genomewide characterization of host-pathogen interactions by ...

Maren Depke
Discussion and Conclusions
Similarly, the expression of extracellular and membrane-bound enzymes, which contribute to
virulence or spreading, was difficult to interpret. Expression of serine proteases (splABCDEF) and
lipase (lip) was increased, whereas the expression of hyaluronate lyase (hysA), a membrane
bound serine protease (htrA), nuclease (nuc), and glutamyl endopeptidase alias V8 peptidase
(sspA) was repressed in internalized staphylococci. Some spl genes were found to be SaeRSregulated
(Rogasch et al. 2006). Thus, the induction of the spl operon fits to the proposed activity
of the SaeRS two-component system.
Gene expression changes which might have influence on cell wall remodeling were also
observed in internalized staphylococci. Surprisingly, this included repression of the dlt operon.
The proteins encoded by dlt participate in incorporation and esterification of D-alanine residues
into the cell wall teichoic acids. In this way, the negative charge of the cell wall structures is
reduced and thus is less attractant for positively charged antimicrobial molecules (Peschel et al.
1999). With the aim to evade the immune response, an induction of dlt expression had been
expected. In parallel, the induction of peptidoglycan hydrolase lytM and staphylococcal secretory
antigen ssaA was recorded (this study), which are also involved in cell wall remodeling (Dubrac et
al. 2007). Repression of dlt and induction of lytM were also observed by Garzoni and coworkers
(Garzoni et al. 2007). Furthermore, the induction of prsA, coding for a foldase involved in
translocation and folding of secreted proteins (Sarvas et al. 2004), was observed in internalized
staphylococci 2.5 h after infection of S9 cells (this study). This regulation was in accordance with
protein abundance data in internalized staphylococci which were determined by Sandra Scharf
(Schmidt et al. 2010). She also found an increased abundance of VraR, regulatory component of
the VraRS two-component system and known to be positively auto-regulated (Kuroda et al.
2003), which led to the examination of vraRS gene expression: vraR (1.8) and vraS (1.6) were not
significantly different in internalized staphylococci (2.5 h) in comparison to control, but the fold
change values indicated a slight trend for induction. Hence, the observed differential expression
of cell wall remodeling related genes was in agreement with the proteome data of Sandra Scharf.
Additionally, genes described to belong to the VraRS regulon (Kuroda et al. 2003) were examined.
Internalized staphylococci induced about 20 % of the published VraRS regulon (Kuroda et al.
2003) at the same time point when a trend of vraRS induction was detected. Since the VraRS
two-component system was suggested to be activated upon cell wall damage or inhibition of cell
wall biosynthesis (Kuroda et al. 2000, Kuroda et al. 2003), the observation of cell wall remodeling
associated gene expression changes might indicate a reaction to stressful influences which
possibly operate on the bacterial cell wall after internalization into the host cell. Another possible
explanation proposed by Garzoni and colleagues (2007) is that the gene induction (e. g. lytM) is
associated with cell division processes.
The gene expression analysis of internalized staphylococci in S9 cells led to the recognition of
changes in the expression of metabolic genes. Purine biosynthesis, tRNA synthetases, and
glycolysis were repressed, whereas gluconeogenesis, TCA cycle enzyme genes, and especially
genes related to amino acid biosynthesis were induced. The induction of amino acid biosynthesis
and TCA cycle genes as well as the repression of purine biosynthesis and tRNA synthetase genes
was clearly confirmed in the proteome analysis of Sandra Scharf. Additionally, transporter genes
were differentially expressed. Induction was observed especially for phosphate transporters, but
also for transporters of phosphonate, methionine, peptide/oligopeptide, sugar/maltose,
osmoprotectants, amino acids, and gluconate. Repression was detected for urea,
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