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Maren Depke Results Pathogen Gene Expression Profiling Comparison of differentially expressed genes [Maren Depke] and proteins with differing abundance [Sandra Scharf] in internalized staphylococci In an experimental setup corresponding to the generation of samples for tiling array analysis, the abundance of proteins in internalized staphylococci was recorded with a combined approach of stable isotope labeling with amino acids in cell culture (SILAC), FACS cell sorting and mass spectrometric analysis (Sandra Scharf, Schmidt et al. 2010). The method, which was applied for this first proteome profiling of in vitro internalized staphylococci, limits the possible protein identification mainly to cytosolic proteins: Secreted proteins were lost during sample preparation, and peptides from membrane associated proteins are difficult to obtain by tryptic digest of whole-cell samples. In the proteomic study, the time points 1.5 h, 2.5 h, 3.5 h, 4.5 h, 5.5 h, and 6.5 h after start of infection were included, i. e. the analysis window was divided into smaller sections than in the tiling array study. In an analysis which included more samples and controls than published in the pilot study, Sandra Scharf identified 648 proteins and obtained a set of 114 proteins with differential abundance. Differential abundance was assigned to proteins when the regulation was observed in at least 2 of 3 biological replicates in at least 1 of 6 analyzed time points, and proteins with contradictory results were excluded. The regulated proteins were normalized in reference to the SILAC-method immanent internal control (heavy isotope labeled peptides) and exhibited an abundance difference when comparing target peptide ratios with the median ratio for all peptides at the corresponding time point. Regulated proteins which were additionally different in abundance in a serum/CO 2 control (without presence of host cells) were excluded. To gain an insight into the comparability of transcriptome and proteome profiling, differentially expressed sequences were compared to regulated proteins. First, up-regulated proteins were compared to the sequences which exhibited increased expression 2.5 h or 6.5 h after start of infection or in both time points (Fig. R.5.11 A). Second, the same comparison was performed for down-regulated proteins and repressed sequences (Fig. R.5.11 B). A B up-regulated proteins in internalized staphylococci down-regulated proteins in internalized staphylococci 53 34 5 2 14 176 96 207 3 0 6 173 121 181 induced sequences in “2.5 h internalization” vs. “1 h serum/CO 2 control” induced sequences in “6.5 h internalization” vs. “1 h serum/CO 2 control” repressed sequences in “2.5 h internalization” vs. “1 h serum/CO 2 control” repressed sequences in “6.5 h internalization” vs. “1 h serum/CO 2 control” Fig. R.5.11: Comparison of differentially expressed sequences in at least one of the two analyzed time points with proteins exhibiting different abundance in internalized staphylococci. A. Up-regulated proteins and induced sequences. B. Down-regulated proteins and repressed sequences. 144
Maren Depke Results Pathogen Gene Expression Profiling In total, the transcripts of 21 up-regulated proteins were increased in at least one of the two analyzed time points. These consisted of 14 proteins, whose genes were induced at both time points, and of 5 and 2 proteins, whose gene expression was increased only at the 2.5 h and 6.5 h time point, respectively. For 6 proteins, whose abundance was reduced, the corresponding gene expression was repressed at both time points, and for further 3 down-regulated proteins the gene expression was only repressed at the 2.5 h time point (Table R.5.6). These numbers result in an overlap of 28 % of up- and 21 % of down-regulated proteins with the corresponding gene expression changes. As the lists of differentially expressed sequences included newly identified transcripts from the tiling array, which were not accessed with mass spectrometry identification, these lists contained many transcriptome-specific results. Table R.5.6: Differentially expressed sequences in internalization samples which exhibit differences in protein abundance and regulation in the same direction. LocusTag gene annotation difference in protein abundance a fold change in comparison to baseline 1 h serum/CO 2 control 2.5 h internalized b 6.5 h internalized b SAOUHSC_01846 acsA acetyl-CoA synthetase, putative + 16.3 4.0 SAOUHSC_01395 asd aspartate-semialdehyde dehydrogenase + 14.1 13.3 SAOUHSC_00086 butA 3-ketoacyl-acyl carrier protein reductase, putative + 2.5 2.2 SAOUHSC_01396 dapA dihydrodipicolinate synthase + 12.5 12.0 SAOUHSC_01398 dapD 2,3,4,5-tetrahydropyridine-2-carboxylate N- succinyltransferase, putative + 9.1 9.8 SAOUHSC_01320 dhoM homoserine dehydrogenase, putative + 2.1 5.4 SAOUHSC_00196 fadB hypothetical protein SAOUHSC_00196 + 84.4 16.6 SAOUHSC_00197 fadD hypothetical protein SAOUHSC_00197 + 100.0 35.8 SAOUHSC_02869 rocA delta-1-pyrroline-5-carboxylate dehydrogenase, putative + 8.0 2.8 SAOUHSC_01418 odhA 2-oxoglutarate dehydrogenase, E1 component + 5.2 2.2 SAOUHSC_01416 odhB 2-oxoglutarate dehydrogenase, E2 component, dihydrolipoamide succinyltransferase + 4.5 2.1 SAOUHSC_00371 hypothetical protein SAOUHSC_00371 + 4.4 2.1 SAOUHSC_01399 hypothetical protein SAOUHSC_01399 + 7.9 8.3 SAOUHSC_02425 hypothetical protein SAOUHSC_02425 + 5.3 2.8 SAOUHSC_01276 glpK glycerol kinase + 2.7 1.7 SAOUHSC_01801 icd, citC isocitrate dehydrogenase, NADP-dependent + 2.1 1.2 SAOUHSC_01972 prsA protein export protein PrsA, putative + 2.0 1.6 SAOUHSC_00767 yfiA hypothetical protein SAOUHSC_00767 + 2.9 1.7 SAOUHSC_00951 hypothetical protein SAOUHSC_00951 + 2.4 1.6 SAOUHSC_01321 thrC threonine synthase + 1.7 5.3 SAOUHSC_00833 hypothetical protein SAOUHSC_00833 + 1.2 2.1 SAOUHSC_01771 hemL glutamate-1-semialdehyde-2,1-aminomutase − -2.2 -2.5 SAOUHSC_01092 pheS phenylalanyl-tRNA synthetase, alpha subunit − -2.5 -3.4 SAOUHSC_01093 pheT phenylalanyl-tRNA synthetase, beta subunit − -2.7 -3.5 SAOUHSC_01010 purC phosphoribosylaminoimidazole-succinocarboxamide synthase − -4.5 -2.2 SAOUHSC_01011 purS phosphoribosylformylglycinamidine synthase, PurS protein − -4.9 -2.3 SAOUHSC_01788 thrS threonyl-tRNA synthetase − -2.1 -2.4 SAOUHSC_01018 purD phosphoribosylamine-glycine ligase − -2.6 -1.1 SAOUHSC_01013 purL phosphoribosylformylglycinamidine synthase II − -3.4 -1.5 SAOUHSC_01012 purQ phosphoribosylformylglycinamidine synthase I − -4.0 -1.9 a Up-regulated protein abundance in internalized staphylococci is indicated by “+”, down-regulated abundance by ”–“. b Differentially regulated genes (significant with p* < 0.05 and with a minimal absolute fold change of 2) are indicated in bold. 145
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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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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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