genomewide characterization of host-pathogen interactions by ...

Maren Depke
Results
Pathogen Gene Expression Profiling
Summing up, the biological replicates exhibited a high reproducibility. Most importantly, the
internalized condition had characteristics very distinct from either the starting condition of
exponential growth phase and the similar early time point controls, but also from the later
controls which were incubated in the presence of serum in 5 % CO 2 -atmosphere without agitation
and from the anaerobic control samples.
Choice of adequate baseline samples for comparison of the experimental conditions:
Time-matched controls are not suitable because of their similarity to stationary growth phase /
stringent response
Many bacterial species are capable of inducing a so-called stringent response with the aim to
adapt their metabolism to situations of nutrient limitation, especially to carbon and amino acid
starvation. The stringent response leads to induced stress resistance, decelerated growth and
alleviated metabolism. The main mediators of the stringent response are pyrophosphorylated
GTP or GDP molecules, which are also called alarmones: guanosine tetraphosphate (ppGpp) and
guanosine pentaphosphate (pppGpp), depending on bacterial species. In the course of the
stringent response, up to one third of the transcriptome can be modulated. The stringent
response has been intensively studied in E. coli, but also S. aureus is capable of such response
(Condon et al. 1995, Crosse et al. 2000, Anderson KL et al. 2006, Wolz et al. 2010).
Entry into stationary growth phase is initiated by a beginning nutrient limitation after
consumption in the exponential phase of growth. Therefore, in stationary phase bacteria’s
stringent response is triggered.
When comparing the gene expression signature of S. aureus RN1HG, which was incubated for
2.5 h in serum-containing infection medium under 5 % CO 2 -atmosphere (37°C), with the signature
of stationary phase time point t 2 , which is defined as 2 h after entry into the stationary phase
(each in comparison to exponentially growing bacteria), a high overlap between both signatures
was recognized. Almost 44 % of sequences in the 2.5 h serum/CO 2 control signature were also
found in the stationary phase signature (Fig. R.5.5; for S. aureus RN1HG stationary phase
response refer to chapter “Growth Media Comparison Study”, page 131). This first hint for
similarities between the controls of later time points in the infection experiment and the
bacterial stationary phase samples provoked a more detailed analysis of expression data of genes
already known to be involved in the stringent response.
Fig. R.5.5:
Comparison of 2.5 h serum/CO 2 control signature
with stationary phase signature.
Each sample condition was compared to its
corresponding baseline samples of exponential
growth in log-transformed space using textbook
one-way ANOVA with Benjamini-Hochberg False
Discovery Rate multiple testing correction, and
p* < 0.05 was regarded as significant. An absolute
fold change cutoff of 2 was applied.
sequences differentially expressed in
the comparison of incubation for
2.5 h in serum-containing medium
and 5% CO 2 atmosphere vs.
exponential growth phase in pMEM
487 418 1005
sequences differentially expressed
between stationary growth phase (t 2 )
and exponential growth phase of
S. aureus RN1HG cultivated in pMEM
Ribosomal proteins are repressed during stringent response (Anderson KL et al. 2006). As the
bacterial metabolism adapts to slower growth and limited energy resources, the cell also
diminishes protein synthesis and therefore does not need to produce further ribosomes.
Reduced expression of 55 genes for ribosomal proteins was observed in late serum/CO 2 controls
(2.5 h and 6.5 h) and in anaerobiosis (Fig. R.5.6 A).
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