genomewide characterization of host-pathogen interactions by ...

S. aureus RN1HG sample conditions and time points
mean intensity of 2 or 3 biological replicates
Maren Depke
Results
Pathogen Gene Expression Profiling
Similarly, tRNA synthetase genes are repressed in stringent response (Anderson KL et al.
2006). Here, repression of these genes was detectable in internalized staphylococci (2.5 h), late
serum/CO 2 controls (2.5 h and 6.5 h) and in samples after 2.5 h anaerobic incubation
(Fig. R.5.6 B). An exception is the isoleucin tRNA synthetase (ileS), which is induced in stringent
response (Anderson KL et al. 2006). But in the 2.5 h and 6.5 h serum/CO 2 controls, this gene
shows a trend of repression similar to the other tRNA synthetases with a fold change of −1.5 and
−1.8, respectively.
Furthermore, translation elongation factor gene expression is known to be suppressed in
stringent response (Anderson KL et al. 2006). Such repression physiologically acts in concert with
repressed ribosomal protein and tRNA synthetase genes and leads to diminished protein
synthesis. In this study, 4 genes for translation elongation factors revealed a trend of repressed
expression in late serum/CO 2 controls (2.5 h and 6.5 h). With p = 0.06, statistical significance was
only slightly missed in the comparison between these two controls and exponential growth
samples (Fig. R.5.6 C).
Stringent response is mediated by the regulator RelA whose transcript lateron is increased in
stringent response conditions (Anderson KL et al. 2006). A strong trend of induction for relA was
observed for the 2.5 h and 6.5 h serum/CO 2 controls. Although relA exhibited significantly
different expression (p* < 0.01) in the comparison of all sequences between the 2.5 h serum/CO 2
control and exponential growth phase samples, the 1.8-fold change did not pass the cutoff level
of 2. On the other hand, in the comparison between the later 6.5 h serum/CO 2 control and
exponential growth phase samples, the fold change amounted to 2.1, but in this comparison
statistical testing was not possible because of limited number of replicates relA at the 6.5 h time point
(n = 2). Nevertheless, relA could be regarded as induced in the late serum/CO 2 controls, because
both time points showed the same trend and all six other experimental samples possessed
almost the same, lower gene expression intensity, which proved relA highly reliable measurement of
the relA expression by the tiling array approach (Fig. R.5.7).
Fig. R.5.7:
Comparison of mean expression
intensities for the stringent response
regulatory gene relA.
A strong trend of relA induction was
visible for the late serum/CO 2 controls
(2.5 h and 6.5 h). Although relA exhibited
significantly different expression (one-way
textbook ANOVA with Benjamini-
Hochberg False Discovery Rate multiple
testing correction and p* < 0.01) in the
comparison between the 2.5 h serum/CO 2
control and exponential growth phase
samples, the 1.8-fold change did not pass
the cutoff level of 2. On the other hand, in
the comparison between the later 6.5 h
serum/CO 2 control and exponential
growth phase samples, the fold change
amounted to 2.1, but in this comparison
statistical testing was not possible
because of limited number of replicates at
the 6.5 h time point (n = 2).
exponential growthOD phase 0.4
1 h serum/CO 2 control 1h CO2
1 non-adh. h non-adherent MOI 25
2.5 h internal. internalized 2.5h
6.5 h internal. internalized 6.5h
2.5 h serum/COCO2 2 control 2.5h
6.5 h serum/COCO2 2 control 6.5h
2.5 h anaerobic anaerobic incubation 2.5h
1.0E+04
10000
2.0E+04
20000
mean intensitySamples
of biological replicates
3.0E+04
30000
From repressed or in trend repressed ribosomal protein, tRNA synthetase, and translation
elongation factor genes and from the strong tendency of relA infection induction experiments it was reasoned medium comparison that the
gene expression signature of late serum/CO 2 controls (2.5 h and 6.5 h) in fact resembled the
stationary phase/stringent response. As internalized staphylococci and the early control samples
did not possess this similarity, it was concluded that the 1 h serum/CO 2 control sample, although
not time-matched, was the most appropriate baseline sample for this infection experiment study.
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