Cell Host & Microbe, Volume 13

Supplemental Information

The Staphylococcal Toxin Panton-Valentine Leukocidin Targets Human C5a Receptors

András N. Spaan, Thomas Henry, Willemien J.M. van Rooijen, Magali Perret, Cédric Badiou, Piet C. Aerts, Johan Kemmink, Carla J.C. de Haas, Kok P.M. van Kessel, François Vandenesch, Gérard Lina, and Jos A.G. van Strijp

Supplementary Figure 1.

Neutro

phils

U937-C

5aR

102

103

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C5aR

Rec

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Binding LukS-PV

TLR2/CD14

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313 nM 0 nM

LukS-PV

Figure S1 (Related to Figure 1). (A) Receptor expression levels of stably transfected cell lines in relation to freshly isolated neutrophils from healthy donors. (B, C) The TLR2/CD14 Receptor complex does not mediate PVL-induced pore formation. (B) No binding of LukS-PV to HEK cells stably co-transfected with TLR2 and CD14. (C) No PVL induced pore formation (concentration PVL 625 nM) in transfected HEK cells. Permeable cells are DAPI-positive cells. Bars express the standard deviation, with n=2. Histograms depict a representative example.

0

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10 100 1000CHIPS (nM)

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llsA C CHIPS

(nM) PVL (nM)

Ratio CHIPS:PVL

Recombinant proteins 500.00 2.34 213.67

5% o/n CCY supernatant 18.65 177.29 0.11

5% o/n IMDM supernatant 24.16 0.29 83.31

5% o/n TSB supernatant 0.19 11.43 0.02

Supplementary Figure 2

B

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Figure S2 (Related to Figure 2). CHIPS is produced in insufficient amounts to compete with PVL for interaction with the C5aR. (A) Pore formation inhibition in U937-C5aR cells by different concentrations of CHIPS (PVL concentration 2.34 nM, incubation time 30 min). (B) Expression over time of CHIPS and LukS-PV by S. aureus USA300-LAC in CCY, IMDM and TSB broth. Detection of proteins by ELISA. (C) Molar ratio of CHIPS versus PVL needed to protect cells from PVL-induced pore formation using recombinant proteins based on panel A. Ratio of CHIPS versus PVL in 5% overnight supernatant as detected in panel B and used in panel C. (D) Pore formation in U937-C5aR cells induced by 5% overnight CCY, IMDM or TSB based bacterial supernatant containing CHIPS and PVL as shown in panel C (incubation time 30 min). (E) Pore formation in PMA-differentiated THP-1 cells induced by 0.2% overnight supernatant of various PVL-positive clinical S. aureus isolates with or without the gene encoding CHIPS (incubation time 180 min). Bars express the standard deviation, with n=3.

C5a Bgr

HumanNeutro

αC5aRIso

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Binding αC5aR antibody

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Supplementary Figure 3

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nts

A B

Figure S3 (Related to Figure 4). Neutrophils from different species express similar levels of C5aR. (A) Fluorescent C5a binding (240 nM) to human, macaque and rabbit neutrophils. (B) Monoclonal antibody binding to the C5aR of different species (human and macaque: mouse anti-human C5aR antibody clone S5/1; rabbit: mouse anti-human C5aR antibody clone 347214; mouse: rat-anti-mouse C5aR clone 10/92). Histograms depict a representative example.

Supplementary Figure 4

HumanC5aR

MouseC5aR

MacaqueC5aR

RabbitC5aR

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nts

Untrans-fected

Receptor expression

Figure S4 (Related to Figure 5 and Experimental Procedures). Receptor expression levels on transient transfected HEK cells. FLAG-tag detection based expression levels of C5aR of different species. Receptor expressing cell populations right of the dashed lines were gated and used for experiments. Histograms depict a representative example.

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HumanC3aR

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Supplementary Figure 5

Figure S5 (Related to Figure 6 and Experimental Procedures). Receptor expression levels on transient transfected HEK cells. (A) HA-tag detection based expression levels of human N-terminal receptors. (B) FLAG-tag detection based expression levels of human C5aR-C3aR chimeras. Receptor expressing cell populations right of the dashed lines were gated and used for experiments. Histograms depict a representative sample.

Table S1 (Related to Experimental Procedures). Bacterial strains used in this study to produce recombinant proteins. All genes were cloned, omitting the region predicted to encode the signal peptide, as determined on the SignalP V3.0 World Wide Web Prediction Server (http://www.cbs.dtu.dk/services/SignalP/). Protein S. aureus strain Reference LukS-PV ATCC 49775 (Perret et al., 2012) LukF-PV ATCC 49775 (Perret et al., 2012) CHIPS Newman (de Haas et al., 2004) CHIPS31-121 Newman (Haas et al., 2005) FLIPr-Like MW2 (Prat et al., 2009)

Table S2 (Related to Experimental Procedures). Primer sequences used in this study to produce plasmids for transient transfections or for qRT-PCR (F: Forward primer; R: Reverse primer; TM: transmembrane domain; N: N-terminus). Restriction enzyme recognition sites are underlined. FLAG tag sequences are double underlined.

Receptor Primers Human C5aR. Accession nr.: NM_001736

Fwd: 5’-CCGGAATTCATGGACTACAAGGACGACGACGACAAGATGAACTCCTTCAATTATACC-3’ Rev: 5’-GCTCTAGACTACACTGCCTGGGTCTTCT-3’

Human C5L2. Accession nr.: NM_001271749

Fwd: 5’-CCGGAATTCATGGACTACAAGGACGACGACGACAAGATGGGGAACGATTCTGTCAGCTACG-3’ Rev: 5’-GCTCTAGACTACACCTCCATCTCCGAGACC-3’

Human C3aR. Accession nr.: BC020742

Fwd: 5’-CGGGATCCATGGACTACAAGGACGACGATGACAAGATGGCGTCTTTCTCTGCTGAGACC-3’ Rev: 5’-GCTCTAGATCACACAGTTGTACTATTTC-3’

Human Chimera N-C3aR / TM-C5aR

Fwd: 5’-CGGGATCCATGGACTACAAGGACGACGATGACAAGATGGCGTCTTTCTCTGCTGAGACC-3’ Rev: 5’-GACCAAGGCCAGGATTACTGGGGGCTCATTCCATG-3’ Fwd: 5’-ATTGAGCCCCCAGTAATCCTGGCCTTGGTCATCTTTG-3’ Rev: 5’-GCTCTAGACTACACTGCCTGGGTCTTCT-3’

Human Chimera N-C5aR / TM-C3aR

Fwd: 5’-CCGGAATTCATGGACTACAAGGACGACGACGACAAGATGAACTCCTTCAATTATACC-3’ Rev: 5’-GACCATGGAGAGAATGTCTGGAACACGCAGCGTGTTAG-3’ Fwd: 5’-TGCGTGTTCCAGACATTCTCTCCATGGTCATTCTC-3’ Rev: 5’-GCTCTAGATCACACAGTTGTACTATTTC-3’

Mouse C5aR (C57BL/6). Accession nr.: BC125643

Fwd: 5’-CCGGAATTCATGGACTACAAGGACGACGACGACAAGATGGACCCCATAGATAACAGCAG-3’ Rev: 5’-GCTCTAGACTACACCGCCTGACTCTTCC-3’

Macaque C5aR (M. fascicularis). Accession nr.: EHH59724

Fwd: 5’-CCGGAATTCATGGACTACAAGGACGACGACGACAAGATGAACTCCTTCAATTATACC-3’ Rev: 5’-GCTCTAGACTACACTGCCTGGGTCTTCT-3’

Rabbit C5aR (N.Z. White). Accession nr.: AF068680

Fwd: 5’-CCGGAATTCATGGACTACAAGGACGACGACGACAAGATGGAAAATAGCACGTACGATTACACC-3’ Rev: 5’-GCTCTAGATCACACCGCTTGGCACTTGTCG-3’

Human N-C5aR Fwd: 5’-TAGTGAAGATCTATGAACTCCTTCAATTATACCACCC-3’ Rev: 5’-AGTACCATCGATGATGTCTGGAACACGCAGCG-3’

Human N-C5L2 Fwd: 5’-TAGTGAAGATCTATGGGGAACGATTCTGTCAGCTACG-3’ Rev: 5’-CCATCGATGGCCACGCGCAGCGGGTCGA-3’

Human C5aR (qRT-PCR)

Fwd: 5’-GGACACTATGGCCCAGAAGA-3’ Rev: 5’-CTGGGAAAGACAGGCAACAT-3’

Human C5aR (qRT-PCR)

Fwd: 5’-CACCCACTCCTCCACCTTTGAC-3’ Rev: 5’-GTCCACCACCCTGTTGCTGTAG-3’

Supplemental Experimental Procedures Quantification of CHIPS and PVL expression in supernatants. Expression of CHIPS and PVL in the supernatant of S. aureus USA300-LAC was measured at multiple time points of growth in casein hydrolysate and yeast extract (CCY) medium (Lipinska et al., 2011; Woodin, 1959), tryptic soy broth (TSB), or Iscove’s Modified Dulbecco’s Medium (IMDM). CHIPS and LukS-PV were detected in supernatant by specific ELISA techniques as described elsewhere (Badiou et al., 2008; Badiou et al., 2010; de Haas et al., 2004). Quantification of PVL expression was based on LukS-PV detection. Clinical S. aureus isolates. Clinical S. aureus strains isolated from patients hospitalized for severe community-acquired pneumonia were collected during prospective studies that were previoulsy described (Gillet et al., 2002; Sicot et al., 2013). The genomic content of the clinical isolates was analysed by commercial microarray (StaphyType, Alere Technologies) as previously described (Albrecht et al., 2011), following manufacturer's instructions to determine the presence or absence of the PVL- and CHIPS-encoding genes. To generate supernatant, single hemolytic colonies were grown overnight in CCY broth (Lipinska et al., 2011; Woodin, 1959). Supernatants were sterilized by a 0.2 μm filter diluted 1/625 before addition onto PMA-differentiated THP-1 macrophages in the presence of propidium iodide (PI) (5 µg/ml). Pore formation was assessed by measuring PI-incorporation on a Tecan fluorimeter after 180 min incubation. Receptor expression determination on neutrophils of different species. Freshly isolated human, macaque and rabbit leukocytes were incubated in a total volume of 50 μL at 5x106 cell/mL with 240 nM FITC-labeled C5a (Bachem). FITC-labeled C5a was produced as decribed before (de Haas et al., 2004; Prat et al., 2009). In a total volume of 50 μL at 5x106 cell/mL, human and macaque leukocytes were incubated with mouse anti-human C5aR antibody clone S5/1 (BioLegend), followed by FITC-conjugated goat-anti-mouse antibody (Dako). Rabbit leukocytes were incubated with PE-conjugated mouse anti-human C5aR antibody clone 347214 (R&D Systems). Murine leukocytes were incubated with Alexa488-conjugated rat-anti-mouse clone 10/92 (AbD SeroTec). Fluorescences were detected by flow cytometry.

Supplemental References:

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