genomewide characterization of host-pathogen interactions by ...
genomewide characterization of host-pathogen interactions by ...
genomewide characterization of host-pathogen interactions by ...
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Maren Depke<br />
Discussion and Conclusions<br />
PATHOGEN GENE EXPRESSION PROFILING<br />
Beside its characteristics as commensal colonizer as well as origin <strong>of</strong> a variety <strong>of</strong> infectious<br />
diseases, S. aureus was identified as one <strong>of</strong> the leading causative organisms <strong>of</strong> pneumonia<br />
besides Streptococcus pneumonia and Haemophilus influenzae (Goto et al. 2009). The <strong>pathogen</strong><br />
is easily transferred to the lung, e. g. <strong>by</strong> aspiration or medical devices, where it encounters<br />
immune cells as well as cells associated with structural and functional aspects <strong>of</strong> the lung like<br />
epithelial cells.<br />
In this study, the human bronchial epithelial cell line S9 was applied to study the <strong>interactions</strong><br />
<strong>of</strong> epithelial cells with S. aureus RN1HG. Here, the analysis <strong>of</strong> the <strong>pathogen</strong> expression pr<strong>of</strong>ile<br />
complements the analysis <strong>of</strong> <strong>host</strong> cell expression patterns, which has been described before.<br />
Similar as in the other studies described in this thesis, also the internalized staphylococci were<br />
monitored in a combined approach <strong>of</strong> transcriptome (Maren Depke) and proteome (Sandra<br />
Scharf) analysis. Internalized staphylococci were extracted from their S9 <strong>host</strong> cells and the<br />
bacterial RNA pr<strong>of</strong>ile was recorded using a tiling array approach. Bacterial intracellular proteins<br />
were monitored and quantified after stable isotope labeling with amino acids in cell culture<br />
(SILAC), FACS-sorting and mass spectrometric analysis.<br />
The advantage <strong>of</strong> the experimental settings in this study was the application <strong>of</strong> complete<br />
bacterial culture with both secreted proteins from supernatant and whole bacterial cells to the<br />
<strong>host</strong> S9 cell culture. Such experimental design was improved after establishment <strong>of</strong> an adapted<br />
cell culture medium named pMEM (Schmidt et al. 2010) which allows reproducible bacterial<br />
growth, but avoids the inoculation <strong>of</strong> <strong>host</strong> cell culture with highly artificial established bacterial<br />
culture media like TBS. Hence, the study <strong>of</strong> <strong>host</strong>-<strong>pathogen</strong> <strong>interactions</strong> in the context <strong>of</strong> all<br />
bacterial factors, membrane-bound and secreted, and additionally without any influence on<br />
bacterial physiology <strong>by</strong> prolonged handling, centrifugation and washing <strong>of</strong> bacteria was<br />
performed using exponential growth phase cultures <strong>of</strong> the rsbU + repaired RN1-derivative strain<br />
S. aureus RN1HG (Herbert et al. 2010).<br />
Additional information on gene expression pattern <strong>of</strong> S. aureus RN1HG during stationary<br />
growth phase in the pMEM medium was available from a second study, which dealt with the<br />
comparison <strong>of</strong> gene expression pattern in different growth media. That study was part <strong>of</strong> an<br />
international co-operation in the settings <strong>of</strong> the EU-IP-FP6-project BaSysBio (LSHG-CT2006-<br />
037469) consortium. Here, proteome analysis was not included.<br />
Both gene expression studies, <strong>of</strong> medium comparison and <strong>of</strong> the infection experiment, applied<br />
custom tiling arrays produced and processed <strong>by</strong> NimbleGen.<br />
First, the knowledge on the gene expression pattern <strong>of</strong> stationary growth phase was used to<br />
determine the most suitable control sample group for the infection experiment study.<br />
In stationary growth phase, nutrients become limited after consumption in the exponential<br />
phase <strong>of</strong> growth, which is the trigger for the so-called stringent response. The stringent response<br />
comprises an adaptation <strong>of</strong> the bacteria’s metabolism to situations <strong>of</strong> nutrient limitation or<br />
starvation, which includes induction <strong>of</strong> stress resistance, decelerated growth and alleviated<br />
metabolism. Many <strong>of</strong> the necessary changes are mediated <strong>by</strong> transcriptional variation (Condon et<br />
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