genomewide characterization of host-pathogen interactions by ...

Maren Depke
Results
Pathogen Gene Expression Profiling
In a similar comparison, up-regulated proteins were compared with repressed transcripts and
vice versa down-regulated proteins with induced transcripts, which resulted in an overlap of 5
(rocD, ldh2, sodM, fabH, SAOUHSC_02150) and 9 (sufC, sufD, spoVG, dps, SAOUHSC_00533,
SAOUHSC_02443, SAOUHSC_02363, SAOUHSC_01854, SAOUHSC_00845) proteins, respectively.
Such contradictory results could be explained in most cases by the following characteristics:
transcript changes do not necessarily influence the protein abundance at the same time
point; the potential time shift between both effects is not defined and may vary between
the different proteins
effects on protein or expression might be outside the analysis window (between start of
infection and first analysis time point of 1.5 h or 2.5 h; after last analysis time point of
6.5 h; between analysis time points especially in the transcriptome analysis)
special protein effects like temporary new synthesis, increased turnover, and other nontranscriptionally
mediated effects
special protein analysis effects: early changes or changes occurring before the first analysis
time point, which stop in the middle of the observation period, might still lead to an
absolute fold change value exceeding 2 at the later time points in cases of stable proteins
protein analysis challenges like low intensity measurements, high variation between
biological replicates, missing data for one of the biological replicates
Expression of genes containing binding sites for Rex, an anaerobiosis regulator and redox
sensor
During in vitro infection/internalization experiments, staphylococci are subjected to changes
in oxygen availability. First, they are shifted from aerobic shaking cultures into cell culture dishes,
second, they are incubated without agitation in a CO 2 -atmosphere optimal for the host cells, and
finally, internalization into eukaryotic host cells might further influence the oxygen availability. To
answer the question whether oxygen limitation has major impact on internalized staphylococci,
the gene expression of anaerobiosis-related genes was analyzed. Very recently, Pagels et al. have
published transcriptome and proteome data of an anaerobic gene expression regulon (Pagels et
al. 2010). Although indirect effects of Rex have also been described, the central regulator Rex, a
redox sensor, acts as transcriptional repressor. In situations of increasing NADH, DNA-binding of
Rex is inhibited, resulting in de-repression of genes which possess a Rex binding site. Pagels et al.
defined 21 genes with such binding site, 19 of which could be mapped to S. aureus NCTC8325
LocusTags (NCBI’s Entrez Genome Gene Plot; http://www.ncbi.nlm.nih.gov/sutils/geneplot.cgi).
For a first impression, the fraction of these genes which were differentially expressed in
staphylococci after 2.5 h of anaerobic incubation, but also in 2.5 h and 6.5 h internalized
staphylococci was determined. In anaerobically incubated staphylococci, 5 Rex binding site
containing genes were found to be regulated (Fig. R.5.12 A), whereas 10 of these genes were
regulated in internalization at both time points and further 3 genes specifically at the time point
of 6.5 h after start of infection (Fig. R.5.12 B). A more detailed analysis revealed that while
induction of these genes in anaerobically incubated staphylococci was expected, the direction of
regulation in internalized staphylococci was clearly opposite (Fig. R.5.12 C). Therefore,
internalization probably did not result in stimuli which were able to inactivate the Rex repressor.
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