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Maren Depke Discussion and Conclusions Similarly, the expression of extracellular and membrane-bound enzymes, which contribute to virulence or spreading, was difficult to interpret. Expression of serine proteases (splABCDEF) and lipase (lip) was increased, whereas the expression of hyaluronate lyase (hysA), a membrane bound serine protease (htrA), nuclease (nuc), and glutamyl endopeptidase alias V8 peptidase (sspA) was repressed in internalized staphylococci. Some spl genes were found to be SaeRSregulated (Rogasch et al. 2006). Thus, the induction of the spl operon fits to the proposed activity of the SaeRS two-component system. Gene expression changes which might have influence on cell wall remodeling were also observed in internalized staphylococci. Surprisingly, this included repression of the dlt operon. The proteins encoded by dlt participate in incorporation and esterification of D-alanine residues into the cell wall teichoic acids. In this way, the negative charge of the cell wall structures is reduced and thus is less attractant for positively charged antimicrobial molecules (Peschel et al. 1999). With the aim to evade the immune response, an induction of dlt expression had been expected. In parallel, the induction of peptidoglycan hydrolase lytM and staphylococcal secretory antigen ssaA was recorded (this study), which are also involved in cell wall remodeling (Dubrac et al. 2007). Repression of dlt and induction of lytM were also observed by Garzoni and coworkers (Garzoni et al. 2007). Furthermore, the induction of prsA, coding for a foldase involved in translocation and folding of secreted proteins (Sarvas et al. 2004), was observed in internalized staphylococci 2.5 h after infection of S9 cells (this study). This regulation was in accordance with protein abundance data in internalized staphylococci which were determined by Sandra Scharf (Schmidt et al. 2010). She also found an increased abundance of VraR, regulatory component of the VraRS two-component system and known to be positively auto-regulated (Kuroda et al. 2003), which led to the examination of vraRS gene expression: vraR (1.8) and vraS (1.6) were not significantly different in internalized staphylococci (2.5 h) in comparison to control, but the fold change values indicated a slight trend for induction. Hence, the observed differential expression of cell wall remodeling related genes was in agreement with the proteome data of Sandra Scharf. Additionally, genes described to belong to the VraRS regulon (Kuroda et al. 2003) were examined. Internalized staphylococci induced about 20 % of the published VraRS regulon (Kuroda et al. 2003) at the same time point when a trend of vraRS induction was detected. Since the VraRS two-component system was suggested to be activated upon cell wall damage or inhibition of cell wall biosynthesis (Kuroda et al. 2000, Kuroda et al. 2003), the observation of cell wall remodeling associated gene expression changes might indicate a reaction to stressful influences which possibly operate on the bacterial cell wall after internalization into the host cell. Another possible explanation proposed by Garzoni and colleagues (2007) is that the gene induction (e. g. lytM) is associated with cell division processes. The gene expression analysis of internalized staphylococci in S9 cells led to the recognition of changes in the expression of metabolic genes. Purine biosynthesis, tRNA synthetases, and glycolysis were repressed, whereas gluconeogenesis, TCA cycle enzyme genes, and especially genes related to amino acid biosynthesis were induced. The induction of amino acid biosynthesis and TCA cycle genes as well as the repression of purine biosynthesis and tRNA synthetase genes was clearly confirmed in the proteome analysis of Sandra Scharf. Additionally, transporter genes were differentially expressed. Induction was observed especially for phosphate transporters, but also for transporters of phosphonate, methionine, peptide/oligopeptide, sugar/maltose, osmoprotectants, amino acids, and gluconate. Repression was detected for urea, 202
Maren Depke Discussion and Conclusions spermidine/putrescine, sodium/glutamate, arginine/ornithine, glycine betaine, amino acid, choline, citrate, xanthine, fructose, and lactate transporters. A good metabolic performance, especially the unaffected ability for amino acid biosynthesis and for the uptake of compounds, is important for the survival of pathogens and has been reported to be linked not only to auxotrophies, but also to virulence. In a mutagenesis study, which was combined with a model of murine systemic infection, 24 genes were identified whose mutation resulted in attenuation of S. aureus virulence. Of these, 9 genes (38 %) were part of purine or amino acid biosynthesis, and 6 genes (25 %) coded for membrane transporters (Benton et al. 2004). Of these virulence associated genes, four genes related to amino acid biosynthesis (lysC, dapB, trpF, asd) and pstS, phosphate transporter, were induced in internalized staphylococci at least in one of the two analyzed time points (this study). Additionally, amino acid biosynthesis genes tyrA and pycA were induced in trend with a significant p-value but a fold change value of less than the cutoff. Few gene expression studies of internalized staphylococci are published until now. One of them has already been cited above in selected details. Garzoni and coworkers analyzed internalized staphylococci in epithelial cells using a microarray approach (Garzoni et al. 2007). Although the general setting resembles that applied in this study, important differences in the experimental procedures as well as in the results can be found between both studies. Most importantly, staphylococcal strain (S. aureus 6850 vs. RN1HG) as well as host cell line (A549 vs. S9) differed. Garzoni et al. mention exactly this topic and conclude that “certain combinations of host cells and bacteria can result in different biological outcomes”, which is clearly supported in the comparison of their and this study. Furthermore, infection took place in a cell culture medium without (Garzoni et al. 2007) or with serum supplement (this study), with washed (Garzoni et al. 2007) or non-washed, exoproteins containing (this study) bacterial suspensions, in a multiplicity of infection (MOI) of 100 for 30 min (Garzoni et al. 2007) or in a MOI of 25 for 1 h (this study). Also RNA preparation and sample processing methods differed: depletion of eukaryotic RNA and amplification step (Garzoni et al. 2007) or utilization of RNA without depletion and amplification since pure bacterial RNA of high quality was available in sufficient amounts (this study). Finally, different arrays were used. Both studies approximately match in the analyzed time points of 2 h/2.5 h and 6 h/6.5 h after infection. The total numbers of differentially expressed genes were higher in the literature reference with 1042 (Garzoni et al. 2007) and 565 (this study, annotated genes) for the early time point and 766 (Garzoni et al. 2007) and 489 (this study, annotated genes) for the later time point, and Garzoni et al. detected a higher fraction of repressed genes whereas this study resulted in a higher number of induced genes. Similar in both studies, comparisons between non-adherent and mock infection samples (staphylococci in infection medium) did not result in the detection of differential gene expression. In a very rough comparison, at least 100 genes were differentially expressed between internalized and control staphylococci in both studies. Garzoni and colleagues describe differential expression of genes or functional groups of genes, which were recognized also in this study, e. g. repression of tRNA synthetase genes, induction of fnbAB, induction of hlgB and lukE, induction of sodA, repression of sspA, induction of lytM, and induction of transporters. Different results were revealed, e. g. clfB repressed (Garzoni et al. 2007) or induced (this study), hla not differentially expressed (Garzoni et al. 2007) or induced (this study), cap operon repressed (Garzoni et al. 2007) or not differentially expressed (this study), TCA cycle 203
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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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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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