Role of Capsule and Suilysin in Mucosal Infection of Complement-Deficient Mice with Streptococcus suis

Maren Seitz,a Andreas Beineke,b Alena Singpiel,a Jörg Willenborg,a Pavel Dutow,c Ralph Goethe,a Peter Valentin-Weigand,a

Andreas Klos,c Christoph G. Baumsa*

Institute for Microbiology, University of Veterinary Medicine Hanover, Hanover, Germanya; Institute for Pathology, University of Veterinary Medicine Hanover, Hanover,Germanyb; Department of Medical Microbiology, Hanover Medical School, Hanover, Germanyc

Virulent Streptococcus suis serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets andhumans. One objective of this study was to elucidate the function of complement in innate immune defense against S. suis. Ex-perimental infection of wild-type (WT) and C3�/� mice demonstrated for the first time that the complement system protectsnaive mice against invasive mucosal S. suis infection. S. suis WT but not an unencapsulated mutant caused mortality associatedwith meningitis and other pathologies in C3�/� mice. The capsule contributed also substantially to colonization of the upperrespiratory tract. Experimental infection of C3�/� mice with a suilysin mutant indicated that suilysin expression facilitated anearly disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface ofca. 40% of S. suis WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity thanon the unencapsulated mutant. Ex vivo multiplication in murine WT and C3�/� blood depended on capsule but not suilysinexpression. Interestingly, S. suis invasion of inner organs was also detectable in C5aR�/� mice, suggesting that chemotaxis andactivation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function ofthe complement system in defense against mucosal S. suis infection. In conclusion, we unequivocally demonstrate here the im-portance of complement against mucosal S. suis serotype 2 infection and that the capsule of this pathogen is also involved in es-cape from complement-independent immunity.

Invasive Streptococcus suis diseases such as septicemia and men-ingitis account for major economic losses in the swine industry.

Furthermore, S. suis is an important zoonotic pathogen, causingmainly meningitis in adult humans (1–3). S. suis is a very diverseorganism, and different serotypes are responsible for morbidity inpiglets. Serotype 2 strains are important worldwide for diseases inpiglets and are by far the leading serotype associated with zoonoticS. suis cases (4).

The complement system is involved in innate and adaptiveimmune responses. It might be activated by three different routes:the classical, the alternative, and the mannose-binding lectinpathways. All three pathways lead to the formation of C3 conver-tases (C3Bp or C4b2a) cleaving C3 into the anaphylatoxin C3aand the most important opsonin, C3b. In addition to opsoniza-tion of bacteria with C3b, the formation of important cytokine-like peptides, in particular C3a and C5a, is a main function of thecomplement system. C5aR is the main receptor for C5a and ishighly expressed by cells of myeloid origin (5). It should be notedthat the impact of the complement system on host defense againstS. suis has not been investigated in vivo. Some virulence factors areknown to be involved in resistance of serotype 2 strains againstkilling by neutrophils in the presence of complete serum (6, 7), butonly a few studies have specifically addressed putative comple-ment evasion strategies of S. suis.

The polysaccharide capsule of S. suis is an essential virulencefactor (8, 9). It protects S. suis against killing by macrophages andneutrophils in vitro. The capsule of serotype 2 strains containssialic acid (10, 11). Surface-associated sialic acid interferes withthe activation of the alternative complement cascade by increasingthe affinity constant of C3b to the complement inhibitor factor H(12, 13). However, S. suis serotype 2 expresses also a cell wall-anchored factor H-binding protein (Fhb) which has been shown

to be crucial for virulence in piglets (14). As an anti-human factorH serum had no effect on the C3b deposition on the surface of theisogenic �Fhb mutant, it was concluded that Fhb is the only factorof S. suis binding human factor H (14).

Suilysin is a cholesterol-dependent pore-forming cytolysin ex-pressed by many virulent S. suis strains (15, 16). Intraperitonealinfections of mice indicated that suilysin expression is essential forvirulence of S. suis in mice (17). However, a sly knockout mutantwas not attenuated in virulence in experimental infection of pig-lets (18). Interestingly, suilysin expression contributes to resis-tance against killing by porcine neutrophils and dendritic cells inthe presence of active but not inactive serum (19, 20). This findingled to the speculation that suilysin leads to reduced complementdeposition on the bacterial surface, as has been demonstrated forpneumolysin (21).

Mice have frequently been used as a model to study the patho-genesis of S. suis diseases using intraperitoneal application (22–

Received 22 January 2014 Returned for modification 9 March 2014Accepted 22 March 2014

Published ahead of print 31 March 2014

Editor: A. Camilli

Address correspondence to Christoph G. Baums, christoph.baums@gmx.de.

* Present address: Christoph G. Baums, Institute for Bacteriology and Mycology,Centre for Infectious Diseases, College of Veterinary Medicine, University ofLeipzig, Leipzig, Germany.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00080-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00080-14

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24). A mucosal mouse model for S. suis meningitis has not untilnow been available. We recently described an intranasal coloniza-tion model for S. suis serotype 2 in C57BL/6J mice (25). Coloni-zation of mucosal surfaces is regarded to be the first step in thepathogenesis of S. suis diseases in piglets. Therefore, it was reason-able to assume that innate immune defense mechanisms pre-vented further progress of invasion in the murine colonizationmodel of a virulent serotype 2 strain. In the present study, weinvestigated the working hypothesis that complement might becrucial for protection against S. suis invasion. Furthermore, weused complement-deficient mice to study the impact of the cap-sule and suilysin on the evasion of complement-independent hostdefense.

MATERIALS AND METHODSMaterials and reagents. Unless stated otherwise, all materials and re-agents were purchased from Sigma (Munich, Germany).

Bacterial strains and growth conditions. S. suis wild-type (WT)strain 10, kindly provided by H. Smith (Lelystad, Netherlands), is a viru-lent serotype 2 strain that expresses suilysin, muramidase-released pro-tein, extracellular factor, opacity factor of S. suis, and the immunoglobulinM-degrading enzyme of S. suis (IdeSsuis) (8, 26, 27). It has been used bydifferent groups successfully for experimental intranasal infections of pig-lets (8, 26) and recently by our group in intranasal infection of mice (25).The unencapsulated isogenic mutant 10cps�EF and the suilysin mutant10�sly were generated in previous studies by insertion of spectinomycinand erythromycin resistance gene cassettes into genes involved in capsulebiosynthesis (cpsE and cpsF) and the suilysin gene of strain 10, respectively(8, 28).

Streptococci were grown on Columbia agar supplemented with 7%sheep blood (Oxoid, Wesel, Germany). For detection of the unencapsu-lated mutant 10cps�EF in the competition experiment, Columbia agarsupplemented with 7% sheep blood and 100 �g of spectinomycin/ml wereused. Cultivation was conducted overnight under aerobic conditions at37°C.

Experimental intranasal infection of mice. For experimental S. suisinfection and as source of murine blood, the following mice strains with aC57BL/6J background were used at an age of 5 weeks: C57BL/6J WTmice (Charles River WIGA, Sulzfeld, Germany), C3-deficient (C3�/�)mice (B6.129S4 –C3tm1Crr/J) (29), and C5aR-deficient (C5aR�/�) mice(B6.129S4 –C5ar1tm1Cge/J) (30). Intranasal infections were conducted asdescribed previously (25), except that a lower dose was used (see below).Briefly, mice were predisposed to infection through application of 5 �l of1% acetic acid (pH 4.0) in each nostril 1 h prior intranasal infection(conducted in anesthesia via inhalation of isoflurane (IsoFlo, Albrecht,Germany). After controlled recovery, mice were infected using additionalanesthesia. Three differently designed infection experiments with C3�/�

and WT mice were conducted. First, C3�/� and WT mice were infectedwith 2 � 108 CFU of either S. suis WT (strain 10), 10cps�EF, or 10�slystrain grown to late exponential growth phase (optical density at 600 nm[OD600] of 0.8) for the determination of Kaplan-Meier diagrams. Theinoculum was applied in two drops of 12.5 �l placed in front of the nos-trils. Mice were monitored for 20 days if they were not killed earlier in thecase of severe clinical signs. Monoinfections were repeated once using,each time, three mice for infection with S. suis 10cps�EF and four mice forinfection with S. suis WT (strain 10) and 10�sly. Second, C3�/� and WTmice were infected with S. suis WT (strain 10) and 10�sly as describedabove but sacrificed 4 days postinfection (dpi) for comparative histolog-ical and bacteriological screenings. Experiments were repeated once, in-cluding 5 (4) mice in each infection group in the first (second) trial.Furthermore, a competitive infection experiment was conducted withC3�/� and WT mice. For this, suspensions of S. suis WT (strain 10) and10cps�EF at 108 CFU/ml for each strain were mixed, and two drops of12.5 �l of this mixture were placed in front of the nostrils. Inoculum

concentrations were verified by plating 10-fold serial dilutions. All mice ofthe competitive infection experiment were sacrificed 2 dpi for determina-tion of bacteriology and pathohistology of inner organs. A further com-petitive infection experiment was carried out the same way but withC5aR�/� mice instead of C3�/� mice. In the competitive infection exper-iments, the following numbers of mice per group were used in the first(second) trials: 5 (4) in WT versus C3�/� and 4 (3) in WT versus C5aR�/�

mice.The animal experiments of the present study were approved by the

Committee on Animal Experiments of the Lower Saxonian State Officefor Consumer Protection and Food Safety (Niedersächsisches Landesamtfür Verbraucherschutz und Lebensmittelsicherheit, LAVES; permits33.14-42502-04-12/0742 and 33.12-42502-04-13/1214). The presentstudy was performed in strict accordance with the principles and recom-mendations outlined in the European Convention for the Protection ofVertebrate Animals Used for Experimental and Other Scientific Purposes(European Treaty Series, no. 123 [http://www.conventions.coe.int/Treaty/en/Treaties/Html/123.htm]) and the German Animal Protection Law(Tierschutzgesetz).

Clinical score. The health status of the mice was examined twice dailyusing a clinical score sheet (25), including weight development, clinicalsigns of general sickness (rough coat, rapid breathing, and dehydration),and clinical signs indicating meningitis (apathy, apraxia) or septicemia(swollen eyes, depression). A cumulative score of 3 to 4 indicated mildclinical signs, a score of 5 to 6 indicated moderate clinical signs, and a score�6 indicated severe clinical signs with specific regard to neural failure.Mice with a cumulative score of �3 were classified as diseased (calculationof morbidity). In the case of severe weight loss (�20%) and/or enduringsevere clinical signs, mice were euthanized for reasons of animal welfareby inhalation of CO2 and blood removal immediately after CO2 inhala-tion.

Histological screening. Necropsy and sampling of organs for bacteri-ological and histological investigations were conducted exactly as de-scribed previously (25). Fibrinosuppurative and purulent lesions werescored as originally described for piglets (26) and subsequently modifiedfor mice (25). The group score � was calculated by dividing the sum of thehighest scores of each animal for any of the investigated organs throughthe number of animals. Rhinitis was not included.

Immunohistochemistry. Formalin-fixed and paraffin-embeddednose and brain tissue was evaluated for the presence of streptococcal an-tigen by immunohistochemistry using the avidin-biotin complex (ABC)method. Sections were deparaffinized in descending series of ethanol andpretreated with sodium-citrate buffer (10 mM, pH 6.0) in the microwave(800 W, 20 min). Primary antibodies consist of rabbit anti-S. suis poly-clonal antibodies (diluted 1:2,000). Binding of secondary goat anti-rabbitantibodies and the formation of the ABC were visualized by a chromogenreaction using 3.3-diaminobenzidine-tetrachloride (Vector Laboratories,Burlingame, CA) (31). For negative controls, primary antibodies werereplaced by rabbit preimmune serum.

Reisolation of S. suis strains from tissue and TNL. Determination ofthe specific bacterial load of each organ and tracheonasal lavage (TNL)was conducted as described previously (25). Briefly, tissue suspensions inphosphate-buffered saline (PBS; pH 7.4) were homogenized, serially di-luted, and plated on blood agar plates (in the case of competitive infectionexperiments with or without spectinomycin). The detection limits for thespecific bacterial loads were 1 CFU/mg of organ and 30 CFU/ml of TNL.The competitive index (CI) was calculated by dividing the specific contentof the test strain (10cps�EF) to the specific content of the WT strain(strain 10) using spectinomycin resistance of strain 10cps�EF for differ-entiation. Typical alpha-hemolytic streptococci were profiled in a S. suismultiplex PCR for the detection of mrp, epf, sly, arcA, gdh, cps1, cps2, cps7,and cps9 (32). Isolates from mice challenged with 10�sly were additionallyinvestigated in a sly-specific PCR according to a described protocol (25).Isolates from mice challenged with 10cps�EF were differentiated in acps2E-specific PCR with the primer pair cps2Efor (TTTCGCACTTTCAA

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GACGTG) and cps2Erev (GGACGGGTACCGACTAGACTC) at a finalconcentration of 0.5 �M.

Flow cytometric analysis of C3 antigen (C3, C3b, and C3c) deposi-tion on the bacterial surface. Bacteria were grown to late logarithmicgrowth phase (OD600 0.8), harvested by centrifugation, washed with PBS(pH 7.4), and resuspended in 10 �l of mouse serum (either serum fromWT mice or serum from C3�/� mice) and incubated for 45 min at 37°C.Bacteria were centrifuged, washed with PBS, and incubated with an fluo-rescein isothiocyanate-conjugated antibody directed against C3c (Dako)at a 1:225 dilution in PBS for 1 h at room temperature. Bacteria werewashed with PBS and then resuspended in PBS with 0.375% formalde-hyde for further flow cytometric analysis.

Fluorescent bacteria were measured using BD Accuri (Becton Dickin-son [BD], 488-nm solid state). Further analysis was performed with BDAccuri C6 software. For each determination, 10,000 events were acquired,and initial analysis of bacterial cells was carried out by dot plot analysis(forward scatter versus sideward scatter) to define the cell population ofinterest. Subsequently, fluorescent bacteria were detected at channel FL-1.The fluorescence index was calculated by multiplying the percentage ofbacteria positive for C3 by the mean fluorescence intensity of the wholebacterial population (33).

Bactericidal assay. Killing of S. suis in blood was investigated in freshlydrawn heparinized blood from either WT or C3�/� mice. Sampling ofblood was conducted according to the recommendations of the GermanSociety for Laboratory Animal Science (Gesellschaft für Versuchst-ierkunde) and the German Veterinary Association for the Protection ofAnimals (Tierärztliche Vereinigung für Tierschutz e. V.) (http://www.gv-solas.de). Stocks of frozen bacterial suspensions including 15% glycerolwere thawed prior to the bactericidal assay, and 1.5 � 103 CFU weremixed with 50 �l of heparinized blood and 10 �l of either RPMI (Gibco/Invitrogen) or porcine hyperimmune serum raised against S. suis WT(strain 10). Incubation was conducted at 37°C for 2 h in a horizontalshaker. The multiplication factor (MF) represents the ratio of CFU afterthe 2 h of incubation to the CFU at time zero.

ELISA for detection of the anaphylatoxin C3a. EDTA plasma wascollected by cardiac puncture from S. suis WT (strain 10)- and 10�sly-infected WT and C3�/� mice sacrificed 4 dpi and, for comparison, fromnoninfected WT and C3�/� mice. For the detection of C3a/C3a-desArg inEDTA plasma by enzyme-linked immunosorbent assay (ELISA), purifiedrat anti-mouse C3a antibody (clone I87-1162; BD), and biotinylated ratanti-mouse C3a (clone I87-419; BD) antibody were used as describedelsewhere (34). As a standard sample, zymosan-activated EGTA plasmawas included in each run. In addition, purified murine C3a (BD Pharmin-gen, Heidelberg, Germany) was used as a standard to calculate actualpeptide concentrations. The specificity of the C3a/C3a-desArg ELISA wasconfirmed using zymosan-activated EGTA plasma from C3�/� mice as anegative control.

Statistical analysis. If not stated otherwise, experiments were per-formed at least five times, and results were expressed as means and stan-dard deviations. Statistical analysis of Kaplan-Meier diagrams was con-ducted with the log-rank test. Differences in bacterial loads of innerorgans and pathohistological, as well as clinical scores, were analyzed withthe Mann-Whitney U-test. The prevalences of meningitis were comparedusing the Fisher exact test. Differences in flow cytometric data and mul-tiplication in murine blood were analyzed with the Student t test. P valuesof �0.05 were considered significant.

RESULTSRole of complement in protective innate immunity of miceagainst S. suis invasion. Kaplan-Meier diagrams for mortality ofWT and C3�/� mice infected intranasally with S. suis serotype 2(WT strain 10) were determined to elucidate the impact of com-plement on host defense against S. suis (Fig. 1A). For this, we usedthe protocol for a recently described intranasal colonizationmodel (25). In agreement with our previous results, none of the

WT mice infected with S. suis WT in the present study died ordeveloped severe signs of disease (Fig. 1A; see also Fig. S1 and S2Aand B in the supplemental material). In contrast, all 8 C3�/� micedied or had to be killed within 5 days after infection because ofsevere clinical signs, such as weight loss greater than 20% (4 of 8)and central nervous dysfunction and lethargy (8 of 8; Fig. 1A; seealso Fig. S2A and B in the supplemental material).

Mortality in C3�/� mice was associated with severe lesions innumerous tissues, including multifocal suppurative meningitisand pneumonia in 4 of 8 animals (Table 1). Accordingly, thegroup pathological score � (ranging from 0 to 5) was high inC3�/� mice infected with the S. suis WT for determination of theKaplan-Meier survival diagram (� 3.1, Table 1). The S. suischallenge strain was detectable in the lungs, spleens, hearts, kid-neys, and brains of all C3�/� mice. The specific bacterial load ofthese organs ranged between 103 and 105 CFU/mg tissue (see Fig.S3A in the supplemental material). Furthermore, S. suis antigenwas detectable by immunohistochemistry inside immune cellsand extracellularly in meningitis lesions of C3�/� mice, demon-strating unequivocally that these lesions were caused by S. suisbacteria which had invaded the central nervous system (Fig. 1B

FIG 1 S. suis serotype 2 causes meningitis and mortality in complement-deficient (C3�/�) but not in wild-type (WT) mice after mucosal infection. (A)Kaplan-Meier diagram for mortality of WT and C3�/� mice intranasally in-fected with 2 � 108 CFU of S. suis WT (strain 10) as described in Materials andMethods. ***, Significant difference between WT and C3�/� mice (P � 0.001;log-rank test). (B) Immunolabeling of extracellular leukocyte-associated bac-teria in the meninges of a C3�/� mouse infected with S. suis WT (strain 10)using an antiserum raised against S. suis serotype 2. Scale bar, 200 �m.

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and, for comparison, see Fig. S4A in the supplemental material).In contrast, S. suis was not recorded in any inner organ of thesurviving WT mice sacrificed 20 dpi with the exception of oneisolate from the kidney (see Fig. S3A in the supplemental mate-rial). Furthermore, S. suis WT was not detectable in the TNL fluidof these WT mice, suggesting that these mice had substantiallyreduced or even eliminated S. suis from the respiratory mucosawithin 20 days after infection (see Fig. S3A in the supplementalmaterial).

The pathological and bacteriological comparisons in the de-scribed experiment were hampered by the fact that all WT micewere sacrificed 20 dpi, substantially later than S. suis WT-infectedC3�/� mice, which died within the first 5 days after experimentalinfection. Therefore, an additional experiment was conducted tocompare histopathological lesions and bacterial loads of inner or-gans of WT and C3�/� mice at the same time point, namely, 4 dpi.None of the WT mice had severe lesions of inner organs, whereas6 of 9 C3�/� mice showed multifocal suppurative meningitis andother pathologies by 4 dpi. The pathological group score � wassignificantly higher in S. suis WT-infected C3�/� mice at 4 dpi incomparison to the respective WT mice (� 4.0 versus � 1.1,respectively [P 0.0017]; Table 2). These results demonstratedthat most of the C3�/� mice, in contrast to the WT mice, devel-oped severe lesions of the inner organs, including the brain, within4 days of mucosal S. suis infection.

The S. suis WT challenge strain was detectable at 4 dpi in 8 of 9C3�/� mice in the spleen and the liver, as well as in the heart, andin 7 of 9 C3�/� mice also in the lung and the brain. The specificbacterial load of these organs ranged between 101 and 105 CFU/mgof tissue (Fig. 2). In contrast, S. suis was less often recorded ininner organs of WT mice by 4 dpi. None of the S. suis WT-infectedWT mice tested positive for the challenge strain in more than oneinner organ, and in the majority of these WT mice (5 of 9) S. suiswas not detected at all in any inner organ. However, the specificbacterial load of S. suis WT in TNL was higher in WT mice com-pared to C3�/� mice at 4 dpi (Fig. 2), indicating that complementdeficiency does not promote initial mucosal colonization of S.suis.

In summary, these results demonstrated that a functional com-plement system is crucial for protection against invasive fatal S.suis infection in an intranasal mouse model. Comparative intra-nasal infection of C3�/� mice with S. suis serotype 2 and respectiveisogenic mutants might be used to identify factors involved incomplement-independent virulence mechanisms, since S. suisWT-infected C3�/� mice developed fatal disease associated withmeningitis and other pathologies.

Immunohistochemical detection of S. suis in the nasal cavityof WT-infected C3�/� mice. Since the upper respiratory tract isthe port of entry for S. suis in our mouse model and also in fieldinfections of piglets, we investigated the localization of S. suis inthe nasal cavity by immunohistochemistry in diseased C3�/� miceinfected with S. suis WT. S. suis antigen was mainly detectable inthe mucus of the nasal cavity (Fig. 3A and B and, for comparison,see Fig. S4B in the supplemental material). Parts of the mucusdisplayed S. suis antigen extracellularly and within leukocytes (Fig.3A). Other regions of the mucus showed immunolabeling of S.suis bacteria without adjacent infiltrating immune cells (Fig. 3B).In addition, S. suis bacteria and leukocytes were detected in necro-tizing lesions of the nasal epithelium (Fig. 3C and, for comparison,see Fig. S4C in the supplemental material). Bacterial antigen was

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not found within or adherent to epithelial cells. In conclusion,immunohistochemical investigation of diseased C3�/� mice indi-cated that S. suis is in these animals either embedded in the mucusor associated with necrotic lesions in the nasal cavity.

Role of the capsule of S. suis in virulence and short-termcolonization in C3�/� mice. Since the capsule of S. suis is an im-portant virulence factor protecting this pathogen against phago-cytosis, we investigated the virulence of an unencapsulated mu-tant of S. suis serotype 2 in C3�/� mice. In contrast to C3�/� miceinfected with WT strain 10, none of the C3�/� mice infected withthe isogenic unencapsulated mutant 10cps�EF died or developedsevere clinical signs (Fig. 4; see also Fig. S2E and F in the supple-mental material). Only one mouse of this group had a clinicalscore of �3, indicating disease. Differences in morbidity and mor-tality of C3�/� mice infected with WT strain 10 and the unencap-sulated mutant were significant (P 0.01 and P 0.002, respec-tively). Severe or moderate fibrinosuppurative lesions were notdetectable in the organs of C3�/� mice infected with the unencap-sulated mutant, leading to a low pathological group score(� 0.8; Table 1).

Invasion and pathology of host tissue was also investigated in afurther experiment, including the killing of all animals by 2 dpi.This experiment was conducted as a competitive infection of WTand C3�/� mice with S. suis WT and the unencapsulated mutant.S. suis WT was detectable in all investigated inner organs of C3�/�

mice (except for the brain of one mouse) with a mean bacterialload above 103 CFU/mg of tissue (Fig. 5A). In contrast, the innerorgans of WT mice were mostly negative for S. suis and in the fewpositive cases were infected with �102 CFU/mg of tissue (Fig. 5A).The unencapsulated mutant was isolated only from the lung of oneC3�/� mouse but not in any other inner organ. However, the unen-capsulated mutant was present in the TNL of 7 of 9 C3�/� mice,although with a significantly reduced bacterial content compared toS. suis WT (5 � 102 � 9 � 102 CFU/ml versus 6 � 105 � 6 � 105

CFU/ml, respectively [P 0.0007]; Fig. 5). The competitive indi-ces for S. suis 10cps�EF versus WT in TNL were 0.00006 �0.00034 and 0.0018 � 0.000174 for WT and C3�/� mice, respec-tively. Differences in these indices for WT and C3�/� mice werestatistically significant (P 0.027). Pathohistological screening ofthe coinfected C3�/� mice but not the coinfected WT mice oftenrevealed moderate or severe suppurative lesions in the lung,spleen, heart, and liver (Table 3). Lesions in the brain were notrecorded (in accordance with the early time point of sacrifice).The pathological group score � of C3�/� mice obtained at 2 dpialready a value of 3.3, which was substantially higher than thescore � for infected WT mice (� 1.4, P 0.059).

In conclusion, different infection experiments demonstratedthat the capsule of S. suis is crucial for virulence in complement-deficient mice, indicating that the capsule is involved also in eva-sion of complement-independent innate immunity. Further-more, a competitive infection experiment showed that theunencapsulated mutant is severely attenuated in short-term colo-nization of the upper respiratory tract in WT and to a lesser extentalso in C3�/� mice in this model.

Role of suilysin in infection of C3�/� mice. We also investi-gated whether suilysin was crucial for complement-independenthost-pathogen interaction, leading to the devastating S. suis infec-tion in C3�/� mice. As shown in Fig. 4 and Table 1, experimentalinfection of C3�/� mice with the isogenic suilysin mutant 10�slyresulted in 88% mortality and severe or moderate fibrinosuppu-T

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rative lesions in numerous inner organs. Accordingly, the patho-histological group score � was high in C3�/� mice infected with10�sly for the determination of the Kaplan-Meier survival dia-gram (� 3.6; Table 1). However, death occurred significantlylater in 10�sly-infected C3�/� mice compared to S. suis WT-in-fected C3�/� mice (on average, 2.8 days [P 0.0367]; Fig. 4).Comparative histopathological screening of mice at 4 dpi revealedthat the lack of suilysin led to a significant lower histopathologicalgroup score at this time point (� 2.3 for 10�sly versus � 4.0for WT [P 0.03]; Table 2). The most striking difference betweenS. suis WT and 10�sly-infected C3�/� mice at 4 dpi was the sig-nificantly higher prevalence of moderate or severe meningitis inthe former (6 of 9 and 0 of 9, respectively [P 0.009]; Table 2).Differences between the two groups of mice were less distinct withregard to specific bacterial loads of different inner organs andTNL. The bacterial loads of kidney and heart were even signifi-cantly higher in 10�sly-infected C3�/� mice at 4 dpi (P 0.046and P 0.015, respectively; Fig. 2). Although the median of thebacterial loads of brain tissue was substantially higher in WT-infected compared to 10�sly-infected C3�/� mice (1,301 � 1,337and 23 � 1,091 CFU/mg of brain, respectively), the differenceswere not significant (P 0.198). In conclusion, suilysin is notcrucial for complement-independent host-pathogen interaction,leading to invasion of host tissue and fatal disease in C3�/� mice,but contributes to an early onset and faster progression of pathol-ogy in particular suppurative meningitis.

Impact of suilysin and capsule expression on complementdeposition on the bacterial surface in murine serum. To better

understand why S. suis serotype 2 infection of WT but not ofC3�/� mice was restricted to the nasal cavity, complement depo-sition on the bacterial surface was investigated in vitro. For this, S.suis strains were analyzed by flow cytometry with an antibodyagainst C3 after incubation in murine sera. Importantly, �40% ofS. suis WT bacteria were positive for C3 (most likely C3b/iC3b)deposition after incubation in WT serum (Fig. 6A). Incubation ofbacteria in serum of C3�/� mice was conducted as a control forthis flow cytometric analysis and confirmed the specificity. Thepercentage of bacteria positive for C3 antigen were significantlyincreased in the unencapsulated mutant 10cps�EF and in the sui-lysin mutant 10�sly compared to the WT (Fig. 6A). Furthermore,the unencapsulated mutant showed a very distinct phenotype withregard to the intensity of C3 antigen deposition on the bacterialsurface, since the mean fluorescence intensity measured for theunencapsulated mutant in this assay was 5-fold higher than thevalues obtained for S. suis WT and the suilysin mutant (Fig. 6B).No significant differences in the intensity of C3 antigen depositionwere found between the S. suis WT strain and the suilysin mutant.In conclusion, murine C3 antigen (most likely C3b/iC3b) is de-posited on the surface of encapsulated S. suis, although the inten-sity is significantly lower than on the surface of an unencapsulatedisogenic mutant.

Impact of suilysin and capsule expression on survival of S.suis in murine blood of WT and C3�/� mice. Since C3 antigendeposition was clearly detectable on the surfaces of S. suis WTbacteria, we comparatively evaluated survival of S. suis WT inblood of WT and C3�/� mice. The MF of S. suis WT was signifi-

FIG 2 Specific bacterial loads of tracheonasal lavage (TNL) and indicated inner organs of WT (�) and C3�/� mice (Œ) at 4 dpi with 2 � 108 CFU of S. suis WT(strain 10 [A]) or its isogenic suilysin mutant (10�sly [B]). Medians are indicated by horizontal bars. Significant differences between WT and C3�/� mice areindicated (*, P � 0.05; **, P � 0.01; ***, P � 0.001), as are significant differences in the bacterial loads of S. suis WT (strain 10) and 10�sly (�, P � 0.05[Mann-Whitney U-test]).

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cantly higher in C3�/� blood than in WT blood (Fig. 7, P 0.028). S. suis WT and also 10�sly showed very high mean MFvalues of 63.7 � 24 and 68.7 � 12, respectively, in C3�/� blood.Differences in multiplication between S. suis WT and the 10�slymutant were neither recorded for C3�/� nor for WT blood. Ad-dition of hyperimmune sera confirmed that murine blood cellswere a priori capable of eliminating S. suis in WT and C3�/� bloodin the presence of specific immunoglobulins under these condi-tions. Interestingly, the unencapsulated mutant showed somemultiplication in C3�/� but not in WT blood in the absence ofspecific immunoglobulin. The MF of the unencapsulated mutant10cps�EF (35.8 � 10) was significantly lower than the MF of S.suis WT and 10�sly in C3�/� blood (P 0.044 and 0.002, respec-tively) and comparable to the MF of S. suis WT and 10�sly in WTblood. In summary, multiplication of S. suis in murine blood exvivo is complement-restricted and depends on capsule but notsuilysin expression in complement-deficient and WT blood.

Role of anaphylatoxins in innate immunity of mice against S.suis invasion. Differences in clinics, pathology, and bacteriologybetween infected C3�/� and WT mice might not only result fromdifferences in opsonization of S. suis but also be a consequence ofdeficient anaphylatoxin formation in C3�/� mice. Therefore, for-mation of the anaphylatoxin C3a in S. suis-infected mice and therole of the anaphylatoxin receptor C5aR in innate immunityagainst S. suis were also investigated. We focused on detection ofthe anaphylatoxin C3a/C3a-desArg in the blood of S. suis-infectedmice, since C5a des-arg is rapidly bound to C5aR and C5L2 andthereby removed from the circulation (both anaphylatoxins, C3aand C5a, are metabolized within seconds in vivo to their des-ar-ginated forms). At 4 days after intranasal infection with S. suis WTand 10�sly, the plasma concentrations of murine C3a in WT micewere 2,617.6 � 1,195 ng/ml and 2,910.5 � 1,269 ng/ml, respec-tively, values significantly higher than in uninfected control mice(940.0 � 736 ng of mC3a/ml [P 0.0035 and 0.0061], respec-tively; see Fig. S5 in the supplemental material). As expected, C3awas not detectable in plasma of C3�/� mice (see Fig. S5 in thesupplemental material). These results showed that the anaphyla-toxin C3a is formed systemically upon infection of WT mice witheither S. suis WT or 10�sly, although moderate or severe inflam-

FIG 3 S. suis antigen in the nasal cavity of diseased C3�/� mice was detectedvia immunohistochemistry mainly in association with mucus but focally alsoin necrotic lesions. (A) The mucus was infiltrated with leukocytes displayingimmunohistochemically labeled S. suis antigen intracellularly. Scale bar, 200�m (inset scale bar, 50 �m). (B) Immunolabeling of S. suis bacteria entrappedin the mucus without infiltrating immune cells. Scale bar, 200 �m. (C) Ne-crotic lesions were observed with numerous immunohistochemically labeledS. suis bacteria and leukocytes positive for S. suis antigen. No obvious differ-ences were observed in this immunohistochemical investigation between S.suis WT (strain 10)- and 10�sly-infected C3�/� mice (panels A and B are froma WT [strain 10]-infected mouse, and panel C is from a 10�sly-infected C3�/�

mouse, but very similar findings were observed in both groups).

FIG 4 Kaplan-Meier diagram for mortality of C3�/� mice intranasally in-fected with 2 � 108 CFU of S. suis WT (strain 10), the isogenic suilysin mutant(10�sly), and the unencapsulated mutant (10cps�EF) as described in Materi-als and Methods. Significant differences between mice infected with S. suisWT, 10�sly, and 10cps�EF are indicated (*, P � 0.05; **, P � 0.01 [log-ranktest]).

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mations of inner organs were not recorded in these mice (with oneexception; Table 2).

C5a mediates chemotaxis of granulocytes and activation of im-mune cells. In order to elucidate the impact of C5a-mediated sig-naling on host defense against S. suis, C5aR�/� mice were infectedin our intranasal model. This was conducted as a competitive in-fection similar to that of C3�/� and WT mice with S. suis WT and10cps�EF, including the sacrifice of all animals at 2 dpi. Similar tothe previous experiment, S. suis was only detectable in inner or-gans of 2 of 7 WT mice. The numbers of S. suis WT bacteria weresignificantly higher in C5aR�/� mice than in WT mice, but incontrast to C3�/� mice with mean values of �103 CFU/mg oftissue (Fig. 8A). The specific bacterial loads in the spleens, livers,hearts, and lungs were significantly lower in C5aR�/� mice than inC3�/� mice, as determined in our previous experiment. Accord-ingly, moderate or severe fibrinosuppurative or purulent lesionswere not recorded in the inner organs of C5aR�/� mice in contrastto C3�/� mice (Table 3). Neutrophilic accumulation of thesplenic red pulp was detected more often in C5aR�/� mice than inWT mice, but differences in the pathological score reflecting fibri-nosuppurative lesions of inner organs were not significant. Mod-erate multifocal suppurative rhinitis was a typical finding of coin-fected WT, C3�/�, and C5aR�/� mice sacrificed at 2 dpi (Table 3).The unencapsulated mutant was not detectable in inner organs ofC5aR�/� mice except for the brain of one mouse. The load of S.suis WT bacteria in the TNL showed no differences betweenC5aR�/� and WT mice (Fig. 8A). However, the unencapsulatedmutant was detectable in TNL of 4 of 7 C5aR�/� mice but only inthe TNL of 1 of 7 WT mice (Fig. 8B).

In conclusion, complement is activated systemically in WTmice infected with S. suis intranasally, and C5aR is involved inprotective innate immunity of these mice preventing high bacte-rial loads of different inner organs at 2 dpi after mucosal infection.However, comparison of the bacteriological and histological re-sults of C3�/� and C5aR�/� infection shows that C5aR signalingis only one of the functions of the complement system involved inprotective immunity against mucosal S. suis infection.

DISCUSSIONInfection of piglets with S. suis might lead to very different states ofthe host: the piglet might either eliminate this pathogen without

showing signs of disease, become an inapparent carrier, or developdifferent types of diseases such as acute meningitis and chronicendocarditis. Only mucosal animal models make it possible toinvestigate all of these different putative outcomes and to identifythe factors which determine whether initial colonization is re-stricted or the first step toward invasive infection. In the presentstudy, we used an intranasal mouse model to investigate for thefirst time the impact of the complement system on the pathogen-esis of S. suis diseases in vivo. The results unequivocally demon-strate that the complement system protects mice against invasivemucosal infection of S. suis. Although similar findings have beenobtained for S. pneumoniae (35, 36), the protective role was sur-prising as S. suis serotype 2 was described to be resistant againstcomplement-dependent killing by dendritic cells and neutrophilsin the absence of specific antibodies (19, 20). Furthermore,Brazeau et al. (37) found that inactivation of serum had no effecton phagocytosis of S. suis by murine macrophages. However, theprotective complement-mediated immunity of WT mice againstS. suis is in agreement with our in vitro findings showing that C3antigen is deposited on the bacterial surface of S. suis WT and thatmultiplication of S. suis is significantly higher in the blood of com-plement-deficient mice.

The unencapsulated mutant was found to be severely attenu-ated in the infection of complement-deficient mice. This result isin accordance with previous in vitro findings of different groupsshowing that unencapsulated S. suis mutants are killed by differentimmune cells in the presence of inactivated serum (19, 20, 28).Interestingly, it has been shown that the capsule inhibits phagocy-tosis by macrophages through destabilization of lipid microdo-mains (38). This complement-independent immune evasionfunction of the capsule might, at least partially, explain the atten-uation of the unencapsulated mutant in complement-deficientmice. Importantly, the results presented here do not exclude thatthe capsule is also crucial for complement evasion. This is sup-ported by the observations that the intensity of C3b deposition onthe bacterial surface is substantially enhanced in the unencapsu-lated mutant, as shown in the present study by flow cytometricanalysis and in a study by Lecours et al. (20) by immunofluores-cence microscopy. Furthermore, multiplication of the unencap-

FIG 5 Reisolation of S. suis WT (strain 10 [A]) and the unencapsulated mutant (10cps�EF [B]) after competitive intranasal infection of WT (�) and C3�/� (Œ)mice at 2 dpi. Each mouse was infected with 108 CFU each of S. suis WT (strain 10) and 10cps�EF (ratio of 1:1). The specific bacterial loads in the TNL and inthe indicated inner organs were determined using the spectinomycin-resistant phenotype of 10cps�EF, as described in Materials and Methods. For each location,one reisolated colony was investigated in a multiplex PCR for profiling of S. suis and in a cps-specific PCR (32). Medians are indicated by horizontal bars.10cps�EF was not detectable in inner organs except for the lungs of one C3�/� mouse. Significant differences between WT and C3�/� mice are indicated (*, P �0.05; **, P � 0.01; ***, P � 0.001), as are significant differences in the specific bacterial loads of S. suis WT (strain 10) and 10cps�EF (�, P � 0.05; ��, P � 0.01;���, P � 0.001 [Mann-Whitney U-test]).

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sulated mutant was found to be higher in C3�/� blood than in WTblood.

In S. pneumoniae the polysaccharide capsule contributes tocolonization of the respiratory tract of WT mice (39). Here, weshowed that an unencapsulated S. suis mutant is also severely at-tenuated in survival in the upper respiratory tract of WT and

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FIG 6 C3 deposition on the surfaces of S. suis WT (strain 10), the unencap-sulated mutant (10cps�EF), and the suilysin mutant (10�sly) in the presenceof naive WT serum. As a control, bacteria were incubated with the sera ofC3�/� mice. Bacteria were incubated for 45 min in sera and analyzed by flowcytometry as described in Materials and Methods. (A) Proportion of bacteriapositive for C3 after incubation for 45 min. (B) Intensity of C3 deposition onthe surfaces of S. suis WT (strain 10), 10�sly, and 10cps�EF strains in sera fromWT mice and, as a control, C3�/� mice. The means and standard deviations offive independent experiments are indicated. Significant differences are indi-cated (*, P � 0.05 [Student t test]).

FIG 7 Survival of S. suis WT (strain 10), 10cps�EF, and 10�sly in murineblood ex vivo (bactericidal assay). The bacterial strains were incubated at 37°Cfor 2 h in blood of WT and C3�/� mice as indicated. The multiplication factor(MF) represents the ratio of CFU after the 2 h of incubation to the CFU at timezero. Porcine hyperimmune serum was added as indicated to demonstrate thatefficient killing of S. suis by protective immunity is a priori possible under theseconditions. Means are indicated by horizontal bars. Significant differences areindicated(*, P � 0.05; **, P � 0.01; ***, P � 0.001 [Student t test]).

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C3�/� mice. However, complement depletion by application ofcobra venom had no significant effect on colonization of the up-per respiratory tract by either WT or unencapsulated pneumo-cocci (39). In contrast, significant differences in competitive indi-ces (S. suis WT versus the unencapsulated mutant) between WTand C3�/� mice were recorded in the present study, which sug-gests that a function(s) of the capsule of S. suis in interaction withthe complement system is important for survival in the upperrespiratory tract in this murine model. An impact of complementon the survival of S. suis in the upper respiratory tract is in accor-dance with the rhinitis found in S. suis-infected mice at 2 dpi andthe association of S. suis with infiltrating leukocytes and focal ne-crotic lesions in the nasal cavity. Further studies are needed toinvestigate whether focal necrosis of the respiratory epithelium iscrucial for host tissue invasion by S. suis in C3�/� mice.

Our findings demonstrated that suilysin expression is not cru-cial for invasive infection of complement-deficient mice leading tofatal diseases. In fact, we observed only 12% survival of C3�/�

mice infected mucosally with 108 CFU of 10�sly, but mortalityoccurred significantly later and histopathological lesions were lesssevere at 4 dpi than in S. suis WT-infected C3�/� mice. In a pre-vious study suilysin expression was found to be essential for S. suisto kill WT BALB/c mice after intraperitoneal application of 107

and 108 CFU (17). One possible reason for the difference in theclinical outcome of the two studies is that expression of suilysin iscrucial for protection against complement-dependent immunity.Accordingly, it has been shown that pneumolysin expression pre-vents C3 deposition on the surface of pneumococci and is crucialfor causing systemic infection in WT mice (21). We also recordeda higher percentage of C3-positive bacteria for the suilysin mutantthan for S. suis WT. However, these differences were not as pro-nounced as one would expect with regard to the substantial dif-ferences in mortality between the two studies. Furthermore, wedid not observe differences in multiplication of WT S. suis and theisogenic suilysin mutant in WT blood. Most likely, the differentapplication routes used in both studies were important for theimpact of suilysin on pathogenesis. Intraperitoneal application ofS. suis in mice in a high doses might lead to cytotoxic effects ofsecreted suilysin. Extensive cytotoxicity might have substantially

determined mortality after intraperitoneal application. In the na-sal cavity, S. suis is mainly surrounded by mucus at the primarysite of infection, as shown via immunohistological investigation.This mucus might be very important for the protection of mucosalsurfaces against the cytotoxic effects of suilysin. Since suilysin ex-pression is also not crucial for virulence of S. suis in experimentalmucosal infections of piglets (without complement deficiency)(18), the results from the intraperitoneal mouse infection shouldbe interpreted with caution.

We have shown here that suilysin contributes to the pathogen-esis of S. suis meningitis, an observation that is in accordance withthe findings of a very recent study comparing sequence type 1 and104 strains (24). Suilysin is known to affect porcine choroid plexusepithelial cells (40), and cytotoxic effects of suilysin have also beenreported for brain microvascular endothelial cells (41, 42). Fur-thermore, suilysin-stimulated release of proinflammatory mole-cules, such as arachidonic acid (43) and interleukin-8 (44, 45),may play an important role in initiating changes in permeabilityand adhesion properties of the blood brain and the blood cerebro-spinal fluid barriers, promoting immune cells to enter the centralnervous system.

Anaphylatoxins might influence pathogenesis in very differentways. Our results show that the anaphylatoxin C3a is formed sys-temically upon mucosal infection of WT mice with S. suis. Fur-thermore, invasion of inner organs was significantly increased inC5aR�/� mice in comparison to WT mice. The latter suggests aprotective role of C5a in mucosal infection with S. suis. C5a-me-diated chemotaxis and activation of immune cells might contrib-ute to the elimination of this pathogen at an early stage of patho-genesis. The high frequency of moderate multifocal suppurativerhinitis in C5aR�/� mice is in agreement with the original pheno-typic characterization of C5aR�/� mice, demonstrating a para-doxical increase in the number of neutrophils recovered from thelungs of Pseudomonas aeruginosa-infected C5aR�/� mice (30).We hypothesize that the infiltrating neutrophils in the nasal cavitywere not efficiently killing S. suis in the case of C5aR deficiency.Interestingly, the genome of S. suis serotype 2 includes an openreading frame encoding a putative C5a protease (NCBI referencesequence YP_006074193.1). Our results suggest that despite this

FIG 8 Reisolation of S. suis WT (strain 10 [A]) and the isogenic unencapsulated mutant (10cps�EF [B]) after competitive intranasal infection of WT (�) andC5aR�/� (o) mice. Each mouse was infected with 108 CFU each of S. suis WT (strain 10) and 10cps�EF (ratio of 1:1). The specific bacterial loads in the TNL andin the indicated inner organs were determined by using the spectinomycin-resistant phenotype of 10cps�EF, as described in Materials and Methods. For eachlocation, a reisolated colony was investigated in a multiplex PCR for profiling of S. suis and in a cps-specific PCR (32). 10cps�EF was not detectable in inner organsexcept for the brain of one C5aR�/� mouse. Medians are indicated by horizontal bars. Significant differences between WT and C5aR�/� mice are indicated (***,P � 0.001), as are the significant differences in the specific bacterial loads of S. suis WT and 10cps�EF (�, P � 0.05; ��, P � 0.01; ���, P � 0.001). Significantdifferences between C3�/� (Fig. 5) and C5aR�/� mice are also indicated (#, P � 0.05; ##, P � 0.01; ###, P � 0.001). Significance was determined using theMann-Whitney U-test.

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putative C5a degradation, sufficient amounts of C5a are func-tional in S. suis-infected mice to protect mice against S. suis inva-sion.

The S. suis serotype 2 strain used here causes meningitis andother pathologies in intranasal infection of normal piglets, i.e.,without complement deficiency (26, 46, 47). In comparison topiglets, WT mice can be regarded as less susceptible to mucosal S.suis infections, as demonstrated here and in our previous study(25). We speculate that these differences might be related to host-specific complement evasion mechanisms. Accordingly, our lab-oratory recently identified an IgM protease cleaving porcine butnot murine IgM (27). Since IgM is part of the innate immuneresponse and an important activator of the classical complementpathway, we are currently investigating IgM cleavage by S. suis as aputative complement evasion mechanism. Furthermore, it is un-known whether the binding of factor H to the surface of S. suisexhibits differences between host species (14). The human patho-gen Neisseria meningitidis only binds human factor H, and trans-genic expression of human factor H substantially predisposes ratsto bacteremia (48, 49).

In the present study, complement-deficient mice were for thefirst time used for S. suis research, including a new intranasal an-imal model for identification of complement-independent viru-lence mechanisms. We anticipate that this research will substan-tially contribute to future studies on the pathogenesis of thisimportant mucosal pathogen.

ACKNOWLEDGMENTS

We thank Hilde E. Smith (Lelystad, Netherlands) for providing S. suisstrains 10 and 10cps�EF. Animal experiments were supported by AnnaKoczula and Jana Seele (both at the Institute for Microbiology, Universityof Veterinary Medicine Hanover).

This study was financially supported by the Deutsche Forschungsge-meinschaft, Bonn, Germany (grant SFB587), and by the NiedersachsenResearch Network on Neuroinfectiology of the Ministry of Science andCulture of Lower Saxony.

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