genomewide characterization of host-pathogen interactions by ...

More documents

Recommendations

Info

Maren Depke Discussion and Conclusions al. 1995, Crosse et al. 2000, Anderson KL et al. 2006, Wolz et al. 2010). In a comparison between the stationary phase gene expression profile and that of the late serum/CO 2 control (each in relation to exponential phase growth) a high overlap was recognized. This included reduced expression of ribosomal protein genes, tRNA synthetase genes, translation elongation factor genes and a clear trend of induction of the stringent response regulator relA as it was reported in literature (Anderson KL et al. 2006). Thus, the gene expression in the late serum/CO 2 controls resembled the stationary phase/stringent response. A corresponding similarity was not observed for the internalized staphylococci as well as for the early control samples. In conclusion from these observations, it was decided that the 1 h serum/CO 2 control sample, although not timematched, was the most appropriate baseline sample for this infection experiment study. Since the shift of bacterial cells from aerobic agitated culture to infection settings in cell culture plates and 5 % CO 2 atmosphere probably resulted in a change of oxygen availability, genes harboring binding sites for Rex, the central anaerobiosis regulator and transcriptional repressor (Pagels et al. 2010), were analyzed to elucidate whether the changes of oxygen availability lead to an anaerobiosis gene expression signature (this study). De-repression of Rex-binding site containing genes was visible during anaerobic incubation, but despite the shift from agitated culture to cell culture plate, internalized samples did not show the same pattern of anaerobic gene expression. Contrarily, many of these Rex binding sites containing genes, which were in trend or significantly induced in anaerobically incubated samples (indicating de-repression after Rex lost its DNA affinity in anaerobic conditions), were in trend or significantly repressed in internalized staphylococci in S9 cells (indicating sustained DNA binding activity of Rex). Thus, it was concluded that internalized staphylococci did not suffer from reduced oxygen availability. One of the first observations during the analysis of the internalized staphylococcal gene expression pattern was that the response regulator gene saeR of the two-component system SaeRS was induced in internalized staphylococci 6.5 h after start of infection and that the sensor component gene saeS was significantly differentially expressed but only did not exceed a fold change of 2. This observation led to a more detailed analysis of the SaeRS regulon in internalized staphylococci. In 2006, Rogasch and coworkers published a transcriptomic and proteomic characterization study of the SaeRS regulon (Rogasch et al. 2006). Of the genes defined to be regulated in that publication, 21 were found to be regulated (mostly increased) in at least one of the two analyzed time points of internalized staphylococci in the study described in this thesis. These included the auto-induced saeR, adhesins, serine protease, toxins, immune-evasive genes and others. Thus, most probably the SaeRS two-component system was activated in internalized staphylococci. The SaeRS system was furthermore studied by DNA array analysis using a saeS gene replacement mutant (Sa371ko) and its parental strain, the clinical isolate S. aureus WCUH29 (Liang et al. 2006). Less than 20 genes (positive influence on coa, fnbB, fnb, efb, hla, hlb, hlgC, saeS, SA1000, which corresponds to SAOUHSC_01110, and others; negative influence on agrA and a gene coding for a hypothetical protein) were observed to be SaeRS dependently regulated. In vivo, the saeS mutant strain resulted in less bacterial load in the kidney compared to the wild type strain in a hematogenous pyelonephritis model of i. v. infected mice (Liang et al. 2006). Interestingly, S. aureus deficient for SaeS exhibited less adhesion to and less invasion in human lung epithelial A549 cells and resulted in a reduced rate of host cell apoptosis which was possibly mediated in parts by reduced expression of hla (Liang et al. 2006). Furthermore, negative influence on adhesion and internalization was documented for efb and SA1000 replacement 198
Maren Depke Discussion and Conclusions mutants. Thus, especially saeRS as regulatory system, but also efb and SA1000 (SAOUHSC_01110) contribute to adhesion and invasion (Liang et al. 2006). Strikingly, these genes were observed to be induced (saeR, efb) or in trend induced (saeS; SAOUHSC_01110; data not shown for SAOUHSC_01110) in internalized staphylococci in the study described in this thesis. Furthermore, also coa, fnbB, hla, and hlgC, determined as SaeRS-dependent by Liang and colleagues, were induced in internalized staphylococci in this study. The final conclusion of Liang et al. stated that “activation of the SaeRS system is required for S. aureus to adhere to and invade epithelial cells”. Supportingly, such activation was also concluded in this study. Nevertheless, the activation SaeRS and induction of saeRS might depend on further experimental aspects which are not defined yet. Infection and gene expression studies by Garzoni and coworkers did not reveal induction but repression of saeR and saeS in S. aureus 6850 upon internalization in human lung epithelial A549 cells (Garzoni et al. 2007). Contrarily, upon phagocytosis by human polymorphonuclear leukocytes the induction of saeR and saeS was observed in different S. aureus strains (Voyich et al. 2005). While adherence of sae mutant and wild type to endothelial cells did not differ, the mutant was less invasive than the wild type (Steinhuber et al. 2003). Further affirmation of the participation of saeS in infection models was derived from mutagenesis studies in combination with murine systemic infection. Here, non-functional saeS led to attenuated virulence (Benton et al. 2004). A different bacterial strain background led to similar observations of reduced virulence in mice (Rampone et al. 1996). Also Goerke et al. identified SaeRS as important regulator which is active in vivo, although the deletion mutant did not yield different bacterial densities compared to the wild type in a device-related infection model (Goerke et al. 2005). Besides the already mentioned saeR and saeS, three other regulators were observed with differential expression in internalized staphylococci (sarT, sarU, rot). Since sarT and sarU are less well characterized and since all these three genes were repressed, the effect of this differential expression cannot easily be rated in the setting of the in vitro infection model. Fittingly to the concluded activity of the SaeRS system in internalized staphylococci, membrane-bound adhesins, which are partly known to be regulated by SaeRS (Liang et al. 2006), were induced (fnbA, fnbB, clfA, clfB). Fibronectin binding proteins (FnBPs) are – together with other surface proteins – important mediators of bacterial adhesion to host cells. They exhibit an even more central position for internalization processes. FnBPs were partly required for adhesion and mainly required for internalization into the bovine mammary gland epithelial cell line, MAC-T (Dziewanowska et al. 1999). The mechanism includes binding of FnBP to β 1 integrins via a bridge formed by fibronectin, but also direct binding of FnBP to host membrane-located Hsp60 (Dziewanowska et al. 2000). Anyway, another study demonstrated variability of fnb genes between different clinical isolates as well as variation in the ability to adhere to fibronectin by the different isolates. Strains which were derived from orthopaedic implant-associated infection showed higher adherence than the other isolates. Community-acquired invasive disease isolates harbored more often two fnb genes than carriage isolates, but the number of fnb genes correlated only to a low extent with the adhesion capacity (Peacock et al. 2000). Fibronectin-binding protein deficiency reduced the colonization of heart tissue in a rat endocarditis model indicating a role of FnBP in heart valve infection. Other tissue samples did not show the same effect (Kuypers/Proctor 1989). In order to avoid compensation of a deleted gene by other, functionally redundant staphylococcal genes, an overexpression study of clfA and fnbA 199
Page 1 and 2:
G E N O M E W I D E C H A R A C T E
Page 3 and 4:
Maren Depke C O N T E N T S GENOMEW
Page 5 and 6:
Maren Depke Z U S A M M E N F A S S
Page 7 and 8:
Maren Depke Zusammenfassung der Dis
Page 9 and 10:
Maren Depke S U M M A R Y O F D I S
Page 11 and 12:
Maren Depke Summary of Dissertation
Page 13 and 14:
Maren Depke I N T R O D U C T I O N
Page 15 and 16:
Maren Depke Introduction Besides op
Page 17 and 18:
Maren Depke Introduction Extracellu
Page 19 and 20:
Maren Depke Introduction with heigh
Page 21 and 22:
Maren Depke Introduction 2005). SaP
Page 23 and 24:
Maren Depke Introduction contains a
Page 25 and 26:
Maren Depke Introduction via fibron
Page 27 and 28:
Maren Depke Introduction cathelicid
Page 29 and 30:
Maren Depke Introduction shock are
Page 31 and 32:
Maren Depke Introduction STUDIES OF
Page 33 and 34:
Maren Depke Introduction are effect
Page 35 and 36:
Maren Depke Introduction cell stimu
Page 37 and 38:
Maren Depke Introduction channel CF
Page 39 and 40:
Maren Depke M A T E R I A L A N D M
Page 41:
Maren Depke Material and Methods Li
Page 44 and 45:
Maren Depke Material and Methods Ki
Page 47 and 48:
Maren Depke Material and Methods GE
Page 49:
Maren Depke Material and Methods Ge
Page 52 and 53:
Maren Depke Material and Methods Ho
Page 54 and 55:
Maren Depke Material and Methods Ho
Page 56 and 57:
Maren Depke Material and Methods Pa
Page 58 and 59:
Page 60 and 61:
Page 63 and 64:
corticosterone [pg/ml plasma] corti
Page 65 and 66:
Maren Depke Results Liver Gene Expr
Page 67 and 68:
lood glucose levels [nmol/l] resist
Page 69 and 70:
LBP [ng/ml] CPR [ng/ml] Maren Depke
Page 71 and 72:
Maren Depke Results Liver Gene Expr
Page 73 and 74:
liver weight [g] Maren Depke Result
Page 75 and 76:
infection rate cfu / 10 mg tissue c
Page 77 and 78:
Maren Depke Results Kidney Gene Exp
Page 79 and 80:
Table R.2.1: Genes displaying stati
Page 81 and 82:
Maren Depke BR1 sign DsigB vs wt BR
Page 83 and 84:
Page 85 and 86:
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Maren Depke Results GENE EXPRESSION
Page 93 and 94:
Maren Depke Results Gene Expression
Page 95 and 96:
log2(ratio C57BL/6 / BALB/c) at IFN
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109:
Page 112 and 113:
Maren Depke Results Host Cell Gene
Page 114 and 115:
Page 116 and 117:
Page 118 and 119:
Page 120 and 121:
Page 122 and 123:
Page 124 and 125:
Page 126 and 127:
Page 128 and 129:
Page 130 and 131:
Page 132 and 133:
Maren Depke Results Pathogen Gene E
Page 134 and 135:
Page 136 and 137:
Page 138 and 139:
S. aureus RN1HG sample conditions a
Page 140 and 141:
Page 142 and 143:
Page 144 and 145:
Page 146 and 147:
Page 148 and 149: mean normalized intensity mean norm
Page 156 and 157: Maren Depke Results Pathogen Gene E
Page 158 and 159: mean normalized intensity Maren Dep
Page 160 and 161: Maren Depke Results Pathogen Gene E
Page 168 and 169: log 2 (ratio) log 2 (ratio) log 2 (
Page 171 and 172: Maren Depke D I S C U S S I O N A N
Page 173 and 174: Maren Depke Discussion and Conclusi
Page 197: Maren Depke Discussion and Conclusi
Page 205: Maren Depke Discussion and Conclusi
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
show all

genomewide characterization of host-pathogen interactions by ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?