Supplementary material

Supplementary methods

Preparation of the extracellular protein fraction for proteome analysis

S. aureus strains were grown in tryptic soy broth (TSB) at 37°C with linear shaking at 100

rpm in a water bath (OLS, Grant Instruments, England). During exponential growth phase

bacteria were pelleted for 15 min at 4°C and 5,200 xg. The supernatant was mixed with TCA

to a final concentration of 10% and incubated at 4°C overnight. Proteins were pelleted via

centrifugation for 1 hour at 4°C and 24,400 g. The protein pellet was washed five times with 1

ml 70% ethanol. After the last washing step, the pellet was incubated for 30 min at 21°C and

600 rpm in a thermomixer (Eppendorf, Germany). Finally, the pellet was washed once with

100% ethanol and dried in a speed vacuum centrifuge for 2 min. Subsequently, protein

pellets were dissolved in a suitable volume of 1x UT buffer (8 M urea and 1 M thiourea) and

incubated for 1 hour at 21°C and 600 rpm in a thermomixer (Eppendorf, Germany). Non

soluble components were pelleted via centrifugation and protein concentration was

determined according to Bradford [1]. 4 µg of protein were reduced and alkylated with

Dithiothreitol and Iodacetamid prior to digestion with Trypsin. The resulting peptide mixture

was purified and desalted using µC18 ZipTip columns and dried in a speed vacuum

centrifuge. Dried peptides were dissolved in LC buffer A (2% ACN and 2% DMSO in water

with 0.1% acetic acid).

Acquisition of tryptic peptides by mass spectrometry

For identification of proteins, the nanoAcquity UPLC (Waters Corporation, Milford, MA, USA)

was coupled to a LTQ-Orbitrap Velos mass spectrometer (ThermoElectron, Germany)

equipped with a nano-ESI source. For nLC separation, peptides were first enriched on a

nanoAcquity C18 pre-column (Waters Corporation, Milford, MA, USA), separated using a

nanoAcquity C18 analytical column (Waters Corporation, Milford, MA, USA). Separation was

achieved via the formation of a linear gradient of 92 min total run time containing LC buffer A

and B (5% DMSO and 0.1% acetic acid in ACN).The percentage of LC buffer A decreased

during the nLC run and the percentage of LC buffer B increased steadily: 1-5% for 2 min, 5-

25% for 63 min, 25-60% for 25 min, 60-99% for 2 min. The peptides were eluted at a flow

rate of 400 nl/min. The eluted peptides were analyzed in a FTMS analyzer, operated in

profile mode and positive polarity. The MS/MS scan was performed in data-dependent

analysis mode and data were acquired in centroid mode with positive polarity. The MS

automatically switched between Orbitrap-MS and LTQ-MS/MS acquisition. Full scan MS

spectra from 300 to 1700 m/z were acquired in the Orbitrap with a resolution of R=30,000.

The methods allowed sequential isolation of maximum 20 most intense ions depending on

the signal intensity. These were subjected for collision induced dissociation (CID)

fragmentation with an isolation width of 2 Da and a target value of 3x104 or with a maximum

ionization time of 100 ms. Target ions already selected for MS/MS were dynamically

excluded for 60 seconds. The ion selection threshold was 2000 counts for MS/MS with an

activation time of 10 ms and normalized activation energy of 35%. Only +2 and +3 ions were

triggered for tandem MS analysis.

Selection of S. aureus strains for the pan proteome database

Tandem-MS spectra were searched against a S. aureus pan proteome FASTA database

consisting of seven S. aureus strains (NCTC8325, USA300_FPR3757, 6850, JH9, TCH60,

MRSA252, and 55/2053). A pan proteome database was chosen because the strains

Cowan1 and LS1 have not been sequenced yet and therefore no strain specific database

was available for these strains. Pre-analysis of the ms-data generated showed that the ms-

data for strain LS1 yielded the highest number of peptide identifications with databases

generated for strains NCTC8325 and USA300_FPR3757 and that the data of strain Cowan1

were represented best by databases generated from sequences of S. aureus strains TCH60,

MRSA252, 55/2053 and JH9. Therefore, these strains were included in the pan proteome

FASTA database.

Prediction of cytoplasmic proteins

To identify cytoplasmic proteins, the prediction of subcellular protein localization PSORT db

version 3 [2] and LocateP version 2 [3] had to classify the proteins as cytoplasmic. Proteins

with “no significant prediction” by PSORT had to be classified as cytoplasmic by LocateP, in

addition no signal peptide had to be predicted by SignalP version 4.1 [4] and no

transmembrane helices by TMHMM [5] .

Supplementary table 1: Bacterial strains used in this study

Strain source referenceS. aureus 6850 Isolated from a patient with complicated S.

aureus bacteremia associated with osteomyelitis and septic arthritis

[6, 7]

S. aureus USA300 Highly successful S. aureus clone that emerged in the community and quickly spread throughout the North American continent to become the leading cause of MRSA infection even in healthcare settings.

[8]

S. aureus LS1 Murine arthritis isolate [9]S. aureus SH1000 Derived from NCTC 8325 [10, 11]

S. aureus Cowan1 Isolated from septic arthritis ATCC 12598S. carnosus TM300 Originally isolated from dry sausage [12]

Supplementary table 2: Host cells used in this studyHost cell Medium Growth conditions and

information on isolation/ for experiment seeded with:

Primary human endothelial cells (HUVEC)

M199 supplemented with 20 mM Hepes (pH 7.4), glutamine (2mM),10% (v/v) human serum, 10% (v/v) newborn calf serum, ECGS (150 µg ml-1), heparin (5 IU ml-1), penicillin (100 IU ml-1), streptomycin (100 µg ml-1)

Isolated by collagenase treatment as described previously [13]grown on fibronectin-coated dishes37°C in a 5% CO2 atmosphere/seeded with 40,000 cells /cm²

Endothelial cell line (Ea.hy926)

DMEM, 10% fetal calf serum (FCS), 1xHAT supplement, penicillin (100 IU ml-1), streptomycin (100 µg ml-1)

37°C in a 5% CO2 atmosphere/seeded with 40,000 cells /cm²

Epithelial cell line (A549)

DMEM, 10% FCS, penicillin (100 IU ml-1), streptomycin (100 µg ml-1)

37°C in a 5% CO2 atmosphere/seeded with 40,000 cells /cm²

Human keratinocyte cell line (HaCaT)

DMEM, 10% FCS, penicillin (100 IU ml-1), streptomycin (100 µg ml-1)

37°C in a 5% CO2 atmosphere/seeded with 15,000 cells /cm²

Primary human osteoblasts (prim HOB)

MEM Alpha Modification and 10 % FCS, penicillin (100 IU ml-1), streptomycin (100 µg ml-1) , L-ascorbic acid 2-phosphate (0.2 mM), β-glycerophosphate disodium salt hydrate (10 mM) and dexamethasone (10 nM)

Generated from normal trabecular bone specimens as described previously [14] with some modifications. Briefly, the bone material was isolated using a sharp spoon. The resulting bone chips were washed with phosphate-buffered saline (PBS) and treated with Trypsin/EDTA (30 min, 37°C). Subsequently, the chips were seeded in medium37°C in a 5% CO2 atmosphere/seeded with 20,000 cells /cm2

Osteoblast cell line (CRL-11372)

DMEM/F-12 and 10% FCS, 1% penicillin (100 IU ml−1), streptomycin (100 µg ml−1) and 0.66% of geneticin

34°C in a 5% CO2 atmosphere/seeded with 30,000 cells /cm²

Fibroblast cell line (CCD-32-SK)

DMEM, 10% FCS, penicillin (100 IU ml-1), streptomycin (100 µg ml-1)

37°C in a 5% CO2 atmosphere/seeded with 25,000 cells /cm²

Supplementary table 3: RT-Primers used in this studyGene name Forward primer Reverse primer

CCL5 CAGTGGCAAGTGCTCCAACC CCATCCTAGCTCATCTCCAAAGAGTCXCL11 CAGAATTCCACTGCCCAAAGG GTAAACTCCGATGGTAACCAGCCIL-6 AGAGGCACTGGCAGAAAACAAC AGGCAAGCTTCCTCATTGAATCCB2M* TGAGTATGCCTGCCGTGTGA AAATGCGGCATCTTCAAACCTGAPDH* GCAAATTTCCATGGCACCGT GCCCCACTTGATTTTGGAGG

*housekeeping genes

Supplementary figure 1

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

HUVEC endothelial cell line

epithelial cell line

keratinocyte cell line

primary human osteoblasts

osteoblast cell line

fibroblast cell line

cfu/

ml

Cowan1

SH1000

************

*

S. aureus invasion is highly dependent on the host cell. Different host cells were infected

with S. aureus strain Cowan1 (MOI50) or SH1000. After 3 h extracellular staphylococci were

removed by washing and lysostaphin treatment. Afterwards, the numbers of viable

intracellular bacteria were determined by lysing host cells, plating the lysates on agar plates

and counting the colonies that have grown on the following day. The values represent the

mean ± SD of 3 independent experiments measured. * P≤0.05, *** P≤0.001, compared to

HUVEC.

0.1

1

10

100

1000

HUVEC endothelial cell line

epithelial cell line

keratinocyte cell line

pHOB osteoblast cell line

fibroblast cell line

norm

aliz

ed fo

ld e

xpre

ssio

n

CXCL-11,24h post infection

Supplementary figure 2A

S. aureus infected host cells become activated and express cytokine CXCL11. After

bacterial invasion of S. aureus 6850 (3 h) extracellular staphylococci were removed by

washing and lysostaphin treatment and infected cells were directly used or incubated with

culture medium for 24 h. To analyse host cell response the changes in the expression of the

chemokine CXCL11 was measured by real-time PCR. Results demonstrate the relative

increase in gene expression, compared to unstimulated cells (control: expression 1). The fold

change represents the normalized expression of each gene to housekeeping genes (B2M

and GAPDH). The values of all experiments represent the means ± SD of at least three

independent experiments performed in duplicates.

0

500

1000

1500

2000

2500

control 24h p.i.

pg/m

l

CCL5

HUVEC

primary human osteoblasts

Supplementary figure 2B

Measurement of chemokine release in cell culture supernatants by enzyme-linked

immunosorbent assay. HUVECs and primary human osteoblasts were infected with S.

aureus 6850 (MOI 50) and incubated for 24 h. The conditioned media were analyzed for

CCL5. The values of all experiments represent the means ± SD of at least three independent

experiments performed in duplicates.

0.4

0.6

0.8

1

1.2

1.4

1.6

0 5 10 15 20

TER

x/TE

R0

time p.i. [h]

control

6850

USA300

LS1

SH1000

Cowan1

00.20.40.60.8

11.21.41.61.8

3 6 9 12 20

TER

x/TE

R0

time p.i. [h]

control6850

USA300

LS1SH1000

Cowan1

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

0 5 10 15 20 25

TER

x/TE

R0

time p.i. [h]

control6850USA300LS1SH1000Cowan1

3.5 h p.i.

6 h p.i.

9 h p.i.0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

3 6 9 12 20

TER

xh/

TER

0h

time p.i. [h]

control

6850

USA300

LS1

SH1000

Cowan1

* * * ******

*

A549

lysostaphin andwash step

TER

x/TE

R0

Supplementary figure 3

A B

C

Cytotoxicity of S. aureus strains is dependent on the strain and the host cell. Epithelial

and keratinocyte cell line layers were infected with different S. aureus strains (MOI50) and

kinetics of bacterial induced cytotoxic effects on the integrity of the host cell layer were

continuously analysed by TER determination. The results represent means +/-SD of a least

three independent experiments. * p≤0.05, ** p≤0.01, one way ANOVA and Dunnett post-test.

(A) Epithelial cell line layer infected with different S. aureus strains. (B) Microscopic images

were taken from epithelial cell line layer 3.5 h, 6 h, and 9 h post infection (p.i.) with S. aureus

6850. (C) Keratinocyte cell line layer infected with different S. aureus strains.

Lysostaphin and wash step

HaCaT

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

Cowan1 SH1000

cfu/

ml

HUVEC

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

Cowan1 SH1000cf

u/m

l

epithelial cell line

day 0 day 7

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

Cowan1 SH1000

cfu/

ml

prim. human osteoblasts

Supplementary figure 4

Persistence of S. aureus Cowan1 and SH1000 in HUVECs, epithelial cells and primary

human osteoblasts. Host cells were infected with S. aureus Cowan1 and SH1000 (MOI50).

The number of viable intracellular persisting bacteria were determined on day 0 (3 h p.i.) and

day 7 by lysing the host cells, plating the lysates on agar plates and counting the colonies

that have grown on the following day. Percentage numbers give the percentage of cfu day 7

in comparison to day 0. The values of all experiments represent the means ± SD of three

independent experiments.

0.12 % 0.1 % 0.004 %

0.029 %

0.15 %

0.73 %

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