genomewide characterization of host-pathogen interactions by ...

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Maren Depke Introduction host as well as pathogen, developed mechanisms to fight or to gain some advantage against the other. The host developed defense mechanisms to either avoid infection or to overcome it, whereas the pathogen gained mechanisms to evade these host defense strategies. One part of the host’s immune defense is the innate immune system, for which the detailed information is encoded in the genome and which has been passed on and further modified and optimized during evolution. The host as a complex organism commands two main levels of such immune defense, non-cellular and cellular. On epithelial barriers, the host deposits antimicrobial peptides, which are often membrane-active and pore-forming and aim to lyse the pathogen during its attempt to enter the body (Schröder 1999). Also enzymes like lyzozyme are secreted and serve a similar function. A very important and complex defense system of cascade-activated components is the complement system (Dunkelberger/Song 2010). The complement system consists of several serum proteins, which interact and build complexes, contain several sequential and interdependent activation steps and enzymatic activities, and finally achieves three main goals: First, in a process called opsonization the labeling of pathogens with a molecular tag to render it recognizable for phagocytosis, and second the lysis of the pathogen by pore-forming components. The third function during activation of the complement is to pass the information of occurring infection to the other immune defense systems. Three activation pathways of the complement system exist (Fig. I.1). The first, called classical pathway, is linked to the non-innate, i. e. adaptive immune system, because it relies in the first step on antibodies, which recognize specific pathogenic structures. Pathogen surface bound antibodies bind and activate the C1 factor of the complement system. The following steps of proteolytic cleavage produce from factors C2 and C4 the smaller “a” and the bigger “b” fragments. C2a and C4b together form the first very important complex during complement activation, the C3-convertase, which cleaves C3 into C3a and C3b. The C3-convertase (via C4b) and C3b will be covalently bound to the pathogen’s cell surface after reaction of an active thioester bond and accomplish opsonization. The second complement activation pathway leads to the same effects, but starts with a different recognition complex including lectins (Fujita et al. 2004). This lectin-activation pathway does not require antibodies bound to the surface of the pathogen. Here, mannose-binding lectin (MBL) or ficolin recognize conserved carbohydrate structures of the pathogen. These molecules belong to the so-called pattern recognition receptors (PRRs), conserved and non-variable receptors for conserved pathogenic structures, which in turn are called pathogen-associated molecular patterns (PAMPs). MBL or ficolin are complexed, among others, with a serine protease (MASP), which achieves the next activation steps of C2 and C4 and formation of the C3- convertase as described before in the classical pathway. The third way of complement activation is named alternative pathway. For activation, it uses the spontaneous hydrolysis of C3 in the plasma to C3(H 2 O), which exposes the molecular site for covalent binding to pathogenic surfaces. In the presence of such surface, C3(H 2 O) binds to it and subsequently complement factor B to C3(H 2 O). Finally, factor D cleaves factor B into the fragments Ba and Bb. The resulting complex of C3(H 2 O)Bb constitutes the initial C3-convertase of the alternative pathway and cleaves further C3 molecules. The resulting C3b fragments again opsonize the pathogen, but also bind factor B and D, and form further C3-convertase complexes C3bBb. At this step, a strong amplification takes place, also when the C3b fragments are derived from activation of the classical and lectin pathway. 14
Maren Depke Introduction Besides opsonization, the complement system aims to lyse the pathogen. This so-called effector function is initiated after formation of C3b by any of the activation pathways. When C3b is complexed with the C3-convertases of classical/lectin or alternative pathway it forms the C5- convertase complex (C2bC4bC3b or C3bBbC3b). The enzymatic function is the cleavage of complement factor C5 into C5b and C5a. Now, C5b initiates the formation of the membrane attack complex (MAC). Sequential binding of factors C6, C7 (membrane binding) and C8 (membrane intrusion) leads to the forming of a pore by several C9 molecules. Such pores finally can lead to the lysis of the pathogen. Because the complement system is a very powerful defense system, which could lead to immense damage to the host itself, it is tightly controlled by many regulatory proteins which especially prevent the activation on the host’s own cellular structures. Fig. I.1: Complement activation pathways. Abbreviations: MBL – mannose-binding lectin; MASP – MBL-associated serine protease; sMAP – small MBL-associated protein; C3(H 2O) – hydrolysed C3 From: Foster 2005. During the activation of the complement cascade, several small peptide fragments, called anaphylatoxins, are produced. C3a, C5a, and to a lesser extent C4a have inflammatory properties. In a localized infection, they lead to increased permeability of blood vessels, induce endothelial adhesion molecules, influence monocytes and neutrophils to increase attachment, and activate mast cells which further enforce the effect with their own mediators. Hence, the small peptides link the non-cellular to the cellular part of the innate immune system. Phagocytes have a central function in the innate immune defense. These cells clear the pathogens from the site of infection and kill them intracellularly. Monocytes from the blood stream migrate into the tissue and develop into long-living macrophages, which reside and are activated in case of an encounter with pathogens. Several organs, especially those at a higher risk to be exposed to pathogens, embody special types of macrophages, e. g. Kupffer cells of the liver. 15
Page 1 and 2: G E N O M E W I D E C H A R A C T E
Page 3 and 4: Maren Depke C O N T E N T S GENOMEW
Page 5 and 6: Maren Depke Z U S A M M E N F A S S
Page 7 and 8: Maren Depke Zusammenfassung der Dis
Page 9 and 10: Maren Depke S U M M A R Y O F D I S
Page 11 and 12: Maren Depke Summary of Dissertation
Page 13: Maren Depke I N T R O D U C T I O N
Page 17 and 18: Maren Depke Introduction Extracellu
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Page 21 and 22: Maren Depke Introduction 2005). SaP
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Page 31 and 32: Maren Depke Introduction STUDIES OF
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Page 39 and 40: Maren Depke M A T E R I A L A N D M
Page 41: Maren Depke Material and Methods Li
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Maren Depke Results Liver Gene Expr
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lood glucose levels [nmol/l] resist
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LBP [ng/ml] CPR [ng/ml] Maren Depke
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Maren Depke Results Liver Gene Expr
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liver weight [g] Maren Depke Result
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infection rate cfu / 10 mg tissue c
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Maren Depke Results Kidney Gene Exp
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Table R.2.1: Genes displaying stati
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Maren Depke BR1 sign DsigB vs wt BR
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Maren Depke Results GENE EXPRESSION
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Maren Depke Results Gene Expression
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log2(ratio C57BL/6 / BALB/c) at IFN
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Maren Depke Results Host Cell Gene
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Maren Depke Results Pathogen Gene E
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S. aureus RN1HG sample conditions a
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mean normalized intensity mean norm
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mean normalized intensity Maren Dep
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log 2 (ratio) log 2 (ratio) log 2 (
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Maren Depke D I S C U S S I O N A N
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Maren Depke Discussion and Conclusi
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Maren Depke References Athanasopoul
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Maren Depke References Byrne GI, Le
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Maren Depke References Demling R. T
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Maren Depke References Foss GS, Pry
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Maren Depke References Hagopian K,
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Maren Depke References Jin T, Bokar
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Maren Depke References Kwan T, Liu
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Maren Depke References Luster AD, U
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Maren Depke References Namiki S, Na
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Maren Depke References Puccetti P.
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Maren Depke References Siewert E, B
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Maren Depke References van den Akke
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Maren Depke References Yang XL, Sch
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Maren Depke A F F I D A V I T / E R
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Maren Depke Acknowledgments Kidney
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