genomewide characterization of host-pathogen interactions by ...

Maren Depke
Material and Methods
Host Cell Gene Expression Pattern in an in vitro Infection Model
cultivation to OD 0.4
experimental time point / h
0 1 2 3 4 5
6 7
infection
1h
lysostaphin treatment
t 0 2.5 h 6.5 h
infected
eukaryotic
samples
FACS for
RNA and
protein
FACS for
RNA and
protein
Fig. M.4.1:
Time line of
infection
experiments
for eukaryotic
host samples
and control
measurements.
eukarotic
control
samples
control
measurements
controlfor
protein
cfu
host cell
vitality
controlfor
RNA and
protein
cfu
host cell
vitality
controlfor
RNA and
protein
cfu
host cell
vitality
Sample harvesting for transcriptome analysis
After co-incubation of staphylococcal with host cells and subsequent lysostaphin treatment,
the S9 cell layer was washed once with Dulbecco’s PBS without Ca 2+ and Mg 2+ (PAA Laboratories
GmbH, Pasching, Austria). Cells were detached with 1 ml trypsin/EDTA (PAA) supplemented with
0.1 µg/ml actinomycin D (Sigma-Aldrich, Steinheim, Germany) and 80 mM sodium azide (Merck
KGaA, Darmstadt, Germany) for some minutes. Trypsin reaction was stopped with 3 ml cell
culture medium eMEM, and cells were pelleted at room temperature for 5 min at 500 x g with
slightly reduced break. The cells were washed once with Dulbecco’s PBS with Ca 2+ and Mg 2+ (PAA)
and resuspended in FlacsFlow (Becton Dickinson Biosciences, San Jose, CA, USA) for sorting of
infected and non-infected host cells. Both solutions were supplemented with 0.025 µg/ml
actinomycin D (Sigma-Aldrich) and 20 mM sodium azide (Merck). Control samples were treated in
the same way.
Protein samples and control measurements [performed by Melanie Gutjahr]
Samples for transcriptome analysis were harvested in parallel with samples for host proteome
analysis (Fig. M.4.1). For protein samples, trypsin, PBS and FacsFlow solutions were not
supplemented with actinomycin D and sodium azide, and control samples were directly lysed in
UT buffer (8 M urea, 2 M thiourea). One additional control without any treatment was included.
For the bacterial starting culture (OD 0.4), the infection mix, and samples after 2.5 h and 6.5 h
of infection (internalized bacteria) viable cell counts were determined by plating on TSB agar and
incubation for 24-48 h at 37°C.
FACS measurements and cell sorting [performed by Petra Hildebrandt], sample harvest and
disruption [performed by Maren Depke]
Cells were sorted into infected (green fluorescence positive) and non-infected (green
fluorescence negative) cells in a biosafety 2 level FACS Aria high-speed cell sorter (Becton
Dickinson Biosciences, San Jose, CA, USA) with 488 nm excitation from a blue Coherent Sapphire
solid state laser at 18 mW. Optical filters were set up to detect the emitted GFP fluorescence at
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