genomewide characterization of host-pathogen interactions by ...

Maren Depke
Results
Pathogen Gene Expression Profiling
Comparison of differentially expressed genes [Maren Depke] and proteins with differing
abundance [Sandra Scharf] in internalized staphylococci
In an experimental setup corresponding to the generation of samples for tiling array analysis,
the abundance of proteins in internalized staphylococci was recorded with a combined approach
of stable isotope labeling with amino acids in cell culture (SILAC), FACS cell sorting and mass
spectrometric analysis (Sandra Scharf, Schmidt et al. 2010). The method, which was applied for
this first proteome profiling of in vitro internalized staphylococci, limits the possible protein
identification mainly to cytosolic proteins: Secreted proteins were lost during sample
preparation, and peptides from membrane associated proteins are difficult to obtain by tryptic
digest of whole-cell samples.
In the proteomic study, the time points 1.5 h, 2.5 h, 3.5 h, 4.5 h, 5.5 h, and 6.5 h after start of
infection were included, i. e. the analysis window was divided into smaller sections than in the
tiling array study. In an analysis which included more samples and controls than published in the
pilot study, Sandra Scharf identified 648 proteins and obtained a set of 114 proteins with
differential abundance. Differential abundance was assigned to proteins when the regulation was
observed in at least 2 of 3 biological replicates in at least 1 of 6 analyzed time points, and
proteins with contradictory results were excluded. The regulated proteins were normalized in
reference to the SILAC-method immanent internal control (heavy isotope labeled peptides) and
exhibited an abundance difference when comparing target peptide ratios with the median ratio
for all peptides at the corresponding time point. Regulated proteins which were additionally
different in abundance in a serum/CO 2 control (without presence of host cells) were excluded.
To gain an insight into the comparability of transcriptome and proteome profiling,
differentially expressed sequences were compared to regulated proteins. First, up-regulated
proteins were compared to the sequences which exhibited increased expression 2.5 h or 6.5 h
after start of infection or in both time points (Fig. R.5.11 A). Second, the same comparison was
performed for down-regulated proteins and repressed sequences (Fig. R.5.11 B).
A
B
up-regulated proteins in
internalized staphylococci
down-regulated proteins in
internalized staphylococci
53
34
5 2
14
176 96
207
3 0
6
173 121
181
induced sequences in
“2.5 h internalization” vs.
“1 h serum/CO 2 control”
induced sequences in
“6.5 h internalization” vs.
“1 h serum/CO 2 control”
repressed sequences in
“2.5 h internalization” vs.
“1 h serum/CO 2 control”
repressed sequences in
“6.5 h internalization” vs.
“1 h serum/CO 2 control”
Fig. R.5.11: Comparison of differentially expressed sequences in at least one of the two analyzed time points with proteins exhibiting
different abundance in internalized staphylococci.
A. Up-regulated proteins and induced sequences. B. Down-regulated proteins and repressed sequences.
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