genomewide characterization of host-pathogen interactions by ...

cfu / S9 cell
cfu / S9 cell
% PI-negative S9 cells
Maren Depke
Results
HOST CELL GENE EXPRESSION PATTERN IN AN
IN VITRO INFECTION MODEL
Staphylococcal cfu per host cell after internalization and host cell vitality [Melanie Gutjahr,
Petra Hildebrandt]
After 2.5 h, an averaged number of 1.3 staphylococcal colony forming units (cfu)/ S9 cell were
found in internalization experiments (data courtesy of Melanie Gutjahr). Although not statistically
significant in the four biological replicates used for this study, a trend of increasing cfu / S9 cell
along with increasing infection time was discernible and led to an internalization mean value of
2.3 cfu / S9 cell 6.5 h after start of infection (Fig. R.4.1 A).
A
S9 infection proteome and transcriptome
4
3
2
1
B
100
80
60
40
20
0
RN1HG GFP RN1HG GFP
infected S9 cells infected S9 cells
2.5 h after start 6.5 h after start
of time infection after start of infection of infection
2.5 h
6.5 h
00
untreated
control
1 2non-sorted3 4 5 sorted 6 7
infected non-infected (GFP – ) infected (GFP + )
2.5 h 6.5 h 2.5 h 6.5 h 2.5 h 6.5 h
S9 cell samples
Fig. R.4.1: Staphylococcal cfu per host cell after internalization and host cell vitality.
A. Staphylococcal viable cell counts per S9 cell in infection experiments. The line indicates mean values per each analyzed time point.
B. Percentages of viable PI – S9 cells in untreated control and after infection with S. aureus RN1HG GFP determined by FACS counting.
Infected samples were analyzed for the two time points in different populations: without sorting or after sorting separately for GFP –
and GFP + S9 cells. Mean values of n = 2 experiments.
Cfu-data: courtesy of Melanie Gutjahr. FACS-data: courtesy of Petra Hildebrandt.
Host cell vitality was determined by propidium iodide (PI) staining and counting in FACS
(n = 2). Although 7.0 % and 12.1 % of cells in the infected, but non-sorted cell suspension were
positive for PI and therefore not viable 2.5 h and 6.5 h after infection, respectively, compared to
3.6 % of PI-positive dead cells in the untreated control, most of the dead, infected cells were
discarded during the sorting procedure (Fig. R.4.1 B). Thus, cell vitality was not influenced in the
subsets of sorted cells 2.5 h after infection (98.7 % in GFP + S9 cells, 99.0 % in GFP – S9 cells). After
6.5 h, a minor drop in viability was recorded for the infected samples (97.3 % in GFP + S9 cells,
98.8 % in GFP – S9 cells; data courtesy of Petra Hildebrandt).
Reproducibility of replicates and clustering of treatment group members
Staphylococcus aureus RN1HG GFP was used to infect S9 cells, a human bronchial epithelial
cell line. The study included samples of two treatments, infected green fluorescence-positive
FACS-sorted S9 cells and control cells that were exposed to medium, and analyzed the two time
points 2.5 h and 6.5 h after start of infection. Hierarchical clustering visualized the grouping of
the array data sets according to their similarity (Fig. R.4.2). Biological replicates of each treatment
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