Mediate Immune Evasion Aureolysin Cleaves Complement C3 To

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

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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 © 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 infection 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 aureolysin in complement escape by S. aureus. Aureolysin is a 301-aa zinc-dependent metalloprotease C3b deposition on S. aureus that belongs to the family of thermolysins (25). Aureolysin was S. aureus strain KV27 was grown to an OD660 of 0.5 in THB and washed in ++ previously shown to have a role in staphylococcal immune escape HEPES buffer (20 mM HEPES, 140 mM NaCl, 5 mM CaCl2, 2.5 mM m as it cleaves the antimicrobial peptide LL-37. In this study, we MgCl2) with 0.1% BSA. Serum was preincubated with 0.5 M BSA or aureolysin for 20 min at 37˚C. Then, we incubated 12.5 3 105 bacteria describe an important role for aureolysin in inhibition of the with the preincubated serum for 30 min while shaking at 900 rpm. Bacteria complement system. were washed with PBS with 0.1% BSA. C3b deposition was detected using mouse anti-human C3d Abs (Quidel) and FITC-conjugated goat anti- mouse IgG (Protos). Fluorescence of 10,000 bacteria was measured by Materials and Methods flow cytometry. C3b deposition with purified components was performed Downloaded from Proteins and sera as described above, but instead of using serum, we incubated bacteria with 0.6 mM C3, 20 nM fD, 50 nM fB, and 60 nM C3b in the presence or Aureolysin, purified from S. aureus supernatant, was purchased from Bio- m Centrum and further purified by gel filtration on a Superdex 75 column absence of 0.5 M aureolysin. (GE Healthcare) using the AktaExplorer system (GE Healthcare). Protease C5a analysis activity was checked by gelatin zymography (26). Purified aureolysin was subjected to SDS-PAGE and verified by mass spectrometry. Heat- S. aureus strain KV27 was grown to an OD660 of 0.5 in THB. Bacteria inactivated aureolysin was made by incubating aureolysin for 20 min at were heat-killed for 30 min at 70˚C and washed in RPMI with 0.1% HSA. 65˚C. SCIN and CHIPS were produced as recombinant proteins as de- Then, 10% serum and 0.5 mM BSA or aureolysin were preincubated for 15 http://www.jimmunol.org/ scribed (17, 27). C3, C3b, and factor B (fB) were purified from human min at 37˚C and subsequently incubated with 2.5 3 106 bacteria at 37˚C plasma as described (28). The purified components factor D (fD), fH, fI, for 30 min while shaking at 600 rpm. Bacteria were centrifuged, and C5a C3a, and C5 were purchased from Quidel. Complement-depleted sera was detected in collected supernatants by calcium mobilization: 10-fold (DC2, DC3, and DC5) were purchased from Quidel. Normal human serum diluted supernatants were added to 7.5 3 104 fluo-4-AM–labeled U937- was obtained from healthy volunteers, who gave informed consent. Heat- C5a receptor cells (U937-C5aR; a generous gift from Prof. Eric Prossnitz, inactivated serum was made by incubating serum for 20 min at 56˚C. University of New Mexico), and the increase of intracellular calcium was measured by flow cytometry. To study specificity for C5a, activation was Bacterial strains and supernatants performed as described above but using 10% C5-depleted serum, supple- Bacterial strains. S. aureus USA300 strain UAMS-1182 (a generous gift mented with or without 10 mg/ml C5. To block C5aR on cells, cells were from Dr. Nina van Sorge, University of California, San Diego), Group B pretreated with 10 mg/ml CHIPS for 5 min at room temperature before use by guest on September 29, 2021 Streptococcus wild-type strain COH1 (29), and the S. aureus clinical in the calcium mobilization assay. isolate (KV27) obtained within the University Medical Center Utrecht were used. All bacteria were cultivated on tryptic soy agar. Complement assays S. aureus aureolysin mutant. The pKOR1-Daur plasmid (30) was trans- Complement ELISAs were performed as described (33) with modifica- duced with phage 80a into USA300 strain UAMS-1182. A markerless aur tions. ELISA plates (Maxisorb; Nunc) were coated overnight with 20 mg/ml deletion was constructed using the pKOR1 knockout protocol as pre- LPS (Salmonella enteritidis; Sigma), 3 mg/ml IgM (Quidel), or 10 mg/ml viously described (31). To construct an aur complementing clone, plasmid mannan (Saccharomyces cerevisiae; Sigma) in 0.1 M sodium carbonate pDB59 (32) was digested with BamHI and EcoRI to remove the P3-YFP buffer, pH 9.6. Plates were blocked with 4% BSA in PBS with 0.05% fusion. An NheI site was introduced by annealing oligonucleotides Tween for 1 h at 37˚C. Samples were diluted in GHEPES++ buffer (20 mM CLM525 (59-GATCCATCGATCGATGCTAGCATTCGATCATG-39) and HEPES, 140 mM NaCl, 0.1% gelatin, 5 mM CaCl2, 2.5 mM MgCl2, 0.05% CLM526 (59-AATTCATGATCGAATGCTAGCATCGATCGATG-39) and Tween). Serum or C2-depleted serum was preincubated with 0.5 mM BSA ligating into the cut pDB59, yielding intermediate plasmid pCM46. The or aureolysin for 20 min at 37˚C and subsequently added to the plates for 1 aur gene was amplified by PCR using oligonucleotides CLM529 (59- h at 37˚C. Deposited C3b and C5b-9 were detected using Abs against C3d GTTGTTGGATCCGTTATCTCACATATTTCAAGCATTG-39)andCLM522 and C6, respectively (Quidel), followed by peroxidase-conjugated goat (59-GTTGTTGCTAGCGGTATACCCCATATATAAGCTG-39) using USA300 anti-mouse IgG (Southern Biotechnology). as template. The PCR product was digested by BamHI and NheI and ligated into pCM46 digested by the same enzymes to yield aur complementing C3 analysis clone pCM47. N-terminal sequencing. C3 (1 mM) was incubated with 0.5 mM aureolysin Supernatants. S. aureus USA300, the isogenic aureolysin mutant, and the for 60 min at 37˚C in HEPES++ buffer. Proteins were subjected to SDS- isogenic aureolysin mutant complemented with aureolysin were cultured PAGE and transferred to a polyvinylidene difluoride membrane. Cleavage overnight in Todd–Hewitt broth (THB) and subsequently diluted to an products were visualized with 0.1% Coomassie blue in 40% methanol, OD660 of 0.05 in fresh THB. Bacteria were grown to an OD660 of 1.5 while excised, and analyzed by N-terminal sequencing (Alphalyse). shaken at 37˚C, and supernatants were collected by centrifugation and Instant blue staining. C3 cleavage was analyzed by incubation of 1 mMC3 passed through an 0.45-mm filter. Collected supernatants were concen- with aureolysin or bacterial supernatants for 1 h at 37˚C in HEPES++ trated 10 times using Amicon Ultra-10 filters (MWCO 10 kDa; Millipore), buffer. The reaction was stopped at different time points by adding sample dialyzed against PBS and stored at 280˚C. Upon usage, bacterial super- buffer containing DTT. Samples were subjected to SDS-PAGE and visu- natants were diluted for functional analysis. alized by instant blue (Gentaur). Phagocytosis and killing Western blotting. C3 cleavage analysis by Western blotting was performed by incubation of 5% serum or 0.6 mM C3 with 0.5 mM aureolysin at 37˚C Phagocytosis assays were performed as described (17). In short, serum was in HEPES++ buffer. The reaction was stopped at different time by adding preincubated with 0.5 mM BSA, SCIN, or aureolysin in RPMI containing sample buffer containing DTT. All samples were subjected to SDS-PAGE 0.1% human serum albumin (HSA) for 15 min at 37˚C. Then, 2.5 3 105 and blotted onto a polyvinylidene difluoride membrane. After blocking freshly isolated human neutrophils and 2.5 3 106 FITC-labeled, heat- with 4% skimmed milk in PBS containing 0.1% Tween, C3 was detected killed S. aureus KV27 were added and incubated for 15 min at 37˚C by a goat anti-human C3 Ab (Protos), followed by a donkey anti-goat IgG while shaking at 600 rpm. The reaction was stopped by adding 1% ice-cold Ab (Jackson) diluted in PBS with 0.1% Tween and 1% skimmed milk. paraformaldehyde in RPMI containing 0.1% HSA. Phagocytosis was an- C3b2SN degradation was analyzed by incubating 0.6 mM C3 with 0.5 mM The Journal of Immunology 6447 aureolysin for 30 min at 37˚C. The reaction was stopped by adding 50 mM positive bacteria (Fig. 1D). Aureolysin strongly prevented bacte- 2SN EDTA. Generated C3b and 0.6 mM C3b were incubated with 5% C3- rial killing indicating that aureolysin helps bacteria to resist depleted serum, and samples were immunoblotted as described above. To complement-mediated killing by neutrophils. In summary, aur- identify the serum component, various combinations of 0.6 mM purified C3, 45 nM fI, 27 nM fH, and 0.5 mM aureolysin were incubated for 30 min eolysin blocks complement-dependent phagocytosis and killing of at 37˚C in HEPES++ buffer and immunoblotted as described above. bacteria by human neutrophils. C3a analysis Aureolysin inhibits C3b deposition and C5a generation S. aureus strain KV27 was washed in RPMI with 0.1% HSA and incubated Complement activation is crucial to phagocytosis because it results with 10% C5-depleted serum, buffer, and/or 0.5 mM aureolysin for 20 min in the labeling of bacteria with opsonins (C3b molecules) that are at 37˚C. Bacteria were centrifuged, and the supernatants were analyzed recognized by phagocyte receptors. To study whether aureolysin by Western blotting as described earlier but using rabbit anti-C3a serum (Calbiochem) and goat anti-rabbit IgG Ab (Southern Biotech). C3a- blocks complement activation, we first analyzed whether aur- mediated neutrophil activation was performed by incubating 0.6 mMC3 eolysin affects opsonization of bacteria with C3b molecules. S. or C3a with 0.5 mM aureolysin in RPMI containing 0.1% HSA for 30 min aureus was incubated with serum in the presence or absence of at 37˚C. Calcium mobilization was measured as described earlier by aureolysin, and surface-bound C3b was detected on the bacterial 3 5 adding 10-fold diluted sample to 1 10 freshly isolated human neu- surface with specific Abs and flow cytometry. Fig. 2A shows that trophils labeled with fluo-4-AM. aureolysin potently blocks the labeling of bacteria with C3b, again indicating that aureolysin is a complement inhibitor. Downstream Results of C3 cleavage, the complement cascade continues with the Aureolysin blocks phagocytosis and killing of bacteria cleavage of C5 into C5a and C5b. C5a binds to the G protein- Downloaded from To test whether aureolysin is involved in complement evasion by coupled C5a receptor (C5aR) on phagocytes, crucial to phagocyte S. aureus, we first studied its activity in a phagocytosis assay. activation and chemotaxis. Recent studies demonstrated an im- Therefore, we incubated fluorescently labeled S. aureus with portant role for C5a in host protection against S. aureus infections isolated human neutrophils in the presence of human serum and (34). To study whether aureolysin blocks C5a formation, we an- aureolysin. BSA and SCIN served as a negative and positive alyzed the formation of C5a during incubation of S. aureus with

control, respectively. Fig. 1A shows that aureolysin potently human serum and aureolysin. Because C5a is released into the http://www.jimmunol.org/ inhibits phagocytosis and that its activity is comparable with that extracellular milieu, we collected supernatants of serum-opson- of the complement inhibitor SCIN. Aureolysin blocks phagocy- ized bacteria and analyzed these supernatants for their potency tosis in a dose-dependent fashion (Fig. 1B). In 10% serum (con- to induce a calcium mobilization in U937 cells transfected with taining ∼0.5 mM C3), the IC50 of aureolysin is ∼0.1 mM implying the C5a receptor (U937-C5aR). To prevent formation of other a molar ratio of aureolysin/C3 of 1:5 at the IC50. To test whether chemoattractants during the incubation of bacteria with serum, the antiphagocytic effect was a result of complement inhibition, we used heat-killed S. aureus. We observed that supernatants of we performed the phagocytosis in complement-inactivated serum serum-opsonized S. aureus increased calcium levels in U937- and found no inhibition by aureolysin; indicating that aureolysin C5aR cells, and this response could be blocked by pretreatment specifically inhibits complement-mediated phagocytosis (Fig. 1C). of cells with the C5aR antagonist CHIPS (Supplemental Fig. 1). by guest on September 29, 2021 To determine whether aureolysin also blocks bacterial killing, Also, supernatants of bacteria incubated with C5-deficient serum we performed killing assays using isolated neutrophils, human could not induce a calcium flux, and this could be restored by serum, and Group B Streptococcus as a model organism for Gram- repletion of serum with purified C5 (Supplemental Fig. 1). Thus,

FIGURE 1. Aureolysin blocks phagocytosis and killing of bacteria. A–C, Phagocytosis of heat-killed S. aureus by human neutrophils in the presence of human serum and aureolysin. A, Aureolysin blocks phagocytosis of bacteria in human serum (aureolysin, BSA, and SCIN at 0.5 mM). B, Dose-dependent inhibition of phagocytosis by aureolysin in 10% serum. C,No phagocytosis inhibition in complement-inactivated serum. D, Aureolysin blocks killing of Group B Strep- tococcus by human neutrophils in 5% serum (BSA, SCIN, and aureolysin at 0.5 mM). All ﬁgures represent mean 6 SE of three separate experiments. For D, the relative survival was calculated by dividing the number of CFUs at each time point by the number of CFUs at time point zero. 6448 S. AUREUS METALLOPROTEASE AUREOLYSIN CLEAVES COMPLEMENT C3

different complement pathways and activation steps can be assessed separately (33). We specifically measured the effect of aureolysin on the CP and LP. The AP could not be investigated because analysis occurs in a buffer with EGTA to chelate calcium ions (35); this interferes with the calcium-dependent proteolytic activity of aureolysin (36). To assess activation of the CP and LP, human serum was incubated with microtiter plates coated with IgM or mannan, respectively. We observed that aureolysin prevents the deposition of C3b and C5b-9 via both pathways (Fig. 3), which is in line with our previous results showing inhibition of C3b deposition and C5a generation on bacteria. The C4b deposition was not inhibited by aureolysin (data not shown), in- FIGURE 2. Aureolysin inhibits complement activation on bacteria. A, dicating specificity for a common molecule in both pathways that Inhibition of C3b deposition on S. aureus by aureolysin. Serum was pre- functions downstream of C4b formation but upstream of C3b incubated with 0.5 mM aureolysin and mixed with bacteria. Deposition of deposition. This suggests that aureolysin targets C3, a common C3b on the bacterial surface was measured by flow cytometry. B, Dose- dependent inhibition of C5a release by aureolysin. Serum (10%) was molecule for all pathways. preincubated with aureolysin and subsequently incubated with heat-killed Aureolysin acts as a C3 convertase bacteria. Release of C5a in bacterial supernatants was measured by a calcium mobilization assay using U937-C5aR cells. All figures represent Because aureolysin targets C3, we studied aureolysin-mediated C3 Downloaded from mean 6 SE of three separate experiments. For B, the relative calcium cleavage. Therefore, we incubated purified C3 with aureolysin (in mobilization was calculated by dividing the fluorescence after stimulation a 1:1 molar ratio) at 37˚C and analyzed C3 cleavage by SDS- by the baseline fluorescence. PAGE. The C3 protein (187 kDa) consists of an a-chain (112 kDa) and a b-chain (75 kDa) that are linked together via disulfide this calcium mobilization assay specifically detects C5a in bac- bonds (37). We observed that aureolysin caused a rapid and spe- a terial supernatants. When aureolysin was added during incubation cific cleavage of the C3 -chain (112 kDa) into a smaller fragment http://www.jimmunol.org/ b of bacteria with serum, we observed a dose-dependent inhibition of around 100 kDa (Fig. 4A). The -chain of C3 was not affected of C5a generation (Fig. 2B). Altogether these data show that by aureolysin. N-terminal sequencing revealed that the aureolysin- a aureolysin is a complement inhibitor that blocks two critical bi- cleaved -chain starts with the amino acid sequence NH2-LDE- ological effects of the complement cascade: opsonization of bac- DII, indicating that aureolysin cleaves C3 between an asparagine teria with C3b and release of the chemoattractant C5a. (N751) and a leucine (L752), which is a typical cleavage site for aureolysin (38, 39). This specific cleavage was also observed at Aureolysin blocks the CP and LP lower molar ratios of aureolysin/C3 (Supplemental Fig. 2). At To study how aureolysin blocks the complement cascade, we tested a ratio of 1:20, we still observe 50% cleavage of C3, which is a 4- its activity in a well-described complement ELISA where the fold lower ratio than the determined IC50 in the phagocytosis by guest on September 29, 2021

FIGURE 3. Aureolysin blocks the CP and LP. Serum was incubated with aureolysin or BSA (0.5 mM), and complement activation via the CP and LP was determined via ELISA. Aureolysin prevents C3b and C5b-9 deposition via the CP (A, C) and the LP (B, D). All data shown represent mean 6 SE of three separate experiments. The Journal of Immunology 6449

FIGURE 4. Aureolysin cleaves C3 into active C3a+SN and active C3b2SN. A, Aureolysin cleaves the a-chain of C3. Purified C3 (1 mM) was incubated with 0.5 mM aureolysin, and cleavage was analyzed by SDS- PAGE and Coomassie staining. B, The C3 cleavage site of aureolysin is 2 aa apart from the C3 convertase cleavage site. N-terminal sequencing results of the C3 a-chain generated by incubation of purified C3 with aureolysin for 60 min at 37˚C. C, C3a+SN is active. Purified C3 and aureolysin were incubated for 30 min at 37˚C and tested for neutrophil activation in a calcium mobilization assay. D,C3b2SN is active. Bacteria were incubated with purified C3 in the presence of buffer, aureolysin, or purified C3 convertase (fD, fB, and C3b) for 30 min at 37˚C. C3b deposition on the bacterial surface was detected by flow cytometry. Data shown in C and D represent the mean 6 SE of three separate experiments. For C, the relative calcium mobilization was calculated by dividing the fluorescence after stimulation by the baseline fluorescence. In D, the relative Downloaded from C3b deposition is the deposition relative to buffer.

assay (Fig. 1B). During activation of C3 by its natural protease, the cleavage products by Western blotting (Fig. 6A). As shown in Fig. C3 convertase, the C3 a-chain is cleaved at position R748-S749; 4A, incubation of aureolysin with purified C3 results in cleavage this cleavage mediates release of the anaphylatoxin C3a (9 kDa) of the C3 a-chain into C3b a92SN within 10 min. In serum, we http://www.jimmunol.org/ and formation of C3b, which can deposit on surfaces via its also observe that aureolysin cleaves the C3 a-chain into a product 2SN thioester domain. The a9-chain of C3b starts with NH2-SNLDE- similar to C3b . However, the generated fragment is completely DII (Fig. 4B). Surprisingly, the cleavage sites of aureolysin and the degraded within 30 min. This indicates that other serum factors C3 convertase are only 2 aa apart; therefore, we named the aur- are involved in further cleavage of C3b2SN. To prove this, we first eolysin cleavage products C3a+SN and C3b2SN. To investigate generated C3b2SN by incubating purified C3 with aureolysin for whether the released C3a+SN is active, we incubated purified C3 20 min at 37˚C and subsequently inactivated aureolysin by adding and aureolysin for 30 min at 37˚C and mixed this with neutrophils EDTA. The sample was then incubated with C3-depleted serum to study calcium mobilization. Fig. 4C shows that aureolysin in- and analyzed by Western blot. Fig. 6B clearly shows that serum by guest on September 29, 2021 deed releases an active C3a molecule by cleavage of C3. Fur- factors mediate the further degradation of C3b2SN. To study thermore, we tested whether C3b2SN can be deposited on bacterial whether serum also degrades natural C3b, we mixed C3b with C3- surfaces by incubating purified C3, aureolysin, and bacteria for 30 depleted serum and obtained similar results. This indicates that min at 37˚C and detecting surface-bound C3b (Fig. 4D). As aureolysin rapidly cleaves C3 into C3b, which is then naturally a control, we cleaved C3 with a C3 convertase by mixing purified degraded by other serum factors. Previously, it has been shown fB, fD, and C3b. Like the C3 convertase, aureolysin increased the that the serum proteins fI and fH are involved in the clearance of C3b deposition on bacteria compared with control. In summary, fluid phase C3b from serum (40). FI is a serine protease that although aureolysin is a complement inhibitor, it specifically cleaves C3b when it is in complex with the cofactor fH (41). To cleaves C3 close to the convertase cleavage site generating active confirm that fI and fH mediate the degradation of aureolysin- C3a+SN and C3b2SN. generated C3b2SN, we incubated purified C3 with aureolysin in the presence of fI and/or fH for 30 min at 37˚C (with C3 and fI, in Complement inhibition by aureolysin depends on its proteolytic concentrations corresponding with 10% serum). As shown in Fig. activity 6C, the C3 a92SN chain generated by aureolysin is further de- To verify that the complement inhibitory effect of aureolysin is due graded in the presence of both fI and fH. Thus, aureolysin col- to its proteolytic capacity, we incubated aureolysin for 20 min at laborates with host factors fI and fH to effectively degrade C3. different temperatures and subsequently analyzed its potency to cleave purified C3 (Fig. 5A). We observed that aureolysin can no Aureolysin degrades C3a in serum longer cleave C3 when preincubated at 65˚C. Subsequently, we To study further the role of aureolysin in C3a generation, we in- tested whether this heat-inactivated form of aureolysin can still cubated bacteria with C5-depleted serum and measured C3a release inhibit C3b deposition and C5a generation (Fig. 5B,5C, re- in the supernatant by calcium mobilization and Western blotting. In spectively). In contrast to untreated aureolysin, heat-inactivated contrast to our findings in Fig. 4C, where aureolysin generates aureolysin (at 65˚C) does not inhibit C3b deposition on bacteria active C3a from purified C3, we observe that aureolysin blocks and release of C5a. This shows that the complement-inhibitory C3a-dependent neutrophil activation in the presence of serum function of aureolysin is a result of its proteolytic activity. (Fig. 7A). Western blotting revealed that C3a is converted to a smaller fragment (C3a9) in the presence of aureolysin and serum Aureolysin collaborates with host factors to inactivate C3b (Fig. 7B). Our finding that aureolysin closely mimics C3 convertases is in sharp contrast with our initial results in serum where aureolysin Aureolysin levels in bacterial supernatants are sufficient for functions as a complement inhibitor. To study whether C3 cleavage cleavage of C3 by aureolysin in serum might be different from purified conditions, To test whether the aureolysin levels in S. aureus supernatant are we incubated aureolysin with serum or purified C3 and detected C3 sufficient and uniquely responsible for cleavage of C3, we used 6450 S. AUREUS METALLOPROTEASE AUREOLYSIN CLEAVES COMPLEMENT C3 Downloaded from

FIGURE 5. Proteolytic activity of aureolysin is important for comple- m ment inhibition. A, Aureolysin (0.5 M) was preincubated for 20 min at http://www.jimmunol.org/ different temperatures and subsequently tested for C3 cleavage by incubation with 1 mM purified C3 for 30 min at 37˚C. Cleavage was analyzed by SDS-PAGE under reducing conditions and Coomassie staining. B, No inhibition of C3b deposition on bacteria by heat-inactivated aureolysin. Serum (5%) was preincubated with 0.5 mM aureolysin, heat-inactivated (65˚C) aureolysin (aureolysin65), or buffer and mixed with bacteria. De- FIGURE 6. Aureolysin inactivates C3 in collaboration with serum position of C3b on the bacterial surface was measured by flow cytometry. components fI and fH. A, Serum-dependent degradation of the C3 a-chain C, No inhibition of C5a generation by heat-inactivated aureolysin. Serum by aureolysin. Human serum (5%) or purified C3 (0.6 mM) was incubated (10%) was preincubated with 0.5 mM aureolysin or heat-inactivated aur- with 0.5 mM aureolysin at 37˚C, and C3 cleavage was analyzed at different eolysin and subsequently added to bacteria. C5a release in the supernatants time points by Western blotting. B, C3b2SN and C3b degradation by hu- by guest on September 29, 2021 was quantified by flow cytometry by measuring the calcium mobilization man serum. C3b2SN and C3b (0.6 mM) were incubated with 5% C3-de- in U937-C5aR cells. Right bar, Cells pretreated with the C5aR antagonist pleted serum at 37˚C, and C3 cleavage products were detected by Western (CHIPS, 10 mg/ml). Data shown in B and C represent the mean 6 SE of blotting. C, fI and fH mediate C3b2SN degradation. Purified C3 (0.6 mM), three separate experiments. In B, the relative C3b deposition is the de- fI (45 nM), and fH (27 nM) were incubated with 0.5 mM aureolysin for 30 position relative to buffer. For C, the relative calcium mobilization was min at 37˚C, and C3 cleavage products were detected by Western blotting. calculated by dividing the fluorescence after stimulation by the baseline fluorescence. Discussion In recent years, our insights into the way bacterial pathogens escape the complement system have grown enormously. S. aureus appears allelic exchange mutagenesis to generate an aureolysin mutant in S. aureus strain USA300, a predominant isolate of community- acquired methicillin-resistant S. aureus (42). Supernatants of wild- type and mutant (Daur) strains were tested for their ability to cleave C3. Whereas supernatants of wild-type USA300 cleaved the a-chain of C3, no cleavage of C3 was observed with supernatants of the aureolysin mutant (Fig. 8). Additionally, heterolo- gous expression of aureolysin on an extrachromosomal plasmid (Daur+aur) restored the C3 cleaving ability of the mutant, indicating that aureolysin is required for C3 cleavage by staphylococcal supernatants. Strikingly, we observed that the cleavage of C3 by aureolysin in the bacterial supernatant was different from purified aureolysin. Aureolysin present in supernatants could fully degrade the C3 a-chain, especially when expressed on a plasmid. Also, the C3 b-chain was cleaved. The remarkable difference in FIGURE 7. Aureolysin inactivates C3a. Heat-killed S. aureus was in- C3 cleavage by supernatants versus purified protease cannot be cubated with C5-depleted serum for 30 min at 37˚C. Supernatants were explained by the protein source, as purified protease was isolated analyzed for their potency to induce calcium mobilization in neutrophils from staphylococcal supernatants. The fact that aureolysin mutant (A) or C3a was detected by Western blotting (B). Data shown represent the did not show any cleavage of C3 strongly suggests that aureolysin mean 6 SE of three separate experiments. In A, the relative calcium collaborates with other proteases in the supernatant to fully de- mobilization was calculated by dividing the fluorescence after stimulation grade C3. by the baseline fluorescence. The Journal of Immunology 6451

FIGURE 8. Aureolysin is required and sufficient for cleavage of C3 by S. aureus supernatant. C3 cleavage by supernatants of S. aureus strain USA300 (Wt), its isogenic aureolysin mutant (Δaur), and the complemented mutant (Δaur+ aur). Undiluted (2), 3-fold concentrated (33), and 10-fold concentrated (103) supernatants of a stationary culture were incubated with 1 mM purified C3 for 1 h at 37˚C in HEPES++. Cleavage was analyzed by SDS-PAGE under reducing conditions and Coomassie staining. to be the “master complement evasion bug” because it produces a large array of small proteins that target different parts of the complement system. These staphylococcal complement inhibitors all have highly specific inactivation mechanisms to block the complement cascade. Whereas some inhibitors specifically bind Downloaded from and inhibit crucial complement enzymes, others sterically hinder important protein–protein interactions. In contrast to S. aureus, other Gram-positive bacteria like Group A streptococci rather use proteases to protect themselves from complement attack (43, 44). For example, streptococcal cysteine protease SpeB degrades C3 to

inhibit bacterial clearance (45), whereas the streptococcal cell- http://www.jimmunol.org/ associated peptidase ScpA cleaves C5a to inhibit neutrophil che- FIGURE 9. Schematic representation of the complement-inhibitory motaxis (46). In this study, we describe that S. aureus also uses its mechanism of aureolysin. A, C3 activation in the absence of aureolysin. proteases to dampen the complement response. Specifically, aur- Fluid-phase C3 is cleaved by C3 convertases (C3bBb) on the bacterial eolysin inactivates the central complement protein C3 and thereby surface. This results in release of C3a and covalent attachment of C3b to blocks important complement-dependent responses such as phago- the bacterial surface via its thioester domain. B, C3 activation in the presence of aureolysin. Aureolysin cleaves C3 in fluid phase into C3b2SN cytosis and neutrophil activation. The molecular mechanism by 2 and C3a+SN. C3b SN is rapidly degraded by host factors fH and fI pre- which aureolysin inactivates C3 is surprising (see Fig. 9 for a +SN venting its deposition on the bacterial surface; C3a is further inacti- schematic overview). In contrast to the streptococcal cysteine vated by aureolysin. protease SpeB that fully degrades C3 (45), aureolysin exerts its by guest on September 29, 2021 function by cleaving C3 at one specific site. Because this site is only 2 aa apart from the C3 convertase cleavage site, aureolysin cleaves C3b after it was deposited on the surface. In contrast, we generates active C3b and C3a and thus functions as a complement observe that aureolysin does not remove C3b from the bacterial activator under purified conditions. Intriguingly, by opening the surface in the absence of serum, indicating that aureolysin and molecule in a C3 convertase manner, the C3b molecule becomes GelE have different mechanisms. vulnerable to proteolytic degradation by host regulators in the Future studies will be needed to address the importance of serum. This inactivation depends on the protease fI and its co- aureolysin in the pathogenesis of S. aureus infections. Next to its factor fH, which forms a binding platform for fI on C3b (40, 41). role in complement escape, aureolysin has other functions by In order for aureolysin to function as a complement inhibitor, it is which it can interact with the host and contribute to bacterial crucial that aureolysin is a secreted protease so it can cleave C3 far virulence. Aureolysin has been shown to contribute to 1) bacterial away from the bacterial surface. In contrast, the C3 convertases of spreading and invasion by activating the fibrinolytic system (49), the complement system are generated on the bacterial surface and 2) resistance to antimicrobial peptides (26), and 3) inhibition of therefore they mediate C3 cleavage close to the bacterial surface. Ig production by lymphocytes (50). Recent studies suggested that This is essential for the covalent attachment of the C3b thiolester aureolysin can be expressed within the phagocytic vacuole after to bacterial proteins/sugars. If C3 is activated further away from phagocytosis of S. aureus (51). Furthermore it was shown that the surface, the thiol ester will react with water in fluid phase and the isogenic aureolysin mutant was more efficiently killed by become subject to degradation by host regulators. Thus, aur- macrophages upon phagocytosis (52). These studies suggest that eolysin will only function as a complement inhibitor when it is aureolysin is not only important to prevent phagocytes from taking secreted. The fact that aureolysin uses host regulators to inactivate up bacteria, but that it also protects bacteria inside the phagocytes complement is in analogy with mechanisms described for other likely through resistance against antimicrobial peptide killing. We bacteria that specifically attract host regulators (mainly fH) to the find that the proteolytic strategy of aureolysin toward complement surface (6, 7, 14, 47). These bacteria use the increased concen- is very efficient, as aureolysin cleaves all C3 in serum into C3b tration of regulators nearby the surface to inhibit complement within minutes, thereby inhibiting the phagocytosis in a nano- activation on the surface. The strategy of aureolysin differs from molar range. Our data with aureolysin mutant bacteria indicate these mechanisms because it uses the regulators to inactivate that the expression levels of aureolysin in the supernatant are complement in fluid phase. Notably, we found that aureolysin sufficient for C3 cleavage. However, aureolysin seems to act in cleaves C3 at the same site as gelatinase E (GelE) from Entero- synergy with other factors from the bacterial supernatant to de- coccus faecalis (48). GelE is also a secreted protease that cleaves grade C3 fully. Because previous studies indicated that aureolysin C3 in a convertase manner. The findings of Park et al. (48) sug- is required for activation of the V8 protease (53), this suggests that gested that GelE consumes the C3 molecule in fluid phase but also the V8 protease may act as a cofactor for aureolysin-mediated 6452 S. AUREUS METALLOPROTEASE AUREOLYSIN CLEAVES COMPLEMENT C3 cleavage of C3. Notably, we observed no direct cleavage of C3 by 19. Bestebroer, J., P. C. Aerts, S. H. Rooijakkers, M. K. Pandey, J. Ko¨hl, J. A. van purified V8 (data not shown). Future studies are needed to reveal Strijp, and C. J. de Haas. 2010. Functional basis for complement evasion by staphylococcal superantigen-like 7. Cell. Microbiol. 12: 1506–1516. the exact collaborative action of staphylococcal proteases on C3 in 20. Postma, B., W. Kleibeuker, M. J. Poppelier, M. Boonstra, K. P. Van Kessel, the context of serum. Data from other bacterial pathogens show J. A. Van Strijp, and C. J. de Haas. 2005. Residues 10-18 within the C5a receptor N terminus compose a binding domain for chemotaxis inhibitory protein of that bacterial proteases are important virulence factors that affect Staphylococcus aureus. J. Biol. Chem. 280: 2020–2027. several different parts of the innate immune system, such as the 21. Zhang, L., K. Jacobsson, J. Vasi, M. Lindberg, and L. Frykberg. 1998. 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