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Maren Depke Results Pathogen Gene Expression Profiling The tiling array approach aimed at the identification of the maximal possible number of transcriptional units − known and newly identified – and therefore included several different growth media in a cooperation between several laboratories. On the other hand, the tiling array analysis could be used to get insights into the stationary phase response in the newly established pMEM medium on transcriptome level under restriction to the already defined transcriptional units and annotated genes. For this purpose, differential expression was assumed for genes regulated in at least one of the two analyzed time points of stationary phase, t 2 and t 4 . Using this application of the array data set, a very clear picture of induced TCA cycle enzyme genes (citZ, citB, citC, sucA, sucB, sucD, sucC, sdhA, sdhB, sdhC; all induced) was revealed. In parallel, reduced expression of glycolysis enzyme genes (pgi, pfkA, pgk, pgm, pykA; all repressed) and induced expression of gluconeogenetic enzyme genes (pckA, gap2, fbp) was observed. This is in accordance with published stationary phase response of S. aureus (Kohler et al. 2005). Furthermore, repression of amino acid biosynthesis pathways was described for the stationary phase. In pMEM, tyryptophan (trpB, trpC, trpD, trpE, trpG), histidine (hisB, hisD, hisG), and aspartate/arginine (argG, argH, SAOUHSC_00150) biosynthesis genes were repressed. But contrarily, induction of lysine (lysC, asd), histidine/glutamate (hutH, hutI, hutU, hutG, rocA), and citruline/ornithine (argF, arg, arcC) biosynthesis genes was visible in the stationary phase samples cultivated in pMEM. Other amino acid biosynthesis genes (leu, ilv, dap, thr, and others) were not significantly differentially expressed. Reduced or even ceased growth diminishes the need for new protein synthesis. Therefore, associated molecules like ribosomal proteins, translation elongation factors, and chaperones do not need to be newly synthesized, which was observed on proteome level for S. aureus COL in TSB (Kohler et al. 2005). This phenomenon is part of the stringent response e. g. to glucose starvation. Here, using pMEM medium, S. aureus RN1HG and transcriptome analysis, only two ribosomal protein genes, rpsD and rpsT, and prmA, a ribosomal protein L11 methyltransferase, were repressed. Only translation elongation factor P (efp) exhibited a fold change of less than −2 in the t 4 samples, but this repression was not significant. Chaperones dnaK, grpE, groEL, and groES exhibited slightly decreased fold change values in the range of −1.4 to −1.9, but the difference was also not significant. Of the Clp complex, expression of subunit X was repressed and of subunit L was induced. Finally, repression of tRNA synthetases is known to be part of the stringent response in S. aureus (Anderson KL et al. 2006). In stationary phase in pMEM, repression of tyrS, leuS, alaS, aspS, hisS, valS, thrS, glyS, proS, ileS, pheS, pheT, gltX, metS/metG, and serS was detected. The other tRNA synthetases except cysS showed a trend of repression with fold change values almost reaching the cutoff (−2). Virulence factors differentially expressed in stationary vs. exponential growth phase In stationary growth phase, differential expression of virulence-associated genes was observed. Many of these genes were regulated at both analyzed time points of stationary phase, t 2 and t 4 . Surface proteins A and G (spa, sasG) were repressed. Other membrane-bound adhesins were induced like clfA, fnbA, and isaB. Secreted adhesins and immunomodulatory molecules efb, chp, and sbi were repressed, whereas ebh and eap were induced. One of the two staphylococcal superoxide dismutases, sodM, was found to be induced. The toxins hla, hlb, hlgC, and hlgB exhibited an increase in expression. In the group of extracellular enzymes, only nuc and htrA were repressed. Contrarily, for secreted lipases geh and lip and for proteases sspB, splC, splB, splA, and aur a higher expression was detected in stationary phase than in exponential growth. The complete cap operon of 15 capsular genes (SAOUHSC_00114 to SAOUHSC_00128) was induced in t 2 samples and still 10 of these genes were also induced in t 4 samples. The biofilm repressor icaR was induced in t 2 samples. Fittingly, two genes of the ica operon, icaB and icaC, were observed to be repressed in both analyzed stationary phase time points. Finally, differential expression of five staphylococcal accessory regulators was detected: sarX and sarT were repressed, while sarV, sarR, and sarZ were induced in stationary phase (Table R.5.3). 134
Maren Depke Results Pathogen Gene Expression Profiling Table R.5.3: Expression of virulence associated genes in stationary phase in comparison to exponential growth in pMEM. LocusTag gene annotation fold change in comparison to baseline exponential growth stationary phase t 2 a stationary phase t 4 a SAOUHSC_00069 spa protein A -7.4 -8.8 SAOUHSC_02798 sasG surface protein G -4.2 -3.4 SAOUHSC_00812 clfA clumping factor 2.8 2.1 SAOUHSC_02803 fnbA fibronectin-binding protein precursor, putative 3.1 2.7 SAOUHSC_02972 isaB immunodominant staphylococcal antigen B 3.2 3.4 SAOUHSC_01114 efb fibrinogen-binding protein -2.7 1.4 SAOUHSC_02169 chp chemotaxis inhibitory protein -4.3 -2.6 SAOUHSC_02706 sbi immunoglobulin G-binding protein Sbi, putative -3.9 -1.7 SAOUHSC_01447 ebh extracellular matrix-binding protein ebh 4.7 2.9 SAOUHSC_02161 eap MHC class II analog protein 2.5 3.9 SAOUHSC_00093 sodM superoxide dismutase, putative 2.1 1.6 SAOUHSC_01121 hla alpha-hemolysin precursor 15.1 36.8 SAOUHSC_02163 hlb hypothetical protein SAOUHSC_02163 1.9 7.2 SAOUHSC_02709 hlgC leukocidin s subunit precursor, putative 16.2 43.6 SAOUHSC_02710 hlgB leukocidin f subunit precursor 11.5 29.6 SAOUHSC_00818 nuc thermonuclease precursor -2.4 -1.4 SAOUHSC_00958 htrA serine protease HtrA, putative -5.7 -6.3 SAOUHSC_00300 geh lipase precursor 10.4 11.1 SAOUHSC_03006 lip lipase 70.4 97.8 SAOUHSC_00987 sspB cysteine protease precursor, putative 2.1 1.8 SAOUHSC_01939 splC serine protease SplC 2.8 5.2 SAOUHSC_01941 splB serine protease SplB 3.1 6.3 SAOUHSC_01942 splA serine protease SplA 2.9 6.0 SAOUHSC_02971 aur aureolysin, putative 2.9 5.0 SAOUHSC_00114 capA capsular polysaccharide biosynthesis protein, putative 7.0 6.4 SAOUHSC_00115 capB capsular polysaccharide synthesis enzyme Cap5B 6.5 5.5 SAOUHSC_00116 capC capsular polysaccharide synthesis enzyme Cap8C 6.0 5.2 SAOUHSC_00117 capD capsular polysaccharide biosynthesis protein Cap5D, putative 5.7 4.9 SAOUHSC_00118 capE capsular polysaccharide biosynthesis protein Cap5E, putative 5.3 4.2 SAOUHSC_00119 capF capsular polysaccharide synthesis enzyme Cap8F 4.8 3.8 SAOUHSC_00120 capG UDP-N-acetylglucosamine 2-epimerase 4.1 3.4 SAOUHSC_00121 capH capsular polysaccharide synthesis enzyme O-acetyl transferase Cap5H, putative 3.9 3.2 SAOUHSC_00122 capI capsular polysaccharide biosynthesis protein Cap5I, putative 3.7 2.9 SAOUHSC_00123 capJ capsular polysaccharide biosynthesis protein Cap5J, putative 3.2 2.3 SAOUHSC_00124 capK capsular polysaccharide biosynthesis protein Cap5K, putative 3.0 2.1 SAOUHSC_00125 capL cap5L protein/glycosyltransferase, putative 2.8 1.9 SAOUHSC_00126 capM capsular polysaccharide biosynthesis protein Cap8M 2.6 1.8 SAOUHSC_00127 capN cap5N protein/UDP-glucose 4-epimerase, putative 2.7 1.8 SAOUHSC_00128 capO cap5O protein/UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase 2.6 1.7 SAOUHSC_00248 lytM peptidoglycan hydrolase, putative -4.2 -2.9 SAOUHSC_00869 dltA D-alanine-activating enzyme -2.5 -2.0 SAOUHSC_00870 dltB dltB protein, putative -2.6 -2.3 SAOUHSC_00872 dltD extramembranal protein -2.6 -2.3 SAOUHSC_02883 ssaA staphylococcal secretory antigen ssaA -5.5 -3.3 SAOUHSC_03001 icaR ica operon transcriptional regulator IcaR, putative 2.3 1.4 SAOUHSC_03004 icaB icaB protein, putative -3.2 -2.1 SAOUHSC_03005 icaC intercellular adhesion protein C, putative -2.6 -2.2 SAOUHSC_00674 sarX staphylococcal accessory regulator X -4.8 -5.1 SAOUHSC_02799 sarT staphylococcal accessory regulator T -8.8 -8.4 SAOUHSC_02532 sarV staphylococcal accessory regulator V 1.7 2.3 SAOUHSC_02566 sarR staphylococcal accessory regulator R 1.9 2.1 SAOUHSC_02669 sarZ staphylococcal accessory regulator Z 2.0 2.4 a Differentially regulated genes (significant with p* < 0.05 and with a minimal absolute fold change of 2) are indicated in bold. 135
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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 Discussion and Conclusi
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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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