genomewide characterization of host-pathogen interactions by ...

Maren Depke
Results
Pathogen Gene Expression Profiling
Comparison of experimental condition groups and assessment of differentially regulated
genes/sequences
The first general data analysis steps had provided insight into the overall similarity of the 23
arrays available after hybridization of samples and controls from the infection experiment.
Biological replicates exhibited good reproducibility. The time-matched serum/CO 2 controls for
the internalized samples built a distinct group of samples. As a more detailed analysis revealed
similarities of these controls with stationary phase/stringent response, it became apparent that
the 1 h serum/CO 2 samples were the most suitable control to use as baseline in this study.
Therefore, the following seven types of comparisons were chosen to elucidate the changes in
gene expression pattern and lateron the physiological reactions of S. aureus RN1HG in the
settings of the in vitro S9 infection model (Fig. R.5.8):
comparison of exponential growth phase samples, which represent the inoculation
condition, with baseline of 1 h serum/CO 2 controls
comparison of non-adherent staphylococci (1 h) with baseline of 1 h serum/CO 2 controls
comparison of 2.5 h internalized samples with baseline of 1 h serum/CO 2 controls
comparison of 6.5 h internalized samples with baseline of 1 h serum/CO 2 controls
comparison of 2.5 h serum/CO 2 controls with baseline of 1 h serum/CO 2 controls
comparison of 6.5 h serum/CO 2 controls with baseline of 1 h serum/CO 2 controls
comparison of 2.5 h anaerobic incubation samples with baseline of 1 h serum/CO 2 controls
inoculation
condition
and early
time point
control
samples
1
1 h
serum/CO 2
control
(3 arrays)
exponential
growth phase
(3 arrays)
2
1h
non-adherent
S. aureus
(3 arrays)
4
3
7
5
6
6.5 h
serum/CO 2
control
(2 arrays)
2.5 h
internalized
S. aureus
(3 arrays)
2.5 h
serum/CO 2
control
(3 arrays)
6.5 h
internalized
S. aureus
(3 arrays)
internalized
samples
2.5 h
anaerobic
incubation
(3 arrays)
late
time point
controls
Fig. R.5.8: Overview on the comparisons between groups in this study that were partly addressed with statistical testing and visualized
with scatter plots.
Baseline for all comparisons was the serum/CO 2 control group of 1 h. The very similar exponential growth phase samples, which
represent the inoculation condition, were compared to this baseline () and the even more similar non-adherent staphylococcal
samples after 1 h (). To elucidate the gene expression signature of internalized bacteria, the 2.5 h () as well as the 6.5 h ()
internalized samples were compared to the samples of 1 h serum/CO 2 control group. For comparison purposes, the late time point
controls were also checked against the early serum/CO 2 control as baseline: 2.5 h serum/CO 2 controls () and samples after 2.5 h of
anaerobic incubation (). The comparison focusing on the difference between 6.5 h and 1 h serum/CO 2 control samples () could
not be performed with statistical testing because of the low number (n = 2) of arrays at the 6.5 h time point.
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