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 Why The JI? Submit online. by guest on October 1, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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