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Maren Depke Discussion and Conclusions in Lactococcus lactis, a bacterium with low pathogenic potential, was performed (Que et al. 2001). Possession of ClfA or FnbA rendered L. lactis as adherent to fibrinogen or fibronectin as the gene-donor S. aureus strain. Furthermore, overexpressing L. lactis strains were able to cause endocarditis in a rat model with a similar minimal infection dose as S. aureus. The authors stated that this overexpression experiment proved the involvement of clfA and fnbA in endocarditis with higher confidence than deletion experiments (Que et al. 2001). Thus, fnb genes have proven to be relevant for adhesion, invasion and in vivo infection situations. Surprisingly, the induction of adhesins was observed after internalization of S. aureus RN1HG into S9 cells (this study). It can be speculated that this induction took place in advance to react to a possible re-liberation from the host cell e. g. after host cell death. In internalized staphylococci, also soluble adhesins were induced (eap, coa, vwb, emp, efb). These are also associated with other functions like immune-evasion. Eap, whose expression is essentially mediated by sae (Harraghy et al. 2005), is one of the examples for a staphylococcal protein which is related to a variety of functions (Harraghy et al. 2003). Eap was reported for example to mediate adhesion to cultured fibroblasts (Hussain et al. 2002), to enhance internalization into human fetal lung fibroblasts and HACAT keratinocytes (Haggar et al. 2003), to impair wound healing via inhibition of angiogenesis (Athanasopoulos et al. 2006), to block Ras activation and signal transduction cascade which leads to anti-angiogenesis (Sobke et al. 2006), to induce pro-inflammatory IL-6 and TNF-α in human peripheral blood mononuclear cells (Scriba et al. 2008), but also to act anti-inflammatorily by inhibition of leukocyte recruitment via blockade of ICAM1 and hence lead to immune evasion (Chavakis et al. 2002, Haggar et al. 2004). Thus, it was not surprising to find induced expression also after internalization into S9 cells (this study). Further immune-evasion related genes were increased in S9-internalized S. aureus RN1HG, e. g. chp, whose gene product CHIPS, chemotaxis inhibitory protein of staphylococci, is known to block the receptor for the chemotactic complement fragment C5a and for bacterialderived formylated peptides (de Haas et al. 2004, Murdoch/Finn 2000). Also induced efb, coding for extracellular fibrinogen-binding protein Efb, interferes with complement action, i. e. opsonization, by inhibition of C3b-binding to bacterial surfaces (Lee et al. 2004a, 2004b). Thus, internalized staphylococci induce certain genes which would benefit their survival in an in vivo situation. In aerobically living cells, reactive oxygen intermediates like hydroxyl radicals (OH•), hydroxide (OH – ), superoxide anion (O – 2 ), or hydrogen peroxide (H 2 O 2 ) occur in normal metabolism and may damage cellular structures like DNA (Fridovich 1978, Imlay/Linn 1988). Phagocytes even produce high amounts of reactive oxygen intermediates during respiratory burst, e. g. superoxide anion by their NADPH-oxidase, as part of their defense and killing strategy (Robinson 2009). S. aureus owns two genes coding for superoxide dismutases: sodA and sodM (Clements et al. 1999, Valderas/Hart 2001). The enzyme participates in the detoxification of reactive oxygen intermediates by catalyzing the reaction of two superoxide anion molecules to oxygen and hydrogen peroxide, of which two molecules are consequently decomposed to oxygen and water by catalase. The complete enzyme constitutes of 2 homo- or heterodimeric subunits. The gene sodM is characteristic for S. aureus since coagulase-negative staphylococci like S. epidermidis do not own this gene or a homologue (Valderas et al. 2002). In the presence of manganese, the internal (methyl viologen) and external (xanthine/xanthine oxidase) generation of O – 2 in S. aureus led to increased expression of sodA and sodM, respectively, but not without the addition of manganese (Karavolos et al. 2003). The observation 200
Maren Depke Discussion and Conclusions of increased sodA and repressed sodM in internalized staphylococci (this study) fits to an independent regulation of both genes. Anyway, the regulation of staphylococcal sod genes has not been intensively studied until now. The analysis of mutants in the regulator genes sigB, agr, or sarA did not influence the sodA expression in experiments of Clements et al. in 1999. Use of a sigB deficient S. aureus strain gave hints for a negative influence of σ B on the expression of sodM (Karavolos et al. 2003). Contradictory to the results of Clements and colleagues, SarA was reported as negative regulator of especially sodM but also sodA expression via its activity as transcriptional repressor since gel-shift analysis and consensus sequence search revealed SarAbinding to the sod promoter regions (Ballal/Manna 2009). The observations of regulatory influence do not completely explain the expression pattern in this study, and further attempts of explanation would be too speculative because of only little existing knowledge on the internalized gene expression regulation. Detoxification of reactive oxygen intermediates and correlated gene expression might have impact on virulence since this detoxification, i. e. evasion of immune defense, can give an advantage to the bacterial cell. Studies from literature give contradictory evidence. Superoxide dismutase did not correlate with lethality in mouse infection but catalase was proposed to enhance virulence using clinical isolates and Wood-46 strain of S. aureus (Mandell 1975). S. aureus 8325-4 sodA mutant did not possess reduced virulence in comparison to wild type strains in a mouse abscess model (Clements et al. 1999). S. aureus SH1000 sodA, sodM, and sodA sodM single and double mutants exhibited reduced virulence in a murine model of subcutaneous infection (Karavolos et al. 2003). The SigB – phenotype of 8325-4, which has been reversed in SH1000 (Kullik/Giachino 1997, Horsburgh et al. 2002b), might have contributed to these observations. The superoxide dismutase gene sodA was reported to be linked to the bacterial acid-adaptive response (Clements et al. 1999). Such function might provide a link to the expression pattern in internalized staphylococci, which probably encounter reduced pH in the phagosome/endo-lysosome. Internalized staphylococci induced two pairs of bicomponent toxins, lukD/lukE, hlgB/hlgC, and alpha-hemolysin, hla. The subunits of the bicomponent toxins were described to be interchangeable and to result in different toxins with distinct properties, which led to the activation of granulocytes (König et al. 1997). Leukocidins target the membrane and lead to disruption of host cells for example of human polymorphonuclear neutrophils by PVL (Colin et al. 1994) or of erythrocytes by a LukF/Hlg combination (Nguyen et al. 2003) and cause severe inflammatory damage in a model of toxin injection into rabbit eyes (Siqueira et al. 1997). LukD/LukE were first characterized by Gravet et al. in 1998. From a set of 429 S. aureus isolates, the prevalence of lukD/lukE was determined as 82 % in blood and 60.5 % in nasal isolates, whereas hlg was detected in almost 100 % of isolates (von Eiff et al 2004). The LukD/LukE combination was shown to result in dermonecrosis after infection of rabbit skin. The dermonecrosis inducing potency of LukD or LukE was reduced when combinations with other toxin subunits were applied. Especially the combinations with HlgB led to reduced activity. HlgB/HlgC possessed lytic activity for erythrocytes, but the LukD/LukE combination did not lead to erythrocyte lysis. Erythrocyte lysis could not be achieved by combining LukD or LukE with other toxin subunits. Finally, human polymorphonuclear leukocytes (granulocytes) could be activated by HlgB/HlgC, LukE/HlgB, LukD/HlgC, and LukD/LukE which decreased potency in the given order (Gravet et al. 1998). Thus, the different toxins have a distinct functional bias as well as potency. The specific effects on S9 cells or in vivo, e. g. in the lung, still need to be determined. 201
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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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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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Page 208 and 209: Maren Depke References Athanasopoul
Page 210 and 211: Maren Depke References Byrne GI, Le
Page 212 and 213: Maren Depke References Demling R. T
Page 214 and 215: Maren Depke References Foss GS, Pry
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.
Page 228 and 229: Maren Depke References Siewert E, B
Page 230 and 231: Maren Depke References van den Akke
Page 232 and 233: Maren Depke References Yang XL, Sch
Page 235: Maren Depke A F F I D A V I T / E R
Page 238 and 239: Maren Depke Acknowledgments Kidney
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