An Alternative Role of C1q in Bacterial : Facilitating pneumoniae Adherence and Invasion of Host Cells This information is current as of October 3, 2021. Vaibhav Agarwal, Jonas Ahl, Kristian Riesbeck and Anna M. Blom J Immunol published online 13 September 2013 http://www.jimmunol.org/content/early/2013/09/13/jimmun

ol.1300279 Downloaded from

Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on October 3, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 13, 2013, doi:10.4049/jimmunol.1300279 The Journal of Immunology

An Alternative Role of C1q in Bacterial Infections: Facilitating Streptococcus pneumoniae Adherence and Invasion of Host Cells

Vaibhav Agarwal,* Jonas Ahl,† Kristian Riesbeck,† and Anna M. Blom*

Streptococcus pneumoniae (pneumococcus) is a major human , which evolved numerous successful strategies to colonize the host. In this study, we report a novel mechanism of pneumococcal–host interaction, whereby pneumococci use a host com- plement C1q, primarily involved in the host-defense mechanism, for colonization and subsequent dissemination. Using -culture assays and confocal microscopy, we observed that pneumococcal surface-bound C1q significantly enhanced pneumococcal adherence to and invasion of host epithelial and endothelial cells. Flow cytometry demonstrated a direct, Ab- independent binding of purified C1q to various clinical isolates of pneumococci. This interaction was seemingly capsule serotype

independent and mediated by the bacterial surface-exposed , as pretreatment of pneumococci with pronase E but not Downloaded from sodium periodate significantly reduced C1q binding. Moreover, similar binding was observed using C1 complex as the source of C1q. Furthermore, our data show that C1q bound to the pneumococcal surface through the globular heads and with the host cell- surface receptor(s)/glycosaminoglycans via its N-terminal collagen-like stalk, as the presence of C1q N-terminal fragment and low m.w. heparin but not the C-terminal globular heads blocked C1q-mediated pneumococcal adherence to host cells. Taken together, we demonstrate for the first time, to our knowledge, a unique function of complement protein C1q, as a molecular bridge between

pneumococci and the host, which promotes bacterial cellular adherence and invasion. Nevertheless, in some conditions, this http://www.jimmunol.org/ mechanism could be also beneficial for the host as it may result in uptake and clearance of the . The Journal of Immunology, 2013, 191: 000–000.

lthough humans have a highly developed defense mech- Pneumococci have evolved numerous successful strategies such anism, during evolution majority of human bacterial as expression of a variety of factors to colonize its host A including the Gram-positive human species (2, 3). These virulence factors function either as adhesins that Streptococcus pneumoniae (the pneumococcus) has developed mul- interact directly with the host cell-surface receptors or use host protein(s) as a molecular bridge between bacteria and the host.

tiple evasion strategies to counteract the host. Pneumococci colonize by guest on October 3, 2021 asymptomatically the human upper . However, These host proteins include extracellular matrix and serum proteins depending upon the host susceptibility, pneumococci can cause mild such as fibronectin, thrombospondin, vitronectin, plasminogen, local infections such as , , as well as severe life- (FH), and C4b-binding protein (C4BP) (4–9). Pneumococcal threatening invasive diseases such as lobar pneumoniae, , and interactions with complement inhibitors FH or C4BP protect (1). The burden of pneumococcal infections is highest bacteria from complement-mediated attacks (6, 10). In addition, among the youngest and elderly population and in patients with association of FH promotes pneumococcal adherence to and in- immunodeficiency. vasion of host cells (4, 7). The human complement system is a major part of the innate immune system and the first line of defense against invading path- ogens (11). Complement system consists of ∼35 proteins, and de- *Medical Protein Chemistry, Department of Laboratory Medicine Malmo¨, Lund University,SE-20502Malmo¨,Sweden;and†Medical Microbiology, Department pending on the initiating agent, it can be classified into three of Laboratory Medicine Malmo¨, Lund University, SE-205 02 Malmo¨, Sweden different pathways (classical, lectin, and alternative). The alternative Received for publication January 28, 2013. Accepted for publication August 15, pathway is activated by autoactivation of the complement compo- 2013. nent C3 and its subsequent deposition on the surface of the path- This work was supported by grants from the Swedish Research Council (K2012-66X- ogen, whereas specific carbohydrates and acetylated ligands on the 14928-09-5), the National Board of Health and Welfare, Foundations of O¨ sterlund, Greta and Johan Kock, King Gustav V’s 80th Anniversary, Knut and Alice Wallen- bacterial surface initiate the lectin pathway. Binding of the first berg, Inga-Britt and Arne Lundberg, and Torsten and Ragnar So¨derberg, the Royal component of complement C1 to the Igs recognizing invading Physiographic Society in Lund, and a grant from the Ska˚ne University Hospital. V.A. pathogens or C-reactive protein (CRP) usually triggers the clas- is the recipient of a postdoctoral stipend from the Swedish Society for Medical Research. The funders had no role in study design, data collection and analysis, sical pathway. All three pathways lead to the formation of the C3 decision to publish, or preparation of the manuscript. and C5 convertases with subsequent deposition of opsonin C3b, Address correspondence and reprint requests to Prof. Anna M. Blom, Department of release of proinflammatory anaphylatoxin C3a and C5a, and finally Laboratory Medicine Malmo¨, Lund University, The Wallenberg Laboratory Floor 4, formation of a lytic membrane attack complex, which disrupts Inga Marie Nilssons Street 53, SE-205 02 Malmo¨, Sweden. E-mail address: anna. [email protected] membrane integrity. Abbreviations used in this article: C4BP, C4b-binding protein; CPS, capsular poly- The complement component C1 is a multimolecular complex 2+ saccharide; CRP, C-reactive protein; FH, factor H; GMFI, geometric mean fluores- composed of C1q, the recognition molecule, and two Ca -depen- cence intensity; GVB++, gelatin-veronal buffer; HBEpC, primary human bronchial dent dimeric serine proteases, C1r and C1s (tetramer C1r -C1s ) epithelial cell; NHS, normal human serum; THY, Todd-Hewitt-Yeast extract broth. 2 2 (12). C1q is a hexamer of three protein subunits—A, B, and C— Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 with molecular masses of 29, 26, and 22 kDa, respectively, and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300279 2 C1q-MEDIATED consists of an N-terminal collagen-like stalk and a C-terminal Table I. Clinical isolates and reference strains of S. pneumoniae used in globular protein heads. C1q, as part of the C1 complex, is central this study to activation of the classical complement pathway, which is initiated upon recognition of Ag-aggregated Ig (IgM and IgG) Fc domains Strains Serotype Site of Isolation Reference No. by the C-terminal globular heads. Moreover, C1q can also recognize S. pneumoniae CRP, serum amyloid P component protein, and pentraxin (13–15). D39 2 This initiates sequential activation of C1q-bound serine protease D39Dcps 2 (33) KR405 3 Blood (50) C1r and C1s, which in turn mediates proteolysis and activation of KR406 3 Blood (50) C4 and C2 and to the formation of C3 and C5 convertases. KR465 3 Blood This study The direct activation of the classical pathway by pneumococci is KR466 3 Blood This study not unique, as direct and Ab-independent binding of C1q was de- KR467 3 Blood This study monstrated also for group B streptococci (16), Salmonella minnesota KR468 3 Nasopharyngeal This study KR469 3 Nasopharyngeal This study (17), Escherichia coli (18, 19), Klebsiella pneumoniae (20), KR470 3 Nasopharyngeal This study Legionella pneumophila (21), and (22). In KR408 4 Blood (51) this study, we have investigated the Ab-independent binding of KR431 4 Blood This study C1q to the respiratory tract pathogen S. pneumoniae. We demon- KR437 4 Blood This study KR440 4 Blood This study strate for the first time, to our knowledge, a unique function of KR409 6A Blood (50) complement protein C1q as a molecular bridge between pneu- KR410 6A Blood (50) mococci and the host that promotes pneumococcal cellular ad- KR430 6B Blood (50) herence and invasion. KR432 6B Blood This study Downloaded from KR471 6B Blood This study Materials and Methods KR472 6B Blood This study KR436 7 Blood (50) Cultivation of pneumococci KR412 7F Blood (50) All pneumococcal strains used in this study, except for NCTC10319 and KR433 7F Blood This study D39, were clinical isolates collected at the Clinical Microbiology labora- KR439 7F Blood This study tory, Ska˚ne University Hospital (Malmo¨, Sweden) (Table I). Pneumococci KR452 7F Blood This study http://www.jimmunol.org/ KR414 8 Blood (51) were cultured on blood agar plates at 37˚C and 5% CO2 or in Todd-Hewitt- broth (Oxoid) supplemented with 0.5% yeast extract (Todd-Hewitt-Yeast KR415 8 Blood (51) 8 21 KR416 9V Blood This study extract broth [THY]) to a density of 5 3 10 CFU ml (OD600 of ∼0.5). The pneumococcal D39 strain deficient in capsular (CPS) KR417 9V Blood (51) was kindly provided by Prof. Sven Hammerschmidt (Department of Ge- KR418 9V Blood (51) netics of Microorganisms, University of Greifswald, Greifswald, Germany). KR419 11 Blood (51) All other bacterial species used in this study (Table II) were cultured on KR443 11 Blood (50) KR420 14 Blood (51) chocolate agar plates at 37˚C and 5% CO2 atmosphere. KR421 14 Blood (51) Proteins and Abs KR422 14 Blood (51) KR423 14 Blood This study by guest on October 3, 2021 C1q was purified from human plasma as described (23). The stalk and head KR425 14 Blood This study regions of C1q were prepared by partial proteolytic digestion of intact C1q KR428 14 Blood This study as described (24, 25). Pepsin (Worthington Biochemicals) was used for KR429 14 Blood This study preparation of the stalk region and purified collagenase ( his- KR435 14 Blood (50) tolyticum, code CLSPA) for preparation of the head region was purchased KR438 14 Blood This study from the same company. C1- and C1q-depleted serum were purchased KR463 14 Blood This study from Complement Technology. Pronase E and sodium periodate (NaIO ) 4 KR426 19F Blood (51) were purchased from Sigma-Aldrich, whereas low m.w. heparin was from KR427 19F Blood (51) the National Institute for Biological Standards & Control. Rabbit anti-human KR473 19F Nasopharyngeal This study C1q and FITC-conjugated C1q Abs from DakoCytomation, whereas poly- KR474 19F Nasopharyngeal This study clonal rabbit Abs against S. pneumoniae were purchased from NordicBiosite. KR475 19F Nasopharyngeal This study Normal human serum (NHS) was prepared from freshly drawn blood KR476 19F Nasopharyngeal This study obtained from six healthy volunteers with informed consent and permission KR477 19F Nasopharyngeal This study of the ethical board of Lund University. The pooled blood was allowed to KR411 23A Blood (50) clot for 30 min at room temperature and then incubated for 1 h on ice. After KR450 23B Blood (50) two centrifugations, the serum fraction was frozen in aliquots and stored at KR454 23B Blood (50) 280˚C or used for preparing IgG-depleted serum. For IgG depletion, fresh NCTC10319 35A serum was passed through a Protein G column (GE Healthcare). The flow- through was analyzed for IgG depletion, and the fractions lacking IgG were pooled and frozen in 280˚C. C1q purified from the plasma was added to the depleted serum to compensate for the C1q that bound to the IgG pneumococci were treated with Pronase E and NaIO4, respectively, as coupled to the protein G column. The concentration of C1q in NHS and IgG- described previously (9). Briefly, bacteria (13 109 CFU/ml) were treated depleted NHS was determined using ELISA. with 100 ml 1 mg/ml Pronase E or 5 ml 1 mg/ml NaIO4, the concentration that does not affect bacterial viability, for 15 min at 37˚C, followed by Flow cytometry analysis of C1q binding to bacteria washing with PBS and used in the C1q binding assay. Finally, bacteria were fixed using 1% paraformaldehyde (Sigma-Aldrich), and the flow Binding of C1q to viable pneumococci or other bacterial species was mea- cytometry analysis was performed using CyFlow space (Partec) to detect sured using flow cytometry. Bacteria (1 3 107) either cultivated in THY the binding of C1q. Bacteria were detected using log-forward and log-side (for pneumococci) or collected directly from culture plates were incubated scatter dot plot, and a gating region was set to exclude debris and larger with purified C1q, at indicated concentrations, in PBS for 1 h at 37˚C. aggregates of bacteria. A total of 15,000 bacteria/events were analyzed for For investigating the binding from serum, bacteria were incubated with fluorescence using log-scale amplifications. The geometric mean fluores- gelatin-veronal buffer (GVB++) (5 mM veronal buffer [pH 7.3], 140 cence intensity (GMFI) was used as a measure for binding activity. mM NaCl, 0.1% porcine gelatin, 1 mM MgCl2, and 0.15 mM CaCl2), NHS, or depleted serum for 1 h at 37˚C. Following incubation, bacteria Cell lines and culture conditions were washed, and bound C1q was detected with FITC-conjugated rabbit anti-human C1q Abs (DakoCytomation). To investigate the role of pneu- Cultivation of host cell lines was performed as described (7). Briefly, human mococcal surface–exposed proteins or bacterial cell glycoconjugates, A549 cells ( alveolar epithelial cells, type II pneumocytes; American The Journal of Immunology 3

Type Culture Collection) were cultured in DMEM (PAA Laboratories) at 30˚C. After washing, bacteria were incubated with C4 (5 mg/ml) in supplemented with 10% of heat-inactivated FCS (Invitrogen), 2 mM glu- a total volume of 50 ml for 20 min at 37˚C. Bacteria were washed and tamine (PAA Laboratories), G (100 units ml21) and streptomycin stained for deposition of C4b using anti-C3c Abs (DakoCytomation) and 21 (0.1 mg ml ; both from Hyclone) at 37˚C under a 5% CO2 atmosphere. Alexa 488–conjugated secondary Abs (Invitrogen) and analyzed by flow Detroit 562 cells, human nasopharyngeal epithelial cells (American Type cytometry. Bacteria treated with 0.25% NHS in sterile GVB++ were used Culture Collection), were cultivated in RPMI 1640 supplemented with 10% as positive control, whereas pneumococci preincubated with only C4 or C1 FCS, 2 mM glutamine, and 1 mM sodium pyruvate (PAA Laboratories). were used as negative control. NCI-H292, human lung epithelial cells (American Type Culture Collection), were cultivated in RPMI 1640 (Hyclone) supplemented with 10% FCS. HUVECs were obtained from Invitrogen and cultivated in M200 media Results supplementedwithlowserumgrowthsupplement (Invitrogen). Primary Binding of C1q to S. pneumoniae human bronchial epithelial cells (HBEpC) were purchased from Promo- Because certain bacterial species interact with C1q in an Ab- Cell and cultured according to the instructions of the manufacturer. The cells were used for experiments between passages 1 and 5. independent fashion, we analyzed whether C1q can also bind S. pneumoniae. Pneumococci (NCTC10319, serotype 35A) were in- Pneumococcal host cell adherence and invasion assay cubated with NHS, and the bacterial lysates were separated by SDS- A549, NCI-H292, or Detroit 562 cells were seeded at a density of 5 3 104 PAGE followed by analysis by Western blot using specific Abs cells/well in plain medium either on 24-well tissue culture plates (Nunc) or against human C1q. Bound C1q was detected in lysates of bacteria glass coverslips (diameter12 mm) when assayed by immunofluorescence that were incubated with serum (Fig. 1A), whereas the signal was and cultivated for 48 h. HBEpC and HUVECs were seeded at a density absent for pneumococci in the absence of serum (Fig. 1A). of 3 3 104 cells/well. Confluent monolayers were washed thoroughly and infected for 3 h with pneumococci in 500 ml respective medium, supple- mented with 1% FCS for A549, Detroit-562, and NCI-H292 cells only Downloaded from (infection medium) at 37˚C using a multiplicity of infection of 25. Bacteria were incubated for 20 min with plasma purified C1q (10 mg) in a total volume of 100 ml infection medium at 37˚C prior to infections. The in- fection assays were carried out in a total volume of 500 ml after adding the bacteria. Postinfection, cells were washed three times with PBS to remove unbound bacteria. The total number of adherent and intracellular recovered bacteria was monitored after detachment and of cells with saponin (1.0% w/v) and plating the bacteria on blood agar. The number of viable http://www.jimmunol.org/ intracellular bacteria was quantified by employing the protection assay (4). Briefly, epithelial or endothelial cells were infected with pneu- mococci (multiplicity of infection of 25) preincubated with or without C1q. After 3 h, the infected cells were washed and further incubated for 1 h with infection medium containing 100 mgml21 gentamicin (Sigma-Aldrich) 21 and 100 units ml penicillin G (Sigma-Aldrich) at 37˚C and 5% CO2 to kill extracellular and nonadherent pneumococci. Invasive and viable pneumo- cocci were recovered from the intracellular compartments of the host cells by a saponin-mediated host cell lysis (1.0% w/v), and the total number of in-

vasive pneumococci was monitored after plating sample aliquots on blood by guest on October 3, 2021 agar plates, followed by colony formation and enumeration. In inhibition ex- periments, infection assays were carried out in the presence of C-terminal globular heads, N-terminal collagen-like stalks, or soluble heparin (Na- tional Institute for Biological Standards & Control) as described (4). Each experiment was repeated at least three times, and results were expressed as mean 6 SD. Fluorescence microscopy Pneumococci attached to host epithelial or endothelial cells were stained using polyclonal pneumococcal Abs (IgG) (Nordic Biosite) in combina- tion with a secondary goat anti-rabbit IgG coupled with Alexa Fluor 488 (green) or Alexa Fluor 546 (red) (Invitrogen) (26). Postinfection, nonspecific binding sites were blocked with 10% FCS, and before incubating, the cells with pneumococcal Abs (1:100), the infected cell layer was thoroughly washed with PBS. Bound Abs were detected with an Alexa Fluor–labeled FIGURE 1. Complement protein C1q binding to pneumococci. (A)Im- goat anti-rabbit Ig conjugate (Invitrogen). The glass coverslips were em- munoblot analysis of the binding of C1q to S. pneumoniae serotype 35A bedded upside down in mounting media (DakoCytomation), sealed with (NCTC10319), incubated in human serum. The samples were separated by nail polish, and stored at 4˚C. A confocal laser scanning microscope (Zeiss SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and ana- LSM 510 META; Carl Zeiss), and the appropriate software was used for lyzed with anti-C1q Abs. C1 deposition on pneumococcal surface. The bac- the image acquisition. teria were incubated with or without 10% NHS, C1q-depleted serum (NHS Binding from serum w/o C1q), and C1q-depleted serum replenished with 10 mg/ml of purified C1q (NHS w/o C1q + 10 mg/ml C1q) (B) or with NHS and IgG-depleted serum For direct recruitment of C1q from serum, pneumococci cultured in THY (C) for 60 min at 37˚C. The bacteria were then washed and incubated with until mid log phase were incubated with NHS (40%) for 60 min at 37˚C in FITC-conjugated anti-C1q Abs followed by flow cytometry analysis. One m a final volume of 100 l. After the incubation, the bacteria were washed to representative dataset from three independent experiments is shown in (B), remove the unbound proteins, and the bacterial pellet was lysed. The whereas the percentage binding of C1q from three independent experi- samples were separated by SDS-PAGE, transferred to a polyvinylidene C difluoride membrane, and submitted to Western blotting using a rabbit ments is shown in ( ). Statistical significance was calculated using Student polyclonal anti-C1q Ab (DakoCytomation) and a secondary peroxidase- t test. (D) Dose-dependent binding of plasma-purified C1q to pneumococci conjugated anti-rabbit IgG Ab (DakoCytomation). as determined by flow cytometry. GMFI is shown as mean of three inde- pendent experiments 6 SD as a measure of C1q binding. One-way ANOVA Deposition of C4b on pneumococci test was performed to calculate statistical difference of C1q binding com- The deposition of C4b on viable pneumococci was measured using flow pared with Ab control. (E) Percentage binding of (5 mg/ml) plasma-purified cytometry. Pneumococcal suspension (50 ml containing 5 3 107 CFU in C1q or C1 complex to pneumococci. Statistical significance was calcu- GVB++) was incubated with or without C1 complex (2 mg/ml) for 20 min lated using Student t test. ***p , 0.001. 4 C1q-MEDIATED PNEUMOCOCCAL INFECTION

Additionally, binding of C1q to pneumococci was investigated from NHS, C1q-depleted serum, and C1q-depleted serum replen- ished with 10 mg/ml of purified C1q. Flow cytometry analysis revealed the binding of C1q to pneumococci from NHS, whereas no such deposition was observed from the C1q-depleted serum (Fig. 1B). However, replenishing the depleted serum with plasma purified C1q restored the binding to the level of NHS (Fig. 1B). Taken together, the data suggest that C1q interacts with pneu- mococci but do not clarify the nature of this interaction, whether it is direct or mediated via the known ligands of C1q such as IgG. Therefore, the binding of C1q from NHS was compared with the binding from IgG-depleted NHS. Surprisingly, depletion of IgG did not affect significantly the binding of C1q when compared with NHS (Fig. 1C). Compared to NHS (100%), the binding from IgG- depleted serum was just moderately reduced to 80.7 6 13.5%, in- dicating that the interaction of C1q with pneumococci from NHS does not require bacterial surface-bound IgGs and could therefore be a direct interaction (Fig. 1C). To confirm this, binding of purified C1q to pneumococci was tested at concentrations ,80 mg/ml present in serum. Flow cytometric analysis indicated a dose-dependent Downloaded from binding of plasma-purified C1q (Fig. 1D). Finally, we compared the binding efficiency of plasma-purified C1q with that of C1q as a part of the C1 complex. As observed, compared with the binding of free C1q (100%), the binding of whole C1 complex to pneumococci was only slightly reduced (74.1 6 17.9%) (Fig. 1E). Taken together, the results suggest that both pu- http://www.jimmunol.org/ rified and as part of C1 complex, C1q binds directly and in an Ab- independent manner with pneumococci. Interference of the pneumococcal capsular with binding of C1q It has been previously shown that the pneumococcal CPS interferes with pneumococcal interaction with host cells and protein (5, 7, 9, 27). To elucidate whether CPS affects the interaction of C1q with pneumococci, we compared the binding of C1q to S. pneumoniae by guest on October 3, 2021 strain D39 (serotype 2) and its isogenic CPS-deficient strain D39Dcps. Flow cytometric analysis indicated a dose-dependent binding of plasma-purified free C1q to the pneumococcal strains used. However, the results suggested a significantly increased bind- ing of C1q to the capsule knockout strain D39Dcps compared with the wild-type strain D39 (Fig. 2A), suggesting that the CPS masks the bacterial surface–exposed ligand(s) of C1q. Characterization of C1q ligand on the pneumococcal surface To characterize the bacterial ligand(s) recognized by C1q, S. pneumoniae were treated with pronase E or sodium periodate fol- lowed by plasma-purified C1q, and the binding was investigated by flow cytometry. Oxidation of pneumococcal glycoconjugates by periodate treatment did not affect C1q binding (Fig. 2B, 2D, 2E). In contrast, proteolytic pretreatment of pneumococci with pronase E significantly reduced the binding of C1q (Fig. 2B, 2C, 2E). These

and periodate treatment of pneumococci on C1q binding. To characterize the bacterial receptor recognized by C1q, S. pneumoniae,13 109 CFU, were treated with or without pronase E (1 mg/ml) or sodium periodate

(NaIO4) (1 mg/ml) for 15 min at 37˚C. The bacteria were washed and FIGURE 2. Pneumococcal surface-exposed proteins but not CPS as the binding of C1q (5 mg/ml) to untreated (none) (B), NaIO4 pretreated ligand for C1q. (A) Effect of CPS on C1q binding to S. pneumoniae. (C), and pronase E (D) pretreated was investigated. Representative flow Pneumococcal wild-type strain D39 and the nonencapsulated D39Dcps cytometry histograms of binding of C1q are shown. (E) The GMFI values were incubated with 0, 5, and 10 mg/ml concentration of purified C1q for of C1q binding to untreated and pretreated pneumococci are shown. 60 min at 37˚C. Binding was analyzed by flow cytometry. Results were Results are presented as the means 6 SD for at least three independent expressed as GMFI (mean 6 SD) from three independent experiments as experiments. One-way ANOVA test was performed to calculate statistical a measure of C1q binding. Statistical significance was calculated using difference with in groups for C1q binding. *p , 0.05 compared with in the two-way ANOVA test. *p , 0.05, ***p , 0.001. Influence of proteolytic absence of C1q. The Journal of Immunology 5

Table I) belonging to 13 different serotypes was analyzed. Flow cytometry analyses demonstrated that although all the clinical isolates bind C1q to variable extents, there is no statistically sig- nificant correlation between the degree of C1q binding and par- ticular serotypes (Fig. 3). Taken together, the data suggest that the binding of C1q is a general mechanism that is not influenced by the capsular serotype but that the capsule does restrict the inter- action as revealed by the CPS-deficient S. pneumoniae D39Dcps (Fig. 2A).

C1q facilitates pneumococcal adhesion and invasion of host FIGURE 3. C1q binding to various clinical isolates of S. pneumoniae. epithelial and endothelial cells Binding of C1q (10 mg/ml) to various clinical isolates, blood isolates, and nasopharyngeal isolates (NP) of S. pneumoniae representing different C1q is primarily involved in the initiation of classical complement serotypes was analyzed by flow cytometry. Results were expressed as pathway by recognizing immune complexes. However, lately there GMFI 3 percentage of gated bacteria as a measure of C1q binding. have been reports suggesting that the complement proteins have additional functions in conjunction to their role in host defense. data suggest that the surface-exposed proteins function as the major For example, upregulation of C1q in the CNS has a novel neu- pneumococcal ligand for C1q. roprotective effect (30). Recently, it has been shown that in ad- dition to other serum factors, C1q is also involved in the entry of Downloaded from C1q binding to pneumococci is serotype independent anthracis spores into epithelial cells (31). Therefore, we The CPSs are essential of pneumococci, and to asked if C1q could have an additional role in pneumococcal–host date, .91 serologically distinct CPSs have been described (28, interactions. 29). However, not all serotypes cause serious infections. There- To elucidate whether pneumococci can exploit the surface-bound fore, we wanted to study whether the binding of C1q is dependent C1q as a molecular bridge between host cells and bacteria, infection on the capsule serotype. Collection of 50 clinical isolates of S. experiments were performed with nasopharyngeal epithelial cells. pneumoniae (42 blood isolates and 8 nasopharyngeal isolates; Pretreatment of pneumococci (NCTC10319) with plasma-purified http://www.jimmunol.org/

FIGURE 4. Pneumococcal surface-bound C1q mediates adhesion and invasion of human epithe- by guest on October 3, 2021 lial cells. (A) Adherence of S. pneumoniae strain NCTC10319 was determined after cell culture in- fection assay on epithelial cells Detroit 562, A549, and NIC H292. The infection assays were con- ducted with or without preincubation of pneumo- cocci with C1q. Results are shown as the fold increase in the adherence of pneumococci that were pretreated with C1q relative to untreated pneumococci (B). Immunofluorescence microscopy of adherent pneumococci. Scale bars, 10 mm. In- vasion and intracellular survival of S. pneumoniae NCTC10319 in host cells Detroit 562 (C), A549 (D), and NCI H292 (E) as determined by the anti- biotic protection assay. (F) C1q-mediated adherence of pneumococcal strain NCTC10319 to HBEpC as determined by cell-culture infection assay. Student t test was performed to calculate statistical differ- ences compared with untreated bacteria. (G)Immu- nofluorescence microscopy of adherent pneumococci to endothelial cells. Scale bars, 10 mm. (H) Invasion and intracellular survival of pneumococci were de- termined by the antibiotic protection assay. Student t test was performed to determine the statistical difference between the groups. Results present the means 6 SD of at least three independent experi- ments. *p , 0.05, **p , 0.01, ***p , 0.001 rela- tive to infections carried out in the absence of C1q. 6 C1q-MEDIATED PNEUMOCOCCAL INFECTION

C1q significantly increased adherence of pneumococci to Detroit 562 there was a tendency for the presence of more intracellular bacteria (Fig. 4A, 4B). Similarly, bacterial adherence was significantly in- when these were treated with C1q (Fig. 6). Taken together, the data creased to A549 and NCI H292 lung epithelial cells (Fig. 4A, 4B). indicate that the number of recovered and viable pneumococci In addition, the internalization of S. pneumoniae into epithelial decreases time-dependently when pneumococci are internalized by cells was quantified. After the infection, the attached bacteria were host cells independent of the presence of C1q. killed by treatment and intracellular pneumococci were Role of C1q globular heads and collagenous tail in recovered. In accordance to the increased adherence, C1q treat- pneumococcal adherence ment significantly increased the number of internalized pneumo- cocci (Fig. 4C–E). To further assess the biological significance of To identify the orientation in which the C1q molecule binds C1q-mediated pneumococcal adherence and invasion, primary pneumococci and thus mediates adherence, cell-culture inhibition human airway epithelial cells were infected for 3 h with S. experiments were performed using purified C-terminal globular pneumoniae pretreated with or without C1q. The presence of C1q heads or the N-terminal collagen-like stalks of C1q as a specific significantly increased the bacterial adherence and invasion of competitor. The Detroit-562 epithelial cells were preincubated with HBEpC (Fig. 4F–H). Furthermore, we tested whether a similar or without C1q collagen-like stalks or C1q globular heads (both at 2 mechanism is functional for endothelial cells. Preincubation of mg/well) prior to infection with pneumococci that had been in- bacteria with C1q significantly enhanced both adherence and in- cubated in the presence or absence of C1q. As observed, the vasion of HUVECs (Fig. 5A–C). Taken together, our infection presence of C1q collagen-like stalks blocked the C1q-mediated experiments showed that C1q acts as a molecular bridge between increase in pneumococcal adherence (Fig. 7A), whereas the pres- host cells and pneumococci and hence facilitates adherence and ence of C1q globular heads showed no inhibition. In conclusion, invasion in both epithelial and endothelial cells. C1q interacts with the pneumococcal surface exposed proteins Downloaded from likely via the C-terminal globular heads, whereas the increase in Intracellular fate of C1q-mediated invasive pneumococci adherence is mediated by the interaction of C1q N-terminal To assess the role of C1q in survival or killing of internalized collagen-like stalk with the host cell-surface receptor(s). pneumococci postinfection, the intracellular fate of pneumococci Influence of heparin on C1q-mediated pneumococcal were analyzed. The A549 lung epithelial cells were infected with adherence to host cells pneumococci pretreated with or without C1q, and postinfection, http://www.jimmunol.org/ extracellular and nonadherent pneumococci were killed by anti- A previous experiment demonstrates the pivotal role played by the biotic treatment. After removing the antibiotics, the viability of N-terminal collagen-like stalk of C1q in facilitating pneumococcal intracellular pneumococci was investigated for the indicated time adherence. Moreover, C1q interacts with heparin via its N-terminal points by plating sample aliquots on blood agar plates. At the time collagen-like stalk (32). Therefore, we investigated the role of point zero, C1q-treatment significantly increased the number of small m.w. heparin on C1q-mediated adherence of pneumococci internalized pneumococci compared with untreated bacteria. Further, to human lung epithelial cells A549. Competitive inhibition ex- a significant decrease in the number of viable intracellular bacteria periments were performed in the presence of soluble heparin (10 was observed for both C1q pretreated and nonpretreated pneumo- mg/ml). Presence of heparin significantly inhibited C1q-mediated cocci with increasing time postinfection. However, at all time points, pneumococcal adherence to epithelial cells, whereas the basal level by guest on October 3, 2021 of adherence in the absence of C1q was not affected (Fig. 7B). To demonstrate that the inhibition observed is not a consequence of altered C1q binding to pneumococci in the presence of heparin, the impact of heparin on C1q binding was analyzed by using heparin as a competitor in a binding experiment. Flow cytometry analysis

FIGURE 5. Pneumococcal surface-bound C1q facilitates adhesion and FIGURE 6. Intracellular fate of C1q-coated internalized pneumococci. invasion of human endothelial cells. (A) C1q-mediated adherence of Intracellular fate of pneumococci as determined by the enumeration of the pneumococcal strain NCTC10319 to HUVECs as determined by cell- viable invasive bacteria. Unbound extracellular bacteria were washed away culture infection assay. One-way ANOVA test was performed to calculate 3 h postinfection, and after killing of extracellular pneumococci by anti- statistical difference compared with untreated bacteria. (B) Immunofluo- biotic (time point 0), the infections were continued for the indicated time rescence microscopy of adherent pneumococci to endothelial cells. Scale points. The results are expressed as CFU recovered per well of 24-well bars, 10 mm. (C) Invasion and intracellular survival of pneumococci were plate (mean 6 SD) obtained from two independent experiments performed determined by the antibiotic protection assay. Student t test was performed in duplicates. Statistical significance between the C1q-treated and un- to determine the statistical difference between the groups. Results present treated pneumococci was calculated using two-way ANOVA test, whereas the means 6 SD of at least three independent experiments. **p , 0.01, one-way ANOVA test was used to calculate statistical significance within ***p , 0.001 relative to infections carried out in the absence of C1q. the respective group. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 7. (A) Role of N-terminal collagen-like stalk region of C1q in mediating pneumococcal adherence to host cells. Adherence of pneumococcal strain NCTC10319 in the presence of C1q to Detroit-562 epithelial cells were followed after preincubation of the epithelial cells with or without C1q stalks (2 mg/well) or C1q heads (2 mg/well) prior to infections. Fold adherence of C1q-coated pneumococci as compared with untreated pneumococci is shown. The results revealed the importance of C1q N-terminal collagen-like stalk in C1q-mediated pneumococcal attachment to host cells as its presence alone inhibited the C1q-mediated increase in pneumococcal adherence. One-way ANOVA test was used to calculate statistical significance. *p , 0.05, **p , 0.01 relative to infections carried out in the absence of C1q. Effect of heparin on C1q-mediated pneumococcal adherence to host cells. (B) The influence of heparin on C1q-mediated pneumococcal adherence to host epithelial A549 cells was determined by cell-culture infection assay using heparin as an in- hibitor. Fold adherence of C1q-coated pneumococci in the absence or presence of heparin is shown. Statistical significance was calculated using one-way ANOVA test. *p , 0.05, **p , 0.01. (C) The effect of heparin on pneumococcal recruitment of C1q was analyzed by flow cytometry after preincubation of 10 mg/ml plasma-purified C1q with indicated amounts of heparin per reaction. Bacterial-bound C1q was determined by flow cytometry, and results were expressed as GMFI of FITC-labeled bacteria. One-way ANOVA test was used to calculate statistical significance. Pneumococcal surface-bound C1 complex is functionally active. Bacteria were preincubated with or without C1 complex (2 mg/ml) for 20 min at 30˚C. After washing, C4 (5 mg/ml) was added and incubated for 20 min at 37˚C. Thereafter, deposited C4/C4b was detected using anti-C4c and Alexa 488–conjugated secondary Ab by flow cytometry. As positive control, bacteria were preincubated with 0.25% NHS, whereas bacteria treated with only C4 or C1 complex were used as negative control. (D)A representative flow cytometry histogram from three independent experiments is shown. (E) The GMFI values of C4/C4b deposition on pneumococci are shown. One-way ANOVA test was used to calculate statistical significance. ***p , 0.001. demonstrated that the acquisition of C1q by pneumococci is not if the C1 complex bound to pneumococci would also be func- influenced by soluble heparin (Fig. 7C). In conclusion, the presence tionally active. Pneumococci were preincubated with purified C1 of heparin did not interfere with pneumococcal acquisition of C1q complex prior to their incubation with purified C4. The functional but blocks the N-terminal collagen-like stalk of C1q, which is in- activity of the bound C1 complex was determined by the ability of volved in C1q-mediated adherence. bound protease within the C1 complex to cleave C4 and subsequent deposition of C4b, which was detected by flow cytometry. Bacteria C1 bound to pneumococcal surface is functionally active preincubated with NHS alone were used as a positive control. A Because our data indicate that C1q interacts via its C-terminal significant level of C4b deposition was observed on the C1 complex globular heads with the pneumococcal surface-exposed proteins and C4-treated pneumococcal surface compared with the only C4 and because activation of the classical complement pathway is or C1 complex–treated pneumococci (Fig. 7D, 7E). Surprisingly, initiated upon binding of the globular heads with the Igs, we tested compared with only C4, a shift in signal was observed on only C1 8 C1q-MEDIATED PNEUMOCOCCAL INFECTION complex–pretreated pneumococci. This could be attributed to some level of nonspecific binding of the detection Abs. Never- theless, the C4b deposition signal obtained in the presence of both C1 and C4 was significantly higher than that of C4 or C1 complex alone. In addition, pneumococci pretreated with NHS, corresponding to ∼10-fold less C1 complex concentration, showed an ∼4-fold in- crease in C4b deposition. However, this enormous increase in C4b deposition could also be due to the naturally occurring anti- pneumococcal Abs as well as complement activation via the lectin pathway. Taken together, our data indicate that the C1 bound to the pneumococcal surface is functionally active and can initiate a basal level of classical complement pathway activation. C1q binding and its effect on host cell adherence of other To test whether, besides pneumococci, other pathogenic bacteria also have the ability to directly bind C1q, we investigated the binding of three clinical isolates of Gram-positive (S. pyogenes) and Gram-negative (E. coli) pathogens to plasma-purified C1q by flow cytometry (Table II). In addition, C1q binding was also Downloaded from analyzed for Moraxella catarrhalis RH4 and nontypeable Hae- mophilus influenzae (NTHi 3655) strains (Table II). Similar to pneumococci, flow cytometry analysis indicated a significant FIGURE 8. Binding of human C1q to other Gram-positive and Gram- A binding of C1q to all the clinical isolates of S. pyogenes and all but negative pathogen and its impact on host cell adherence. ( ) Binding of C1q (10 mg/ml) to various clinical isolates of S. pyogenes and E. coli and one of the E. coli isolates (Fig. 8A). In addition, we demonstrated M. catarrhalis and nontypeable H. influenzae was analyzed by flow http://www.jimmunol.org/ for the first time, to our knowledge, the direct and Ab-independent cytometry. Results were expressed as GMFI 3 percentage of gated bac- binding of C1q to M. catarrhalis RH4 and nontypeable H. influ- teria as a measure of C1q binding. One-way ANOVA test was used to enzae (Fig. 8A). calculate statistical significance. (B) Adherence of other Gram-positive and To determine whether these other bacterial species can also use Gram-negative pathogens was determined after cell-culture infection assay C1q as a bridging molecule to facilitate adherence to host cells, on A549 lung epithelial cells. The infection assays were conducted with or cell-culture infection assays were carried out using A549 cells. The without preincubation of pathogens with C1q. Results are shown as the presence of C1q did not enhance the number of host cell–associated fold increase in the adherence of pathogen that were pretreated with C1q bacteria, as determined by counting the CFU obtained after plat- relative to untreated bacteria. Statistical significance was calculated using , ting the total number of cell-associated and recovered intracellular two-way ANOVA test. ***p 0.001. bacteria (Fig. 8B). Taken together, the data indicate that even by guest on October 3, 2021 though the direct binding of C1q is a generalized host defense mechanism of pneumococcal–host interaction, in which pneu- mechanism against the invading pathogens, its ability to function mococci use a host protein, C1q, primarily involved in the host- as a bridging molecule that facilitates bacterial host cell coloni- defense mechanism (complement system), for colonization and zation is rather specific for pneumococci (Fig. 9). subsequent internalization of host cells. We demonstrate that C1q interacts directly in an Ab-independent manner with pneumococci Discussion and functions as a bridging molecule, facilitating pneumococcal Colonization of the host is a critical step in the pathogenesis of any infectious agent. Therefore, one of the most promising strategies in controlling pathogens is to have a detailed understanding of the molecular mechanism of interaction and identification as well as selective targeting of the essential colonization factor(s) that facilitates colonization and subsequent dissemination. The pneu- mococcus is a major human pathogen, affecting both adults and children, and has developed multiple strategies such as expression of adhesins or acquirement of various host proteins to facilitate colonization (2–5, 7–9, 33). In this study, we describe a novel

Table II. Other Gram-positive and Gram-negative bacterial strains used in this study

Strains Reference No. KR714 E. coli This study KR715 E. coli This study KR716 E. coli This study FIGURE 9. Schematic model of the C1q-mediated pneumococcal ad- KR717 S. pyogenes This study herence and invasion of host cells. C1q interacts via its C-terminal globular KR718 S. pyogenes This study heads with pneumococcal surface-exposed proteins and with host cell- KR719 S. pyogenes This study surface receptor(s)/glycosaminoglycans via its N-terminal collagen-like M.c. RH4 M. catarrhalis RH4 (52) stalk, thereby facilitating pneumococcal adherence and invasion of host NTHi 3655 Nontypeable H. influenzae (53) cells. The Journal of Immunology 9 adherence to and invasion of host epithelial and endothelial cells result in higher availability of uncovered bacterial surface pro- (Fig. 9). teins, which would otherwise be masked by the CPS. Our data are Complement is a crucial part of innate immunity, protecting us in agreement with the study of group B streptococci, in which the from infections and cleaning the body from unwanted debris such target is probably not the CPS, but rather protein(s) (48). Indeed, as dying cells. C1q plays an important role during the activation of we could not find any significant difference in C1q binding among the classical complement pathway by recognizing the Ag–Ab the tested clinical isolates of pneumococci, representing common complex via its C-terminal globular heads. In case of pneumo- serotypes causing infections. This suggests that the binding of C1q coccal infections, the activation of the classical pathway has been to pneumococci is a general phenomenon independent of the cap- suggested to be a major host-defense mechanism (34). Moreover, sule serotype; however, one role of the capsule seems to protect the classical pathway activities have been detected in the lung and the bacteria against direct deposition of C1q on their surface. airway epithelium (35, 36). Apart from the naturally occurring Abs, Some of the recently published studies have indicated that the acute-phase proteins such as CRP and serum amyloid P component complement proteins have functions in addition to their role in host protein that bind both S. pneumoniae and C1q have been sug- defense. For example, C1q alone promotes ingestion of apoptotic gested to play a role in complement-mediated immunity against cells by , modulates inflammation, and promotes neu- this pathogen (37–39). Additionally, a new mechanism of comple- ronal survival (30, 49). Similarly, complement inhibitor FH bound ment activation has been identified involving C-type lectin receptor to the surface of pneumococci plays a dual role in pneumococcal SIGN-R1, present on the surface of macrophages and interacting infection. On one hand, it promotes pneumococcal adherence and with pneumococcal polysaccharides and C1q (40). Thus, com- invasion of mucosal surfaces, while on the other hand, in invasive plement plays an important role against pneumococcal infection infection, it improves survival by inhibiting complement activa- both at the early stages of colonization as well as in the bacteremic tion (4, 7). Consistent with this, we showed that C1q bound to the Downloaded from phase of infection. However, pneumococci have evolved numer- pneumococcal surface significantly enhanced the adherence and ous strategies for attenuation or escaping such attacks, and these internalization of host epithelial and endothelial cells. A some- mechanisms are one of the key determinants for their survival what similar role for C1q has been suggested in the entry of spores within the human host. These include recruitment of complement of B. anthracis into epithelial cells, where C1q is involved in inhibitors such as C4BP or FH, which inhibit the classical and linking of spores surface protein BclA with host cell receptors via alternative pathway activation, respectively (6, 7, 41), or the ex- its collagen-like stalk region (31). However, the C1q did not aug- http://www.jimmunol.org/ pression of thick capsule, which not only inhibits the deposition of ment the host cell adherence of other Gram-positive and -negative C3b but also complement-mediated opsonophagocytosis (42). bacteria, as has been the case for pneumococci. Furthermore, our There have been reports describing direct binding of C1q to data show that C1q bind to the surface of pneumococci through pathogens, independent of the presence of recognizing Abs or other the globular head region and with the host cell-surface receptor(s) ligands (16–22). Accordingly, direct and Ab-independent binding via its N-terminal collagen-like stalk, as the presence of C1q N- of C1q was observed for clinical isolates of E. coli, S. pyogenes, terminal fragment and not the C-terminal globular heads blocked M. catarrhalis, and nontypeable H. influenzae. Therefore, we tested C1q-mediated pneumococcal adherence to host cells. Additionally, whether a similar Ab-independent C1q binding is also relevant in C1q-mediated adherence of pneumococci to host cells was inhibited by guest on October 3, 2021 pneumococci, and if yes, does it have any additional function dis- by heparin. Taken together, the data suggest that the interaction of tinct from its conventional role in host defense. Indeed, we show C1q-coated pneumococci is mediated by the interaction of the direct and dose-dependent binding of plasma-purified C1q to heparin-binding site on the N-terminal collagen-like stalk region pneumococci. In agreement with this, depletion of IgG from NHS with cell-surface receptor(s) such as glycosaminoglycans (32). It did not significantly diminish the interaction of C1q with pneumo- is indeed intriguing, whether under in vivo conditions; C1 bound cocci, suggesting that the presence of specific anti-pneumococcal to pneumococcal surface would favor complement activation or Abs may not be pivotal for the basal level of classical complement C1q-mediated bacterial colonization and internalization. Because pathway initiation but rather to augment the effect. In conjunction, complement is an important arm of innate immunity, it is highly even though C4b deposition on pneumococci in the presence of likely that the protective role of C1q resulting in complement ac- purified C1 complex and C4 was detected, it was significantly tivation and opsonization of the pathogen would be preferred. lower compared with NHS alone. Another role of this direct C1q- However, considering the fact that pneumococci have other mech- pneumococcal interaction may be to influence the complement- anisms to circumvent the complement attack, they can simulta- mediated immune responses at initial stages of infection, perhaps neously afford to use C1q as a bridging molecule for colonization immediately following pulmonary exposure to pneumococci. and internalization of host cells. Taken together, C1q has a dual role The C-terminal globular head region of C1q functions as a pattern- in pneumococcal infections, one as complement activator/opsonin recognition molecule and mediates binding to various molecules and second as a bridging molecule. In line with this, our data sug- exposed on the surface of apoptotic cells such as DNA and cal- gest that the C1q-mediated pneumococcal uptake by host cells may reticulin (43, 44) and thus acts as a bridging molecule between also promote the intracellular clearance of bacteria, thus facilitating apoptotic cells and APCs such as macrophages and dendritic cells host protection. Most likely, both outcomes are possible, but under (45–47). Although, a number of C1q ligands have been charac- certain conditions, one will prevail. However, to dissect whether terized on apoptotic cells, the list is relatively short in the case of and when the direct C1q binding mainly facilitates pneumococcal bacterial pathogens. The only reported bacterial ligand of C1q is pathogenesis or promotes host protection, additional in vivo studies the OmpK36 porin of K. pneumoniae (20), whereas still unchar- are necessary. acterized outer membrane proteins have been suggested to be the In conclusion, C1q interacts directly even in the absence of ligands for other bacterial species (16, 17, 19, 21, 22). In accor- specific Abs to S. pneumoniae. This binding results from the in- dance, in this study, we showed that C1q interacts via its C-terminal teraction between the bacterial surface-exposed protein(s) and C- globular heads primarily with the pneumococcal surface-exposed terminal globular head of C1q, as is evident by the fact that C1 protein(s). To corroborate this, higher C1q binding was observed bound to pneumococci is functionally active. In contrast, the en- in a D39Dcps strain deficient in CPS as compared with the wild- hanced bacterial adherence and invasion of host cells is mediated type D39 strain, as the deletion of the capsule locus might by the N-terminal collagen-like stalk, which interacts with the host 10 C1q-MEDIATED PNEUMOCOCCAL INFECTION cell-surface receptor(s)/glycosaminoglycans. Our study demon- pathway by Klebsiella pneumoniae outer membrane proteins. Infect. Immun. 61: 852–860. strates that complement protein C1q, which is primarily involved 21. Mintz, C. S., P. I. Arnold, W. Johnson, and D. R. Schultz. 1995. Antibody- in the initiation of the classical complement pathway, indeed has independent binding of complement component C1q by Legionella pneumo- an auxiliary role as a molecular bridge and hence mediates pneu- phila. Infect. Immun. 63: 4939–4943. 22. Koroleva, I. V., A. G. Sjo¨holm, and C. Schale´n. 1998. Binding of complement mococcal adherence to host cells. subcomponent C1q to Streptococcus pyogenes: evidence for interactions with the M5 and FcRA76 proteins. FEMS Immunol. Med. Microbiol. 20: 11–20. 23. Tenner, A. J., P. H. Lesavre, and N. R. Cooper. 1981. Purification and radio- Acknowledgments labeling of human C1q. J. Immunol. 127: 648–653. We thank the Clinical Microbiology laboratory at Labmedicine Ska˚ne 24. Paˆques, E. P., R. Huber, H. Priess, and J. K. Wright. 1979. Isolation of the (Malmo¨, Sweden) for providing clinical isolates. We thank Prof. Sven globular region of the subcomponent q of the C1 component of complement. Hammerschmidt (Department of Genetics of Microorganisms, University Hoppe Seylers Z. Physiol. Chem. 360: 177–183. 25. Reid, K. B., and R. R. Porter. 1976. Subunit composition and structure of sub- of Greifswald, Greifswald, Germany) for providing the capsule-less mutant component C1q of the first component of human complement. Biochem. J. 155: of pneumococcal D39 strain. 19–23. 26. Agarwal, V., and S. Hammerschmidt. 2009. Cdc42 and the phosphatidylinositol 3-kinase-Akt pathway are essential for PspC-mediated internalization of Disclosures pneumococci by respiratory epithelial cells. J. Biol. Chem. 284: 19427–19436. The authors have no financial conflicts of interest. 27. Hammerschmidt, S., S. Wolff, A. Hocke, S. Rosseau, E. Mu¨ller, and M. Rohde. 2005. Illustration of pneumococcal polysaccharide capsule during adherence and invasion of epithelial cells. Infect. Immun. 73: 4653–4667. 28. Henrichsen, J. 1995. Six newly recognized types of Streptococcus pneumoniae. References J. Clin. Microbiol. 33: 2759–2762. 1. Cartwright, K. 2002. Pneumococcal disease in western Europe: burden of dis- 29. Park, I. H., D. G. Pritchard, R. Cartee, A. Brandao, M. C. Brandileone, and ease, antibiotic resistance and management. Eur. J. Pediatr. 161: 188–195. M. H. Nahm. 2007. Discovery of a new capsular serotype (6C) within serogroup 2. Bergmann, S., and S. Hammerschmidt. 2006. Versatility of pneumococcal sur- 6ofStreptococcus pneumoniae. J. Clin. Microbiol. 45: 1225–1233. Downloaded from face proteins. Microbiology 152: 295–303. 30. Benoit, M. E., and A. J. Tenner. 2011. Complement protein C1q-mediated 3. Kadioglu, A., J. N. Weiser, J. C. Paton, and P. W. Andrew. 2008. The role of neuroprotection is correlated with regulation of neuronal gene and microRNA Streptococcus pneumoniae virulence factors in host respiratory colonization and expression. J. Neurosci. 31: 3459–3469. disease. Nat. Rev. Microbiol. 6: 288–301. 31. Xue, Q., C. Gu, J. Rivera, M. Ho¨o¨k, X. Chen, A. Pozzi, and Y. Xu. 2011. Entry 4. Agarwal, V., T. M. Asmat, S. Luo, I. Jensch, P. F. Zipfel, and S. Hammerschmidt. of spores into epithelial cells is mediated by the spore surface 2010. Complement regulator Factor H mediates a two-step uptake of Strepto- protein BclA, integrin a2b1 and complement component C1q. Cell. Microbiol. pneumoniae by human cells. J. Biol. Chem. 285: 23486–23495. 13: 620–634.

5. Bergmann, S., A. Lang, M. Rohde, V. Agarwal, C. Rennemeier, C. Grashoff, 32. Almeda, S., R. D. Rosenberg, and D. H. Bing. 1983. The binding properties of http://www.jimmunol.org/ K. T. Preissner, and S. Hammerschmidt. 2009. Integrin-linked kinase is required human complement component C1q. Interaction with mucopolysaccharides. J. for vitronectin-mediated internalization of Streptococcus pneumoniae by host Biol. Chem. 258: 785–791. cells. J. Cell Sci. 122: 256–267. 33. Jensch, I., G. Ga´mez, M. Rothe, S. Ebert, M. Fulde, D. Somplatzki, 6. Dieudonne´-Vatran, A., S. Krentz, A. M. Blom, S. Meri, B. Henriques-Normark, S. Bergmann, L. Petruschka, M. Rohde, R. Nau, and S. Hammerschmidt. 2010. K. Riesbeck, and B. Albiger. 2009. Clinical isolates of Streptococcus pneumo- PavB is a surface-exposed adhesin of Streptococcus pneumoniae contributing niae bind the complement inhibitor C4b-binding protein in a PspC allele- to nasopharyngeal colonization and airways infections. Mol. Microbiol. 77: dependent fashion. J. Immunol. 182: 7865–7877. 22–43. 7. Hammerschmidt, S., V. Agarwal, A. Kunert, S. Haelbich, C. Skerka, and 34. Brown, J. S., T. Hussell, S. M. Gilliland, D. W. Holden, J. C. Paton, P. F. Zipfel. 2007. The host immune regulator factor H interacts via two contact M. R. Ehrenstein, M. J. Walport, and M. Botto. 2002. The classical pathway is the sites with the PspC protein of Streptococcus pneumoniae and mediates adhesion dominant complement pathway required for innate immunity to Streptococcus to host epithelial cells. J. Immunol. 178: 5848–5858. pneumoniae infection in mice. Proc. Natl. Acad. Sci. USA 99: 16969–16974.

8. Pracht, D., C. Elm, J. Gerber, S. Bergmann, M. Rohde, M. Seiler, K. S. Kim, 35. Sarma, V. J., M. Huber-Lang, and P. A. Ward. 2006. Complement in lung dis- by guest on October 3, 2021 H. F. Jenkinson, R. Nau, and S. Hammerschmidt. 2005. PavA of Streptococcus ease. Autoimmunity 39: 387–394. pneumoniae modulates adherence, invasion, and meningeal inflammation. Infect. 36. Watford, W. T., A. J. Ghio, and J. R. Wright. 2000. Complement-mediated host Immun. 73: 2680–2689. defense in the lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 279: L790–L798. 9. Rennemeier, C., S. Hammerschmidt, S. Niemann, S. Inamura, U. Za¨hringer, 37. Agrawal, A., A. K. Shrive, T. J. Greenhough, and J. E. Volanakis. 2001. To- and B. E. Kehrel. 2007. Thrombospondin-1 promotes cellular adherence of pology and structure of the C1q-binding site on C-reactive protein. J. Immunol. gram-positive pathogens via recognition of peptidoglycan. FASEB J. 21: 3118– 166: 3998–4004. 3132. 38. Yuste, J., M. Botto, S. E. Bottoms, and J. S. Brown. 2007. Serum amyloid P aids 10. Zipfel, P. F., T. Hallstro¨m, S. Hammerschmidt, and C. Skerka. 2008. The com- complement-mediated immunity to Streptococcus pneumoniae. PLoS Pathog. 3: plement fitness factor H: role in human diseases and for immune escape of 1208–1219. pathogens, like pneumococci. 26(Suppl 8): I67–I74. 39. Yuste, J., A. Sen, L. Truedsson, G. Jo¨nsson, L. S. Tay, C. Hyams, 11. Ricklin, D., G. Hajishengallis, K. Yang, and J. D. Lambris. 2010. Comple- H. E. Baxendale, F. Goldblatt, M. Botto, and J. S. Brown. 2008. Impaired ment: a key system for immune surveillance and homeostasis. Nat. Immunol. opsonization with C3b and of Streptococcus pneumoniae in sera 11: 785–797. from subjects with defects in the classical complement pathway. Infect. Immun. 12. Naff, G. B., J. Pensky, and I. H. Lepow. 1964. The Macromolecular Nature of the 76: 3761–3770. First Component of Human Complement. J. Exp. Med. 119: 593–613. 40. Kang, Y. S., Y. Do, H. K. Lee, S. H. Park, C. Cheong, R. M. Lynch, 13. Bottazzi, B., V. Vouret-Craviari, A. Bastone, L. De Gioia, C. Matteucci, G. Peri, J. M. Loeffler, R. M. Steinman, and C. G. Park. 2006. A dominant complement F. Spreafico, M. Pausa, C. D’Ettorre, E. Gianazza, et al. 1997. Multimer for- fixation pathway for pneumococcal polysaccharides initiated by SIGN-R1 mation and ligand recognition by the long pentraxin PTX3. Similarities and interacting with C1q. Cell 125: 47–58. differences with the short pentraxins C-reactive protein and serum amyloid P 41. Lu, L., Y. Ma, and J. R. Zhang. 2006. Streptococcus pneumoniae recruits component. J. Biol. Chem. 272: 32817–32823. complement factor H through the amino terminus of CbpA. J. Biol. Chem. 281: 14. Bristow, C. L., and R. J. Boackle. 1986. Evidence for the binding of human 15464–15474. serum amyloid P component to Clq and Fab gamma. Mol. Immunol. 23: 1045– 42. Hyams, C., E. Camberlein, J. M. Cohen, K. Bax, and J. S. Brown. 2010. The 1052. Streptococcus pneumoniae capsule inhibits complement activity and neutrophil 15. Volanakis, J. E., and M. H. Kaplan. 1974. Interaction of C-reactive protein phagocytosis by multiple mechanisms. Infect. Immun. 78: 704–715. complexes with the complement system. II. Consumption of guinea pig com- 43. Paı¨dassi, H., P. Tacnet-Delorme, T. Lunardi, G. J. Arlaud, N. M. Thielens, and plement by CRP complexes: requirement for human C1q. J. Immunol. 113: 9–17. P. Frachet. 2008. The lectin-like activity of human C1q and its implication in 16. Eads, M. E., N. J. Levy, D. L. Kasper, C. J. Baker, and A. Nicholson-Weller. DNA and apoptotic cell recognition. FEBS Lett. 582: 3111–3116. 1982. Antibody-independent activation of C1 by type Ia group B streptococci. J. 44. Paı¨dassi, H., P. Tacnet-Delorme, M. Verneret, C. Gaboriaud, G. Houen, K. Duus, Infect. Dis. 146: 665–672. W. L. Ling, G. J. Arlaud, and P. Frachet. 2011. Investigations on the C1q- 17. Stemmer, F., and M. Loos. 1985. Evidence for direct binding of the first com- calreticulin-phosphatidylserine interactions yield new insights into apoptotic ponent of complement, C1, to outer membrane proteins from Salmonella min- cell recognition. J. Mol. Biol. 408: 277–290. nesota. Curr. Top. Microbiol. Immunol. 121: 73–84. 45. Fraser, D. A., A. K. Laust, E. L. Nelson, and A. J. Tenner. 2009. C1q differ- 18. Tenner, A. J., R. J. Ziccardi, and N. R. Cooper. 1984. Antibody-independent C1 entially modulates phagocytosis and cytokine responses during ingestion of activation by E. coli. J. Immunol. 133: 886–891. apoptotic cells by human monocytes, macrophages, and dendritic cells. J. 19. Aubert, B., S. Chesne, G. J. Arlaud, and M. G. Colomb. 1985. Antibody- Immunol. 183: 6175–6185. independent interaction between the first component of human complement, 46. Fraser, D. A., K. Pisalyaput, and A. J. Tenner. 2010. C1q enhances microglial C1, and the outer membrane of Escherichia coli D31 m4. Biochem. J. 232: clearance of apoptotic neurons and neuronal blebs, and modulates subsequent 513–519. inflammatory cytokine production. J. Neurochem. 112: 733–743. 20. Albertı´, S., G. Marque´s, S. Camprubı´, S. Merino, J. M. Toma´s, F. Vivanco, and 47. Ogden, C. A., A. deCathelineau, P. R. Hoffmann, D. Bratton, B. Ghebrehiwet, V. J. Benedı´. 1993. C1q binding and activation of the complement classical V. A. Fadok, and P. M. Henson. 2001. C1q and mannose binding lectin en- The Journal of Immunology 11

gagement of cell surface calreticulin and CD91 initiates macropinocytosis and 51. Agarwal, V., A. Kuchipudi, M. Fulde, K. Riesbeck, S. Bergmann, and A. M. Blom. uptake of apoptotic cells. J. Exp. Med. 194: 781–795. 2013. Streptococcus pneumoniae endopeptidase O (PepO) is a multifunctional 48. Butko, P., A. Nicholson-Weller, and M. R. Wessels. 1999. Role of complement plasminogen- and fibronectin-binding protein, facilitating evasion of innate im- component C1q in the IgG-independent opsonophagocytosis of group B strep- munity and invasion of host cells. J. Biol. Chem. 288: 6849–6863. tococcus. J. Immunol. 163: 2761–2768. 52. Singh, B., A. M. Blom, C. Unal, B. Nilson, M. Morgelin, and K. Riesbeck. 2010. 49. Pisalyaput, K., and A. J. Tenner. 2008. Complement component C1q inhibits Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface beta-amyloid- and serum amyloid P-induced neurotoxicity via caspase- and protein A2 and confers complement-inhibitory activity. Mol. Microbiol. 75: calpain-independent mechanisms. J. Neurochem. 104: 696–707. 1426–1444. 50. Agarwal, V., S. Hammerschmidt, S. Malm, S. Bergmann, K. Riesbeck, and 53. Jalalvand, F., Y. C. Su, M. Morgelin, M. Brant, O. Hallgren, G. Westergren- A. M. Blom. 2012. Enolase of Streptococcus pneumoniae binds human com- Thorsson, B. Singh, and K. Riesbeck. 2013. Haemophilus influenzae protein F plement inhibitor C4b-binding protein and contributes to complement evasion. mediates binding to laminin and human pulmonary epithelial cells. J. Infect. Dis. J. Immunol. 189: 3575–3584. 207: 803–813. Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021