Classical and Lectin Pathways Blocks Complement Activation Via the Alkaline Protease Pseudomonas Aeruginosa

Pseudomonas aeruginosa Alkaline Protease Blocks Complement Activation via the Classical and Lectin Pathways

This information is current as Alexander J. Laarman, Bart W. Bardoel, Maartje Ruyken, of September 28, 2021. Job Fernie, Fin J. Milder, Jos A. G. van Strijp and Suzan H. M. Rooijakkers J Immunol 2012; 188:386-393; Prepublished online 30 November 2011;

doi: 10.4049/jimmunol.1102162 Downloaded from http://www.jimmunol.org/content/188/1/386

Supplementary http://www.jimmunol.org/content/suppl/2011/11/30/jimmunol.110216 Material 2.DC1 http://www.jimmunol.org/ References This article cites 44 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/188/1/386.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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Pseudomonas aeruginosa Alkaline Protease Blocks Complement Activation via the Classical and Lectin Pathways

Alexander J. Laarman,1 Bart W. Bardoel,1 Maartje Ruyken, Job Fernie, Fin J. Milder, Jos A. G. van Strijp, and Suzan H. M. Rooijakkers

The complement system rapidly detects and kills Gram-negative bacteria and supports bacterial killing by phagocytes. However, bacterial pathogens exploit several strategies to evade detection by the complement system. The alkaline protease (AprA) of Pseudomonas aeruginosa has been associated with bacterial virulence and is known to interfere with complement-mediated lysis of erythrocytes, but its exact role in bacterial complement escape is unknown. In this study, we analyzed how AprA interferes with complement activation and whether it could block complement-dependent neutrophil functions. We found that AprA potently blocked phagocytosis and killing of Pseudomonas by human neutrophils. Furthermore, AprA inhibited opsonization of bacteria with C3b and the formation of the chemotactic agent C5a. AprA speciﬁcally blocked C3b deposition via the classical and lectin Downloaded from pathways, whereas the alternative pathway was not affected. Serum degradation assays revealed that AprA degrades both human C1s and C2. However, repletion assays demonstrated that the mechanism of action for complement inhibition is cleavage of C2. In summary, we showed that P. aeruginosa AprA interferes with classical and lectin pathway-mediated complement activation via cleavage of C2. The Journal of Immunology, 2012, 188: 386–393.

he innate immune system rapidly detects and kills invading C2a (70 kDa), which remains attached to C4b, forming the C3 http://www.jimmunol.org/ bacteria via different mechanisms, such as TLRs and the convertase complex (C4b2a). The smaller C2b domain (30 kDa) is T complement system. TLRs are expressed on immune cells released into the surrounding area of inflammation (the literature and detect an enormous variety of microorganisms via recognition is inconsistent regarding the nomenclature of C2 fragments; in this of highly conserved microbial molecular patterns, such as flagellin article we use C2a and C2b for the 70- and 30-kDa fragments, via TLR5 and LPS via TLR4 (1). Upon ligand binding, TLRs respectively). Activation of the LP occurs in a similar fashion. trigger an intracellular signaling cascade that leads to production Recognition of microbes occurs via the mannose-binding lectin of proinflammatory cytokines and activation of phagocytes. The (MBL) or ficolins that bind to repeating molecular patterns on complement system is a proteolytic cascade of plasma proteins, microbial surfaces (3, 4). MBL and ficolins are complexed to the which results in direct killing of certain microorganisms and ef- MBL-associated protease (MASP), which functionally resembles by guest on September 28, 2021 ficient bacterial recognition by phagocytes. The complement C1s because it cleaves C4 and C2 to induce formation of the C3 system consists of three distinct pathways: the classical pathway convertase complex. In the AP, microbial surfaces are recognized (CP), lectin pathway (LP) and alternative pathway (AP). All three by properdin (5), which initiates assembly of the AP C3 con- recognition pathways culminate in the formation of C3 convertase vertase (C3bBb) on the surface. The AP also serves as an am- enzymes that mediate deposition of C3b on foreign surfaces (2). plification loop to enhance the concentration of C3b on the The CP is initiated by binding of C1q to bacterium-bound Abs or surface. Cleavage of C3 by convertases is critical to opsonization repeating molecular patterns on bacteria. This binding initiates of the bacterial surface with C3b, which is recognized by com- activation of the C1q-attached protease C1s that can cleave C4 plement receptors on phagocytes and supports phagocytosis and into C4b, which covalently binds the microbial surface. The C4b killing. Further downstream in the cascade, formation of C5 protein is bound by C2, forming the proconvertase C4bC2. The convertases and cleavage of C5 result in generation of the potent C1s protease then cleaves C2 into the larger protease fragment anaphylatoxin C5a and the formation of C5b-9, the membrane attack complex (MAC) that can directly kill certain Gram-negative bacteria (6). Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands The Gram-negative bacteria Pseudomonas aeruginosa lives in 1A.J.L. and B.W.B. contributed equally to this work. water and soil and acts as an opportunistic pathogen in humans Received for publication July 25, 2011. Accepted for publication October 24, 2011. and plants. In healthy individuals, P. aeruginosa is efficiently killed via the innate immune system; however it causes chronic A.J.L., B.W.B., J.A.G.v.S., and S.H.M.R. are supported by grants of The Netherlands Organization for Scientific Research (TOP and VIDI). infections in immunocompromised patients. For instance, cystic Address correspondence and reprint requests to Dr. S.H.M. Rooijakkers, Department fibrosis (CF) patients suffer from chronic infections caused by P. of Medical Microbiology, University Medical Center Utrecht, PO G04.614, Heidel- aeruginosa that cannot be eradicated after colonization. Strains berglaan 100, 3584 CX Utrecht, The Netherlands. E-mail address: s.h.m.rooijakkers@ isolated from CF patients rapidly adapt to the environment in the umcutrecht.nl lung, resulting in dramatic genetic and morphological changes (7). The online version of this article contains supplemental material. Infection by P. aeruginosa is complicated by its inherent resis- Abbreviations used in this article: AP, alternative pathway; AprA, alkaline protease; CCP, complement control protein; CF, cystic fibrosis; CP, classical pathway; fB, tance to several classes of antibiotics and acquisition of resistance factor B; HSA, human serum albumin; LB, Luria–Bertani; LP, lectin pathway; genes via mobile genetic elements (8). In addition, biofilm for- MAC, membrane attack complex; MASP, mannose-binding lectin-associated prote- mation enhances the resistance to antibiotics in the CF lung (9). ase; MBL, mannose-binding lectin; vWFA, von Willebrand factor A. Remarkably, the strains isolated from CF patients are generally Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 sensitive to direct complement killing, which is different from www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102162 The Journal of Immunology 387 strains isolated from non-CF patients. These serum-resistant strains ous important complement functions hampering the bacterial consume more C5b-9 complement components and have less clearance by human neutrophils. Furthermore, by using biological C3b deposited on their surface (10). assays with more physiological relevant protease concentrations, Bacterial survival in the host is accompanied by the acquisition we uncovered that AprA specifically blocks the CP and LP and of different immune-evasive strategies. These strategies involve predominantly cleaves complement protein C2. alteration of the bacterial surface, such as production of a cap- sule or immune-modulating membrane proteins, or secretion of soluble immune-evasion proteins, such as proteolytic factors. P. Materials and Methods aeruginosa secretes a number of toxins and virulence factors, Proteins, sera, and bacterial strains like elastase and alkaline protease (AprA). Elastase cleaves a C3 was purified from human plasma, as described (18). The purified wide variety of host proteins, such as collagen, IgG, and comple- components C1, C2, and C4 were purchased from Quidel. C1s was ob- ment proteins (11). AprA is a 50-kDa zinc metalloprotease that tained from R&D, and C2-depleted serum was purchased from Sigma. degrades several components of the host immune system, such as Normal human serum was obtained from healthy volunteers, who gave complement C1q and C3 (12), or cytokines, like IFN-g and informed consent. AprA and AprI of P. aeruginosa PAO1 were expressed and isolated from Escherichia coli, as described (14). Briefly, both proteins TNF-a (13). In addition, AprA prevents TLR5 activation via were expressed with a hexa-histidine tag in E. coli BL21 (Invitrogen). the cleavage of monomeric flagellin (14). AprA is secreted via its Recombinant proteins were isolated under denaturing conditions using own type I secretion system, which is encoded by three genes a His trap column (GE Healthcare). AprA was refolded by dilution in 50 upstream of the aprA gene. P. aeruginosa also encodes a highly mM Tris-HCl (pH 9) containing 0.8 M L-arginine and 1 mM CaCl2. Refolding of AprI was performed by dilution in PBS. The His-tag of AprI Downloaded from specific inhibitor of AprA named AprI, which is translocated to was removed using enterokinase (Invitrogen). Both proteins were .95% the periplasmic space according to its signal sequence. The role pure, as assessed by SDS-PAGE and Instant Blue staining. Wild-type P. of AprA in pseudomonal virulence has been illustrated in a aeruginosa PAO1 was obtained from the Genome Sciences Manoil labo- Drosophila infection model (15). AprA contributes to the per- ratory (University of Washington), and GFP-labeled PAO1 (19) was kindly sistence of Pseudomonas entomophila infection and protects provided by J.M. Beekman (University Medical Center Utrecht). against antimicrobial peptides produced by the innate immune Hemolytic assays system of Drosophila. Furthermore, AprA was found to be ex- http://www.jimmunol.org/ pressed in human CF patients infected with P. aeruginosa (16, The CP hemolytic assay was performed, as previously described (20), with minor modifications. Serum was preincubated with 22, 66, and 200 nM 17). AprA or 200 nM AprA plus 1000 nM AprI for 30 min at 37˚C. Subse- In a previous study, Hong and Ghebrehiwet (12) showed that quently, opsonized (anti-sheep IgM) sheep erythrocytes (Alsever) were AprA cleaves complement components C1q and C3 and inhibits incubated with AprA-treated serum in Veronal-buffered saline containing lysis of sheep erythrocytes via MAC. However, because P. aeru- 0.5 mM CaCl2 and 0.25 mM MgCl2. After 30 min at 37˚C, samples were centrifuged, and the absorbance of the supernatants at 405 nm was mea- ginosa is generally resistant to direct lysis via MAC, we wondered sured. The AP hemolytic assay was performed, as described above, using whether cleavage of complement proteins by AprA contributes to rabbit erythrocytes (Alsever) with Veronal-buffered saline containing 5 pseudomonal immune evasion. We found that AprA blocks vari- mM MgCl2 and 10 mM EGTA. by guest on September 28, 2021

FIGURE 1. Secreted AprA concentration sufﬁcient to inhibit complement activity. A, Overnight culture of P. aeruginosa (PAO1) was diluted 10 times in IMDM. After 2, 4, and 6 h, supernatant was collected and analyzed for AprA production by Western blot using a polyclonal Ab against AprA. B, Serum was preincubated with different concentrations of AprA (nM) or together with 1000 nM AprI for 30 min at 37˚C. Subsequently, E. coli K12 (1 3 105 CFU/ml) in RPMI-HSA was added to treated serum for 1 h at 37˚C. Number of surviving bacteria/ml (CFU/ml) was determined by serial dilutions. Data represent mean 6 SEM of three independent experiments. C, Serum was preincubated with AprA, with or without AprI (1000 nM), for 30 min at 37˚C. Treated serum was mixed with opsonized sheep erythrocytes and incubated for 1 h at 37˚C. Erythrocytes were pelleted, and OD of supernatant at 405 nm was measured. Data are expressed as relative lysis compared with lysed erythrocytes and represent mean 6 SEM of three independent experiments. 388 COMPLEMENT INHIBITION BY P. AERUGINOSA

E. coli killing AprA concentration, and Y is the percentage of phagocytosis (for the AprA curve, bottom = 9.38 and top = 102.5, R2 = 0.8979). E. coli K12 was cultured in Luria–Bertani (LB) medium overnight at 37˚C. For neutrophil-killing assays, P. aeruginosa was grown to an OD660 of The next day, the overnight culture was diluted 20 times in LB medium and 0.5 in LB medium and subsequently washed in RPMI 1640 containing grown to an OD660nm of 0.5. Bacteria were washed with RPMI 1640 0.1% HSA. Then, 5% serum was preincubated with buffer, 200 nM AprA, supplemented with 0.05% human serum albumin (HSA). Human pooled or 200 nM AprA plus 1000 nM AprI for 30 min at 37˚C. Subsequently, serum was incubated with different concentrations of AprA, with or 2.5 3 105 P. aeruginosa and 8.5 3 106 neutrophils were added and incu- without 1000 nM AprI, for 30 min at 37˚C in RPMI-HSA. Subsequently, bated at 37˚C. A sample was taken at different time points, and neutrophils 3 5 bacteria were added (1 10 /ml) and incubated for 1 h at 37˚C. CFU were were lysed with Milli-Q water. Surviving bacteria were enumerated by determined by plating serial dilutions on tryptic soy agar + 5% sheep blood. plating serial dilutions on LB agar. Phagocytosis and killing C3b deposition on P. aeruginosa Phagocytosis assays were performed, as described (21). In short, serum was An overnight culture of P. aeruginosa strain PAO1-GFP in LB medium preincubated with buffer, 200 nM AprA, or 200 nM AprA plus 1000 nM was washed in Veronal-buffered saline containing 0.5 mM CaCl ,0.25mM AprI in RPMI 1640 containing 0.1% HSA for 30 min at 37˚C. Then, 2.5 3 2 5 3 6 MgCl2, and 0.1% BSA. Serum was preincubated with different concen- 10 freshly isolated human neutrophils and 2.5 10 GFP-labeled, heat- trations of AprA, with or without AprI, for 30 min at 37˚C. Then, 2.5 3 killed P. aeruginosa were added and incubated for 15 min at 37˚C while 106 bacteria were incubated with the preincubated serum for 30 min while shaking at 600 rpm. The reaction was stopped by adding 1% ice-cold shaking at 900 rpm. Bacteria were washed with PBS with 0.1% BSA. C3b paraformaldehyde in RPMI 1640 containing 0.1% HSA. Phagocytosis deposition was detected using mouse anti-human C3b Abs (WM-1; was analyzed using the FACSCalibur (Becton Dickinson). Neutrophils American Type Culture Collection) and allophycocyanin-conjugated goat were gated on the basis of forward- and side-scatter properties. Fluores- anti-mouse IgG (Protos). Median ﬂuorescence of 10,000 bacteria was cence intensity of 10,000 gated neutrophils was determined, and phago-

measured by ﬂow cytometry. Downloaded from cytosis was expressed as the percentage of these cells containing ﬂuorescently labeled bacteria (calculated using BD CellQuest Pro soft- C5a analysis ware). In another experiment, different concentrations of AprA were incubated with 5% serum in RPMI 1640 containing 0.1% HSA for 30 min at An overnight culture of P. aeruginosa strain PAO1 in LB medium was washed 37˚C. The IC50 value was calculated with GraphPad Prism software using in RPMI 1640 with 0.1% HSA. Ten-percent serum and 200 nM AprA or the sigmoidal dose-response function and the equation Y = bottom + 200 nM AprA plus 1000 nM AprI were preincubated for 30 min at 37˚C (top 2 bottom)/(1+10^[logEC502X]) where X is the logarithm of the and subsequently incubated with 5 3 107 bacteria at 37˚C for 30 min while http://www.jimmunol.org/

FIGURE 2. AprA inhibits complement-dependent effector functions important for P. aeruginosa. A and

B, Phagocytosis of P. aeruginosa PAO1-GFP by hu- by guest on September 28, 2021 man neutrophils in the presence of human serum and AprA. A, Serum was preincubated with buffer, AprA (200 nM), or AprA (200 nM) and AprI (1000 nM). B, Dose-dependent inhibition of phagocytosis by AprA in 5% serum with an IC50 value of 99.6 nM AprA (calculated with GraphPad Prism software using the sigmoidal dose-response function; R2 = 0.8979). C, Inhibition of C3b deposition on P. aeruginosa by AprA. Serum was preincubated with buffer or different concentrations of AprA (nM) and mixed with bacteria. Deposition of C3b on the bacterial surface was quantified by flow cytometry, measuring the median fluorescence of 10,000 bacteria. The relative C3b deposition is the fluorescence relative to buffer. D, Inhibition of C5a release by AprA. Serum (10%) was preincubated with AprA, with or without AprI, and subsequently incubated with bacteria. Release of C5a in bacterial supernatants was measured by a calcium-mobilization assay using U937-C5aR cells. E, AprA blocks killing of P. aeruginosa by human neutrophils in 5% serum (AprA at 200 nM and AprI at 1000 nM). All figures represent mean 6 SEM of three independent experiments. For D, the relative calcium mobilization was calculated by dividing the fluorescence after stimulation by the baseline fluorescence. For E, the relative survival was calculated by dividing the number of CFU by the number of CFU at time 0. The Journal of Immunology 389

FIGURE 3. AprA inhibits C3b deposition via the CP and LP. Serum was incubated with different concentrations of AprA, and complement activation via the CP, LP, and AP was determined via ELISA. AprA prevents C3b deposition via the CP (A) and the LP (B), but not the AP (C). No inhibition of C4b deposition was observed via the CP (D)orLP(E). All ﬁgures represent mean 6 SEM of three independent experiments. Downloaded from http://www.jimmunol.org/

shaking at 600 rpm. Bacteria were centrifuged, and C5a was detected in Analysis of complement factor cleavage in serum was performed by collected supernatants by calcium mobilization: 10-fold–diluted supernatants Western blotting. Three-percent serum was incubated with different con- were added to 5 3 104 Fluo-4-AM–labeled U937-C5aR cells (a generous gift centrations of AprA at 37˚C in PBS. The reaction was stopped by adding from Prof. Eric Prossnitz, University of New Mexico, Albuquerque, NM), Laemmli sample buffer containing DTT. All samples were subjected to and the increase in intracellular calcium was measured by flow cytometry. SDS-PAGE and blotted onto a polyvinylidene difluoride membrane. After blocking with 4% skimmed milk in PBS containing 0.1% Tween, C1s was Complement assays detected by a mouse anti-human C1s Ab (Quidel), followed by HRP- labeled goat anti-mouse; C2, C3, and factor B (fB) were detected by by guest on September 28, 2021 Complement ELISAs were performed, as described (22), with modifications. goat anti-human C2 (Quidel), goat anti-human C3 (Protos), and goat anti- m ELISA plates (MaxiSorp; Nunc) were coated overnight with 20 g/ml LPS human fB (Quidel), respectively, followed by HRP-labeled donkey anti- (Salmonella enteriditis;Sigma),3mg/ml IgM (Quidel), or 10 mg/ml man- goat (Jackson); and C4 was detected by biotin-labeled chicken anti-human Saccharomyces cerevisiae nan ( ;Sigma)in0.1Msodiumcarbonatebuffer C4 (Abcam), followed by HRP-labeled streptavidin (Southern Biotech). (pH 9.6). Plates were blocked with 4% BSA in PBS with 0.05% Tween for All Ab incubations were diluted in PBS with 0.1% Tween and 1% skim- 1 h at 37˚C. For the CP and LP, samples were diluted in Veronal-buffered med milk. ECL (GE Healthcare) was used for final signal detection. saline containing 0.5 mM CaCl2,0.25mMMgCl2, 0.1% gelatin, and 0.05% Tween. For the AP, samples were diluted in Veronal-buffered saline containing 5 mM MgCl2, 10 mM EGTA, 0.1% gelatin, and 0.05% Tween. Results Serum or C2-depleted serum was mixed with different concentrations of Physiological AprA levels inhibit complement-mediated lysis AprA or AprA together with AprI and subsequently added to the plates for 1 h at 37˚C. Deposited C3b and C4b were detected using Abs against C3d To study the functional relevance of AprA secretion by P. aeru- (WM-1–digoxigenin-labeled) and C4d (Quidel), respectively, followed by ginosa on the complement system, we first estimated AprA con- peroxidase-conjugated sheep anti-digoxigenin or a goat anti-mouse IgG centrations in culture supernatants. Supernatants were collected at (Southern Biotechnology), respectively. For the repletion experiment, IgM- coated plates were used, and the assay was performed, as described above. In short, 1% serum was preincubated with 200 nM AprA for 30 min at 37˚C. Then, 1000 nM AprI was added together with C1 (2 nM), C1s (3 nM), C2 (2 nM), C3 (67 nM), or C4 (20 nM), and the mixtures were added to the plate for 1 h at 37˚C. C3b deposition was detected, as described above. C2 cleavage C2 was incubated with different concentrations of AprA for 30 min at 37˚C in Veronal-buffered saline containing 0.5 mM CaCl2 and 0.25 mM MgCl2. Proteins were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Cleavage products were visualized with 0.1% Coo- massie blue in 50% methanol, excised, and analyzed by N-terminal sequencing (Alphalyse).

Western blotting FIGURE 4. Serum (5%) was preincubated with 200 nM AprA for 30 min at 37˚C. A, Treated serum was incubated with rabbit erythrocytes for The P. aeruginosa strain PAO1 was cultured overnight in LB medium and 1 h at 37˚C. B, Treated serum was mixed with opsonized sheep erythrocytes subsequently diluted 10 times in IMDM (Invitrogen). Bacterial supernatant was collected after 2, 4, and 6 h. The presence of AprA in supernatant of P. and incubated for 1 h at 37˚C. Erythrocytes were pelleted, and OD of aeruginosa was analyzed by Western blotting using a protein G-purified supernatant at 405 nm was measured. Data are expressed as relative lysis polyclonal rabbit anti-AprA Ab (Genscript), followed by a goat anti-rabbit compared with water-treated erythrocytes and represent mean 6 SEM of IgG Ab (Southern Biotech). Bands were quantified using ImageJ software. three independent experiments. 390 COMPLEMENT INHIBITION BY P. AERUGINOSA different time points and analyzed for AprA expression by on neutrophils. Activation of the C5a receptor induces intracel- Western blotting using an Ab against AprA (Fig. 1A). The levels lular calcium mobilization and chemotaxis of the neutrophil to the of AprA in the supernatant were semiquantified by comparison site of inflammation. We incubated P. aeruginosa with serum and with fixed concentrations of recombinant AprA (Supplemental studied the release of C5a into the supernatant by measuring the Fig. 1). The AprA concentrations in culture supernatants were supernatant-induced calcium mobilization on Fluo-3-AM–labeled between 8 nM (2-h culture) and 208 nM (∼10 mg/ml; 6-h super- U937 cells transfected with the C5a receptor (specificity of this natant). Because AprA was described to inhibit complement- assay is presented in Supplemental Fig. 2). When bacteria were dependent hemolysis via MAC, we repeated the assay to con- incubated with AprA, supernatants could not induce calcium firm AprA activity. AprA inhibited the lysis of Ab-sensitized mobilization, meaning that there was no active C5a in the pres- sheep erythrocytes, a CP-mediated hemolysis assay, which was ence of AprA (Fig. 2D). Because AprA also blocks formation of restored in the presence of AprI (Fig. 1B). Furthermore, we tested C5b-9 (Fig. 1B,1C), we believe that AprA does not specifically different concentrations of AprA in a serum-killing assay using E. cleave C5a but prevents activation of C5 into C5a and C5b. Be- coli as a model. In contrast to P. aeruginosa strain PAO1, E. coli cause AprA also blocks C3b deposition, it seems likely that it K-12 is not serum resistant and is lysed at low serum concen- inhibits complement activation upstream of C3. To verify that trations as a result of incorporation of the lytic MAC. Indeed, AprA also inhibits the killing of P. aeruginosa by neutrophils, we AprA inhibited the complement-dependent lysis with an estimated incubated neutrophils with bacteria and serum and determined the IC50 of 30 nM, which is within the concentration range detected percentage of survival after 15 min. After 15 min, 80% of the in the supernatant (Fig. 1C). The natural inhibitor AprI blocks bacteria were killed. AprA inhibited the killing of P. aeruginosa, proteolytic activity of AprA (23), thereby allowing complement- but not completely, showing a killing of 40% after 15 min. This Downloaded from dependent lysis of E. coli. Thus, AprA blocks complement- effect was restored in the presence of AprI (Fig. 2E). In summary, dependent lysis in a concentration range similar to AprA levels AprA inhibited all complement-dependent effector functions, in P. aeruginosa supernatant. which allowed P. aeruginosa to escape neutrophil killing. AprA inhibits complement-dependent neutrophil functions AprA inhibits complement activation via the CP and LP important for P. aeruginosa killing

To further study the effect of AprA on the complement system, http://www.jimmunol.org/ Because many P. aeruginosa strains are resistant to direct com- we dissected the individual pathways using pathway-specific complement lysis (24), we investigated whether AprA facilitates re- plement ELISAs (22). The CP, LP, and AP were specifically in- sistance to other complement processes that are critical to duced in the presence of serum using the coatings IgM, mannan, neutrophil functioning. To study whether AprA blocks comple- and LPS, respectively. The level of C3b deposition was detected ment-dependent phagocytosis, we incubated GFP-labeled P. aer- with specific Abs. AprA strongly inhibited C3b deposition of the uginosa with human neutrophils in the presence of human serum CP (Fig. 3A) and LP (Fig. 3B) at all tested serum concentrations. and AprA. AprA blocked the phagocytosis compared with buffer Inhibition of the AP was not observed at AprA concentrations up or AprA supplemented with AprI (Fig. 2A), and its activity was to 66 nM (Fig. 3C). The AP ELISA was not inhibited at higher dose dependent (Fig. 2B). The IC50 of AprA is 100 nM, which is concentrations of 2000 nM AprA (data not shown). Because of by guest on September 28, 2021 in the relevant range produced by P. aeruginosa. Incubation of P. technical limitations, we could not detect AP-mediated C3b de- aeruginosa with serum results in the deposition of C3b molecules position at serum concentrations ,10%. We used a more sensitive on the bacterial surface, which are recognized by complement hemolytic complement assay to compare the effect of AprA in the receptors on phagocytes. To study whether AprA inhibits the C3b AP and CP at similar serum concentrations. Fig. 4A shows that deposition, we incubated bacteria with serum and detected 200 nM AprA did not block AP-mediated hemolysis in 5% serum; surface-bound C3b using specific Abs and flow cytometry. AprA however, 200 nM AprA blocked CP-mediated hemolysis (Figs. partially inhibited C3b deposition on Pseudomonas at comparable 1B,4B). As mentioned above, C3b deposition via the CP and LP is concentrations as observed for phagocytosis of P. aeruginosa (Fig. mediated by C3 convertases, bimolecular complexes that consist 2C). Further along the complement cascade, C5 is cleaved into the of the protease C2a loosely attached to surface-bound C4b. C4b potent anaphylatoxin C5a that is recognized by the C5a receptor and C2a are formed by cleavage of C4 and C2 by the C1 or MBL–

FIGURE 5. AprA depletes the limiting factor C2. A, AprA degrades C2 and C1s. Different concentrations of AprA were incubated with 3% serum for 30 min at 37˚C. C1s, C2, C3, C4, and fB were detected by Western blotting. B, C2 is the limiting factor. Serum (1%) was preincubated with 200 nM AprA for 30 min at 37˚C. Then, 0.2 mM AprI was added to stop the AprA cleavage, together with C1, C1s, C2, C3, or C4. Complement activation via the CP was determined by ELISA by detecting C3b deposition. For B, the data represent mean 6 SEM of three independent experiments. Blots are representatives of three separate experiments. The Journal of Immunology 391

and LP upstream of C3 and downstream of C4 activation in the complement cascade. AprA depletes the limiting factor C2 Many complement factors that are involved in the CP and LP induce C3b deposition; however, only two factors (C2 and C3) are involved after C4b deposition and before C3b deposition. To identify the critical factor that is cleaved by AprA, we incubated human serum with AprA for 30 min at 37˚C. The complement factors C2, C3, and C4 were analyzed by Western blotting. As controls, C1s and fB were detected, which are crucial components of the CP and AP, respectively. AprA efﬁciently cleaved C2, whereas C3, C4, and fB were not affected (Fig. 5A). In addition, cleavage of C1s by AprA was observed; however, this was less efﬁcient than C2 cleavage. We used the CP complement ELISA to discriminate the limiting factor that was cleaved by AprA. First, we incubated serum with AprA and inactivated the protease by adding the irreversible inhibitor AprI. Then, treated serum was

used in the CP ELISA supplemented with the different puriﬁed Downloaded from complement factors. Only the serum that was supplemented with puriﬁed C2 restored the complement activation, whereas AprA- treated serum supplemented with C1, C1s, or C3 showed no activity (Fig. 5B). Thus, although AprA also cleaves C1s, the cleavage of C2 determines inhibition of complement activation.

AprA cleaves C2 http://www.jimmunol.org/ C2 is a 100-kDa protein that consists of three complement control protein (CCP) regions, a von Willebrand factor A (vWFA) domain, and a peptidase domain. The cleavage of C2 by its natural proteases C1s and MASP results in formation of C2b (CCP1–3) and C2a (vWFA and peptidase domain) (Fig. 6B,6C). For the formation of a functional C3 convertase (C4b2a), full-length C2 ﬁrst binds to C4b and is subsequently cleaved. C2b is then released, while C2a

stays attached to C4b. The C4b2a convertase has a very short half- by guest on September 28, 2021 life due to the irreversible dissociation of C2a. C2 cleavage by AprA was further analyzed by SDS-PAGE and Coomassie staining. After incubating purified C2 with AprA, we observed three cleavage products of ∼75, ∼40, and ∼30 kDa, which were sent for FIGURE 6. AprA cleaves C2. A, C2 cleavage by AprA. Purified C2 was N-terminal sequencing (Fig. 6A). Cleavage product 1 starts with incubated with different concentrations of AprA, and cleavage was ana- NH2-IQIQRS, which is only 1 aa different from C2a (starts with lyzed by SDS-PAGE and Coomassie staining. Cleavage products 1, 2, and NH2-KIQIQR). At higher protease concentrations, an increase in 3 were sent for N-terminal sequencing. B, Sequence of human C2; the cleavage product 2 is observed, meaning that AprA secondarily obtained N-terminal sequences of cleavage products 1, 2, and 3 are cleaves between the vWFA and peptidase domain. Next to C2a, underlined. C, Schematic representation of C2. The domains and the AprA cleaves C2b between CCP1 and CCP2. These data indicated cleavage site for C1s, MASP, and AprA are indicated. that AprA efficiently interferes with CP and LP activation via the degradation of C2 (Fig. 7). MASP complexes. To study at what level AprA affects these pathways, we also analyzed the C4b deposition. In contrast to C3b Discussion deposition, AprA did not block C4b deposition in the CP and LP Bacterial pathogens adopt many strategies to counteract the re- (Fig. 3D,3E). This indicated that AprA specifically blocks the CP dundant attack of the human host defense. For instance, bacteria

FIGURE 7. Schematic representation of the complement-inhibitory mechanism of AprA. A, In the CP, fluid-phase C4 and C2 are cleaved by C1s (MASP for the LP) on the bacterial surface. This results in the formation of the C3 convertase (C4b2a), which cleaves C3, resulting in C3b deposition on the bacterial surface. B, Complement activation in the presence of AprA. C2 is cleaved by AprA into fluid-phase C2a and C2b; subsequently, further cleavage of both C2a and C2b occurs. Because fluid-phase C2a cannot bind to C4b, an active C3 convertase cannot be generated. In this way, AprA prevents C3b deposition on the bacterial surface and downstream complement effector functions. 392 COMPLEMENT INHIBITION BY P. AERUGINOSA secrete many proteins that specifically interact with the comple- termination of AprA concentration in vivo is complicated by the ment system, which can either be proteolytic or steric interactions fact that AprA is produced locally in the bacterial environment (25, 26). In the defense against Gram-negative bacteria, the and is subsequently diluted or degraded by host proteases in complement system directly destroys cells by inserting the pore- clinical samples. In a rat pouch model, only small amounts of forming MAC into the bacterial membrane (27). Although all injected recombinant AprA could be recovered after 6 h (39), Gram-positive bacteria are protected from MAC lysis by their suggesting that production of AprA in vivo is much greater than thick peptidoglycan layer, some Gram-negative bacteria have also measured. In this article, we showed that AprA cleaves C2 in become resistant to direct complement attack by altering their concentrations similar to levels secreted by P. aeruginosa in surface properties (28). The Gram-negative bacteria P. aeruginosa functionally relevant time incubations. However, increasing AprA does this by lengthening the O-Ag side chains of LPS in the outer concentrations or incubation times allow cleavage of more com- membrane (24). However, resisting the MAC is likely not suffi- plement factors, such as C3 and C4 (data not shown). A previous cient for Gram-negative pathogens to fully overcome complement- article also showed that AprA cleaved C3 and C1q, but high mediated immune clearance. Complement also labels these bacteria concentrations of AprA (400 nM) and long incubation times (20 h) with C3b to support phagocytic uptake and generates C5a to attract were used (12). Western blotting experiments showed that AprA phagocytes to the site of infection. In vivo studies showed that (at 200 nM) effectively cleaved C1s in serum within 30 min (Fig. complement receptor 3 (29, 30) and the C5a receptor (31–33) on 5A). However, the functional-repletion assay (Fig. 5B) clearly neutrophils are important for protection against P. ae ru gi nos a lung showed that only C2 restored C3b deposition through the CP, infection. In this study, we showed that P. aeruginosa also inhibited suggesting that C1s is still active. Possibly, C1s is cleaved in other steps in the complement cascade by secretion of the metal- a manner that does not block its function. This could be similar to Downloaded from loprotease AprA. AprA blocks CP and LP activation and, thereby, natural activation of C1s, which is mediated by the protease C1r prevents C3b-dependent uptake of Pseudomonas by neutrophils and that cleaves the zymogen C1s into its active form, which can then C5a-dependent neutrophil activation. Previously, it was shown that cleave C4. Thus, by cleaving C1s, AprA may have paradoxically AprA inhibits MAC-dependent lysis of erythrocytes (12). In this enhanced CP activity up until the stage of C4b deposition. Indeed, study, we determined how AprA contributes to pseudomonal re- we observed that C4b deposition in the CP was increased in the

sistance against complement effector functions relevant to host presence of AprA. However, because AprA cleaves C2, we found http://www.jimmunol.org/ clearance of this bacterium. Furthermore, we identified the molec- that it blocked the CP at the level of C3b. The cleavage of C2 is in ular mechanism to be cleavage of C2. line with our findings that AprA does not affect the AP, because AprA is the first bacterial protease demonstrated to cleave C2. activation of this route is not dependent on C2. The AprA con- Because C2 concentrations in serum (0.25 mM) are low compared centrations used for cleavage of C2 were in a similar range as with other complement molecules (C3 at 6.8 mM, C4 at 2.1 mM), previous studies examining AprA cleavage of flagellin and other this molecule is a vulnerable target for proteolytic degradation. human immune proteins (11, 14). Interestingly, we found that AprA cleaves C2 very close to the Thus, we showed that AprA blocks activation of the CP and cleavage site of its natural proteases C1s and MASP. Strikingly, LP by degrading C2. Thereby P. aeruginosa evades complement- this is similar to the Staphylococcus aureus metalloprotease aur- mediated phagocytosis and killing by neutrophils. Other known by guest on September 28, 2021 eolysin, which cleaves the C3 molecule 2 aa apart from the natural complement-modulating proteins of P. aeruginosa are: the intra- protease-cleavage site (34). This suggests that the natural cleavage cellular elongation factor tuf that binds factor H (40), elastase that site in these proteins is an accessible part of the protein that makes degrades C3 (41), and protease IV that degrades C1q and C3 (42). it vulnerable for cleavage by bacterial proteases. Fig. 7 provides As observed for other bacterial pathogens (26), complement re- a schematic overview of how we think AprA blocks complement sistance factors in P. aeruginosa are probably as redundant as activation. The formation of the C4b2a convertase is a multistep the complement system itself. Especially because P. aeruginosa process (35). First, C2 binds surface-bound C4b and is subse- strains from CF patients are sensitive to complement C5b-9 lysis quently cleaved into C2a, forming the active convertase. Because in vitro (43, 44), we expect this bacterium to have multiple mol- of its short half-life, C2a is released from the complex and cannot ecules that counteract complement activation and enable bacterial reassociate with C4b. The fluid-phase cleavage of C2 by AprA survival in the human host. prevents formation of the proconvertase C4bC2 and an active C4b2a complex. At present, it is not clear whether the additional Acknowledgments cleavage sites of AprA in C2 (between CCP1 and CCP2 and be- We thank Eric Prossnitz for providing the U937-C5aR cells and Jeffrey tween the vWFA and peptidase domain) are functionally relevant. Beekman for providing GFP-labeled PAO1. Although some residues of the AprA-cleavage sites in C2 can also be found in factor B (the protein with the greatest homology to Disclosures C2), we did not observe any cleavage of purified fB (data not The authors have no financial conflicts of interest. shown) or fB in serum (Fig. 5). We expect that structural differ- ences between fB and C2 could explain the difference in acces- sibility of the cleavage site. However, the lack of structural data on References C2 makes it difficult to speculate about this point. 1. Kawai, T., and S. Akira. 2010. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11: 373–384. Production of AprA by P. aeruginosa differs among strains and 2. Gros, P., F. J. Milder, and B. J. Janssen. 2008. Complement driven by confor- is highly dependent on culture conditions (36). This is illustrated mational changes. Nat. Rev. Immunol. 8: 48–58. by the enhanced production of AprA by culturing in the presence 3. Gasque, P. 2004. Complement: a unique innate immune sensor for danger sig- nals. Mol. Immunol. 41: 1089–1098. of sputum from CF patients (37). P. aeruginosa produces detect- 4. Endo, Y., M. Takahashi, and T. Fujita. 2006. Lectin complement system and able levels of AprA in vivo, as measured in samples obtained from pattern recognition. Immunobiology 211: 283–293. patients with corneal infections (38) and in sputum isolated from 5. Kemper, C., J. P. Atkinson, and D. E. Hourcade. 2010. Properdin: emerging roles CF patients (16, 17). The observed in vivo concentrations tend to of a pattern-recognition molecule. Annu. Rev. Immunol. 28: 131–155. 6. Klos, A., A. J. Tenner, K. O. Johswich, R. R. Ager, E. S. Reis, and J. Ko¨hl. 2009. be in a different range than observed in P. aeruginosa overnight The role of the anaphylatoxins in health and disease. Mol. Immunol. 46: 2753– cultures and the concentrations used in our study. However, de- 2766. The Journal of Immunology 393

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