genomewide characterization of host-pathogen interactions by ...

Maren Depke
Results
Pathogen Gene Expression Profiling
The tiling array approach aimed at the identification of the maximal possible number of
transcriptional units − known and newly identified – and therefore included several different
growth media in a cooperation between several laboratories. On the other hand, the tiling array
analysis could be used to get insights into the stationary phase response in the newly established
pMEM medium on transcriptome level under restriction to the already defined transcriptional
units and annotated genes. For this purpose, differential expression was assumed for genes
regulated in at least one of the two analyzed time points of stationary phase, t 2 and t 4 .
Using this application of the array data set, a very clear picture of induced TCA cycle enzyme
genes (citZ, citB, citC, sucA, sucB, sucD, sucC, sdhA, sdhB, sdhC; all induced) was revealed. In
parallel, reduced expression of glycolysis enzyme genes (pgi, pfkA, pgk, pgm, pykA; all repressed)
and induced expression of gluconeogenetic enzyme genes (pckA, gap2, fbp) was observed. This is
in accordance with published stationary phase response of S. aureus (Kohler et al. 2005).
Furthermore, repression of amino acid biosynthesis pathways was described for the stationary
phase. In pMEM, tyryptophan (trpB, trpC, trpD, trpE, trpG), histidine (hisB, hisD, hisG), and
aspartate/arginine (argG, argH, SAOUHSC_00150) biosynthesis genes were repressed. But
contrarily, induction of lysine (lysC, asd), histidine/glutamate (hutH, hutI, hutU, hutG, rocA), and
citruline/ornithine (argF, arg, arcC) biosynthesis genes was visible in the stationary phase
samples cultivated in pMEM. Other amino acid biosynthesis genes (leu, ilv, dap, thr, and others)
were not significantly differentially expressed. Reduced or even ceased growth diminishes the
need for new protein synthesis. Therefore, associated molecules like ribosomal proteins,
translation elongation factors, and chaperones do not need to be newly synthesized, which was
observed on proteome level for S. aureus COL in TSB (Kohler et al. 2005). This phenomenon is
part of the stringent response e. g. to glucose starvation. Here, using pMEM medium, S. aureus
RN1HG and transcriptome analysis, only two ribosomal protein genes, rpsD and rpsT, and prmA, a
ribosomal protein L11 methyltransferase, were repressed. Only translation elongation factor P
(efp) exhibited a fold change of less than −2 in the t 4 samples, but this repression was not
significant. Chaperones dnaK, grpE, groEL, and groES exhibited slightly decreased fold change
values in the range of −1.4 to −1.9, but the difference was also not significant. Of the Clp
complex, expression of subunit X was repressed and of subunit L was induced. Finally, repression
of tRNA synthetases is known to be part of the stringent response in S. aureus (Anderson KL et al.
2006). In stationary phase in pMEM, repression of tyrS, leuS, alaS, aspS, hisS, valS, thrS, glyS,
proS, ileS, pheS, pheT, gltX, metS/metG, and serS was detected. The other tRNA synthetases
except cysS showed a trend of repression with fold change values almost reaching the cutoff (−2).
Virulence factors differentially expressed in stationary vs. exponential growth phase
In stationary growth phase, differential expression of virulence-associated genes was
observed. Many of these genes were regulated at both analyzed time points of stationary phase,
t 2 and t 4 . Surface proteins A and G (spa, sasG) were repressed. Other membrane-bound adhesins
were induced like clfA, fnbA, and isaB. Secreted adhesins and immunomodulatory molecules efb,
chp, and sbi were repressed, whereas ebh and eap were induced. One of the two staphylococcal
superoxide dismutases, sodM, was found to be induced. The toxins hla, hlb, hlgC, and hlgB
exhibited an increase in expression. In the group of extracellular enzymes, only nuc and htrA
were repressed. Contrarily, for secreted lipases geh and lip and for proteases sspB, splC, splB,
splA, and aur a higher expression was detected in stationary phase than in exponential growth.
The complete cap operon of 15 capsular genes (SAOUHSC_00114 to SAOUHSC_00128) was
induced in t 2 samples and still 10 of these genes were also induced in t 4 samples. The biofilm
repressor icaR was induced in t 2 samples. Fittingly, two genes of the ica operon, icaB and icaC,
were observed to be repressed in both analyzed stationary phase time points. Finally, differential
expression of five staphylococcal accessory regulators was detected: sarX and sarT were
repressed, while sarV, sarR, and sarZ were induced in stationary phase (Table R.5.3).
134