Staphylococcus aureus Metalloprotease Aureolysin Cleaves Complement C3 To Mediate Immune Evasion This information is current as Alexander J. Laarman, Maartje Ruyken, Cheryl L. Malone, of September 29, 2021. Jos A. G. van Strijp, Alexander R. Horswill and Suzan H. M. Rooijakkers J Immunol 2011; 186:6445-6453; Prepublished online 18 April 2011; doi: 10.4049/jimmunol.1002948 Downloaded from http://www.jimmunol.org/content/186/11/6445 Supplementary http://www.jimmunol.org/content/suppl/2011/04/18/jimmunol.100294 Material 8.DC1 http://www.jimmunol.org/ References This article cites 56 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/186/11/6445.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • 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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Staphylococcus aureus Metalloprotease Aureolysin Cleaves Complement C3 To Mediate Immune Evasion Alexander J. Laarman,* Maartje Ruyken,* Cheryl L. Malone,† Jos A. G. van Strijp,* Alexander R. Horswill,† and Suzan H. M. Rooijakkers* Complement is one of the first host defense barriers against bacteria. Activated complement attracts neutrophils to the site of in- fection and opsonizes bacteria to facilitate phagocytosis. The human pathogen Staphylococcus aureus has successfully developed ways to evade the complement system, for example by secretion of specific complement inhibitors. However, the influence of S. aureus proteases on the host complement system is still poorly understood. In this study, we identify the metalloprotease aureolysin as a potent complement inhibitor. Aureolysin effectively inhibits phagocytosis and killing of bacteria by neutrophils. Furthermore, we show that aureolysin inhibits the deposition of C3b on bacterial surfaces and the release of the chemoattractant C5a. Cleavage analyses show that aureolysin cleaves the central complement protein C3. Strikingly, there was a clear difference Downloaded from between the cleavages of C3 in serum versus purified conditions. Aureolysin cleaves purified C3 specifically in the a-chain, close to the C3 convertase cleavage site, yielding active C3a and C3b. However, in serum we observe that the aureolysin-generated C3b is further degraded by host factors. We pinpointed these factors to be factor H and factor I. Using an aureolysin mutant in S. aureus USA300, we show that aureolysin is essential and sufficient for C3 cleavage by bacterial supernatant. In short, aureolysin acts in synergy with host regulators to inactivate C3 thereby effectively dampening the host immune response. The Journal of Immu- nology, 2011, 186: 6445–6453. http://www.jimmunol.org/ he complement system is a proteolytic cascade of plasma proteins. Human plasma contains several soluble regulators that proteins, which is crucial to the host defense against in- accelerate the dissociation of C3 convertases and/or promote in- T vading bacteria (1–3). Recognition of bacteria via the activation of C4b and C3b (C4 binding protein, factor I [fI], and classical pathway (CP) or lectin pathway (LP) results in formation factor H [fH]). Further, host cells express membrane-bound of the C3 convertase complex (C4b2a) that cleaves C3, the central complement regulators that dissociate C3 convertases (mem- molecule of the complement system. Cleavage of C3 by con- brane cofactor protein; decay accelerating factor) or prevent in- vertases is a critical event in complement activation, as it leads to tegration of the C5b-9 complex (CD59). by guest on September 29, 2021 release of the anaphylatoxin C3a and deposition of C3b on the Bacterial pathogens have acquired strategies to resist various bacterial surface. C3b molecules effectively opsonize the bacte- parts of the complement system. Different bacterial complement rium and facilitate a series of events: 1) activation of the alter- evasion strategies have been identified: 1) production of a capsule native pathway (AP) to amplify the cleavage of C3 via AP C3 to prevent complement recognition, 2) attraction of host comple- convertases (C3bBb), 2) formation of the C5 convertases gener- ment regulators to the bacterial surface, or 3) secretion of proteins ating C5b-9 complexes (membrane attack complex) and the potent that inactivate complement proteins (6–8). Among these secreted anaphylatoxin C5a that attracts and activates phagocytes, 3) factors we find a number of proteases that can cleave complement phagocytosis via recognition of C3b by complement receptors on (9). Staphylococcus aureus is a human pathogen that causes a wide neutrophils, and 4) Ag presentation (4, 5). To protect host cells, range of infections including endocarditis, pneumonia, sepsis, the complement cascade is tightly regulated by various regulatory and toxic shock syndrome (10). During the past decade, the pre- valence and pathological problems of S. aureus increased due *Medical Microbiology, University Medical Center Utrecht, 3584 CX Utrecht, The to the emergence of antibiotic-resistant strains. Its success as a Netherlands; and †Department of Microbiology, Roy J. and Lucille A. Carver College human pathogen also depends on the production of many viru- of Medicine, University of Iowa, Iowa City, IA 52242 lence factors. For instance, S. aureus expresses various surface Received for publication August 31, 2010. Accepted for publication February 23, proteins that promote adhesion to extracellular matrices (micro- 2011. bial surface components recognizing adhesive matrix molecules; This work was supported by grants from The Netherlands Organization for Scientific Ref. 11), colonization, or biofilm formation (12). Further, S. au- Research (NWO-TOP and NWO-Vidi to A.J.L., J.A.G.v.S., and S.H.M.R.), by fund- ing from the European Molecular Biology Organization (to S.H.M.R.), and by Award reus uses both secreted and surface proteins, which are known to AI078921 from the National Institute of Allergy and Infectious Diseases (to A.R.H.). promote lysis of host cells (13) or block the host immune response Address correspondence and reprint requests to Mr. Alexander J. Laarman, Depart- (7, 14, 15). In S. aureus, a large arsenal of complement evasion ment of Medical Microbiology, University Medical Center Utrecht, PO G04.614, molecules have been identified. These include secreted factors that Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail address: a.laarman@ umcutrecht.nl block several steps of the complement cascade: staphylococcal The online version of this article contains supplemental material. superantigen-like protein 10 inhibits CP activation (16); staphylo- Abbreviations used in this article: AP, alternative pathway; CHIPS, chemotaxis in- coccal complement inhibitor (SCIN) (17), extracellular fibrinogen- hibitory protein of S. aureus; CP, classical pathway; fB, factor B; fD, factor D; fH, binding protein, and extracellular complement-binding protein factor H; fI, factor I; GelE, gelatinase E; HSA, human serum albumin; LP, lectin block C3/C5 conversion by convertases (18); staphylococcal pathway; SCIN, staphylococcal complement inhibitor; THB, Todd–Hewitt broth. superantigen-like protein 7 inhibits C5 conversion (19); and Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 chemotaxis inhibitory protein of S. aureus (CHIPS) inhibits C5a- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002948 6446 S. AUREUS METALLOPROTEASE AUREOLYSIN CLEAVES COMPLEMENT C3 dependent neutrophil chemotaxis by binding to the C5a receptor alyzed by flow cytometry (FACSCalibur; Becton Dickinson) measuring (C5aR) (20). Next to these secreted molecules, S. aureus also fluorescence of 10,000 gated neutrophils. The complement-independent produces several surface proteins that interact with the comple- phagocytosis assay was performed as described above, but instead of normal human serum, we used heat-inactivated serum. For bacterial killing ment system: staphylococcal IgG-binder binds both IgG and C3 to assays, Group B Streptococcus was grown to an OD660 of 0.5 in THB and prevent classical and alternative complement activation (21, 22), subsequently washed in RPMI containing 0.1% HSA. Then, we pre- and clumping factor A and the iron-regulated surface determinant incubated 5% serum with 0.5 mM BSA, SCIN, or aureolysin for 20 min at 3 5 3 6 protein H promote degradation of opsonic C3b (23, 24). 37˚C. Subsequently, 2.5 10 Group B Streptococcus and 8.5 10 neutrophils were added and incubated at 37˚C. At different time points Even though S. aureus produces a number of proteases, their a sample was taken, and neutrophils were lysed with Milli-Q. Surviving role in bacterial complement evasion is not known. We studied bacteria were enumerated by plating serial dilutions on Todd–Hewitt agar. a role for the metalloprotease