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Classical and Lectin PathwaysBlocks Complement Activation via the

Alkaline ProteasePseudomonas aeruginosa

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

ol.1102162http://www.jimmunol.org/content/early/2011/11/30/jimmun

published online 30 November 2011J Immunol 

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The Journal of Immunology

Pseudomonas aeruginosa Alkaline Protease BlocksComplement 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

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: 000–000.

Theinnate immune system rapidly detects and kills invadingbacteria via different mechanisms, such as TLRs and thecomplement system. TLRs are expressed on immune cells

and detect an enormous variety of microorganisms via recognitionof highly conserved microbial molecular patterns, such as flagellinvia TLR5 and LPS via TLR4 (1). Upon ligand binding, TLRstrigger an intracellular signaling cascade that leads to productionof proinflammatory cytokines and activation of phagocytes. Thecomplement system is a proteolytic cascade of plasma proteins,which results in direct killing of certain microorganisms and ef-ficient bacterial recognition by phagocytes. The complementsystem consists of three distinct pathways: the classical pathway(CP), lectin pathway (LP) and alternative pathway (AP). All threerecognition pathways culminate in the formation of C3 convertaseenzymes that mediate deposition of C3b on foreign surfaces (2).The CP is initiated by binding of C1q to bacterium-bound Abs orrepeating molecular patterns on bacteria. This binding initiatesactivation of the C1q-attached protease C1s that can cleave C4into C4b, which covalently binds the microbial surface. The C4bprotein is bound by C2, forming the proconvertase C4bC2. TheC1s protease then cleaves C2 into the larger protease fragment

C2a (70 kDa), which remains attached to C4b, forming the C3convertase complex (C4b2a). The smaller C2b domain (30 kDa) isreleased into the surrounding area of inflammation (the literatureis inconsistent regarding the nomenclature of C2 fragments; in thisarticle we use C2a and C2b for the 70- and 30-kDa fragments,respectively). Activation of the LP occurs in a similar fashion.Recognition of microbes occurs via the mannose-binding lectin(MBL) or ficolins that bind to repeating molecular patterns onmicrobial surfaces (3, 4). MBL and ficolins are complexed to theMBL-associated protease (MASP), which functionally resemblesC1s because it cleaves C4 and C2 to induce formation of the C3convertase complex. In the AP, microbial surfaces are recognizedby properdin (5), which initiates assembly of the AP C3 con-vertase (C3bBb) on the surface. The AP also serves as an am-plification loop to enhance the concentration of C3b on thesurface. Cleavage of C3 by convertases is critical to opsonizationof the bacterial surface with C3b, which is recognized by com-plement receptors on phagocytes and supports phagocytosis andkilling. Further downstream in the cascade, formation of C5convertases and cleavage of C5 result in generation of the potentanaphylatoxin C5a and the formation of C5b-9, the membraneattack complex (MAC) that can directly kill certain Gram-negativebacteria (6).The Gram-negative bacteria Pseudomonas aeruginosa lives in

water and soil and acts as an opportunistic pathogen in humansand plants. In healthy individuals, P. aeruginosa is efficientlykilled via the innate immune system; however it causes chronicinfections in immunocompromised patients. For instance, cysticfibrosis (CF) patients suffer from chronic infections caused by P.aeruginosa that cannot be eradicated after colonization. Strainsisolated from CF patients rapidly adapt to the environment in thelung, resulting in dramatic genetic and morphological changes (7).Infection by P. aeruginosa is complicated by its inherent resis-tance to several classes of antibiotics and acquisition of resistancegenes via mobile genetic elements (8). In addition, biofilm for-mation enhances the resistance to antibiotics in the CF lung (9).Remarkably, the strains isolated from CF patients are generallysensitive to direct complement killing, which is different from

Department of Medical Microbiology, University Medical Center Utrecht, 3584 CXUtrecht, The Netherlands

1A.J.L. and B.W.B. contributed equally to this work.

Received for publication July 25, 2011. Accepted for publication October 24, 2011.

A.J.L., B.W.B., J.A.G.v.S., and S.H.M.R. are supported by grants of The NetherlandsOrganization for Scientific Research (TOP and VIDI).

Address correspondence and reprint requests to Dr. S.H.M. Rooijakkers, Departmentof Medical Microbiology, University Medical Center Utrecht, PO G04.614, Heidel-berglaan 100, 3584 CX Utrecht, The Netherlands. E-mail address: s.h.m.rooijakkers@umcutrecht.nl

The online version of this article contains supplemental material.

Abbreviations used in this article: AP, alternative pathway; AprA, alkaline protease;CCP, complement control protein; CF, cystic fibrosis; CP, classical pathway; fB,factor B; HSA, human serum albumin; LB, Luria–Bertani; LP, lectin pathway;MAC, membrane attack complex; MASP, mannose-binding lectin-associated prote-ase; MBL, mannose-binding lectin; vWFA, von Willebrand factor A.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102162

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strains isolated from non-CF patients. These serum-resistant strainsconsume more C5b-9 complement components and have lessC3b deposited on their surface (10).Bacterial survival in the host is accompanied by the acquisition

of different immune-evasive strategies. These strategies involvealteration of the bacterial surface, such as production of a cap-sule or immune-modulating membrane proteins, or secretion ofsoluble immune-evasion proteins, such as proteolytic factors. P.aeruginosa secretes a number of toxins and virulence factors,like elastase and alkaline protease (AprA). Elastase cleaves awide variety of host proteins, such as collagen, IgG, and comple-ment proteins (11). AprA is a 50-kDa zinc metalloprotease thatdegrades several components of the host immune system, such ascomplement C1q and C3 (12), or cytokines, like IFN-g andTNF-a (13). In addition, AprA prevents TLR5 activation viathe cleavage of monomeric flagellin (14). AprA is secreted via itsown type I secretion system, which is encoded by three genesupstream of the aprA gene. P. aeruginosa also encodes a highlyspecific inhibitor of AprA named AprI, which is translocated tothe periplasmic space according to its signal sequence. The roleof AprA in pseudomonal virulence has been illustrated in aDrosophila infection model (15). AprA contributes to the per-sistence of Pseudomonas entomophila infection and protectsagainst antimicrobial peptides produced by the innate immunesystem of Drosophila. Furthermore, AprA was found to be ex-pressed in human CF patients infected with P. aeruginosa (16,17).In a previous study, Hong and Ghebrehiwet (12) showed that

AprA cleaves complement components C1q and C3 and inhibitslysis of sheep erythrocytes via MAC. However, because P. aeru-ginosa is generally resistant to direct lysis via MAC, we wonderedwhether cleavage of complement proteins by AprA contributes topseudomonal immune evasion. We found that AprA blocks vari-

ous important complement functions hampering the bacterialclearance by human neutrophils. Furthermore, by using biologicalassays with more physiological relevant protease concentrations,we uncovered that AprA specifically blocks the CP and LP andpredominantly cleaves complement protein C2.

Materials and MethodsProteins, sera, and bacterial strains

C3 was purified from human plasma, as described (18). The purifiedcomponents C1, C2, and C4 were purchased from Quidel. C1s was ob-tained from R&D, and C2-depleted serum was purchased from Sigma.Normal human serum was obtained from healthy volunteers, who gaveinformed consent. AprA and AprI of P. aeruginosa PAO1 were expressedand isolated from Escherichia coli, as described (14). Briefly, both proteinswere expressed with a hexa-histidine tag in E. coli BL21 (Invitrogen).Recombinant proteins were isolated under denaturing conditions usinga His trap column (GE Healthcare). AprA was refolded by dilution in 50mM 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 AprIwas removed using enterokinase (Invitrogen). Both proteins were .95%pure, as assessed by SDS-PAGE and Instant Blue staining. Wild-type P.aeruginosa PAO1 was obtained from the Genome Sciences Manoil labo-ratory (University of Washington), and GFP-labeled PAO1 (19) was kindlyprovided by J.M. Beekman (University Medical Center Utrecht).

Hemolytic assays

The CP hemolytic assay was performed, as previously described (20), withminor modifications. Serum was preincubated with 22, 66, and 200 nMAprA or 200 nM AprA plus 1000 nM AprI for 30 min at 37˚C. Subse-quently, opsonized (anti-sheep IgM) sheep erythrocytes (Alsever) wereincubated with AprA-treated serum in Veronal-buffered saline containing0.5 mM CaCl2 and 0.25 mM MgCl2. After 30 min at 37˚C, samples werecentrifuged, and the absorbance of the supernatants at 405 nm was mea-sured. The AP hemolytic assay was performed, as described above, usingrabbit erythrocytes (Alsever) with Veronal-buffered saline containing 5mM MgCl2 and 10 mM EGTA.

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.

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E. coli killing

E. coli K12 was cultured in Luria–Bertani (LB) medium overnight at 37˚C.The next day, the overnight culture was diluted 20 times in LB medium andgrown to an OD660nm of 0.5. Bacteria were washed with RPMI 1640supplemented with 0.05% human serum albumin (HSA). Human pooledserum was incubated with different concentrations of AprA, with orwithout 1000 nM AprI, for 30 min at 37˚C in RPMI-HSA. Subsequently,bacteria were added (13 105/ml) and incubated for 1 h at 37˚C. CFU weredetermined by plating serial dilutions on tryptic soy agar + 5% sheep blood.

Phagocytosis and killing

Phagocytosis assays were performed, as described (21). In short, serum waspreincubated with buffer, 200 nM AprA, or 200 nM AprA plus 1000 nMAprI in RPMI 1640 containing 0.1% HSA for 30 min at 37˚C. Then, 2.5 3105 freshly isolated human neutrophils and 2.5 3 106 GFP-labeled, heat-killed P. aeruginosa were added and incubated for 15 min at 37˚C whileshaking at 600 rpm. The reaction was stopped by adding 1% ice-coldparaformaldehyde in RPMI 1640 containing 0.1% HSA. Phagocytosiswas analyzed using the FACSCalibur (Becton Dickinson). Neutrophilswere gated on the basis of forward- and side-scatter properties. Fluores-cence intensity of 10,000 gated neutrophils was determined, and phago-cytosis was expressed as the percentage of these cells containingfluorescently labeled bacteria (calculated using BD CellQuest Pro soft-ware). In another experiment, different concentrations of AprA were in-cubated with 5% serum in RPMI 1640 containing 0.1% HSA for 30 min at37˚C. The IC50 value was calculated with GraphPad Prism software usingthe sigmoidal dose-response function and the equation Y = bottom +(top 2 bottom)/(1+10^[logEC502X]) where X is the logarithm of the

AprA concentration, and Y is the percentage of phagocytosis (for the AprAcurve, bottom = 9.38 and top = 102.5, R2 = 0.8979).

For neutrophil-killing assays, P. aeruginosa was grown to an OD660 of0.5 in LB medium and subsequently washed in RPMI 1640 containing0.1% HSA. Then, 5% serum was preincubated with buffer, 200 nM AprA,or 200 nM AprA plus 1000 nM AprI for 30 min at 37˚C. Subsequently,2.5 3 105 P. aeruginosa and 8.5 3 106 neutrophils were added and incu-bated at 37˚C. A sample was taken at different time points, and neutrophilswere lysed with Milli-Q water. Surviving bacteria were enumerated byplating serial dilutions on LB agar.

C3b deposition on P. aeruginosa

An overnight culture of P. aeruginosa strain PAO1-GFP in LB mediumwas washed in Veronal-buffered saline containing 0.5 mM CaCl2, 0.25 mMMgCl2, and 0.1% BSA. Serum was preincubated with different concen-trations of AprA, with or without AprI, for 30 min at 37˚C. Then, 2.5 3106 bacteria were incubated with the preincubated serum for 30 min whileshaking at 900 rpm. Bacteria were washed with PBS with 0.1% BSA. C3bdeposition was detected using mouse anti-human C3b Abs (WM-1;American Type Culture Collection) and allophycocyanin-conjugated goatanti-mouse IgG (Protos). Median fluorescence of 10,000 bacteria wasmeasured by flow cytometry.

C5a analysis

An overnight culture of P. aeruginosa strain PAO1 in LB medium was washedin RPMI 1640 with 0.1% HSA. Ten-percent serum and 200 nM AprA or200 nM AprA plus 1000 nM AprI were preincubated for 30 min at 37˚Cand subsequently incubated with 53 107 bacteria at 37˚C for 30 min while

FIGURE 2. AprA inhibits complement-dependent

effector functions important for P. aeruginosa. A and

B, Phagocytosis of P. aeruginosa PAO1-GFP by hu-

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 dif-

ferent 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 fluo-

rescence. For E, the relative survival was calculated

by dividing the number of CFU by the number of

CFU at time 0.

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shaking at 600 rpm. Bacteria were centrifuged, and C5a was detected incollected supernatants by calcium mobilization: 10-fold–diluted supernatantswere added to 53 104 Fluo-4-AM–labeled U937-C5aR cells (a generous giftfrom Prof. Eric Prossnitz, University of New Mexico, Albuquerque, NM),and the increase in intracellular calcium was measured by flow cytometry.

Complement assays

Complement ELISAs were performed, as described (22), with modifications.ELISA plates (MaxiSorp; Nunc) were coated overnight with 20 mg/ml LPS(Salmonella enteriditis; Sigma), 3 mg/ml IgM (Quidel), or 10 mg/ml man-nan (Saccharomyces cerevisiae; Sigma) in 0.1 M sodium carbonate buffer(pH 9.6). Plates were blocked with 4% BSA in PBS with 0.05% Tween for1 h at 37˚C. For the CP and LP, samples were diluted in Veronal-bufferedsaline containing 0.5 mM CaCl2, 0.25 mMMgCl2, 0.1% gelatin, and 0.05%Tween. For the AP, samples were diluted in Veronal-buffered saline con-taining 5 mM MgCl2, 10 mM EGTA, 0.1% gelatin, and 0.05% Tween.Serum or C2-depleted serum was mixed with different concentrations ofAprA or AprA together with AprI and subsequently added to the plates for1 h at 37˚C. Deposited C3b and C4b were detected using Abs against C3d(WM-1–digoxigenin-labeled) and C4d (Quidel), respectively, followed byperoxidase-conjugated sheep anti-digoxigenin or a goat anti-mouse IgG(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 at37˚C. Then, 1000 nM AprI was added together with C1 (2 nM), C1s (3nM), C2 (2 nM), C3 (67 nM), or C4 (20 nM), and the mixtures were addedto 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˚Cin Veronal-buffered saline containing 0.5 mM CaCl2 and 0.25 mM MgCl2.Proteins were subjected to SDS-PAGE and transferred to a polyvinylidenedifluoride membrane. Cleavage products were visualized with 0.1% Coo-massie blue in 50% methanol, excised, and analyzed by N-terminal se-quencing (Alphalyse).

Western blotting

The P. aeruginosa strain PAO1 was cultured overnight in LB medium andsubsequently diluted 10 times in IMDM (Invitrogen). Bacterial supernatantwas collected after 2, 4, and 6 h. The presence of AprA in supernatant of P.aeruginosa was analyzed by Western blotting using a protein G-purifiedpolyclonal rabbit anti-AprA Ab (Genscript), followed by a goat anti-rabbitIgG Ab (Southern Biotech). Bands were quantified using ImageJ software.

Analysis of complement factor cleavage in serum was performed byWestern blotting. Three-percent serum was incubated with different con-centrations of AprA at 37˚C in PBS. The reaction was stopped by addingLaemmli sample buffer containing DTT. All samples were subjected toSDS-PAGE and blotted onto a polyvinylidene difluoride membrane. Afterblocking with 4% skimmed milk in PBS containing 0.1% Tween, C1s wasdetected by a mouse anti-human C1s Ab (Quidel), followed by HRP-labeled goat anti-mouse; C2, C3, and factor B (fB) were detected bygoat anti-human C2 (Quidel), goat anti-human C3 (Protos), and goat anti-human fB (Quidel), respectively, followed by HRP-labeled donkey anti-goat (Jackson); and C4 was detected by biotin-labeled chicken anti-humanC4 (Abcam), followed by HRP-labeled streptavidin (Southern Biotech).All Ab incubations were diluted in PBS with 0.1% Tween and 1% skim-med milk. ECL (GE Healthcare) was used for final signal detection.

ResultsPhysiological AprA levels inhibit complement-mediated lysis

To study the functional relevance of AprA secretion by P. aeru-ginosa on the complement system, we first estimated AprA con-centrations in culture supernatants. Supernatants were collected at

FIGURE 3. AprA inhibits C3b deposition via

the CP and LP. Serum was incubated with dif-

ferent 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) or LP (E). All figures represent mean 6SEM of three independent experiments.

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

1 h at 37˚C. B, 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 water-treated erythrocytes and represent mean 6 SEM of

three independent experiments.

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different time points and analyzed for AprA expression byWestern blotting using an Ab against AprA (Fig. 1A). The levelsof AprA in the supernatant were semiquantified by comparisonwith fixed concentrations of recombinant AprA (SupplementalFig. 1). The AprA concentrations in culture supernatants werebetween 8 nM (2-h culture) and 208 nM (∼10 mg/ml; 6-h super-natant). Because AprA was described to inhibit complement-dependent hemolysis via MAC, we repeated the assay to con-firm AprA activity. AprA inhibited the lysis of Ab-sensitizedsheep erythrocytes, a CP-mediated hemolysis assay, which wasrestored in the presence of AprI (Fig. 1B). Furthermore, we testeddifferent concentrations of AprA in a serum-killing assay using E.coli as a model. In contrast to P. aeruginosa strain PAO1, E. coliK-12 is not serum resistant and is lysed at low serum concen-trations as a result of incorporation of the lytic MAC. Indeed,AprA inhibited the complement-dependent lysis with an estimatedIC50 of 30 nM, which is within the concentration range detectedin the supernatant (Fig. 1C). The natural inhibitor AprI blocksproteolytic activity of AprA (23), thereby allowing complement-dependent lysis of E. coli. Thus, AprA blocks complement-dependent lysis in a concentration range similar to AprA levelsin P. aeruginosa supernatant.

AprA inhibits complement-dependent neutrophil functionsimportant for P. aeruginosa killing

Because many P. aeruginosa strains are resistant to direct com-plement lysis (24), we investigated whether AprA facilitates re-sistance to other complement processes that are critical toneutrophil functioning. To study whether AprA blocks comple-ment-dependent phagocytosis, we incubated GFP-labeled P. aer-uginosa with human neutrophils in the presence of human serumand AprA. AprA blocked the phagocytosis compared with bufferor AprA supplemented with AprI (Fig. 2A), and its activity wasdose dependent (Fig. 2B). The IC50 of AprA is 100 nM, which isin the relevant range produced by P. aeruginosa. Incubation of P.aeruginosa with serum results in the deposition of C3b moleculeson the bacterial surface, which are recognized by complementreceptors on phagocytes. To study whether AprA inhibits the C3bdeposition, we incubated bacteria with serum and detectedsurface-bound C3b using specific Abs and flow cytometry. AprApartially inhibited C3b deposition on Pseudomonas at comparableconcentrations as observed for phagocytosis of P. aeruginosa (Fig.2C). Further along the complement cascade, C5 is cleaved into thepotent anaphylatoxin C5a that is recognized by the C5a receptor

on neutrophils. Activation of the C5a receptor induces intracel-lular calcium mobilization and chemotaxis of the neutrophil to thesite of inflammation. We incubated P. aeruginosa with serum andstudied the release of C5a into the supernatant by measuring thesupernatant-induced calcium mobilization on Fluo-3-AM–labeledU937 cells transfected with the C5a receptor (specificity of thisassay is presented in Supplemental Fig. 2). When bacteria wereincubated with AprA, supernatants could not induce calciummobilization, meaning that there was no active C5a in the pres-ence of AprA (Fig. 2D). Because AprA also blocks formation ofC5b-9 (Fig. 1B, 1C), we believe that AprA does not specificallycleave C5a but prevents activation of C5 into C5a and C5b. Be-cause AprA also blocks C3b deposition, it seems likely that itinhibits complement activation upstream of C3. To verify thatAprA also inhibits the killing of P. aeruginosa by neutrophils, weincubated neutrophils with bacteria and serum and determined thepercentage of survival after 15 min. After 15 min, 80% of thebacteria were killed. AprA inhibited the killing of P. aeruginosa,but not completely, showing a killing of 40% after 15 min. Thiseffect was restored in the presence of AprI (Fig. 2E). In summary,AprA inhibited all complement-dependent effector functions,which allowed P. aeruginosa to escape neutrophil killing.

AprA inhibits complement activation via the CP and LP

To further study the effect of AprA on the complement system,we dissected the individual pathways using pathway-specific com-plement ELISAs (22). The CP, LP, and AP were specifically in-duced in the presence of serum using the coatings IgM, mannan,and LPS, respectively. The level of C3b deposition was detectedwith specific Abs. AprA strongly inhibited C3b deposition of theCP (Fig. 3A) and LP (Fig. 3B) at all tested serum concentrations.Inhibition of the AP was not observed at AprA concentrations upto 66 nM (Fig. 3C). The AP ELISA was not inhibited at higherconcentrations of 2000 nM AprA (data not shown). Because oftechnical limitations, we could not detect AP-mediated C3b de-position at serum concentrations ,10%. We used a more sensitivehemolytic complement assay to compare the effect of AprA in theAP and CP at similar serum concentrations. Fig. 4A shows that200 nM AprA did not block AP-mediated hemolysis in 5% serum;however, 200 nM AprA blocked CP-mediated hemolysis (Figs.1B, 4B). As mentioned above, C3b deposition via the CP and LP ismediated by C3 convertases, bimolecular complexes that consistof the protease C2a loosely attached to surface-bound C4b. C4band 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 experi-

ments. Blots are representatives of three separate ex-

periments.

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MASP complexes. To study at what level AprA affects thesepathways, we also analyzed the C4b deposition. In contrast to C3bdeposition, AprA did not block C4b deposition in the CP and LP(Fig. 3D, 3E). This indicated that AprA specifically blocks the CP

and LP upstream of C3 and downstream of C4 activation in thecomplement cascade.

AprA depletes the limiting factor C2

Many complement factors that are involved in the CP and LPinduce C3b deposition; however, only two factors (C2 and C3) areinvolved after C4b deposition and before C3b deposition. Toidentify the critical factor that is cleaved by AprA, we incubatedhuman serum with AprA for 30 min at 37˚C. The complementfactors C2, C3, and C4 were analyzed by Western blotting. Ascontrols, C1s and fB were detected, which are crucial componentsof 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 lessefficient than C2 cleavage. We used the CP complement ELISA todiscriminate the limiting factor that was cleaved by AprA. First,we incubated serum with AprA and inactivated the protease byadding the irreversible inhibitor AprI. Then, treated serum wasused in the CP ELISA supplemented with the different purifiedcomplement factors. Only the serum that was supplemented withpurified C2 restored the complement activation, whereas AprA-treated serum supplemented with C1, C1s, or C3 showed no ac-tivity (Fig. 5B). Thus, although AprA also cleaves C1s, thecleavage of C2 determines inhibition of complement activation.

AprA cleaves C2

C2 is a 100-kDa protein that consists of three complement controlprotein (CCP) regions, a von Willebrand factor A (vWFA) domain,and a peptidase domain. The cleavage of C2 by its natural proteasesC1s and MASP results in formation of C2b (CCP1–3) and C2a(vWFA and peptidase domain) (Fig. 6B, 6C). For the formation ofa functional C3 convertase (C4b2a), full-length C2 first binds toC4b and is subsequently cleaved. C2b is then released, while C2astays attached to C4b. The C4b2a convertase has a very short half-life due to the irreversible dissociation of C2a. C2 cleavage byAprA was further analyzed by SDS-PAGE and Coomassie stain-ing. After incubating purified C2 with AprA, we observed threecleavage products of ∼75, ∼40, and ∼30 kDa, which were sent forN-terminal sequencing (Fig. 6A). Cleavage product 1 starts withNH2-IQIQRS, which is only 1 aa different from C2a (starts withNH2-KIQIQR). At higher protease concentrations, an increase incleavage product 2 is observed, meaning that AprA secondarilycleaves between the vWFA and peptidase domain. Next to C2a,AprA cleaves C2b between CCP1 and CCP2. These data indicatedthat AprA efficiently interferes with CP and LP activation via thedegradation of C2 (Fig. 7).

DiscussionBacterial pathogens adopt many strategies to counteract the re-dundant attack of the human host defense. For instance, bacteria

FIGURE 7. Schematic representation of the com-

plement-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.

FIGURE 6. AprA cleaves C2. A, C2 cleavage by AprA. Purified C2 was

incubated with different concentrations of AprA, and cleavage was ana-

lyzed by SDS-PAGE and Coomassie staining. Cleavage products 1, 2, and

3 were sent for N-terminal sequencing. B, Sequence of human C2; the

obtained N-terminal sequences of cleavage products 1, 2, and 3 are

underlined. C, Schematic representation of C2. The domains and the

cleavage site for C1s, MASP, and AprA are indicated.

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secrete many proteins that specifically interact with the comple-ment system, which can either be proteolytic or steric interactions(25, 26). In the defense against Gram-negative bacteria, thecomplement system directly destroys cells by inserting the pore-forming MAC into the bacterial membrane (27). Although allGram-positive bacteria are protected from MAC lysis by theirthick peptidoglycan layer, some Gram-negative bacteria have alsobecome resistant to direct complement attack by altering theirsurface properties (28). The Gram-negative bacteria P. aeruginosadoes this by lengthening the O-Ag side chains of LPS in the outermembrane (24). However, resisting the MAC is likely not suffi-cient for Gram-negative pathogens to fully overcome complement-mediated immune clearance. Complement also labels these bacteriawith C3b to support phagocytic uptake and generates C5a to attractphagocytes to the site of infection. In vivo studies showed thatcomplement receptor 3 (29, 30) and the C5a receptor (31–33) onneutrophils are important for protection against P. aeruginosa lunginfection. In this study, we showed that P. aeruginosa also inhibitedother steps in the complement cascade by secretion of the metal-loprotease AprA. AprA blocks CP and LP activation and, thereby,prevents C3b-dependent uptake of Pseudomonas by neutrophils andC5a-dependent neutrophil activation. Previously, it was shown thatAprA inhibits MAC-dependent lysis of erythrocytes (12). In thisstudy, we determined how AprA contributes to pseudomonal re-sistance against complement effector functions relevant to hostclearance of this bacterium. Furthermore, we identified the molec-ular mechanism to be cleavage of C2.AprA is the first bacterial protease demonstrated to cleave C2.

Because C2 concentrations in serum (0.25 mM) are low comparedwith other complement molecules (C3 at 6.8 mM, C4 at 2.1 mM),this molecule is a vulnerable target for proteolytic degradation.Interestingly, we found that AprA cleaves C2 very close to thecleavage site of its natural proteases C1s and MASP. Strikingly,this is similar to the Staphylococcus aureus metalloprotease aur-eolysin, which cleaves the C3 molecule 2 aa apart from the naturalprotease-cleavage site (34). This suggests that the natural cleavagesite in these proteins is an accessible part of the protein that makesit vulnerable for cleavage by bacterial proteases. Fig. 7 providesa schematic overview of how we think AprA blocks complementactivation. The formation of the C4b2a convertase is a multistepprocess (35). First, C2 binds surface-bound C4b and is subse-quently cleaved into C2a, forming the active convertase. Becauseof its short half-life, C2a is released from the complex and cannotreassociate with C4b. The fluid-phase cleavage of C2 by AprAprevents formation of the proconvertase C4bC2 and an activeC4b2a complex. At present, it is not clear whether the additionalcleavage sites of AprA in C2 (between CCP1 and CCP2 and be-tween the vWFA and peptidase domain) are functionally relevant.Although some residues of the AprA-cleavage sites in C2 can alsobe found in factor B (the protein with the greatest homology toC2), we did not observe any cleavage of purified fB (data notshown) 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 onC2 makes it difficult to speculate about this point.Production of AprA by P. aeruginosa differs among strains and

is highly dependent on culture conditions (36). This is illustratedby the enhanced production of AprA by culturing in the presenceof sputum from CF patients (37). P. aeruginosa produces detect-able levels of AprA in vivo, as measured in samples obtained frompatients with corneal infections (38) and in sputum isolated fromCF patients (16, 17). The observed in vivo concentrations tend tobe in a different range than observed in P. aeruginosa overnightcultures and the concentrations used in our study. However, de-

termination of AprA concentration in vivo is complicated by thefact that AprA is produced locally in the bacterial environmentand is subsequently diluted or degraded by host proteases inclinical samples. In a rat pouch model, only small amounts ofinjected recombinant AprA could be recovered after 6 h (39),suggesting that production of AprA in vivo is much greater thanmeasured. In this article, we showed that AprA cleaves C2 inconcentrations similar to levels secreted by P. aeruginosa infunctionally relevant time incubations. However, increasing AprAconcentrations or incubation times allow cleavage of more com-plement factors, such as C3 and C4 (data not shown). A previousarticle also showed that AprA cleaved C3 and C1q, but highconcentrations of AprA (400 nM) and long incubation times (20 h)were used (12). Western blotting experiments showed that AprA(at 200 nM) effectively cleaved C1s in serum within 30 min (Fig.5A). However, the functional-repletion assay (Fig. 5B) clearlyshowed that only C2 restored C3b deposition through the CP,suggesting that C1s is still active. Possibly, C1s is cleaved ina manner that does not block its function. This could be similar tonatural activation of C1s, which is mediated by the protease C1rthat cleaves the zymogen C1s into its active form, which can thencleave C4. Thus, by cleaving C1s, AprA may have paradoxicallyenhanced CP activity up until the stage of C4b deposition. Indeed,we observed that C4b deposition in the CP was increased in thepresence of AprA. However, because AprA cleaves C2, we foundthat it blocked the CP at the level of C3b. The cleavage of C2 is inline with our findings that AprA does not affect the AP, becauseactivation of this route is not dependent on C2. The AprA con-centrations used for cleavage of C2 were in a similar range asprevious studies examining AprA cleavage of flagellin and otherhuman immune proteins (11, 14).Thus, we showed that AprA blocks activation of the CP and

LP by degrading C2. Thereby P. aeruginosa evades complement-mediated phagocytosis and killing by neutrophils. Other knowncomplement-modulating proteins of P. aeruginosa are: the intra-cellular elongation factor tuf that binds factor H (40), elastase thatdegrades C3 (41), and protease IV that degrades C1q and C3 (42).As observed for other bacterial pathogens (26), complement re-sistance factors in P. aeruginosa are probably as redundant asthe complement system itself. Especially because P. aeruginosastrains from CF patients are sensitive to complement C5b-9 lysisin vitro (43, 44), we expect this bacterium to have multiple mol-ecules that counteract complement activation and enable bacterialsurvival in the human host.

AcknowledgmentsWe thank Eric Prossnitz for providing the U937-C5aR cells and Jeffrey

Beekman for providing GFP-labeled PAO1.

DisclosuresThe authors have no financial conflicts of interest.

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