysaccharide (LPS) contains various Lewis blood group antigens such as Lewis x (Lex) and Lewis y (Ley). Additional bacterial adherence modes, which are independent of the BabA and/or SabA adhesins, could possibly be mediated by Lex interactions. Adherence of a clinical isolate and its corresponding Lex mutant to human gastric mucosa with various gastric pathologies was studied in situ. The results suggest that H. pylori LPS plays a distinct but minor role in promotion of bacterial adhesion. Taken together, the results suggest mechanisms for continuous selection of H. pylori strains, involving capacity to adapt to changes in the local environment such as shifts in cell differentiation and associated glycosylation patterns. Adherence of H. pylori is dependent on both the BabA and the SabA adhesin. Multi-step dependent attachment mechanisms may direct the microbes to distinct ecological niches during persistent infections, driving the chronic inflammation processes further toward the development of peptic ulcer disease and/or malignant transformation. Key words: H. pylori, BabA, adhesin, Lewis b, MUC5AC, sialyl-dimeric-Lewis x, chronic inflammation, SabA, Lewis x, LPS. 8 Print & Media Introduction 1. A brief history of H. pylori Infection In 1982, Barry Marshall and Robin Warren managed to culture Helicobacter pylori from stomach biopsies. The material came from patients with gastritis, but their initial reports suggested that gastric inflammation and peptic ulcers might be the consequences of bacterial infections were initially met with considerable skepticism by the medical community (Marshall & Warren, 1984). Isolation of H. pylori (or Campylobacter pylori as it was first named) was a significant scientific result, but still did not establish whether the bacteria were the cause of the inflammation with which they were associated, or whether they occurred as a result of it. However, the scientific field has rapidly moved forward during the last two decades of H. pylori research, and today we know that merely 10% of infected people develop serious illness such as gastritis, peptic ulcer disease and gastric cancer, i.e. the majority (90%) of people infected with H. pylori suffer no symptoms related to their infection In 1994, the National Institutes of Health (NIH) concluded that there is a strong association between H. pylori and peptic ulcer disease and the International Agency for Research on Cancer (IARC), part of the World Health Organization (WHO), classified H. pylori as a class I carcinogen. The pathophysiology of this infection can be understood better by considering five central concepts;