Interaction of Helicobacter pylori with glycosylated salivary proteins [Elektronische Ressource] / vorgelegt von Anke Walz

Anke Walz

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• Gesundheit

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• Publié par: universitat_regensburg
• Publié le: 01 janvier 2006
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

Interaction of Helicobacter pylori
with Glycosylated Salivary Proteins




Dissertation zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer. nat.)
der Fakultät Chemie/Pharmazie
der Universität Regensburg









vorgelegt von
Anke Walz
aus Regensburg
August 2006
























Promotionsgesuch eingereicht am: 31. August 2006

Datum der mündlichen Prüfung: 10. Oktober 2006

Die Arbeit wurde angeleitet von: Prof. Dr. Stefan Ruhl

Prüfungsausschuss: Vorsitzender: Prof. Dr. A. Buschauer
Erstgutachter: Prof. Göpferich
Zweitgutachter: Dr. S. Ruhl
Drittprüfer: Prof. J. Heilmann
Meinen Eltern
und
Thomas
gewidmet in Liebe und Dankbarkeit
Table of Contents

Chapter 1 Introduction..…………...............................................…....................7

Chapter 2 The Aims of this Thesis....................................................................41

Chapter 3 Establishment of Two-Dimensional Gel Electrophoresis of Human
Saliva................................................................................................45

Chapter 4 Proteome Analysis of Glandular Parotid and Submandibular-
sublingual Saliva in Comparison to Whole Human Saliva by Two-
dimensional Gel Electrophoresis.....................................................63

Chapter 5 Identification and Characterization of Binding Properties of
Helicobacter pylori by Glycoconjugate Array.................................85

Chapter 6 A Novel Approach for Identification of Receptors in Human
Saliva for the BabA and SabA Adhesins of Helicobacter pylori
by Bacterial Overlay in Combination with Proteomics
Techniques................….…….........................................................107

Chapter 7 Applicability of the Overlay Technique to Commensal Bacteria of
the Oral Cavity Using Streptococcus gordonii and Actinomyces
naeslundii as Probes.............….......................................................129

Chapter 8 Summary and Conclusion...............................................................147

Appendices List of Abbreviations......................................................................155
Supplementary Material to Chapter 4.............................................157 entary Material to Chapter 6.............................................164
Curriculum Vitae............................................................................165
List of Publications.........................................................................167
Acknowledgements.........................................................................171
Chapter 1 Introduction





Chapter 1




Introduction:
Helicobacter pylori
A Gastric Pathogen on its Way Through the Oral Cavity

- 7 - Chapter 1 Introduction

Abstract
Helicobacter pylori (H. pylori) resides in the human stomach and is the primary cause for
severe gastric diseases such as gastritis, peptic ulcers and even gastric cancer. It is generally
accepted that H. pylori is acquired by oral ingestion and several studies provided evidence
that H. pylori may be present in the human oral cavity. Therefore an interaction of this
bacterium with components of saliva seems likely. The subject of this thesis was the
“Interaction of H. pylori with glycosylated salivary proteins”. It ought to be investigated
whether the mechanisms of adhesin-receptor interactions known from the stomach are also
found for interactions with salivary glycoproteins. Thus, this Introduction chapter describes
the pathogenesis of H. pylori and, in particular, the interaction of H. pylori adhesins with
corresponding receptor structures. Moreover, glycoproteins from saliva that could serve as
putative receptors for these adhesins as well as the current knowledge about H. pylori in the
oral cavity are summarized.

- 8 - Chapter 1 Introduction

General Introduction to Helicobacter pylori
History
The story began at the end of the 1980s with a fortuitous observation by J. Robin Warren, an
australian pathologist at the Royal Perth Hospital. While examining routine biopsies from
dyspeptic patients he observed a large number of curved and spiral-shaped bacteria. Why had
stomach acid not destroyed these organisms? One explanation became obvious. They were
closely associated with the thick gastric mucus layer. J. R. Warren had observed these
organisms only in inflamed tissues. This observation led him to study this question in more
detail. With his colleague Barry J. Marshall he tried to culture the bacteria. Their attempts
were not successful until they forgot some culture plates over Easter holidays. In these five
days bacterial colonies had emerged (reviewed in [1]). They named these bacteria
Campylobacter pyloridis because of their morphologic similarity to other bacteria of the
Campylobacter genus and published their observations [2]. Because Warren and Marshall had
found C. pyloridis in the majority of patients with gastric ulcers, they reasoned that peptic
ulcer disease is associated with the occurrence of these bacteria in the stomach. As an
experimental test Marshall himself drank culture material with C. pyloridis and, as expected,
developed dyspeptic illness. In addition, the spiral-shaped bacteria could be detected in his
gastric biopsies. The infection vanished without treatment. Some years later a successful
antiobiotic therapy was possible, and, thus, eradication of C. pyloridis. Because C. pyloridis
did not really fit into the Campylobacter genus, they renamed these microbes into
Helicobacter pylori (H. pylori) in 1989 [3]. Approximately 20 years after their discovery of
H. pylori and of its role in peptic ulcer diseases Warren and Marshall were awarded with the
Nobel prize in Physiology or Medicine [4].
Microbiology
Three morphologic shapes have been described for H. pylori. Normally, this bacterium
appears spiral-shaped or curved, but it assumes a rod-like shape on solid medium and coccoid
forms after prolonged culture [5]. It is gram-negative and requires a microaerophilc
atmosphere with about 5% O and 5-10% CO . H. pylori cells are 2.5 to 5.0 µm long and 0.5 2 2
to 1.0 µm wide. They have four to six unipolar flagella (Fig. 1), which are essential for
bacterial motility and thus for infection [5]. H. pylori survives in the acidic environment of the
stomach because it produces high levels of urease, in order to buffer the pH in its immediate
vicinity. Urease hydrolyses urea into NH and CO , in this way creating a neutral layer around 3 2

- 9 - Chapter 1 Introduction

the bacterial surface. Because urease-defective bacteria can not colonize the stomach, it is
obvious that urease is indispensable as a colonization factor. Despite of its capability to
survive in strong acid, H. pylori has to leave the stomach lumen, in order to prevent its
transport into the intestine. Using its flagella H. pylori swims towards the viscous mucus layer
that covers the epithelial lining of the gastric mucosa, guided by chemotactic factors including
urea and bicarbonate. Like a corkscrew the bacterium bores into the mucus layer, finally
reaching the surface of the gastric epithelium. This behavior is a second colonization factor,
because non-motile mutants are not able to colonize the stomach (reviewed in [6]).

Fig. 1: Curved Helicobacter pylori with unipolar
flagella. Source: [7]


H. pylori-associated diseases
Because of its very good adaptation to the hostile environment of the human stomach H.
pylori establishes a lifelong chronic infection after a first infection early in life. The initial
colonization with H. pylori causes an acute inflammatory response (acute gastritis) that is
accompanied by an infiltration of the gastric mucosa by neutrophils. The degree of mucosal
damage correlates with the neutrophil infiltration. Today it is accepted that a H. pylori
infection is the primary cause of active chronic (type B) gastritis in humans [8].
Most infected people are asymptomatic initially but they may develop peptic ulcer
diseases including gastric or duodenal ulcers with a risk of 10%. Almost all duodenal ulcers
and 70% of gastric ulcers are attributable to H. pylori infection. This is supported by the fact
that the recurrence rate of peptic ulcer diseases is noticeably reduced after the successful
treatment of a H. pylori infection [9, 10].
A long-term consequence of gastric ulcer could be the development of
adenocarcinoma [11] and mucosa-associated lymphoid tissue (MALT) lymphoma [12].
Therefore, H. pylori was designated as a class I carcinogen by the World Health Organization
in 1994 [13]. Although the interrelation of H. pylori and gastric cancer is known, only a small
fraction of colonized individuals develop this malignancy. This lead to the assumption that