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mean normalized intensity Maren Depke Results Pathogen Gene Expression Profiling At the early internalization time point, several genes of purine and pyrimidine nucleotide biosynthesis pathways were repressed. Amino acid biosynthesis genes exhibited increase of expression in both analyzed internalization time points. Furthermore, genes coding for respiration chain components were induced in expression in internalized staphylococci. In internalized staphylococci, several tRNA synthetase genes and some glycolytic enzyme genes were observed to be repressed. Finally, transporters and permeases for sugar molecules, phosphate, oligopeptides, and other substances were differentially expressed (Fig. R.5.23 A, B). Serum/CO 2 control samples featured in some aspects similar changes in metabolic enzyme’s gene expression pattern, e. g. amino acid biosynthesis, glycolysis, tRNA synthetases. On the other hand, aerobic respiration, sugar uptake phosphotransferase system, and some enzymes of peptidoglycan biosynthesis were repressed in this group. Anaerobic incubation instead did not provoke changes in expression of glycolytic enzymes or respiration chain components (Fig. R.5.23 C, D). Subsequently to the first approach of BIOCYC pathway mapping, selected genes were analyzed in more detail. Here, the example of purine biosynthesis was chosen. Although not all genes were significantly differentially expressed, the gene expression changes were highly similar in the complete pur operon and guaAB, which are separately encoded but are also involved in purine biosynthesis. Five genes (purA, purC, purE, purK, purS) were differentially expressed in both analyzed internalization time points, six genes (purD, purF, purL, purQ, guaA, guaB) were differentially expressed 2.5 h after start of infection. Three genes, purH, purM, and purN, exhibited fold change values between −2.6 and −2.8, but were not significant in statistical testing. Finally, purB was significant in the statistical test in both analyzed time points of internalization, but did not pass the minimal absolute fold change cutoff of 2. In the 2.5 h serum/CO 2 control, differential expression occurred only for purA and in trend for purB, whereas almost all genes exhibited fold change values around −3 to −4 in the 6.5 h serum/CO 2 control. Exceptions were guaA and guaB, whose fold change values were still around 1, and the repressor gene purR, which did not exhibit expression changes in any of the analyzed experimental conditions (Fig. R.5.24, Fig. R.5.25). A fold change in comparison to baseline 1 h serum/CO 2 control S. aureus RN1HG sample conditions and time points purA purB purC purD purE purF purH purK purL purM purN purQ purR purS guaA guaB exponential growth phase -1.1 -1.0 1.2 1.3 1.2 1.1 1.2 1.2 1.1 1.1 1.2 1.1 -1.2 1.0 -1.0 -1.1 2.5 h internalized -4.8 -1.6 -4.5 -2.6 -6.2 -3.0 -2.6 -5.4 -3.4 -2.8 -2.6 -4.0 1.1 -4.9 -2.0 -2.3 6.5 h internalized -4.8 -1.6 -2.2 -1.1 -3.4 -1.2 -1.2 -2.9 -1.5 -1.2 -1.1 -1.9 1.3 -2.3 -1.3 -1.4 2.5 h serum/CO 2 control -6.4 -1.6 1.0 1.1 1.0 -1.1 -1.0 1.1 -1.0 -1.0 1.0 1.0 1.3 -1.1 1.4 1.3 6.5 h serum/CO 2 control -28.2 -2.7 -3.2 -3.4 -3.8 -4.1 -4.3 -3.1 -4.0 -3.9 -3.6 -4.0 1.1 -3.9 1.1 -1.0 2.5 h anaerobic incubation -2.1 -1.7 1.4 1.1 1.7 1.3 1.2 1.5 1.3 1.3 1.2 1.5 -1.1 1.1 1.0 1.0 Fig. R.5.24: Purine biosynthesis gene expression. A. Overview on fold change values relative to the baseline sample “1 h serum/CO 2 control” and on significance in statistical group comparisons. The sample “6.5 h serum/CO 2 control” could not be included in statistical testing because of small group size (n = 2). B. Overview on mean normalized gene expression intensity. After the global normalization by inter-chip scaling and detrending, each individual gene has been normalized to the expression level of the baseline sample “1 h serum/CO 2 control” (1 h co.). Although not being continuous data, values were depicted as line graphs instead of bar charts for better facility of inspection. (exp. − exponential growth phase; 1 h co. – 1 h serum/CO 2 control; 2.5 h int. − 2.5 h internalization; 6.5 h int. − 6.5 h internalization; 2.5 h co. − 2.5 h serum/CO 2 control; 6.5 h co. − 6.5 h serum/CO 2 control; 2.5 h anae. − 2.5 h anaerobic incubation) B 1.00 0.10 0.01 exp. 1 h co. 2.5 h int. 6.5 h int. 2.5 h co. 6.5 h co. 2.5 h anae. S. aureus RN1HG sample conditions and time points purA purB purC purD purE purF purH purK purL purM purN purQ purR purS guaA guaB 158
Maren Depke Results Pathogen Gene Expression Profiling 2 0 -2 -4 -6 -8 01014 purF 2 0 -2 -4 -6 -8 01018 purD 2 0 -2 -4 -6 -8 01016 purN 2 0 -2 -4 -6 -8 01013 purL 2 0 -2 -4 -6 -8 01012 purQ 2 0 -2 -4 -6 -8 01011 purS 2 0 -2 -4 -6 -8 01015 purM 2 0 -2 -4 -6 -8 2 0 -2 -4 -6 -8 01009 purK 01008 purE 2 0 -2 -4 -6 -8 01010 purC 2 0 -2 -4 -6 -8 02126 purB 2 0 -2 -4 -6 -8 00467 purR 2 0 -2 -4 -6 -8 01017 purH 2 0 -2 -4 -6 -8 00019 purA 2 0 -2 -4 -6 -8 02126 purB AMP PRPP I-5‘- GMP PRPP : 5-phosphoribosyl-1-pyrophosphate I-5‘- : inosine-5‘-phosphate AMP : adenosine-5‘-monophosphate GMP : guanosine-5‘-monophosphate 00374 guaB 2 0 -2 -4 -6 -8 00375 guaA 2 0 -2 -4 -6 -8 Fig. R.5.25: Purine biosynthesis pathway and gene expression. Pathway reactions and gene LocusTags were extracted from omics-viewer(s) of BIOCYC (BIOCYC, SRI International, CA, USA, http://biocyc.org/expression.html). Arrows represent reactions and dots substrates/products when these are not specially named. Numbers next to enzyme reactions will result in the LocusTag of the gene when combined with “SAOUHSC_*” instead of the wildcard character. Bar charts indicate fold change values of internalized samples in reference to the baseline sample of 1 h serum/CO 2 control. Values for the 2.5 h internalized samples are depicted in light gray (first column) and those of the 6.5 h internalized samples in dark gray (second column). A third analysis of metabolic gene expression involved the KEGG pathway maps (KEGG: Kyoto Encyclopedia of Genes and Genomes). Already BIOCYC pathway mapping had revealed changes in amino acid biosynthesis and glycolysis. Corresponding pathways and additionally pathways of TCA and urea cycle were selected in KEGG specific for S. aureus NCTC8325. The strain’s genes, which were mapped by KEGG to the pathways, were extracted and checked for differential expression in at least one time point after internalization in S9 cells. The biosynthesis pathways for asparagine, histidine, glutamine, serine, tryptophan, leucine, valine, isoleucine, lysine, and threonine were induced to a large extent (Fig. R.5.26). A fraction of associated genes were not included in the lists of differentially expressed genes. A further analysis of these genes identified some of them to be either significant in statistical testing without passing the minimal absolute fold change cutoff or to pass that cutoff without reaching statistical significance. These expression changes were rated as trend, which in many cases further confirmed the findings of expression changes in the metabolic pathways. In addition to induced expression of amino acid biosynthesis genes, induction of four TCA cycle enzyme genes was observed, which was even more substantiated by a trend of increase for further four genes. Contrarily, especially genes encoding enzymes of irreversible glycolysis reactions were repressed. Finally, genes coding for enzymes of anabolic reactions during gluconeogenesis were induced in internalized staphylococci. Using energy, their gene products reverse the reactions whose equilibrium is normally set to the side of glucose degradation products. Repressed tRNA synthetase gene expression has already been mentioned before. The synthetase genes for tyrosine, leucine, threonine, phenylalanine, serine, aspartate, and histidine (tyrS, leuS, thrS, pheS, pheT, serS, aspS, hisS) were repressed in at least one time point of internalization, many of them even in both (Fig. R.5.27 A). Further genes (ileS, alaS, metS, asnS, gltX, trpS) exhibited a trend of repression in at least one time point of internalization with p* < 0.05, but an absolute fold change of less than 2 (Fig. R.5.27 B). Three genes (valS, glyS, cysS) behaved contrarily and possessed a trend of induction in one time point of internalization (Fig. R.5.27 C). Finally, the remaining three tRNA synthetase genes (argS, proS, lysS) showed no change in expression (Fig. R.5.27 D). 159
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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 2005). SaP
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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 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 GE
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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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log2(ratio C57BL/6 / BALB/c) at IFN
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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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