genomewide characterization of host-pathogen interactions by ...

Maren Depke
Results
Pathogen Gene Expression Profiling
This difference could not be assessed with statistical testing because only two arrays were
available for the 6.5 h control. Thus, in a second step, all sequences which possessed an absolute
fold change equal or greater than 2 in the comparison between 6.5 h and 1 h serum/CO 2 control
were excluded from the subsets generated in the first step. This procedure was performed for
induced and repressed genes separately and resulted in 45 sequences specifically induced after
2.5 h, 37 sequences specifically induced at both time points, 34 sequences specifically induced
after 6.5 h, 98 sequences specifically repressed after 2.5 h, 75 sequences specifically repressed at
both time points, and 43 sequences specifically repressed after 6.5 h in samples of internalized
staphylococci (Fig. R.5.35). Most interestingly, these lists included besides newly identified
transcripts also genes which were described above in the context of virulence or metabolic
genes, e. g. clfB, coa, phoP, prsA, glpK (2.5 h, induced), fnbB, vwb, ssaA, phoU, pstA, pstB, pstC,
pstS (2.5 h and 6.5 h, induced), chp, lukD, lukE, hla, efb, emp (6.5 h, induced), icaD, icaC, rot, nuc,
sspA, urea transporter SAOUHSC_02557, ureF (2.5 h, repressed), sarU (6.5 h, repressed).
New transcripts identified with the tiling array approach which are regulated in S9 cell
internalized staphylococci
All sequences of the tiling arrays were included in the statistical testing to identify the
internalization specific gene expression signature. Therefore, the resulting lists of differentially
expressed genes contained newly identified transcripts: 200 sequences for the 2.5 h and 138
sequences for the 6.5 h time point which were significant in ANOVA with p* < 0.05 and exhibited
a minimal absolute fold change of 2 (Table R.5.5).
When comparing these lists, 100 transcripts were differentially expressed at both time points
(Fig. R.5.36 A). These consisted of 67 induced and 33 repressed sequences (Fig. R.5.36 B).
A
B
100 100 38
2.5 h
repressed
repressed 6.5 h
induced
35 18
induced
new transcripts differentially
expressed in the comparison
“2.5 h internalization” vs.
“1 h serum/CO 2 control”
new transcripts differentially
expressed in the comparison
“6.5 h internalization” vs.
“1 h serum/CO 2 control”
65
33
67
20
Fig. R.5.36: Comparison of newly identified transcripts in the 2.5 h and 6.5 h signatures of internalized staphylococci.
Both internalized samples were compared to the baseline of 1 h serum/CO 2 control with statistical testing and multiple testing
correction (p* < 0.05), and a minimal absolute fold change cutoff of 2 was applied. The comparison of differentially expressed newly
identified transcripts at the 2.5 h and 6.5 h time point was performed for all regulated transcripts (A) and for induced and repressed
transcripts separately (B).
Although these 200 and 138 newly identified transcripts were known to be differentially
expressed in at least one of the two internalized samples in comparison to the 1 h serum/CO 2
control, they were expected to form further subgroups e. g. dependent on the direction of
regulation. Therefore, a k-means clustering was applied to a ratio data set (experimental
condition normalized to the 1 h serum/CO 2 control) consisting of the five experimental conditions
of non-adherent (1 h), internalized (2.5 h and 6.5 h), and serum/CO 2 control (2.5 h and 6.5 h)
staphylococci.
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