genomewide characterization of host-pathogen interactions by ...

Maren Depke
Results
Pathogen Gene Expression Profiling
In vitro Infection Experiment Study
Reproducibility of replicates and clustering of experimental condition groups
Staphylococcus aureus RN1HG or RN1HG GFP were used to infect S9 cells, a human bronchial
epithelial cell line. The study included samples from the four time points 0 h (start of
experiment), 1 h, 2.5 h and 6.5 h after start of infection, and included besides internalized
samples also control samples from inoculation condition of exponential growth phase,
serum/CO 2 controls, non-adherent staphylococci, and an anaerobic incubation control, although
not every sample condition was represented in every time point (see Material and
Methods / In vitro Infection Experiment Study / “Cell culture infection model” and “Bacterial
control samples”, pages 57 and 58). Hierarchical clustering visualized the grouping of the array
data sets according to their similarity (Fig. R.5.4). In an upper clustering level, grouping into three
major classes clearly revealed the separation of main experimental groups: 1) internalized
staphylococci, 2) controls of the later time points 2.5 h and 6.5 h, and 3) exponential growth
phase samples, which represent the inoculation sample condition, and controls of the early time
point 1 h (Fig. R.5.4, red dashed line). A lower level of clustering separated the individual sample
conditions and time points (Fig. R.5.4, orange dashed line). Here, biological replicates for 2.5 h
internalized, 6.5 h internalized, 2.5 h serum/CO 2 control, 6.5 h serum/CO 2 control, 2.5 h
anaerobic incubation and exponential growth phase samples were arranged together. Samples of
1 h serum/CO 2 control and 1 h non-adherent staphylococci were an exception: Because they
were very similar, the clustering did not lead to a complete segmentation of biological replicates
but additionally grouped 2 samples of the two different conditions (Fig. R.5.4, orange dashed line,
lower part). These two control groups were not only similar to each other, but additionally still
featured a high similarity to the inoculation condition of exponential growth phase.
S. aureus RN1HG sample conditions and time points
internalized
2.5 h internalized
6.5 h internalized
controls of
later time
points 2.5 h
and 6.5 h
2.5 h serum/CO 2 control
6.5 h serum/CO 2 control
2.5 h anaerobic incubation
exponential
growth
phase and
controls of
early time
point 1 h
exponential growth phase
1 h serum/CO 2 control
1 h non-adherent
1 h serum/CO 2 control
1 h non-adherent
Fig. R.5.4: Hierarchical clustering of 23 tiling array data sets from in vitro infection experiment.
The following clustering algorithms were applied on z-score-transformed data: Agglomerative clustering with average linkage using
using cosine correlation as similarity measure. All sequences were included in the cluster analysis. The red dashed line indicates
grouping into three major classes: 1) internalized staphylococci, 2) controls of the later time points 2.5 h and 6.5 h, and 3) exponential
growth phase samples, which represent the inoculation sample condition, and controls of the early time point 1 h. A lower level of
clustering (orange dashed line) separated the individual sample conditions and time points. Here, biological replicates for 2.5 h
internalized, 6.5 h internalized, 2.5 h serum/CO 2 control, 6.5 h serum/CO 2 control, 2.5 h anaerobic incubation and exponential growth
phase samples were arranged together. Samples of 1 h serum/CO 2 control and 1 h non-adherent staphylococci were an exception:
Because they were very similar, the clustering did not lead to a complete segmentation of biological replicates, but additionally
grouped 2 samples of the two different conditions (orange dashed line, lower part).
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