genomewide characterization of host-pathogen interactions by ...

mean normalized intensity
Maren Depke
Results
Pathogen Gene Expression Profiling
At the early internalization time point, several genes of purine and pyrimidine nucleotide
biosynthesis pathways were repressed. Amino acid biosynthesis genes exhibited increase of
expression in both analyzed internalization time points. Furthermore, genes coding for
respiration chain components were induced in expression in internalized staphylococci. In
internalized staphylococci, several tRNA synthetase genes and some glycolytic enzyme genes
were observed to be repressed. Finally, transporters and permeases for sugar molecules,
phosphate, oligopeptides, and other substances were differentially expressed (Fig. R.5.23 A, B).
Serum/CO 2 control samples featured in some aspects similar changes in metabolic enzyme’s gene
expression pattern, e. g. amino acid biosynthesis, glycolysis, tRNA synthetases. On the other
hand, aerobic respiration, sugar uptake phosphotransferase system, and some enzymes of
peptidoglycan biosynthesis were repressed in this group. Anaerobic incubation instead did not
provoke changes in expression of glycolytic enzymes or respiration chain components
(Fig. R.5.23 C, D).
Subsequently to the first approach of BIOCYC pathway mapping, selected genes were analyzed
in more detail. Here, the example of purine biosynthesis was chosen. Although not all genes were
significantly differentially expressed, the gene expression changes were highly similar in the
complete pur operon and guaAB, which are separately encoded but are also involved in purine
biosynthesis. Five genes (purA, purC, purE, purK, purS) were differentially expressed in both
analyzed internalization time points, six genes (purD, purF, purL, purQ, guaA, guaB) were
differentially expressed 2.5 h after start of infection. Three genes, purH, purM, and purN,
exhibited fold change values between −2.6 and −2.8, but were not significant in statistical testing.
Finally, purB was significant in the statistical test in both analyzed time points of internalization,
but did not pass the minimal absolute fold change cutoff of 2. In the 2.5 h serum/CO 2 control,
differential expression occurred only for purA and in trend for purB, whereas almost all genes
exhibited fold change values around −3 to −4 in the 6.5 h serum/CO 2 control. Exceptions were
guaA and guaB, whose fold change values were still around 1, and the repressor gene purR,
which did not exhibit expression changes in any of the analyzed experimental conditions
(Fig. R.5.24, Fig. R.5.25).
A
fold change in comparison to
baseline 1 h serum/CO 2 control
S. aureus
RN1HG
sample
conditions
and time
points
purA purB purC purD purE purF purH purK purL purM purN purQ purR purS guaA guaB
exponential growth phase -1.1 -1.0 1.2 1.3 1.2 1.1 1.2 1.2 1.1 1.1 1.2 1.1 -1.2 1.0 -1.0 -1.1
2.5 h internalized -4.8 -1.6 -4.5 -2.6 -6.2 -3.0 -2.6 -5.4 -3.4 -2.8 -2.6 -4.0 1.1 -4.9 -2.0 -2.3
6.5 h internalized -4.8 -1.6 -2.2 -1.1 -3.4 -1.2 -1.2 -2.9 -1.5 -1.2 -1.1 -1.9 1.3 -2.3 -1.3 -1.4
2.5 h serum/CO 2 control -6.4 -1.6 1.0 1.1 1.0 -1.1 -1.0 1.1 -1.0 -1.0 1.0 1.0 1.3 -1.1 1.4 1.3
6.5 h serum/CO 2 control -28.2 -2.7 -3.2 -3.4 -3.8 -4.1 -4.3 -3.1 -4.0 -3.9 -3.6 -4.0 1.1 -3.9 1.1 -1.0
2.5 h anaerobic incubation -2.1 -1.7 1.4 1.1 1.7 1.3 1.2 1.5 1.3 1.3 1.2 1.5 -1.1 1.1 1.0 1.0
Fig. R.5.24: Purine biosynthesis gene expression.
A. Overview on fold change values relative to the baseline
sample “1 h serum/CO 2 control” and on significance in statistical
group comparisons. The sample “6.5 h serum/CO 2 control” could
not be included in statistical testing because of small group size
(n = 2).
B. Overview on mean normalized gene expression intensity.
After the global normalization by inter-chip scaling and
detrending, each individual gene has been normalized to the
expression level of the baseline sample “1 h serum/CO 2 control”
(1 h co.). Although not being continuous data, values were
depicted as line graphs instead of bar charts for better facility of
inspection.
(exp. − exponential growth phase; 1 h co. – 1 h serum/CO 2
control; 2.5 h int. − 2.5 h internalization; 6.5 h int. − 6.5 h
internalization; 2.5 h co. − 2.5 h serum/CO 2 control; 6.5 h co. −
6.5 h serum/CO 2 control; 2.5 h anae. − 2.5 h anaerobic
incubation)
B
1.00
0.10
0.01
exp.
1 h
co.
2.5 h
int.
6.5 h
int.
2.5 h
co.
6.5 h
co.
2.5 h
anae.
S. aureus RN1HG sample conditions and time points
purA
purB
purC
purD
purE
purF
purH
purK
purL
purM
purN
purQ
purR
purS
guaA
guaB
158