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Maren Depke Discussion and Conclusions KIDNEY GENE EXPRESSION PATTERN IN AN IN VIVO INFECTION MODEL The SigB regulon contains several virulence associated genes, and expression of this regulon in general promotes adhesion and reduces production of extracellular toxins (Bischoff et al. 2004). While regulation of SigB activity has already been studied in vitro (Senn et al. 2005), its impact on in vivo physiology, e. g. in infection models, is still matter of debate. The aim of this study was the analysis of the relevance of SigB-dependent gene expression regulation for the adaptation of the host transcriptome in murine infection. In an i. v. murine infection model the two Staphylococcus aureus strains RN1HG and its isogenic sigB mutant were used to infect mice with comparable infection doses. In mouse kidney, the infection resulted in similar infection rates of approximately 1.0E+06 cfu/10 mg of tissue. The role of SigB has already been analyzed in different in vivo infection models. A general observation of these studies was that sigB deletion mutants and their parental strain can accomplish comparable bacterial loads. In the clinical isolate WCUH29 the allelic replacement of sigB did not lead to differing bacterial load in organs or wounds after testing original strain and isogenic mutant in different types of infection models (Nicholas et al. 1999). Also Chan et al. (1998) observed similar numbers of cfu in a skin abcess model, but the results might have been compromised by the fact that the group used a sigB deletion in the 8325-4 background which itself harbors an inactivating mutation in rsbU, a gene for a SigB regulatory phosphatase (Kullik/Giachino 1997). Horsburgh et al. published in 2002 the construction of S. aureus SH1000, a variant of strain 8325-4, in which the rsbU gene had been repaired. SH1000 and 8325-4 resulted in the recoverage of similar numbers of cfu in a murine subcutaneous skin abcess model (Horsburgh et al. 2002b). Contrarily, another study reported a significantly different bacterial burden in kidney after i. v. infection with S. aureus 8325-4 (RsbU − ), SH1000 (RsbU + ), and a sigB knockout mutant of SH1000 (SigB − ). In this setting, the repair of rsbU leads to an increased cfu count 14 days after inoculation (Jonsson et al. 2004). In the study described in this thesis, almost equal mean values of infection rate were observed for S. aureus RN1HG and its isogenic sigB mutant, a further confirmation of literature data indicating similar virulence of sigB deficient and positive S. aureus strains. In order to gain a much more detailed view of the host reaction to both strains, kidney gene expression profiles were compared using Affymetrix GeneChip arrays. In a total number of 19 infected samples, highly similar gene expression profiles were recorded 4 or 5 days after infection with RN1HG or its sigB derivative. Even with this sophisticated analysis no difference in the expression pattern in murine kidney after infection with the two strains was observed, indicating that SigB indeed does not influence the host response. However, the comparison to sham-infected samples revealed a rather strong reaction of the murine host to infection, bacterial accumulation and proliferation in the kidney. Several immune response pathways were induced on transcriptional level indicating a vivid activation of the immune response. This applies first to pathways of the innate immune system: Pattern recognition receptors (PRR) like toll-like receptors (TLR) and complement system components 178
Maren Depke Discussion and Conclusions were induced and strengthened the first line of defense against the pathogen. Second, several immune response signaling pathways exhibited increased gene expression of their members, e. g. proinflammatory pathways of IFN, IL-6, and TREM1 signaling, and several proinflammatory cytokines were also induced, e. g. TNFα, IL-6, RANTES/Ccl5, MCP-1/Ccl2, MCP-3/Ccl7, and MIP- 1α/Ccl3. Members of IL-10 signaling, like the IL-10 receptor α-chain (Il10ra) and the signal transducer STAT3, were induced, too. This pathway aims at limitation of the cellular proinflammatory response. But as the ligand IL-10 was not induced and STAT3 is also part of the IL-6 signaling, it is not clear whether the IL-10 signaling was active at the time point of analysis or whether it was only prepared to receive signals in a later phase of infection. As third example, the infected samples exhibited a distinct transcriptional induction of several members of the presentation pathways for intracellular and extracellular antigens via MHC molecules. The comparison of infection and sham infection additionally revealed metabolic disturbances shifting to both anabolic and catabolic direction of metabolism. This might indicate a high extent of infection and illness that impairs organ function, nutrition supply and possibly oxygen availability. The comparison of S. aureus infected samples with non-infected controls proved the strong reaction of the host to infection. But in the model described in this thesis the host reaction does not differ depending on the strain used for infection. Until now, the role of sigB in infections has not been completely clarified. There is evidence for an influence of sigB on biofilm formation. Attachment and microcolony formation are the two central steps in the early biofilm formation. In an in vitro biofilm formation model (using polystyrene microtiter plates) it was shown that increased sigB expression leads to increased attachment and to increased microcolony formation with increased size of staphylococcal autoaggregates (Bateman et al. 2001). In detail, biofilm formation is mediated by polysaccharide intercellular adhesin/PIA, an extracellular adhesin of staphylococci composed of poly-N-acetylglucosamine/PNAG. Although highly conserved in staphylococcal strains, the ica operon, coding for enzymes responsible for PIA synthesis, is expressed in vitro only in a few strains. For the mucosal isolate MA12 it was proven that a sigB insertion mutant (sigB::ermB) lost the biofilm formation ability after osmotic stress (3 % NaCl) compared to the wild type strain. Additionally, it was demonstrated under osmotic stress conditions that the mutant exhibited strongly reduced ica expression compared to the wild type (Rachid et al. 2000). Even more, a major part of ica expression and biofilm formation was proven to be mediated by SarA. It is known that SigB induces sarA expression, but it has not been demonstrated yet whether the influence on ica expression is an indirect effect of SigB or whether SarA acts independently of SigB in this context. Interestingly, experiments predict the existence of a SigBactivated factor, which either might degrade PIA or repress its synthesis. This factor in turn is repressed by SarA (Valle et al. 2003). Complementary information regarding the influence of sigB knock-out on biofilm formation and virulence is available from device-associated staphylococcal infection models in mice. When MA12 and its isogenic sigB mutant were applied to a central venous catheter (CVC) and later to the blood stream, both strains were able to form multilayered biofilms inside the catheter. Therefore, sigB is not essential for biofilm formation. On the other hand, there were structural differences between the biofilms of both strains: The mutant’s biofilm was lacking certain extracellular substance. SigB is thus proposed to affect the ability of spreading or release from adhesive sites/biofilms. In the quantitation of total bacterial burden in the inner organs, a 179
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G E N O M E W I D E C H A R A C T E
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Maren Depke C O N T E N T S GENOMEW
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Maren Depke Z U S A M M E N F A S S
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Maren Depke Zusammenfassung der Dis
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Maren Depke S U M M A R Y O F D I S
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Maren Depke Summary of Dissertation
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Maren Depke I N T R O D U C T I O N
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Maren Depke Introduction Besides op
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Maren Depke Introduction Extracellu
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Maren Depke Introduction with heigh
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Maren Depke Introduction 2005). SaP
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Maren Depke Introduction contains a
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Maren Depke Introduction via fibron
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Maren Depke Introduction cathelicid
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Maren Depke Introduction shock are
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Maren Depke Introduction STUDIES OF
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Maren Depke Introduction are effect
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Maren Depke Introduction cell stimu
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Maren Depke Introduction channel CF
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Maren Depke M A T E R I A L A N D M
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Maren Depke Material and Methods Li
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Maren Depke Material and Methods Ki
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Maren Depke Material and Methods GE
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Maren Depke Material and Methods Ge
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Maren Depke Material and Methods Ho
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Maren Depke Material and Methods Ho
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Maren Depke Material and Methods Pa
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corticosterone [pg/ml plasma] corti
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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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Page 210 and 211: Maren Depke References Byrne GI, Le
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Page 216 and 217: Maren Depke References Hagopian K,
Page 218 and 219: Maren Depke References Jin T, Bokar
Page 220 and 221: Maren Depke References Kwan T, Liu
Page 222 and 223: Maren Depke References Luster AD, U
Page 224 and 225: Maren Depke References Namiki S, Na
Page 226 and 227: 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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