genomewide characterization of host-pathogen interactions by ...

Maren Depke
Discussion and Conclusions
HOST CELL GENE EXPRESSION PATTERN IN AN
IN VITRO INFECTION MODEL
Several different cell types of mammalian hosts are in the risk of encounter with pathogens
depending of the entry and manifestation site of infection. These are immune cells as well as cells
associated with structural and functional aspects of the tissue. In this study, the human bronchial
epithelial cell line S9 (American Type Culture Collection ATCC, Manassas, VA, USA; www.atcc.org;
S9 cell ATCC number CRL-2778) served as a model system to study the reaction of epithelial
cells to infection. With regard to the identification of S. aureus as one of the leading causative
organisms of pneumonia (Goto et al. 2009), this species was employed as model pathogen. More
specific, S. aureus RN1HG, a rsbU + repaired RN1-derivative strain (Herbert et al. 2010) with a
SigB-positive phenotype, was chosen.
The infection setting involved bacterial cultivation in an adapted cell culture medium (Schmidt
et al. 2010), which allowed inoculation of eukaryotic host cell cultures with a fraction of complete
bacterial culture. This experimental setup allowed the study of host cell reactions to the influence
of both bacterial factors, its secreted proteins from supernatant and the membrane-bound
factors, which both interact during infection and contribute to the success of the bacterial cells. It
also avoided potentially disturbing influences of bacterial cell handling like that occurring during
centrifugation and washing.
In a combined approach of transcriptome (Maren Depke) and proteome (Melanie Gutjahr)
analysis, the host reaction to infection and bacterial internalization was recorded. Two time
points, 2.5 h and 6.5 h after start of infection, were selected for sampling. Because only about
50 % of host cells in infected cell culture plates harbor internalized staphylococci after infection,
the fraction of non-infected cells was removed by FACS-sorting, which was feasible because a
GFP-expressing S. aureus RN1HG strain has been used. The remaining infected S9 cells were
compared to medium control cells.
In the comparison of infected S9 cell proteome and transcriptome signatures, a considerable
time shift of cellular reaction between both analysis levels was evident. In samples of the first
time point 2.5 h, approximately half the number of proteins differed in abundance between
infected and control cells in comparison to the number of regulated proteins at the later time
point of 6.5 h. Conversely, for the transcriptome analysis, the number of differentially expressed
genes at the first time point amounted to about 3.5 % of the number of regulated genes at the
second time point. Thus, the reaction on proteome level started to a stronger extent between
inoculation and first analysis time point 2.5 h, whereas the transcriptional reaction most
articulately started between the first (2.5 h) and the second (6.5 h) time point.
The cellular reaction to infection in this experimental setting seems to lead first to protein
abundance changes independent from mRNA changes, which on the one hand probably rely on
the already existing messengers and on the other hand might represent post-transcriptional,
translational and degradation/turnover processes as reduction in protein abundance prevailed at
the early time point. Differentially expressed genes and regulated proteins did not overlap at the
first time point 2.5 h after start of infection. Two genes, IFIT2 and IFIT3, were induced at both the
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