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Differential Interaction of the Staphylococcal Toxins Panton−Valentine Leukocidin and γ -Hemolysin CB with Human C5a Receptors

This information is current as András N. Spaan, Ariën Schiepers, Carla J. C. de Haas, of October 1, 2021. Davy D. J. J. van Hooijdonk, Cédric Badiou, Hugues Contamin, François Vandenesch, Gérard Lina, Norma P. Gerard, Craig Gerard, Kok P. M. van Kessel, Thomas Henry and Jos A. G. van Strijp J Immunol 2015; 195:1034-1043; Prepublished online 19 June 2015; Downloaded from doi: 10.4049/jimmunol.1500604 http://www.jimmunol.org/content/195/3/1034 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2015/06/19/jimmunol.150060 Material 4.DCSupplemental References This article cites 46 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/195/3/1034.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Interaction of the Staphylococcal Toxins Panton–Valentine Leukocidin and g-Hemolysin CB with Human C5a Receptors

Andra´s N. Spaan,*,†,‡ Arie¨n Schiepers,* Carla J. C. de Haas,* Davy D. J. J. van Hooijdonk,* Ce´dric Badiou,†,‡,x Hugues Contamin,{ Franc¸ois Vandenesch,†,‡,x,‖ Ge´rard Lina,†,‡,x,‖ Norma P. Gerard,#,** Craig Gerard,# Kok P. M. van Kessel,* Thomas Henry,†,‡,x and Jos A. G. van Strijp*

Staphylococcus aureus is well adapted to the human host. Evasion of the host phagocyte response is critical for successful infection. The staphylococcal bicomponent pore-forming toxins Panton–Valentine leukocidin LukSF-PV (PVL) and g-hemolysin CB

(HlgCB) target human phagocytes through interaction with the complement receptors C5aR1 and C5aR2. Currently, the appar- Downloaded from ent redundancy of both toxins cannot be adequately addressed in experimental models of infection because mice are resistant to PVL and HlgCB. The molecular basis for species specificity of the two toxins in animal models is not completely understood. We show that PVL and HlgCB feature distinct activity toward neutrophils of different mammalian species, where activity of PVL is found to be restricted to fewer species than that of HlgCB. Overexpression of various mammalian C5a receptors in HEK cells confirms that cytotoxicity toward neutrophils is driven by species-specific interactions of the toxins with C5aR1. By taking advantage of the species-specific engagement of the toxins with their receptors, we demonstrate that PVL and HlgCB differentially http://www.jimmunol.org/ interact with human C5aR1 and C5aR2. In addition, binding studies illustrate that different parts of the receptor are involved in the initial binding of the toxin and the subsequent formation of lytic pores. These findings allow a better understanding of the molecular mechanism of pore formation. Finally, we show that the toxicity of PVL, but not of HlgCB, is neutralized by various C5aR1 antagonists. This study offers directions for the development of improved preclinical models for infection, as well as for the design of drugs antagonizing leukocidin toxicity. The Journal of Immunology, 2015, 195: 1034–1043.

taphylococcus aureus is one of the most common causes of vaccines has increased. Despite promising results in preclinical bacterial infections in humans worldwide (1). S. aureus models, a recent vaccine candidate failed in clinical trials (3). by guest on October 1, 2021 S causes various diseases ranging from superficial skin and The pathogen S. aureus is well adapted to the human host. Many soft tissue infections to severe invasive diseases. The emergence of of the pathogen’s virulence factors show different specificities across hospital-acquired and community-associated methicillin-resistant S. mammalian species frequently used during preclinical in vivo studies aureus (MRSA) strains has now become a global problem. As no (4). As a result, the contribution to pathophysiology of many of these new antibiotic agents are expected to be released in the near future virulence factors cannot be investigated in an integrated model for (2), interest in the development of alternative therapeutics and infection. More importantly, the potential of these virulence factors as vaccine or drug targets cannot be assessed accurately. Phagocytes play a crucial role in the host defense against *Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX infections with S. aureus (4, 5). However, S. aureus has evolved Utrecht, the Netherlands; †Centre International de Recherche en Infectiologie, Uni- versite´ Lyon 1 and Ecole Normale Supe´rieure de Lyon, 69007 Lyon, France; multiple strategies to evade the human immune system. A key ‡Inserm, Unite´ 1111, 69007 Lyon, France; xCentre National de la Recherche Scien- mechanism of S. aureus to repel attack by host phagocytes is the tifique, Unite´ Mixte de Recherche 5308, 69007 Lyon, France; {Cynbiose, 69280 Marcy l’Etoile, France; ‖Centre National de Re´fe´rence des Staphylocoques, Hospices production of cytolytic toxins (6). Staphylococcal leukocidins are Civils de Lyon, 69007 Lyon, France; #Ina Sue Perlmutter Laboratory, Division of bicomponent pore-forming toxins that perforate the host cell plasma Pulmonary Medicine, Department of Pediatrics, Boston Children’s Hospital, membrane (7). Based on chromatography elution profiles, the in- Department of Medicine, Harvard Medical School, Boston, MA 02115; and **Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215 dividual leukocidin subunits are designated S (slow) or F (fast) (8). Received for publication March 13, 2015. Accepted for publication May 22, 2015. Initial binding of the S-component to the surface of the target cell allows secondary binding of the F-component (9). This sub- This work was supported in part by grants from the European Commission (222718 to C.B., G.L., and F.V.), the Agence Nationale de la Recherche (to G.L., F.V., and T.H.), sequently results in the assembly of lytic pore-forming hetero- the Foundation Finovi (to T.H.), and the National Institute of Allergy and Infectious octamers (10). The genome of human S. aureus isolates can Diseases/National Institutes of Health (HL051366 to C.G.). encode up to five leukocidin toxins: Panton–Valentine leukocidin Address correspondence and reprint requests to Prof. Jos A.G. van Strijp, University LukSF-PV (PVL) (11), g-hemolysin AB and g-hemolysin CB Medical Center Utrecht, Department of Medical Microbiology, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands. E-mail address: [email protected] (HlgCB) (12, 13), LukED (14), and LukAB (also known as LukGH) The online version of this article contains supplemental material. (15, 16). The leukocidin protein components are closely related, and Abbreviations used in this article: CHIPS, chemotaxis inhibiting protein of S. aureus; the amino acid sequence of the S-components of PVL (LukS-PV) ECL, extracellular loop; HEK, human embryonic kidney (cell); HlgCB, g-hemolysin and HlgC shows highest identity (81%) (17). Of the different CB; LukS-PV, S-components of PVL; MRSA, methicillin-resistant S. aureus; PVL, staphylococcal leukocidins, the cytotoxic activity of PVL was the Panton–Valentine leukocidin LukSF-PV. first to be described in detail (11). Although rare in methicillin- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 susceptible S. aureus isolates, the genes encoding PVL are over- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500604 The Journal of Immunology 1035 represented in epidemic community-associated MRSA strains (18). Cell isolation Although numerous epidemiological studies suggest a relation be- Human neutrophils, obtained from healthy volunteers, macaque (Macaca tween PVL and severe invasive disease (19, 20), the role of PVL fascicularis), and rabbit (New Zealand White) neutrophils were isolated by during infection is still not fully elucidated. The controversy re- Ficoll/Histopaque centrifugation (31). Neutrophils from cows (Holstein garding PVL is mostly caused by species-specific differences in Frisian) were isolated using Percoll (1.09176 g/l) centrifugation. Mouse susceptibility of phagocytes toward PVL (21). The HlgCB-encoding (C57BL/6) leukocytes were obtained from bone marrow, and immune cells were collected as described (28). Unless specified otherwise, all in vitro genes are present in almost all human S. aureus isolates (22, 23). experiments with cells were performed using RPMI 1640 (Invitrogen) Near-universal prevalence and consistent toxin expression by vari- supplemented with 0.05% human serum albumin (Sanquin), with cell ous S. aureus strains distinguish HlgCB from the other leukocidins concentrations adjusted to 5 3 106 cell/ml. (24). However, the contribution to pathophysiology is not well Cell lines and transfections established because mouse neutrophils are resistant to HlgCB (24). The molecular basis for species specificity of both PVL and HlgCB HEK293T cells [a human embryonic kidney (HEK) cell line] were used for in different animal models is incompletely understood. overexpression of wild-type and chimeric receptors. The genes encoding the C5a receptors of all mammalian species investigated reside on a single exon. For all leukocidins, recent identification of the myeloid host Amplifications were performed on QUICK-Clone cDNA of human bone receptors revealed a specific molecular adaptation to the human host marrow or mouse liver (BD Biosciences Clontech), macaque, rabbit, and cow (18, 24–28). The S-components of PVL, HlgAB, HlgCB, and LukED, peripheral blood using PfuTurbo DNA polymerase (Stratagene). N terminus each target specific receptors belonging to the family of complement and ECL exchange chimeric receptors were constructed using overlap ex- and chemokine receptors. This family of receptors shares a seven- tension PCR, as described elsewhere (33). The receptor sequences were amplified and fused together in multiple PCR steps. The 59-and39-terminal transmembrane spanning architecture, with the N terminus and three

primers contained, respectively, EcoRI and NotI restriction sites, allowing, Downloaded from extracellular loops (ECLs) exposed to the extracellular milieu (29). respectively, for specific ligation into the multiple cloning sites of the The involvement of specific host receptors in pore formation is a new pcDNA3.1 vector (Invitrogen) (28) or the bicistronic expression plasmid concept, and molecular mechanisms of the multistep process of pore pIRESPuro3 (Clontech) (24). To confirm surface expression of the receptors on transiently transfected cells during binding studies of LukS-PV, an formation are only partially understood. PVL and HlgCB both target N-terminal FLAG-tag (DYKDDDDK) was placed after the first methionine. the complement component C5a receptors C5aR1 (C5aR, CD88) and An additional methionine was placed directly after the FLAG-tag to keep the C5aR2 (C5L2, GPR77) (24, 28). Sharing of receptors by the closely N-terminal sequence intact. Expression vectors, primer designs, restriction related leukocidins PVL and HlgCB suggests redundancy. However, sites, accession numbers, and primer combinations used in this study are listed http://www.jimmunol.org/ experimental investigation of this apparent redundancy is seriously in Supplemental Table I (wild-type full-length receptors), Supplemental Table II (primer designs used for chimeric receptors), and Supplemental hindered by the lack of appropriate animal models for infection (7). Table III (primer combinations used for C5aR1 or chimeric receptors). The development of improved animal models for infection is urgently Cloning of rabbit C5aR2 resulted in an amino acid sequence slightly divergent needed to address this issue. from the annotated sequence. All wild-type amino acid sequences used in this In an attempt to better understand the process leading to pore study are aligned in Fig. 2. HEK293T cells stably transfected with a plasmid encoding human C5aR2 were donated by Peter N. Monk (Sheffield University formation by PVL and HlgCB, we investigated the interactions of Medical School, Sheffield, U.K.) (34). both toxins with their receptors in more detail. By taking advantage Cells were either transiently or stably transfected, as described elsewhere of the species-specific interaction of the leukocidins with the (24, 28). Briefly, cells were transfected with 4 mg DNA and 5 ml Lip- by guest on October 1, 2021 receptors, we demonstrate that PVL and HlgCB differentially ofectamine 2000 (Life Technologies), according to the manufacturer’s interact with C5aR1 and C5aR2. In addition, we illustrate that protocol. For transient transfections, cells were harvested after 24 h with 0.05% trypsin/0.53 mM EDTA. For stable transfections, selection pressure different parts of C5aR1 are involved in the initial binding of the was applied using 1 mg/ml puromycin in DMEM with 10% FCS, and cells S-components and the subsequent formation of lytic toxin pores. were subcloned after several weeks. We show that toxicity of PVL, but not of HlgCB, can be neutralized The expression of receptors was confirmed using 10 mg/ml mAb by various C5aR1-antagonists. Together, these findings highlight (PE-conjugated mouse anti-human C5aR1 clone S5/1, 1:50, BioLegend; FITC-conjugated rat anti-mouse C5aR1 clone 20/70, 1:50, BioLegend; PE- the implications for our understanding of pore formation, the conjugated mouse anti-human C5aR2 clone 1D9-M12, 1:50, BioLegend) or development and application of improved preclinical models for 240 nM FITC-conjugated C5a (Bachem) where possible. Specific suscepti- infection, and the development of drugs antagonizing leukocidin bility toward PVL and/or HlgCB toxicity of cells stably transfected with toxicity. plasmids encoding wild-type C5a receptors from different mammalian species was interpreted as a confirmation of receptor expression. In the case of resistance of cells toward both PVL and HlgCB toxicity, stable expression of Materials and Methods wild-type C5a receptors was confirmed (Supplemental Fig. 1A, 1B). Owing Ethics statement to inability to bind mAbs or C5a, expression of chimera Mouse C5aR1 Human ECL1+2+3 and chimera Mouse C5aR2 Human ECL1+3 on stably Human neutrophils were isolated after informed consent was obtained from transfected cells could not be verified. The expression of the remaining all donors in accordance with the Declaration of Helsinki. For this, approval chimeric receptors was verified similarly (Supplemental Fig. 1C–P). was obtained from the medical ethics committee of the University Medical Center Utrecht, the Netherlands. Experiments involving animals were Cell permeability assays reviewed and approved by the animal ethics committees of Lyon, France (CECCAPP, protocol number ENS_2012_033), or Utrecht, the Netherlands Cells were exposed to recombinant toxins in a volume of 50 ml for 30 min at (protocol number DEC.2012.II.09.136). 37˚C. Human, macaque, rabbit, and cow neutrophils, and cell lines, were subsequently analyzed by flow cytometry and gated for forward and side Recombinant protein production and purification scatters. At least 10,000 cells per sample were analyzed. For graphical presentation, pore formation was defined as the percentage of cells positive S. aureus strains used to amplify coding sequences of toxins and bacterial for intracellular staining by DAPI. proteins include strain ATCC 49775 for LukS-PVand LukF-PV (30), strain After treatment with FcgR block, mouse leukocytes were stained with Cagnant for HlgC and HlgB (30), strain Newman for chemotaxis inhibiting mAbs directed toward CD11b (clone M1/70, 1:400; BD), Ly6G (clone 1A8, protein of S. aureus (CHIPS) (31), and strain MW2 for FLIPr-Like (32). 1:100; BD), and Ly6C (clone AL-21, 1:200; BD). After exposure to toxins, Coding sequences were cloned and expressed as described elsewhere (30–32). mouse cells were incubated on ice with the fixable viability dye eFluor 780 For the bicomponent pore-forming toxins, single protein-coding sequences (1:1000, eBiosciences). Cell viability of mouse neutrophils was analyzed by omitting the predicted signal peptide were cloned and transformed into flow cytometry in the presence of Flow-Count Fluorospheres (BD) and de- Escherichia coli M15 or BL21 pLys. Polyhistidine-tagged proteins were fined as eFluor 780–negative cells relative to buffer-treated cells. isolated from supernatant of E. coli cell lysates on Ni-nitrilotriacetic acid For C5aR1-competition experiments, human neutrophils were pre- columns (QIAGEN). incubated for 10 min at room temperature with CHIPS (710 nM), mouse 1036 PVL AND HlgCB DIFFERENTIALLY TARGET C5aR1 AND C5aR2 anti-human C5aR1 mAb (clone S5/1, AbD SeroTec; 67 nM), NDT 9513727 HEK cells overexpressing the respective orthologs of human (Tocris, R&D Systems; 10 mM), W-54011 (Calbiochem, Merck Millipore; C5aR1 and C5aR2 for their susceptibility to these two toxins. 10 mM), PMX 205 (Tocris, R&D Systems; 10 mM), or AS-65122 In line with susceptibility of neutrophils, only cells expressing (AnaSpec; 10 mM). Concentrations of C5aR1 antagonists used are based on efficacy titration for PVL and HlgCB. human or rabbit C5aR1 were susceptible toward PVL. As illus- Analysis of pore formation in transiently transfected HEK293T cells was trated in Fig. 1B, cells expressing macaque, cow, or mouse C5aR1 corrected for the fraction of receptor-positive cells. Transiently transfected or were fully resistant toward pore formation induced by PVL. In stably transfected cells were reciprocally compared in functional experiments. accordance with the results obtained with primary cells, cells Equimolar concentrations of toxin protein components were used. expressing different mammalian C5a receptors, with the exception Binding assays of mouse C5aR1, were susceptible toward HlgCB-induced toxicity (Fig. 1B). PVL was cytotoxic toward cells expressing human or For binding experiments with LukS-PV, cells transiently transfected with plasmids encoding receptors with an N-terminal FLAG tag (28) were rabbit C5aR2, whereas mouse and cow C5aR2 were incompatible prelabeled with a mouse anti-FLAG mAb (clone M2, 1:500; Sigma- with PVL (Fig. 1C). In contrast to the resistance of neutrophils, Aldrich), followed by PE-labeled goat–anti-mouse Ab (1:80, Dako). For HEK cells expressing macaque C5aR2 were susceptible to pore experiments with HlgC, stably transfected cells were directly used for formation induced by PVL (Fig. 1C). Just as in humans (28), subsequent binding. After incubation with the protein on ice for 30 min in a volume of 50 ml, cells were washed and incubated with an FITC- expression of C5aR2 on macaque neutrophils is very low com- conjugated mouse anti-his mAb (1:80; LifeSpan Biosciences). Binding pared with expression of C5aR1 (Supplemental Fig. 1Q). Simi- was detected using flow cytometry, and $10,000 cells were analyzed. larly to C5aR1, screening of C5aR2 with HlgCB showed broad Analysis of binding to transiently transfected cells was limited to receptor- species compatibility, again with the exception of mouse C5aR2 positive cells. Binding of the S-components was expressed as the per- (Fig. 1C). Cells transfected with plasmids encoding PVL- and centage of mean fluorescence in relation to saturated binding of the Downloaded from S-component to human C5aR1 at 31 nM. HlgCB-incompatible receptors expressed equal levels of receptors compared with cells transfected with plasmids encoding PVL- or Quantification of leukocyte receptor expression levels HlgCB-compatible receptors (Supplemental Fig. 1A, 1B). Freshly isolated macaque and human neutrophils were incubated in a total Jointly, these analyses demonstrate that the mouse is a poor volume of 50 mlat53 106 cells/ml on ice with 10 mg/ml mouse anti-human model for investigating both PVL and HlgCB as a virulence factor chemokine receptor mAbs for C5aR1 (clone S5/1; AbD SeroTec) or C5aR2 owing to a molecular incompatibility of the toxins with its re- (clone 1D9-M12; BioLegend), followed by PE-conjugated goat–anti-mouse spective receptors. Furthermore, these data show that although PVL http://www.jimmunol.org/ Ab (1:80; Dako). Samples were analyzed using flow cytometry. At least 10,000 cells were analyzed, and cells were gated for forward and side and HlgCB target the complement receptors C5aR1 and C5aR2, scatters. Ab binding was quantified by calibration to defined Ab binding both toxins do so in a species-dependent differential manner. capacity units using QIFIKIT (Dako) and corrected for isotype controls. Different domains of C5aR1 define human specificity of PVL Graphical and statistical analyses and HlgCB Flow cytometric data were analyzed with FlowJo (TreeStar Software). Although C5aR1 and C5aR2 are relatively conserved among Statistical analyses were performed using Prism (GraphPad Software). different mammalian species, amino acid sequence identities vary from 60 to 93% when compared with the human receptors (Fig. 2A, by guest on October 1, 2021 Results 2B). In an attempt to define the regions of the receptors respon- PVL and HlgCB feature distinct species specificity sible for the observed species-specific toxin–receptor interaction, Some leukocidins exert cytotoxic activity in a species-specific we tested cells expressing chimeric C5a receptors for suscepti- manner (21, 24, 26, 28). For PVL, species-specific toxicity is well bility toward PVL and HlgCB. Because most amino acid sequence described (21). Although PVL and HlgCB have shared receptors, divergence between the C5a receptors of the different mammalian species-specific susceptibility of primary cells toward HlgCB is not species is clustered in the N termini and ECLs (Fig. 2C, 2D), we completely understood. To compare species specificity of HlgCB focused on the extracellular domains of the respective receptors. with that of PVL, we tested toxin-mediated cell permeability in Although macaque C5aR1 is most closely related to human freshly isolated neutrophils from different mammalian species, us- C5aR1, this receptor is incompatible with PVL (Figs. 1B, 2C). ing a membrane-impermeant fluorescent dye. Cells from mouse and Therefore, we used macaque and human C5aR1 as chimeric rabbit were investigated, as both mammals are frequently used in templates to investigate the importance of the different extracel- preclinical experimental models of S. aureus infections (21). In lular receptor domains for interaction with PVL on receptor- addition, macaque neutrophils were investigated because this pri- expressing HEK cells. Receptor expression was similar for all mate is closely related to humans. Finally, cow neutrophils were constructs tested (Supplemental Fig. 1C–L). For PVL, exchange included, as this species is a natural host to S. aureus (35). of the N terminus of human C5aR1 with macaque or mouse N- As expected, PVL was active only toward human and rabbit terminal C5aR1 did not result in loss of susceptibility (Fig. 3A). neutrophils, but not toward neutrophils from macaque, cow, and Exchange of the individual human ECL2 or ECL3 with those of mouse (Fig. 1A). However, HlgCB was active toward neutrophils macaque resulted in loss of susceptibility to PVL; however, re- from all species tested, except for mice (Fig. 1A). placement of ECL1 showed no effect (Fig. 3B). Because the These data identify that the rabbit is the only compatible species amino acid sequence of human and macaque ECL1 is nearly for establishing the contribution toward staphylococcal patho- identical (Fig. 2C), we additionally tested a chimeric human physiology of both PVL and HlgCB in vivo. Furthermore, the C5aR1 with mouse ECL1. Cells expressing this receptor were divergent susceptibility of neutrophils from different mammalian resistant to pore formation induced by PVL, just as were cells species toward PVL and HlgCB suggests a differential interaction expressing a human C5aR1 chimera with mouse ECL2 or ECL3 of both toxins with their receptors. (Fig. 3C). Of interest, although replacement of human ECL2 with macaque ECL2 is sufficient to prevent PVL toxicity, the sequences PVL and HlgCB differentially interact with C5aR1 and C5aR2 of ECL2 differ in only a few amino acids (Fig. 2C). Thus, we in a species-dependent manner introduced single amino acid mutations in the ECL2 of human To further investigate the molecular interaction of PVL and HlgCB C5aR1 following the macaque sequence. Cells expressing a P183S with neutrophils from different mammalian species, we screened mutant human C5aR1 were resistant to pore formation induced by The Journal of Immunology 1037

FIGURE 1. The differential interaction of PVL and HlgCB with the C5a receptors C5aR1 and C5aR2 defines the distinct species specificity featured by both toxins. (A) Pore formation in freshly isolated neutrophils from different mammalian species after incubation with PVL or HlgCB shows differential species-specific cytotoxicity of PVL and HlgCB. (B and C) Pore formation following treatment with PVL or HlgCB was monitored in HEK cells transfected with plasmids encoding (B) C5aR1 or (C) C5aR2 of different mammalian species. Pore formation was defined as the percentage of cells positive for intracellular staining by DAPI. Bars indicate SD, with n = 3. For enhanced graphical representation, the dashed lines indi-

cate background pore formation in untransfected HEK cells Downloaded from at 61 nM toxin. http://www.jimmunol.org/

PVL (Fig. 3D), highlighting the critical role of C5aR1-ECL2 in HlgCB (Fig. 4D). Introduction of both human ECL1 and ECL3 human-specific cytotoxicity of PVL. In addition, mutation of the did not further enhance susceptibility of cells toward HlgCB. human amino acid cassette sequence SHDK to the respective These data indicate that specificity of HlgCB for human C5aR1 is macaque sequence NNDT resulted in loss of susceptibility toward mostly defined by ECL3 and ECL1. PVL. Introduction of the individual human ECL2 or ECL3 in Together, these findings demonstrate that PVL and HlgCB by guest on October 1, 2021 macaque C5aR1 did not restore susceptibility of cells toward PVL differentially interact with C5aR1 in a complex manner. Results (Fig. 3E). Cells expressing a macaque C5aR1 chimera with both with chimeric receptors suggest that both PVL and HlgCB interact human ECL2 and ECL3 showed a partially restored susceptibility with multiple extracellular domains of C5aR1. Whereas human toward PVL, just as did cells expressing a mouse C5aR1 chimera C5aR1 specificity of PVL is critically driven by ECL2, human with human ECL1, 2, and 3 (Fig. 3F). This observation suggests C5aR1 specificity of HlgCB is mostly determined by ECL3 and that, in addition to the extracellular domains, other parts of the ECL1. Incomplete functional restoration of macaque and mouse receptor are involved in the interaction with PVL as well. C5aR1 chimeric receptors toward PVL and HlgCB indicate that, in Mouse C5aR1 is the most divergent receptor from the panel of addition to the extracellular domains, other domains of the receptor mammalian C5a receptors investigated, and it is the only HlgCB- are likely involved in the interaction with both toxins as well. incompatible C5aR1 (Figs. 1B, 2C). Thus, we used human and mouse C5aR1 as chimeric templates to study the interaction be- PVL and HlgCB differentially interact with human C5aR2 tween HlgCB and C5aR1. Similar to PVL, exchange of the N In addition to C5aR1, we tested cells expressing chimeric C5aR2 terminus of human C5aR1 with mouse N-terminal C5aR1 did not receptors for susceptibility toward PVL and HlgCB. Mouse C5aR2 is result in loss of susceptibility of cells toward HlgCB (Fig. 4A). In the most divergent receptor from the mammalian C5aR2 receptors contrast to PVL, introduction of mouse ECL2 in human C5aR1 tested, and the only PVL- and HlgCB-incompatible C5aR2 did not affect susceptibility of cells toward HlgCB (Fig. 4B). In- (Fig. 1C). The N terminus of mouse C5aR2 is most divergent troduction of mouse ECL1 or ECL3 in human C5aR1 resulted in compared with the amino acid sequence of other mammalian spe- partial and complete loss of cell susceptibility toward HlgCB- cies (Fig. 2D). Thus, we used human and mouse C5aR2 as chimeric induced pore formation, respectively (Fig. 4B). ECL3 of mouse templates to study the interaction of PVL and HlgCB with receptor- C5aR1 is highly divergent from the human amino acid sequence expressing HEK cells. Receptor expression was similar for all (Fig. 2C). Introduction of single amino acid mutations in the constructs tested (Supplemental Fig. 1L–P). ECL3 of human C5aR1 following the mouse sequence did not Exchange of the N terminus of human C5aR2 with the N terminus completely abolish susceptibility of cells toward HlgCB (Fig. 4C), of mouse C5aR2 resulted in a complete and partial loss of sus- indicating that the interaction between HlgCB and human C5aR1 ceptibility for PVL and HlgCB, respectively (Fig. 5A, 5B). Although involves a combination of multiple amino acid residues in ECL3. introduction of mouse ECL1 and ECL3 into human C5aR2 caused As expected based on the studies with loss of function chimeric a complete loss of susceptibility of cells toward PVL (Fig. 5C), receptors, introduction of human ECL1 in mouse C5aR1 was in- none of the mouse ECLs substantially affected susceptibility toward sufficient to result in susceptibility of cells toward HlgCB HlgCB toxicity (Fig. 5D). Introduction of mouse ECL2 into human (Fig. 4D). However, introduction of human ECL3 in mouse C5aR2 only moderately affected susceptibility of cells toward both C5aR1 resulted in a substantial restoration of susceptibility toward PVL (Fig. 5C). Introduction of individual human ECLs, or all three 1038 PVL AND HlgCB DIFFERENTIALLY TARGET C5aR1 AND C5aR2

FIGURE 2. The complement receptors C5aR1 and C5aR2 are highly conserved between different mammalian species. (A and B) Amino acid substitutions per 100 aas of (A)C5aR1 and (B) C5aR2 of different mam- Downloaded from malian species related to human C5aR1 and C5aR2, with a schematic representation of the seven-transmembrane spanning architecture of C5aR1 and C5aR2. (C and D) Amino acid residue alignments of the (C)C5aR1 and (D) C5aR2 of different mamma- http://www.jimmunol.org/ lian species. Extracellular domains are highlighted, and amino acids divergent from the human sequence are shaded. Amino acid sequence alignments were generated by ClustalW alignment, using Lasergene MegAlign software (DNAStar). by guest on October 1, 2021

human ECLs together, into mouse C5aR2 was insufficient to restore the cell membrane, the role of the receptors during the process of susceptibility toward PVL and HlgCB (Fig. 5E, 5F). Additional binding, oligomerization, and pore formation itself has not been fully introduction of the human N terminus did not result in PVL and elucidated. This fact led us to investigate, in addition to pore for- HlgCB susceptibility either (Fig. 5E, 5F). mation, the binding of LukS-PV and HlgCB (HlgC) to an exemplary These findings indicate that PVL and HlgCB interact with human selection of HEK cells overexpressing chimeric C5a receptors. C5aR2 in a different manner. As the introduction of all human Fig. 6A confirms that LukS-PV cannot bind to HEK cells extracellular domains in mouse C5aR2 did not result in suscep- expressing macaque C5aR1, in contrast to human C5aR1 (28). In tibility of cells toward both toxins, other parts of the receptor are line with the loss of pore-forming capacity of PVL (Fig. 6B), clearly also involved in the interaction with the toxins. introduction of macaque ECL2 in human C5aR1 resulted in a complete loss of detectable binding of LukS-PV (Fig. 6A). Al- S-component binding to the receptor does not necessarily allow though a replacement in macaque C5aR1 with human ECL2 did subsequent pore formation not restore the pore-forming ability of PVL (Fig. 6B), binding of Recently, it was identified that staphylococcal leukocidins act via cell LukS-PV to cells expressing this chimeric receptor was similar surface receptors (24–26, 28). Although expression of the specific when compared with wild-type human C5aR1 (Fig. 6A). Simi- receptor is a prerequisite for the bicomponent toxins to form pores in larly, HlgC could not bind to HEK cells expressing mouse C5aR1 The Journal of Immunology 1039 Downloaded from http://www.jimmunol.org/ FIGURE 3. ECL2 of C5aR1 drives human specificity of PVL. Pore formation by PVL in transfected HEK cells expressing (A) macaque or mouse N-terminal chimeric receptors with human C5aR1 as the core, (B) macaque ECL exchange chimeric receptors with human C5aR1 as the core, (C) mouse ECL exchange chimeric receptors with human C5aR1 as the core, (D) ECL2 mutant receptors with human C5aR1 as the core, (E) human ECL exchange chimeric receptors with macaque C5aR1 as the core, and (F) human ECL exchange chimeric receptors with mouse C5aR1 as the core. For graphical support, parts of the receptors are colored black for human domains, red for macaque domains, and green for mouse domains in the schematic representations. Pore formation was defined as the percentage of cells positive for intracellular staining by DAPI. Bars indicate SD, with n = 3–5. For the highest toxin concentrations, statistical significance is displayed as ns. **p , 0.01, ***p , 0.005 using two-way ANOVA with Bonferroni posttest correction for multiple comparison.

(Fig. 6C). Partial restoration of susceptibility toward HlgCB cy- resulted in a nearly complete loss of susceptibility toward HlgCB by guest on October 1, 2021 totoxicity in cells expressing a mouse C5aR1 with human ECL3 toxicity (Fig. 6D), HlgC binding was still observed (Fig. 6C). (Fig. 6D) was associated with suboptimal binding of HlgC when These data show, as an example, that binding of the S-component compared with cells expressing wild-type human C5aR1 to the speciﬁc receptor itself is not necessarily sufﬁcient to allow (Fig. 6C). Although introduction of mouse ECL3 in human C5aR1 subsequent pore formation.

FIGURE 4. ECL3 of C5aR1 drives human specificity of HlgCB. Pore formation by HlgCB in transfected HEK cells expressing (A) mouse N-terminal chimeric receptors with human C5aR1 as the core, (B) mouse ECL exchange chimeric receptors with human C5aR1 as the core, (C) ECL3 mutant receptors with human C5aR1 as the core, and (D) human ECL exchange chimeric receptors with mouse C5aR1 as the core. For graphical support, parts of the receptors are colored black for human domains and green for mouse domains in the schematic representations. Pore formation was defined as the percentage of cells positive for intracellular staining by DAPI. Bars indicate SD, with n = 3–5. For the highest toxin concentrations, statistical significance is displayed as ns. *p , 0.05, **p , 0.01, ***p , 0.005 using two-way ANOVAwith Bonferroni posttest correction for multiple comparison or Student t test where appropriate. 1040 PVL AND HlgCB DIFFERENTIALLY TARGET C5aR1 AND C5aR2 Downloaded from http://www.jimmunol.org/

FIGURE 5. PVL and HlgCB differentially interact with the extracellular domains of C5aR2. Pore formation by PVL and HlgCB in transfected HEK cells expressing (A and B) mouse N-terminal chimeric receptors with human C5aR2 as the core, (C and D) mouse ECL exchange chimeric receptors with human C5aR2 as the core, and (E and F) human ECL exchange chimeric receptors with mouse C5aR2 as the core. For graphical support, parts of the receptors are colored black for human domains and green for mouse domains in the schematic representations. Pore formation was deﬁned as the percentage of cells positive for intracellular staining by DAPI. Bars indicate SD, with n = 3–5. For the highest toxin concentrations, statistical signiﬁcance is displayed as ns. *p , 0.05, ***p , 0.005 using two-way ANOVA with Bonferroni posttest correction for multiple comparison or Student t test where appropriate.

Toxicity of PVL, but not of HlgCB, is neutralized by specific Together, these data show that although PVL and HlgCB both by guest on October 1, 2021 C5aR1 antagonists target the human C5a receptors, only toxicity of PVL can be an- C5aR1 is considered the major host receptor for PVL, and C5aR1 tagonized in vitro by C5aR1 competition on human neutrophils. competition interferes with PVL toxicity on human neutrophils This observation provides further evidence that both toxins dif- (28). Because PVL and HlgCB target the human C5a receptors in ferentially interact with human C5aR1. a specific but divergent manner, we investigated whether toxicity of PVL and HlgCB toward human neutrophils can be equally Discussion neutralized by different C5aR1 antagonists in vitro. Susceptibility of host cells to staphylococcal leukocidins is de- As observed before, pretreatment of neutrophils with CHIPS termined by surface expression of specific receptors (24–28). Both (31), which binds human C5aR1, significantly shifted the EC50 of PVL and HlgCB interact with their shared receptors, C5aR1 and PVL (Fig. 7A, 7D). However, CHIPS could not neutralize toxicity C5aR2, in a host-adapted manner (24, 28). C5aR1 is highly of HlgCB (Fig 7A, 7D). The staphylococcal protein FLIPr-Like, expressed on phagocytic cells. Compared with C5aR1, expression targeting two unrelated receptors, did not confer protection to of C5aR2 on the surface of neutrophils is very low (28, 40, 41). neutrophils for either of the toxins. Pretreatment of neutrophils Although macaque C5aR2 is compatible with PVL, macaque with a mAb, directed toward the N terminus of human C5aR1 neutrophils are resistant to the toxin. Incompatibility of macaque (clone S5/1), resulted in a minimal shift of the EC50 for PVL, but, C5aR1 with PVL apparently drives resistance of macaque neu- again, not for HlgCB (Fig 7B, 7D). In the next step, we tested the trophils toward the toxin. For this reason, the abundantly potency of different commercially available C5aR1 antagonists to expressed C5aR1 can be considered the major host receptor for neutralize PVL and HlgCB toxicity. NDT 9513727 and W-54011 PVL, and presumably for HlgCB as well. are small molecules with potent C5aR1 antagonistic properties Although PVL and HlgCB both target C5aR1 and C5aR2, the (36, 37). PMX 205 and AS-65122 are C5a-mimetic peptides toxins interact with these receptors in a divergent manner. Although blocking the binding of C5a (38, 39). Although pretreatment of multiple receptor domains are involved in the interaction with the human neutrophils with all compounds resulted in 5- to 10-fold toxins, human C5aR1 specificity is mostly defined by ECL2 for increases in EC50 values for PVL, none of the compounds reduced PVL, and ECL3 and ECL1 for HlgCB. Although HlgCB tolerates susceptibility of cells toward HlgCB (Fig 7C, 7D). Remarkably significant variations in the C5a receptors, receptor compatibility of and in contrast to the other C5aR1-antagonists tested, both small PVL is more restricted. Tolerance of receptor variations by HlgCB molecules NDT 9513727 and W-54011 significantly enhanced is illustrated by a substantial restoration of cytotoxic susceptibility susceptibility to HlgCB, and pretreatment of neutrophils with of mouse C5aR1 chimera with human ECL3, which still harbors these molecules resulted in 5-fold decreased EC50 values for significant amino acid sequence divergence in the other extracel- HlgCB (Fig. 7C, 7D). lular domains when compared with human C5aR1. In contrast to The Journal of Immunology 1041 Downloaded from http://www.jimmunol.org/

FIGURE 6. S-component binding to the receptor does not necessarily allow subsequent pore formation. (A and B) Binding of LukS-PV and pore formation by PVL in HEK cells expressing wild-type human C5aR1, macaque C5aR1, or chimeric receptors with exchange of ECL2. (A) Binding of LukS-PV expressed in relation to saturated binding of LukS-PV to human C5aR1 as observed at 31 nM. (B) Pore formation by PVL (204 nM). (C and D) Binding of HlgC and pore formation by HlgCB in HEK cells expressing wild-type human C5aR1, mouse C5aR1, or chimeric receptors with exchange of ECL3. (C) Binding of HlgC expressed in relation to saturated binding of HlgC to human C5aR1 as observed at 31 nM. (D) Pore formation by HlgCB (24 nM). For graphical support, parts of the receptors are colored black for human domains, red for macaque domains, and green for mouse domains in the schematic representations. Pore formation was deﬁned as the percentage of cells positive for intracellular staining by DAPI. Bars indicate SD, with n = 3–5. For , ,

binding studies at the highest protein concentration and for pore formation, statistical significance is displayed as **p 0.01, *** p 0.005 using two-way by guest on October 1, 2021 ANOVA with Bonferroni posttest correction for multiple comparison. this observation, restricted receptor compatibility of PVL is re- mation by PVL and HlgCB potentially involves conformational flected by the incompatibility of PVL with macaque C5aR1, which changes of toxin and receptor, which are mediated by the trans- has high amino acid sequence identity with human C5aR1. S. membrane regions of the receptors. Whether the interaction be- aureus is well adapted to the human host, and high-affinity tween receptor and toxin is limited to the S-component only or protein–protein interactions are human specific for many of its directly involves the F-component as well remains to be deter- virulence factors (4). Whereas HlgCB is core-genome encoded mined. (22, 23), PVL is encoded on a mobile genetic element (42). It is The contribution of PVL and HlgCB to staphylococcal patho- possible that PVL is a result of the adaptation of S. aureus to the physiology is not completely understood. Our data underscore human host. previous observations that the mouse is a suboptimal mammalian For PVL and HlgCB, multiple extracellular domains of both species to investigate PVL and HlgCB in vivo (21, 24). Use of C5aR1 and C5aR2 are involved during the formation of lytic pores. rabbits or transgenic mice offers opportunities for future studies to However, different parts of the receptor mediate initial S- explore the relative contribution to pathophysiology of both tox- component binding and subsequent pore formation of the leuko- ins. Despite many attempts over decades, a safe and effective cidin, as illustrated by LukS-PV and HlgC for C5aR1. This finding vaccine against S. aureus is still not available. Again, use of ap- suggests the occurrence of conformational changes of receptors and propriate animal models for staphylococcal infections is essential toxins during the process of binding, hetero-oligomerization, and for future vaccine development. Owing to the increasing resis- pore formation. Introduction of human extracellular domains into tance of S. aureus to traditional antimicrobial agents, innovative macaque and mouse C5aR1 only partially restored susceptibility of therapeutics are urgently needed. One approach is the develop- cells toward both PVL- and HlgCB toxicity. For C5aR2, no gain of ment of toxin-neutralizing mAbs (43). The complement and function could be achieved in a similar approach. These findings chemokine receptors, which are highly expressed on phagocytic imply that interactions of PVL and HlgCB with C5aR1 and C5aR2 cells and targeted by the staphylococcal leukocidins, are, poten- not only are mediated by the extracellular domains of the receptors tially, host candidate drug targets (44, 45). The divergent inter- but involve other parts of the receptors as well. The ligand of the action of PVL and HlgCB with the C5a receptors is illustrated by C5a receptors, the complement component C5a, is known to in- the differential competitive potency of various C5aR1 antagonists teract with the receptor N termini and ECLs, but also with binding on human neutrophils. Although several C5aR1-binding mole- pockets formed by hydrophobic residues of the receptor trans- cules reduced the toxicity of PVL, none of the molecules tested membrane regions (29). The multifaceted sequence of pore for- had a protective effect on HlgCB cytotoxicity. Although CHIPS 1042 PVL AND HlgCB DIFFERENTIALLY TARGET C5aR1 AND C5aR2

FIGURE 7. Specific C5aR1 antagonists protect human neutrophils from cytotoxicity induced by PVL, but not HlgCB. Pore formation inhibition in human neu- Downloaded from trophils by preincubation with (A) the staphylococcal proteins CHIPS (710 nM) or FLIPr-Like (833 nM); (B) the mouse anti-human C5aR1 mAb clone S5/1 or IgG2a-k isotype control (both 67 nM); or (C) the C5aR1-antagonists NDT 9513727, W-54011, PMX 205, or AS-65122 (all 10 mM). (D) Pore formation http://www.jimmunol.org/ inhibition expressed as EC50 for PVL and HlgCB. Concentrations of C5aR1 antagonists used in this study are based on efficacy titration for PVL. Pore formation was defined as the percentage of cells positive for intracellular staining by DAPI. Bars indicate SD, with n = 5–8. For comparison of EC50 values, statistical significance is displayed as *p , 0.05, ***p , 0.005 using two-way ANOVA with Bonferroni posttest correction for multiple comparison. by guest on October 1, 2021

antagonizes the proinflammatory responses of neutrophils induced HlgCB (47). Our data provide a proof of concept for the protective by sublytic concentrations of both PVL and HlgCB (24, 28), at potential of small C5aR1-antagonizing molecules against PVL lytic concentrations, CHIPS could only antagonize PVL but not toxicity. Future additional screening of other compounds is needed HlgCB. The molar ratios between HlgCB and CHIPS likely de- to identify molecules antagonizing cytotoxicity of both PVL and termine the competitive potency of the antagonist. Intriguingly, HlgCB. From there, the efficacy of molecules blocking the toxin– two small-molecule C5aR1 antagonists substantially potentiated receptor interaction could be investigated in improved animal susceptibility of neutrophils toward HlgCB. In line with this models for infection. finding, others have recently observed an amplification of calcium mobilization induced by sublytic concentrations of HlgCB in the Acknowledgments presence of one of these antagonists (46). Likely, distinctive in- We thank Marie Daoud El-Baba (IUT Lyon 1, Lyon, France), Fabrice terference of the various C5aR1 antagonists with the conforma- Taborik (Cynbiose, Marcy l’Etoile, France), and Manouk Vrieling (UMC tional state of the receptor affects the susceptibility of cells toward Utrecht, Utrecht, the Netherlands) for providing rabbit, macaque, and bo- The Journal of Immunology 1043 vine blood, and Kaila Bennett and Ron Gorham (University Medical Cen- 25. Alonzo, F., III, L. Kozhaya, S. A. Rawlings, T. Reyes-Robles, A. L. DuMont, ter Utrecht) for critically reviewing the manuscript. D. G. Myszka, N. R. Landau, D. Unutmaz, and V. J. Torres. 2013. CCR5 is a receptor for Staphylococcus aureus leukotoxin ED. Nature 493: 51–55. 26. DuMont, A. L., P. Yoong, C. J. Day, F. Alonzo, III, W. H. McDonald, Disclosures M. P. Jennings, and V. J. Torres. 2013. Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1. The authors have no financial conflicts of interest. Proc. Natl. Acad. Sci. USA 110: 10794–10799. 27. Reyes-Robles, T., F. Alonzo, III, L. Kozhaya, D. B. Lacy, D. Unutmaz, and V. J. Torres. 2013. Staphylococcus aureus leukotoxin ED targets the chemokine References receptors CXCR1 and CXCR2 to kill leukocytes and promote infection. Cell 1. Lowy, F. D. 1998. Staphylococcus aureus infections. N. Engl. J. Med. 339: 520– Host Microbe 14: 453–459. 532. 28. Spaan, A. N., T. Henry, W. J. van Rooijen, M. Perret, C. Badiou, P. C. Aerts, 2. DeLeo, F. R., M. Otto, B. N. Kreiswirth, and H. F. Chambers. 2010. Community- J. Kemmink, C. J. de Haas, K. P. van Kessel, F. Vandenesch, et al. 2013. The associated methicillin-resistant Staphylococcus aureus. 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