Clinical Isolates of Streptococcus pneumoniae Bind the Complement Inhibitor C4b-Binding Protein in a PspC Allele-Dependent Fashion

Antoine Dieudonné-Vatran, Stefanie Krentz, Anna M. Blom, Seppo Meri, Birgitta Henriques-Normark, Kristian Riesbeck and Barbara Albiger
J Immunol June 15, 2009, 182 (12) 7865-7877; DOI: https://doi.org/10.4049/jimmunol.0802376

Abstract

The complement system constitutes an important component of the innate immune system. To colonize their host and/or to cause disease, many pathogens have evolved strategies to avoid complement-mediated bacterial lysis and opsonophagocytosis. In this study, using a collection of 55 clinical isolates of Streptococcus pneumoniae, we demonstrate for the first time that pneumococci bind the complement inhibitor C4b-binding protein (C4BP). C4BP binding seems to be restricted to certain serotypes such as serotype 4, 6B, 7F, and 14, of which the strains of serotype 14 are the strongest binders. We show that bacteria-bound C4BP retains its functional activity and down-regulates the activation of the classical pathway. Thus, this major respiratory pathogen may escape immune recognition and eradication by the complement system. Furthermore, we show that C4BP binding varies between strains but is dependent on the expression of pneumococcal surface protein C, PspC of group 4. The study of the distribution of group 4 pspC locus shows that most of high-binder serotype 14 isolates harbor an allelic variant of group 4 pspC. Using PspC-negative mutant strains, we identified a new allelic variant of PspC (PspC4.4) as a major ligand for C4BP, revealing a new function for this important pneumococcal virulence factor. Thus pneumococci exploit host C4BP for complement evasion in a PspC allele-dependent manner.

The complement system is part of the first line of host innate immune defense. It is composed of >30 soluble and membrane-bound proteins that upon microbial recognition initiate a regulated cascade of reactions. There are three pathways of complement activation: 1) the classical pathway, 2) the alternative pathway, and 3) the lectin pathway. Activation of the classical pathway is initiated by C1q binding to, for example, lipopolysaccharides or Fc regions of Abs on bacterial surfaces. The alternative and lectin pathways are activated directly by bacterial surface components in the absence of Ab-Ag complexes. All three pathways lead to the formation of the C3 convertase with subsequent cleavage into C3a (anaphylatoxin) and C3b (opsonin). This results in either opsonization and phagocytosis of the pathogen or in the formation of the bacteriolytic membrane attack complex (MAC).3 To avoid overconsumption and attack against host cells, while targeting microbial surfaces, the complement activation needs to be tightly regulated. This regulation occurs via both soluble (factor I, factor H, FHL-1, C4b-binding protein (C4BP)) and membrane-bound (CRIg, CD35, CD46, CD55, and CD59) complement regulatory proteins.

C4BP is the major soluble inhibitor of the classical and lectin pathways. It inhibits the formation and accelerates the decay of the C3 convertase (C4bC2a) (1, 2). It is a 570-kDa glycoprotein composed of eight polypeptide chains (seven α-chains and one β-chain) arranged in a spider-like structure. Both the α-chains and β-chain are composed of repeating domains of ∼60 aa each, known as complement control protein (CCP) domains or short consensus repeats (SCRs). The physiologic concentration range of C4BP in normal human serum (NHS) is 200–300 μg/ml. Most complement proteins are produced in the liver, but production of complement factors, particularly of C1q and C7, and inhibitors also occurs in the lungs by alveolar macrophages and epithelial cells (3, 4). Upon inflammation, plasma exudates can also leak complement proteins into the respiratory tract.

To colonize the host and/or to cause disease, microbes have to overcome recognition and clearance by the immune system, and they have evolved a plethora of strategies to evade the complement system. One such strategy is to bind C4BP, which will prevent the activation of the classical pathway on the bacterial surface. Indeed, many pathogens (Escerichia coli K1, Bordetella pertussis, Neissseria meningitidis, Neissseria gonorrhoeae, Moraxella catarrhalis, Haemophilus influenzae, Streptococcus pyogenes, Borrelia recurrentis, and Borrelia duttonii) have been shown to avoid deleterious effects of complement activation by binding C4BP (5, 6, 7, 8, 9, 10, 11).

A recent report from the World Health Organization and the United Nations Children’s Fund showed that pneumonia kills more children <5 years of age than any other infectious diseases worldwide (AIDS, malaria, tuberculosis, measles). Thus, >2 million children die annually, mainly in developing countries from pneumonia (12). The major causative agent is the bacterium Streptococcus pneumoniae (also called pneumococcus). Pneumococci are encapsulated Gram-positive bacteria with 91 different capsules or serotypes identified so far. Pneumococci cause invasive pneumococcal diseases (IPD) such as sepsis and meningitis, which kill and leave severe sequelae in children worldwide. Additionally, pneumococci are among the most common bacterial causes of middle ear infections (otitis media). The nasopharynx of small children constitutes the normal habitat for pneumococci and asymptomatic carriage may be found in up to 70% of healthy children <2 years of age. Carriage is thought to facilitate horizontal gene transfer between strains and also represents a prerequisite for the spread of this pathogen in the community. A period of nasopharyngeal colonization usually occurs before and during the development of IPD (13).

Pneumococci express many virulence factors helping the bacteria both to inhibit complement deposition and to resist opsonophagocytosis. Important virulence factors are, for example, the polysaccharide capsule, the cytotoxin pneumolysin, and the pneumococcal surface protein, PspC. The capsule may reduce the amount of bound C3b and restrict the access of professional phagocytes for cell-bound C3b, which hampers opsonophagocytosis (14). Pneumolysin contributes to complement resistance by quenching complement away from the pneumococcal surface (15). The PspC protein family can inhibit C3b deposition, but also bind the host complement inhibitor factor H, leading to inhibition of the alternative pathway activation (16, 17, 18). Recently, the pneumococcal histidine triad (Pht) was shown to be required for inhibition of complement deposition on the pneumococcal surface through the recruitment of complement factor H (19). Therefore, complement resistance is a major contributor to pneumococcal virulence and pathogenesis.

In this study, we investigated whether clinical isolates of S. pneumoniae bind the complement inhibitor C4BP as mechanism of immune evasion. We also investigated the relative contribution of the polysaccharide capsule, pneumolysin, and PspC in C4BP-mediated immune evasion. We show that pneumococcal isolates bind C4BP to various degrees and that this interaction is dependent on a new allelic variant (PspC4.4) of PspC but not on capsular serotype or pneumolysin expression. Thus, we demonstrate that the pneumococcus exploits host C4BP for complement evasion in a PspC allele-dependent manner.

Materials and Methods

Bacterial strains and culture conditions

All pneumococcal strains, except TIGR4 and D39, used in this study are clinical pneumococcal isolates obtained from the Swedish Institute for Infectious Disease Control, Stockholm (Table I⇓) (20, 21). The clinical isolates were selected to represent common serotypes and different clones as assessed by serotyping and multilocus sequence typing (MLST) (22). TIGR4 is a clinical encapsulated isolate of serotype 4 sequenced by the Institute for Genomic Research (TIGR) (American Type Culture Collection BAA-334; www.jcvi.org). The isogenic variants of TIGR4 expressing different capsular types, that is, serotypes 6B (TIGR4:64), 7F (TIGR4:74), 14 (TIGR4:144), or 19F (TIGR4:194), and their parental capsule donors 619, 703, 1401, and 1902, respectively, were a gift from Prof. M. Lipsitch from Harvard School for Public Health (Boston, MA) (23). BBH18 and NTHi506 are clinical isolates of M. catarrhalis and non-typeable H. influenzae (8, 10).

View this table:
Table I.

Strains used in this study and carriage of group 4 pspC allele among clinical isolatesa

For in vitro experiments, bacteria were grown overnight from frozen stocks on blood agar or chocolate agar plates at 37°C and 5% CO2. Colonies were taken directly from plates in PBS to OD600 nm of 0.5 (1 × 108 bacteria/ml). Appropriate dilutions were made to obtain the desired concentration. The concentration was retrospectively confirmed by viable counting on blood agar plates.

Multilocus sequence typing

MLST was performed as previously described (22). In brief, internal fragments of the seven housekeeping genes (aroE, gdh, gki, recP, spi, xpt, and ddl) were amplified by PCR from bacterial genomic DNA and sequenced. Sequences were compared with existent sequences in the MLST database (http://spneumoniae.mlst.net). After sequence alignment analyses, a sequence type (ST) was attributed.

Construction of S. pneumoniae pneumolysin-negative, capsule-negative, and PspC-negative strains using PCR ligation mutagenesis

Flanking regions upstream and downstream of the target gene (SP1923, ply; capsule operon, cps; or SP2190, pspC) were amplified from TIGR4 chromosomal DNA using specific primers (Table II⇓). Each fragment was ∼1 kb and contained restriction sites for either ApaI (5′ end of upstream fragment, underlined in the primer sequence) or BamHI (3′ end of downstream fragment, underlined in the primer sequence). The PCR products were digested with corresponding restriction enzymes, purified, and ligated with either the Km-rpsL cassette, Janus (24), or the erythromycin cassette from the shuttle vector pMU1328 (25) amplified using specific primers, creating a PCR product with ApaI and BamHI termini (Table II⇓). The ligation was performed using equal molar amounts of upstream fragment, downstream fragment, and either erythromycin or Janus fragment. The ligation product was then used to transform the TIGR4 wild-type (wt) strain or into the recipient pneumococcal strain. The recipient pneumococcal strains were grown in 5 ml of C+Y medium to an OD620 nm of 0.2, and 2.5 μl of competence stimulatory peptide (CSP)1 (EMRLSKFFRDFILQRKK) and/or CSP2 (EMRISRIILDFLFLRKK) was added to 200 μl of the log-phase culture (CSP1 and CSP2 were a gift from Prof. D. Morrison, University of Illinois, Chicago, IL). Ten microliters of the ligation mix was added to the competent cells and the mixture was incubated successively on ice for 10 min, at 31°C for 30 min, and finally at 37°C for 2 h. The transformation mixture was then plated on blood agar plates containing kanamycin (400 μg/ml) and/or erythromycin (1 μg/ml) and incubated overnight at 37°C, 5% CO2. Several independent transformants were selected. They were verified by PCR and by sequencing with control primers. Pneumolysin-negative strains were checked for the production of pneumolysin by Western blot using a polyclonal anti-pneumolysin antiserum (provided by Prof. T. J. Mitchell, University of Glasgow, Glasgow, U.K.). PspC-negative strains were also checked for the production of PspC by Western blot using rabbit polyclonal anti-PspC antiserum (gift from Prof. E. Tuomanen, St. Jude Children Research Hospital, Memphis, TN).

View this table:
Table II.

Primers used for construction of capsule, pneumolysin, and pspC mutants and for screening of pspC locusa

Sequencing of pspC allelic variant, pspC4.4, and nucleotide sequence accession number

Using primers located in the upstream and downstream flanking regions of the pspC gene (SP2189/SP2191), the pspC gene of BHN79 strain was amplified by PCR. The PCR fragment was subsequently sequenced by a primer walking strategy using BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer’s instructions. New primers were designed from the obtained sequences until full sequence was completed. The nucleotide sequence of the pspC gene of BHN79 strain is assigned GenBank accession no. EU881702 (www.ncbi.nlm.nih.gov/nuccore/195963558).

Cloning and expression of pspC allelic variant, pspC3.4, as a His 6-tagged fusion protein

The full-length pspC3.4 gene (excluding the leader peptide and the stop codon) was amplified with high-fidelity pfu DNA polymerase (Fermentas/Life Sciences) from TIGR4 chromosomal DNA using primers BamStartPspC and SalStopPspC (Table II⇑). The resulting PCR product with BamHI and SalI termini was cloned into pET21b vector and expressed into E. coli BL21 (DE3)pLysS (Novagen). Recombinant PspC3.4 was expressed as His6-tagged fusion protein and was purified by HisPur Cobalt Resin purification kit according to the manufacturer’s instructions (Pierce/Thermo Fisher Scientific). The purified protein was run on SDS-PAGE for correct size and also checked by Western blot using rabbit polyclonal anti-PspC antiserum.

Proteins and Abs

Human C4BP was purified from human plasma (26). rC4BP was expressed in human kidney cells 293 and purified using affinity chromatography with a mAb directed against the α-chain of C4BP (27). The recombinant C4BP mutants lacking individual CCPs (ΔCCP1–8) were constructed and expressed as previously described (28). C3b-like (C3met) and C4b-like (C4met) molecules were prepared by incubation of C3 and C4 with 100 mM methylamine (pH 7.6) for 1 h at 37°C and subsequent dialysis against 100 mM Tris-HCl and 150 mM NaCl (pH7.5). Both C3met and C4met are functionally equivalent to C3b and C4b and will be referred to as such. Purified factor H (FH), factor I (FI), the secondary peroxidase-conjugated anti-mouse IgG Ab, and the polyclonal goat anti-C3 IgG were purchased from Sigma-Aldrich. The polyclonal rabbit anti-C4c IgG, the secondary peroxidase-conjugated swine anti-rabbit IgG Ab, and the peroxidase-conjugated anti-goat IgG Ab were purchased from Dako. The polyclonal rabbit anti-human C4BP and the murine monoclonal against CCP1 and CCP4 of C4BP (mAb104 and mAb67, respectively) were a gift of Prof. B. Dahlbäck (Lund University, Malmö, Sweden).

Direct binding assay with 125I-C4BP and with 125I-FH

Fifty micrograms of C4BP from plasma and purified FH were labeled with 0.05 mol of iodine per mol of protein using chloramine-T method. Bacteria were grown overnight on blood agar plates and were washed once and resuspended to OD600 nm of 0.5 in PBS with 1% BSA. Bacteria (1010) were incubated for 1 h at 37°C with 1.5 μg/ml 125I-labeled C4BP in PBS with 1% BSA. In the inhibition assays, unlabelled C4BP (0–1500 nM), prothrombin (0–1000 nM), recombinant PspC3.4 (0–1500 nM), heparin (0–10 mg/ml), NaCl (0–500 mM), and C4met (0–100 nM) were added to the reaction with 125I-labeled C4BP and incubated for 1 h at 37°C. Then, bacterial-bound 125I-C4BP was separated from unbound 125I-C4BP by centrifugation through 20% sucrose columns for 3 min at 10,000 rpm. Both bound and unbound 125I-C4BP to bacteria was measured in a gamma counter.

Serum absorption of C4BP

NHS pool was prepared from the blood of five healthy volunteers. The blood was allowed to clot for 30 min at room temperature (RT), followed with 1 h of incubation on ice. After centrifugation the sera were pooled, aliquoted, and stored at −70°C. Bacteria were grown overnight on blood agar plates and were washed once and resuspended in gelatin veronal buffer (GVB; 142 mM NaCl, 1.8 mM sodium barbital, 3.3 mM barbituric acid, 0.1% fish gelatin (pH 7.4)). Bacteria (108) were incubated for 30 min at 37°C with 30% NHS or GVB. The unbound proteins were removed with five washes with PBS. The bacterial pellet was resuspended in 30 μl of 0.1 M glycine (pH 2.5) and was incubated 20 min at 37°C to elute the bound proteins. The bacteria were centrifuged and the supernatant was recovered and neutralized with 30 μl of 1 M Tris-HCl (pH 9.5). Ten microliters of the supernatants were run on a gradient 4–12% NuPAGE Tris gel (Invitrogen) under nonreducing conditions. The proteins were transferred to a polyvinylidene difluoride membrane and submitted to Western blotting using a mouse monoclonal anti-C4BP Ab (mAb 104) directed against CCP1 from C4BP and a secondary peroxidase-conjugated anti-mouse IgG Ab.

Flow cytometry

Bacteria were grown overnight on blood agar plates and washed once and resuspended to OD600 nm of 0.5 in PBS with 1% BSA. Bacteria (108, 107, and 106) were incubated for 1 h at 37°C with 80 μg/ml plasma C4BP, 50 μg/ml rC4BP, 50 μg/ml rC4BP (ΔCCP1–8), 50% NHS (containing ∼100–150 μg/ml C4BP), or PBS with 1% BSA. After three washes, the samples were incubated for 1 h either with a polyclonal rabbit anti-C4BP serum or with anti-C4c Ab. After an additional three washes with PBS, incubation with FITC-conjugated swine anti-rabbit Ab was performed. The samples were processed by flow cytometry using the FACSCalibur.

ELISA for C4BP binding to S. pneumoniae

To validate C4BP binding to S. pneumoniae, a saturation assay was performed by whole-cell ELISA. Ninety-six-well plates were coated either with bacterial suspension (107) and air dried at room temperature or with purified recombinant PspC3.4 (5 μg/well) and incubated overnight at 4°C. The plates were subsequently blocked with 200 μl of PBS-3% fish gelatin (Sigma-Aldrich) for 2 h at RT before 50 μl of different concentrations of purified C4BP were added to each well for 1 h at RT. Then the plates were incubated 1 h at RT with 50 μl of mouse anti-C4BP (mAB67) diluted 1/5000, washed three times, and incubated with 50 μl of secondary peroxidase-conjugated anti-mouse IgG Ab diluted 1/5000 for 1 h at RT. Finally, the plates were developed using tetramethylbenzidine substrate reagent set (BD Biosciences) for 30 min before determining the OD450 using a microtiter plate reader.

Functional assay for cofactor activity of cell-bound C4BP

The cofactor activity of C4BP bound to pneumococci was analyzed by measuring FI-mediated degradation of C3met (C3b) and C4met (C4b). Bacteria were grown overnight on blood agar plates and washed once and resuspended to OD600 nm of 0.5 in PBS. Pneumococci (5 × 107) were incubated with plasma-purified C4BP (80 μg/ml) for 1 h at 37°C with gentle agitation. After extensive washing with PBS, C3met/C4met (20 μg/ml) and FI (50 μg/ml) were added to the bacteria and the mixture was incubated for 1 h at 37°C with gentle agitation. The cells were sedimented by centrifugation at 14,000 × g for 10 min and the supernatants were mixed with sample buffer (Invitrogen). The samples were then subjected to SDS-PAGE under reducing conditions and transferred to a polyvinylidene difluoride membrane (Invitrogen). C3met degradation products were evaluated by detection of the α-chain cleavage fragments of 76 kDa and 43 kDa by using polyclonal goat anti-C3 IgG (Sigma-Aldrich) at a final dilution of 1/1000 and a secondary peroxidase-conjugated anti-goat IgG Ab. C4met degradation was evaluated by detection of the degradation of the α-chain using a polyclonal rabbit anti-C4c IgG at a final dilution of 1/500 and a secondary peroxidase-conjugated anti-rabbit IgG Ab. The polyclonal rabbit anti-C4c IgG recognizes C4, C4b, and C4c but not C4d.

Results

Pneumococci bind the complement inhibitor C4BP

To investigate whether S. pneumoniae binds human C4BP, we incubated 26 clinical isolates of S. pneumoniae of different serotypes with radiolabeled plasma-purified C4BP. The two laboratory strains D39 and TIGR4 were included as well as M. catarrhalis (BBH18) and nontypeable H. influenzae (NTHi506), which were used as positive controls because they were previously shown to bind C4BP (8, 10). We observed that pneumococci bound C4BP with binding percentages ranging from 0.4% to 27.4%. The strains could be divided in three groups: 1) strong binders with binding rates ranging from 25.3% to 27.4%, 2) medium or intermediate binders with rates between 11.2% and 18.7%, and 3) weak or nonbinders with rates between 0.4% and 9.9% (Fig. 1⇓A). None of the pneumococcal strains tested bound C4BP as efficiently as NTHi506 or BBH18 (61.1 ± 1.5% and 69.9 ± 5%, respectively). Interestingly, both D39 and TIGR4 were weak binders (2.5 ± 0.3% and 4.9 ± 0.6%, respectively) (Fig. 1⇓A, indicated with stars). Several strains of serotype 4, 6B, and 7F exhibited intermediate binding to C4BP. We also observed that all isolates (n = 4) of serotype 14 tested were either strong (n = 3) or intermediate binders (n = 1).

FIGURE 1.
FIGURE 1.

Binding of pC4BP to clinical isolates of S. pneumoniae. A, A total of 28 pneumococcal strains of different serotypes (1, 2, 3, 4, 6B, 7F, 9V, 14, 19F) including TIGR4 (serotype 4) and D39 (serotype 2), a strain of M. catarrhalis BBH18, and a strain of nontypeable H. influenzae (NTHi506) were incubated with 1.5 μg/ml purified plasma 125I-C4BP. The binding was defined as a ratio between bound radioactivity and total amount of added radioactivity. Binding varies between 0.4% and 27.4% for pneumococcal strains. The mean values of at least three independent experiments are shown, and each circle represents an individual strain. The binding for TIGR4 and D39 is highlighted with star symbols. Binding was considered weak below 10%, medium or intermediate between 10% and 20%, and strong above 20%. B, C4BP binding to encapsulated and unencapsulated strains. C4BP binding to four isogenic capsular variants of TIGR4 harboring either serotypes 6B (TIGR4:64), 7F (TIGR4:74), 14 (TIGR4:144), or 19F (TIGR4:194) capsules to their parental capsule donor strains (619, 703, 1401, 1902) and to capsule-deficient mutants (BHN78Δcps and BHN79Δcps) was compared with C4BP binding to wt TIGR4, BHN78, and BHN79, respectively. Binding for isogenic capsular variants of TIGR4 ranges between 3% and 11.1%, while the binding for unencapsulated strains and their isogenic parents varies between 27.5% and 66.9%. The mean values of at least three independent experiments are shown. Binding below 10% was considered weak. C4BP binding rates were analyzed by a nonparametric Mann-Whitney test. ∗∗, p < 0.01. C, C4BP binding to serotype 14 strains belonging to different clonal lineages. Binding varies between 7.7% and 59.1%. The mean values of at least three independent experiments are shown. Binding was considered weak below 10%, medium or intermediate between 10% and 20%, and strong above 20%.

To study whether C4BP binding is capsule-dependent or whether the serotypes 4, 6B, 7F, and 14 capsule have biochemical or structural properties that allow C4BP binding as compared with other capsular serotypes, we analyzed the C4BP binding capacity of isogenic variants of TIGR4 expressing different capsular types (gift from Dr. M. Lipsitch, Harvard School for Public Health, Boston, MA), that is, serotypes 6B (TIGR4:64), 7F (TIGR4:74), 14 (TIGR4:144), or 19F (TIGR4:194), respectively, as well as of isogenic capsule deletion mutants (Δcps) of two selected serotype 14 strains, BHN78 and BHN79. When we compared the C4BP binding capacity between TIGR4 and its isogenic capsular strains, we observed that all the isogenic capsular variants exhibited the same weak C4BP binding as compared with wt TIGR4 (Fig. 1⇑B). All parental capsule donor strains, including the serotype 14 parental isolate, also showed weak C4BP binding. Furthermore, we observed that the loss of capsule significantly increased C4BP binding, demonstrating that the polysaccharide capsule hinders rather than promotes the binding (Fig. 1⇑B). Thus, we conclude that it is unlikely that C4BP binds to a specific sugar moiety of the polysaccharide capsule.

To study the genetic relatedness between clinical isolates (or clonality), sequence-based methods such as multilocus sequence typing are used. Using MLST, we found that of the four serotype 14 strains tested (Fig. 1⇑A), three isolates (BHN78, BHN79, and BHN84) belonged to the same clone or ST (i.e., ST124), while BHN83 belonged to an unrelated clone ST555. To investigate whether C4BP binding correlates with clonal properties, we selected 24 additional clinical isolates of serotype 14 belonging to different clonal clusters. We chose six and eight isolates from the two major invasive clones, ST9 and ST124, respectively, as well as 10 strains from unrelated minor clones. We tested these strains for C4BP binding and observed that all isolates but three exhibited strong or intermediate binding capacities with rates between 7.7% and 59.2% (Fig. 1⇑C and Table I⇑). This confirmed our previous observation (Fig. 1⇑A) that most serotype 14 strains are able to bind C4BP to various degrees as compared with strains from other serotypes. Furthermore, we also observed that the strength to which the isolates bind C4BP seemed to correlate with clonal properties, as all isolates belonging to the clone ST124 were strong binders (Fig. 1⇑C). Taken together, our data show that the strength to bind C4BP is dependent on one or more genetic properties present in specific clonal types of pneumococci.

Next, we selected two strains BHN79 and TIGR4, a strong and a weak binder, respectively, and confirmed C4BP binding by flow cytometry using a polyclonal anti-C4BP antiserum (Fig. 2⇓A). Both strains were incubated with 80 μg/ml plasma-purified C4BP (pC4BP) or with PBS (Fig. 2⇓A). pC4BP was deposited in high amounts on the surface of BHN79 and significantly less C4BP was bound to TIGR4 (Fig. 2⇓A). C4BP deposition on BHN79 depended on the quantity of bacteria used with the maximum binding occurring at a dose of 106-107 bacteria (data not shown). We also investigated whether BHN79 is able to recognize and bind C4BP in whole human serum. We performed absorption assays by incubating the bacteria with 30–50% NHS and assessed C4BP binding by either flow cytometry (Fig. 2⇓A) or Western blotting (Fig. 2⇓B). Since pneumococci have a thick and rigid cell wall, they are resistant to lysis by MAC. Both TIGR4 and BHN79 survived in 50% NHS for over 1 h as determined by colony forming units (data not shown). Moreover, our data show that C4BP was absorbed from whole human serum by strain BHN79 in contrast to TIGR4 (Fig. 2⇓). Taken together, our data show that pneumococci are able to bind C4BP also from serum but binding efficiency varies between different strains. Interestingly, we also observed that serum C4BP binding to BHN79 was weaker as compared with pC4BP (Fig. 2⇓A), suggesting that the efficiency of C4BP binding to bacteria might be influenced by the presence of other serum proteins such as FH.

FIGURE 2.
FIGURE 2.

C4BP binding to TIGR4 and BHN79 strains. A, Representative flow cytometry histograms of C4BP binding to TIGR4 and BHN79 strains. Bacteria (107) were incubated with either 80 μg/ml pC4BP (dark gray line), 50% NHS (light gray line), or PBS (black line). The binding was detected using a polyclonal rabbit anti-C4BP serum and FITC-conjugated swine anti-rabbit Ab. B, Pneumococcal strain BHN79 but not TIGR4 recruits C4BP from NHS. Bacteria (108) were incubated with either 30% NHS or GVB buffer. Bound proteins were eluted from bacterial surfaces, run onto SDS-PAGE, and submitted to Western blot using a monoclonal anti-C4BP Ab (mAb104) and a secondary peroxidase-conjugated anti-mouse IgG Ab. Lane 1, 1% NHS; lane 2, BHN79 in absence of NHS; lane 3, BHN79 in presence of NHS; lane 4, TIGR4 in absence of NHS; and lane 5, TIGR4 in presence of NHS.

Binding of C4BP to pneumococci is specific and dose-dependent

We tested the specificity of the C4BP binding to BHN79 strain by incubating BHN79 with increasing amounts of rC4BP in a whole-cell ELISA (Fig. 3⇓A). BHN79 bound C4BP in a dose-dependent manner, reaching saturation at 100–200 μg/ml (Fig. 3⇓A). This concentration correlated with the physiologic concentration range of C4BP in NHS, which is 200–300 μg/ml. In contrast, TIGR4 bound significantly less C4BP (Fig. 3⇓A).

FIGURE 3.
FIGURE 3.

Characterization of the interaction between pC4BP and BHN79. A, Binding of C4BP to BHN79 is saturable and dose-dependent. Bacteria (106) or 5 μg of purified recombinant PspC3.4 (recPspC3.4) was immobilized in microtiter plates and consequently incubated with rC4BP. Bound C4BP was detected by ELISA with mAbs (mAb67). The mean values of triplicates from one representative experiment are shown. B–D, Inhibition of 125I -C4BP binding with increasing amount of competitors. BHN79 strain (108) was coincubated with 1.5 μg/ml 125I plasma C4BP and increasing concentrations of cold recombinant C4BP (0.1–1500 nM), prothrombin (0.1–1000 nM), heparin (0–10 mg/ml), C4met (0–100 nM). Prothrombin was used as negative control. The binding was determined as a ratio between bound radioactivity and total amount of added radioactivity. The mean values of at least three independent experiments are shown. E, Effect of salt on the binding of C4BP to BHN79 strain. Increasing amounts of NaCl (0–500 mM) were added to phosphate buffer containing 150 mM NaCl. The binding was determined as a ratio between bound radioactivity and total amount of added radioactivity. The mean values of at least three independent experiments are shown. F, Localization of the pneumococcal-binding site on C4BP using rC4BP deletion mutants. Bacteria (107) were incubated either with 50 μg/ml wt rC4BP or with recombinant mutant C4BP lacking individual CCP domains (ΔCCP1–8). The binding was detected with flow cytometry using a polyclonal rabbit anti-C4BP serum and FITC-conjugated swine anti-rabbit Ab. The mean fluorescence intensity (MFI) of at least three independent experiments is shown. C4BP binding rates were analyzed by ANNOVA test. ∗, p < 0.05.

To confirm specificity of C4BP binding, BHN79 strain was incubated with increasing amounts of unlabeled rC4BP or human prothrombin in addition to 125I-C4BP in a competition assay (Fig. 3⇑B). rC4BP inhibited the binding of 125I-C4BP, whereas prothrombin did not affect the binding (Fig. 3⇑B). Our data demonstrate that the interaction between pneumococci and C4BP is specific and dose-dependent.

Inhibition of C4BP binding to pneumococci by C4b, heparin, and salt

To further characterize the structural properties of the interaction between plasma C4BP and BHN79, we performed competition experiments using heparin and C4met (Fig. 3⇑, C and D) (1). Both heparin and C4met are ligands of C4BP and bind to the CCP1-CCP3 domains of the C4BP α-chain. Increasing concentrations of either heparin or C4met inhibited C4BP binding to BHN79 (Fig. 3⇑, C and D).

Using increasing amounts of NaCl, we demonstrated that the interaction between C4BP and BHN79 was salt sensitive as an addition of 50 mM NaCl to physiological ionic strength buffer resulted in a 58% reduction in C4BP binding (Fig. 3⇑E). Thus, we have shown that BHN79 in addition to binding to plasma purified C4BP (Figs. 1⇑ and 2⇑) also bound rC4BP, which is exclusively composed of the α-chain (Fig. 3⇑F) (27). Using eight mutant rC4BP proteins, each lacking individual CCP domains, we highlighted the importance of CCP2-CCP3 region for the interaction (Fig. 3⇑F). It seems also that to some extent CCP8 might play a role in the interaction. These data suggest that the interaction between pneumococci and C4BP is ionic and probably mediated by the CCP2-CCP3 region of the C4BP α-chain.

Bacteria-bound C4BP retains its functional activity

C4BP acts as a cofactor for the serine protease FI to inactivate both fluid phase and cell-bound C4b, and thereby it inhibits the classical complement pathway. C4met is composed of three chains, α-, β-, and γ-chains of 97, 75, and 33 kDa, respectively (Fig. 4⇓, lane 8). In the presence of both soluble purified FI and C4BP, the α-chain of C4met becomes entirely degraded (Fig. 4⇓, lane 1).

FIGURE 4.
FIGURE 4.

Analysis of bacterial-bound C4BP cofactor activity by C4met degradation. Bacteria (108) were coincubated with C4met and C4BP in presence of FI. Degradation of the α-chain of C4met was analyzed by Western blot using a polyclonal rabbit anti-C4c IgG and a secondary peroxidase-conjugated anti-rabbit IgG Ab. As positive control, C4met was coincubated with FI, and C4BP (lane 1), TIGR4 (lane 2), TIGR4ΔpspC (lane 3), BHN79 (lane 4), and BHN79ΔpspC (lane 5) were coincubated with FI, C4BP, and C4met; TIGR4 (lane 6) and BHN79 (lane 7) were coincubated with C4met in absence of C4BP and FI; the three chains of C4met are visualized in lane 8.

To investigate whether bacteria-bound C4BP could retain its functional activities, we performed C4met degradation assays (Fig. 4⇑). TIGR4 and BHN79 were first incubated with C4BP, and after removal of unbound C4BP, bacteria were incubated with FI and C4met. In negative controls, either FI or C4BP were omitted (data not shown). C4BP retained its cofactor activity when bound to BHN79, as demonstrated by the almost total degradation of C4met α-chain (Fig. 4⇑, lane 4). In contrast, insufficient amounts of C4BP bound to TIGR4 did not allow C4met cleavage (Fig. 4⇑, lane 2). The degradation of C4met was not a result of a proteolytic or cofactor activity from the bacteria themselves, as shown by a control experiment, where bacteria without bound C4BP were incubated with C4met alone (Fig. 4⇑, lanes 6 and 7) or with C4met and FI (data not shown).

C4BP also acts to some extent as a cofactor for FI in degradation of soluble and surface-bound C3b. C3b is composed of two chains, α′- and β-chains of 110 and 75 kDa, respectively, and when degraded by FI in association with C4BP, the α′-chain is cleaved into two fragments. C4BP bound to BHN79 retained its cofactor activity as demonstrated by the appearance of the degradation fragments of the C3met α′-chain of appropriate sizes (data not shown).

A new allelic variant of PspC is a ligand for C4BP

PspC is a pneumococcal surface-exposed protein that binds several host proteins (i.e., secretory IgA (sIgA), plateletactivating factor receptor (PAF-R), and polymeric IgG receptor (pIgR)) and more specifically complement proteins such as C3 and FH (16, 17, 29, 30, 31, 32). To study whether PspC is also involved in pneumococcal binding to C4BP, we engineered pspC deletion mutants of TIGR4 and BHN79 strains T4ΔpspC and BHN79ΔpspC, respectively. There was no difference in growth fitness between wt and mutant strains as determined by growth curves in vitro (data not shown). Since PspC is one of the known pneumococcal ligand of FH, we tested both pspC mutants for FH binding in a direct binding assay with 125I-purified FH (Fig. 5⇓A) and in an absorption assay with NHS (Fig. 5⇓B). We confirmed that the loss of PspC expression on the bacterial surface correlated with a reduced binding of FH (Fig. 5⇓, A and B). When we tested our isogenic pairs for C4BP binding in direct binding (Figs. 3⇑A and 5⇓C), we observed that the deletion of PspC in BHN79ΔpspC resulted in a significant reduction of C4BP binding (Figs. 3⇑A and 5⇓C) and of C4met cleavage (Fig. 4⇑, lane 5). Additionally, we performed an absorption assay with NHS and assessed C4BP binding to BHN79 and its isogenic mutant BHN79ΔpspC by flow cytometry (Fig. 5⇓D). We observed that the shift to the right is lost in the pspC-negative strain, confirming the loss of C4BP binding by BHN79ΔpspC. However, no difference was observed with TIGR4 and its isogenic pspC mutant (Figs. 3⇑A, 4⇑, lanes 2 and 3, and 5⇓C). Furthermore, we observed that binding of serum C4BP to BHN79 was weaker as compared with binding of serum FH (Fig. 5⇓, B and D). This is in agreement with our previous observation (Fig. 2⇑A) that the efficiency of C4BP binding to bacteria might be influenced by the presence of other serum proteins such as FH. We also tested isogenic mutants of BHN79 and TIGR4 deleted in pneumolysin (Δply), a cytotoxin known to inhibit complement by inhibiting C3 deposition, but found no difference between wt and isogenic ply mutant strains with respect to C4BP binding (data not shown).

FIGURE 5.
FIGURE 5.

Effect of pspC mutations on FH and C4BP binding. pspC isogenic mutants of TIGR4 and BHN79 were engineered by insertion replacement with an erythromycin resistance cassette. The binding was defined as a ratio between bound radioactivity and total amount of added radioactivity. FH and C4BP binding rate were analyzed by a nonparametric Mann-Whitney test. ∗∗, p < 0.01. A, Binding of 125I-FH to pspC mutants was compared with wt strains. B, Representative flow cytometry histograms of FH binding to BHN79 and BHN79ΔpspC. Bacteria (107) were incubated with either 50% NHS (gray line) or PBS (black line). M represents the histogram marker for the positive population; MFI, mean fluorescence intensity. C, Binding of 125I-C4BP to pspC mutants of TIGR4, BHN79, and BHN84 strains. D, Representative flow cytometry histograms of C4BP binding to BHN79 and BHN79ΔpspC. Bacteria (107) were incubated with either 50% NHS (gray line) or PBS (black line). M represents the histogram marker for the positive population; MFI, mean fluorescence intensity. E, PCR amplification of pspC locus of TIGR4 (lanes 1 and 3) and BHN79 (lanes 2 and 4) using either primers of the upstream and downstream flanking regions SP2189/SP2191 (lanes 1 and 2) or specific primers of pspC group 4 LU9/LU10 (lanes 3 and 4); lane 7, 1-kb DNA ladder (Invitrogen). F, Schematic representation of PspC3.4 and PspC4.4. The overall percentage of identity at the protein level is 46.5%. The leader peptide (LP) is conserved (37 aa). PspC4.4 harbors a single R2-like domain, which exhibits a 51.8% identity with the R2 domain of PspC3.4. The C-terminal anchor of PspC4.4 is composed of 10 choline-binding domain (CBD) repeats as compared with 8 CBD for PspC3.4. Localization of primers LU9 and LU10 is shown. LU10 has two mismatches against pspC3.4 sequence. P, Proline-rich region; R1 and R2, R1 and R2 domains.

Although the pspC locus varies in size and in sequence, it is located at the same chromosomal position in all pneumococcal strains tested (33). The pspC locus of TIGR4 is located between two open reading frames annotated SP2189 and SP2191. PCR amplification using primers located in the upstream and downstream flanking regions of the pspC gene (SP2189/SP2191) showed that the pspC gene product of BHN79 is larger than the pspC gene product of TIGR4 (Fig. 5⇑D, lanes 1 and 2). We sequenced the pspC gene of BHN79 and showed that it had a size of 2814 bp (GenBank accession no. EU881702; www.ncbi.nlm.nih.gov/nuccore/195963558) as compared with 2082 bp for TIGR4, confirming that PspC of BHN79 is of larger size than PspC of TIGR4.

Allelic variants of PspC can be divided into 11 groups, and PspC of TIGR4 belongs to group 3. It is the fourth sequenced member of this group and is hence designated as PspC3.4 (Fig. 5⇑, E and F). PspC of the serotype 2 strain D39 (PspC3.1) is the archetype of the group 3. However, sequence and PCR analysis showed that the pspC gene of BHN79 belonged to group 4 (data not shown and Fig. 5⇑D, lanes 3 and 4). This group contains three sequenced members, PspC4.1, PspC4.2, and PspC/SP14-BS69 (GenBank accession nos. AF145044, AF154033, and ZP01827960; www.ncbi.nlm.nih.gov) (33). Thus, PspC of BHN79 is the fourth sequenced member of this family and is therefore referred to as PspC4.4 (Fig. 5⇑E). When we compared PspC3.4 (TIGR4) and PspC4.4 (BHN79), we only found an overall percentage of identity of 46.5% at the protein level. Both PspC3.4 and Psp4.4 express a conserved 37-aa leader peptide. Like all members of group 4, PspC4.4 harbors a single R2-like domain, which exhibits a 51.8% identity with the R2 domain of PspC3.4. The C-terminal anchor of PspC4.4 is composed of 10 choline-binding domain repeats as compared with 8 choline-binding domain repeats for PspC3.4 (Fig. 5⇑D). Using purified recombinant PspC3.4, we showed that there is no direct interaction with C4BP (Fig. 3⇑A). We also performed a heterologous competition assay using increasing amounts of recombinant PspC3.4 to inhibit BHN79 binding to C4BP and we showed that even the highest concentration of PspC3.4 did not inhibit the interaction between C4BP and BHN79 (data not shown). Taken together, our data suggest that pneumococcal C4BP binding occurs in a PspC allele-dependent manner.

Interestingly, both PspC4.1 and PspC/SP14-BS69 have been sequenced from serotype 14 isolates, V26 and SP14-BS69 strains, respectively (33). Moreover, SP14-BS69 belongs to the clonal lineage of ST124. PspC4.2 has been sequenced from a serotype 6 strain, G100, and the pspC locus from this strain contains two pspC genes (pspC4.2 and pspC10.1) in tandem separated by an insertion sequence (33). Sequence comparisons showed that PspC4.1, PspC/SP14-BS69, and PspC4.4 clustered together, while PspC4.2 is more distant (Fig. 6⇓A). Therefore, we studied the distribution of group 4 pspC allele among our strain collection and investigated whether the expression of group 4 pspC alleles correlates with C4BP binding. PCR amplification using primers (LU9/LU10) designed to specifically amplify group 4 pspC locus showed that group 4 pspC alleles are polymorphic, with size of the resulting PCR fragments ranging between 1.1 and 2.6 kb (Table I⇑). This is in agreement with the high allelic variability between strains. We also found that group 4 pspC alleles were mostly present in strains able to bind C4BP. All ST124 and ST9 isolates of serotype 14 (Fig. 6B) as well as isolates of serotype 4, 6B, and 7F (Table I⇑) with intermediate binding to C4BP harbored a copy of group 4 pspC gene. However, no PCR product could be amplified from seven strains of serotype 14 using either LU9/LU10 primers or other specific primers of pspC4.4 (Table I⇑ and data not shown). Three of these strains, BHN181, BHN183, and 1401, exhibited a weak C4BP binding, which could be explained by the absence of a group 4 pspC allele. Four strains (BHN180, BHN405, BHN406, and BHN407) lacking a group 4 pspC allele were intermediate to high binders, suggesting that either PspC from another group could bind C4BP as well or that these strains express another C4BP ligand. Interestingly, strains of serotype 1, which are weak C4BP binders, contained a shorter variant of group 4 pspC gene (Table I⇑), suggesting that the shorter variant might have lost the C4BP binding site. Taken together, our data show that certain subtypes of PspC may be potential ligands for C4BP.

FIGURE 6.
FIGURE 6.

Distribution of group 4 pspC allele. A, Phylogenetic tree of all the allelic variants of PspC of group 4 PspC4.1, PspC4.2, PspC SP14-BS69, and Psp4.4 using Megalign program (DNAStar Lasergene). B, PCR amplification of pspC locus of serotype 14 isolates using primers LU9/LU10. Mw, m.w. 1-kb DNA ladder (Invitrogen).

To confirm the importance of the expression of PspC4.4 for C4BP binding, we created an additional isogenic pspC deletion mutant of the ST124 isolate BHN84, BHN84ΔpspC. We tested this mutant for C4BP binding and we observed that the deletion of PspC in the BHN84 strain also resulted in a significant reduction of C4BP binding (Fig. 5⇑B), confirming that the PspC4.4 allelic variant expressed by pneumococcal strains of ST124 acts as a specific receptor for C4BP.

Bacteria-bound C4BP down-regulates the activation of the classical pathway

Activation of the classical pathway results in surface deposition of C4b, which in the presence of C4BP and FI is degraded to C4c and C4d. Thus, if the classical pathway is down-regulated by C4BP, high amounts of C4c will be detected. We selected two serotype 14 isolates, BHN79 and BHN182, a strong and a weak C4BP binder, respectively. Bacteria were incubated with 50% NHS and we estimated C4b degradation by measuring C4c by flow cytometry (Fig. 7⇓). Less C4c was detected in association with BHN182 as compared with BHN79 (Fig. 7⇓A). Since we demonstrated that the PspC4.4 allelic variant is a specific receptor for C4BP, we also compared C4b degradation between BHN79 and its isogenic PspC mutant BHN79ΔpspC (Fig. 7⇓B). The absence of PspC4.4 resulted in a reduced C4b degradation as less C4c was detected. Our results show that the ability to bind C4BP affects C4b degradation, which in turn will affect bacterial virulence.

FIGURE 7.
FIGURE 7.

Pneumococcal C4b degradation in normal human serum. Bacteria (107) were incubated with 50% NHS. C4c was detected using an anti-C4c serum IgG. M represents the histogram marker for the positive population; MFI, mean fluorescence intensity. A, Representative flow cytometry histograms of C4c. BHN182, gray line; BHN79, black line. B, Representative flow cytometry histograms of C4c. BHN79ΔpspC, gray line; BHN79, black line.

Discussion

Both the classical and the alternative pathways of the complement are crucial players in the innate immunity response to pneumococci (34). Clinical and experimental studies have shown that patients with deficiencies in various components of the complement system are at higher risk to develop IPD (35). A Swedish study showed that >50% of patients with C2 deficiency had one or more episodes of infection (sepsis, meningitis, and pneumonia) with encapsulated bacteria mainly pneumococci, while 40% of C3-deficient patients had an increased susceptibility to pneumococcal infections (36, 37). Similarly, complement-deficient mice (C1q, C3, C4) developed severe and rapid septicemia after pneumococcal infections (34, 38, 39). Additionally, a new mechanism of classical pathway activation was recently identified whereby the pneumococcal polysaccharide capsule interacts with the C-type lectin receptor SIGN-R1 and C1q, allowing assembly of a C3 convertase without the need for Abs or factor B (40, 41). SIGN-R1-deficient mice are also more susceptible to lethal pneumococcal infections (42). Thus, complement plays a crucial role against pneumococcal infections both at the early stages of colonization and pneumonia as well as at the later bacteremic phase of the infection. Nevertheless, despite these responses, pneumococci can cause severe infections, indicating that it has evolved efficient complement evasion strategies.

Many pathogens have developed common strategies to evade complement activation. One such strategy is the acquisition of fluid-phase complement inhibitor, such as C4BP. Indeed, binding to C4BP renders two common respiratory bacteria, M. catarrhalis and nontypeable H. influenzae, resistant to serum-mediated killing and is responsible for S. pyogenes resistance to phagocytosis. It is likely that S. pneumoniae uses the same strategy to evade complement activation. In the present study, we investigated whether S. pneumoniae could also bind C4BP.

Using a collection of clinical isolates representing common serotypes and clones causing invasive disease, we showed that several pneumococcal isolates bound strongly C4BP. This is in contrast to previous studies where no significant C4BP binding to pneumococci was found (43, 44). Hence, our report is the first to show a binding between C4BP and pneumococci. Interestingly, several strains of serotype 4, 6B, and 7F exhibited intermediate binding to C4BP, but most strains of serotype 14 tested showed a strong binding of C4BP. While the pneumococcus can express up to 91 different capsular polysaccharide types, only ∼20 serotypes are responsible for >80% of IPD worldwide in all age groups (13, 45, 46). The serotype 14 is a common serotype among invasive isolates around the world, especially in young children. It accounted for nearly 30% of the invasive isolates collected from Canadian children between 1991 and 2004 and 25% of the invasive isolates recovered in German children between 1999 and 2003 (47, 48). Similarly, serotype 14 isolates accounted for 10% of the invasive isolates collected from the Stockholm area in 1997, and this serotype has also been shown to spread and expand successfully in Sweden (21). Furthermore, two major clones of serotype 14 as identified by MLST, ST9 and ST124, have been shown to spread around the world causing a major part of invasive diseases in children. We therefore investigated whether C4BP binding correlated specifically with the expression of the serotype 14 capsule and/or with specific clonal properties. Using isogenic serotype 6B, 7F, 14, and 19F capsular variants of TIGR4 (23), we demonstrated that C4BP binding was not dependent on the capsule type but rather on the expression of other virulence determinants. Moreover, the presence of the capsule significantly decreased C4BP binding, indicating that the capsule interferes with the binding as previously seen in binding studies with sIgA or FH (16, 30). Similar observations were done with unencapsulated N. meningitidis strains as compared with encapsulated ones (9). We also observed that while C4BP binding was strong or intermediate for most of serotype 14 strains tested, a strong binding correlated mostly with the clone ST124. This is in agreement with epidemiological studies, which have shown that not only capsular types are important for disease outcome but that also other genetic properties such as specific virulence factors associated with genetically related strains or clonal clusters may play an important role for pathogenicity (21, 49).

Most binding interactions between microbes and C4BP have been found to be either hydrophobic or ionic (5, 6, 7, 8, 9, 10, 50, 51, 52, 53, 54). In contrast to S. pyogenes, we demonstrated that the interaction between the pneumococcal strain BHN79 and C4BP was ionic in nature, as it could be inhibited using increasing concentrations of NaCl. The ionic interaction is presumably mediated by a patch of positively charged amino acids on the interface of CCP1 and CCP2 of the α-chain. This indicates that these two domains of the α-chain are important for C4BP binding to the pneumococcus. Using C4met and heparin, two C4BP ligands, whose binding sites have been mapped to CCP1–3 (1), and deletion mutants of individual CCP, we confirmed the importance of the CCP2–3 region for pneumococcal binding to C4BP. This is similar to most other bacterial pathogens previously investigated. Only M. catarrhalis and H. influenzae bind via CCP7 as well (8, 10). Although, the pneumococcus binds via the same site as C4b, we showed that C4BP retained its inhibitory function and cofactor activity when bound to the bacteria. Since C4BP is a polymer of seven identical α-chains, it can bind simultaneously C4b and bacteria.

The pneumococcus expresses multiple virulence factors, and among them the pneumococcal surface protein C family (also called CbpA, Hic, PbcA, SpsA, and SP2190) has been extensively studied. PspC surface proteins are expressed by 75% of all pneumococcal clinical isolates. Most PspC proteins are choline-binding proteins, although a few variants, such as Hic expressed by serotype 3 strains, are anchored to the cell wall by the typical LPXTG motif (33, 55). Even though there is a high variability at the sequence level and allelic variants of PspC are divided into 11 groups, all PspC proteins have a common organization (33, 55). PspC contributes to virulence in colonization and systemic mouse models (56, 57, 58, 59, 60). PspC is a multifunctional protein that binds to the secretory component of pIgR to promote adherence and invasion of epithelial cells (61). It also binds soluble host factors such as sIgA and IgM as well C3 and FH (62). Pneumococcal binding to C3 and FH help in the evasion of the complement system. Pneumococcal recognition of multiple serum proteins probably influences the efficiency of C4BP binding and could explain the lower efficiency of BHN79 to bind serum C4BP as compared with pC4BP.

We identified a new allelic variant of PspC belonging to the group 4, PspC4.4, and we showed that the loss of expression of PspC4.4 protein led to the loss of C4BP binding. We also found that the presence of pspC alleles of group 4 correlated with the ability to bind C4BP. Interestingly, all serotype 14 strains except four are high C4BP binders and 72% of them harbor a group 4 pspC allele, including ST124 isolates. Furthermore, of the three PspC of group 4 found by sequence database searches, two were from serotype 14 isolates and one of these isolates SP14-BS69 belongs to the clone, ST124. Hence, PspC of group 4 is widely expressed by the clone ST124, which could suggest that the expression of PspC group 4 might give a selective advantage to the serotype 14 strains in general and more specifically to the clone ST124. We also observed a high polymorphism among the group 4 pspC alleles in size. Given the high variability of PspC, it is possible that some variants lack some of the virulence phenotypes associated with this protein, which would explain the variation in degrees of binding by the different isolates. Although high binders, few isolates of serotype 14 did not express a PspC of group 4, suggesting that a second ligand also could promote binding of C4BP on the bacterial surface, for example, a PspC of another group or an unrelated protein.

Several pneumococcal surface proteins have been described to bind to complement inhibitors and to reduce the amounts of C3b deposition on their surfaces (63). For instance, pneumococcal surface protein PspA can inhibit the formation of the C3 convertase (64), and pneumolysin, an intracellular cytotoxin released during autolysis, can reduce the opsonic capacity by depleting C3 binding (15). However, using deletion mutants of pneumolysin, we demonstrated that C4BP binding correlated specifically with the expression of PspC and not of pneumolysin.

Pneumococcal adherence to epithelial cells is dependent on the expression of several adhesins such as PspC and pili encoded by the newly discovered rlrA pathogenicity island (65). Of interest, C4BP binding does not correlate to pilus expression, since none of the serotype 14 strains tested harbors the rlrA operon (B. Henriques-Normark, unpublished observation).

PspC promotes pneumococcal adherence and invasion of epithelial cells by binding to the pIgR, but also the interaction with FH was shown recently to mediate pneumococcal adherence to epithelial cells in vitro and to enhance invasion of mouse lungs in vivo (66, 67). C4BP binding was shown to enhance the adhesion of the yeast Candida albicans to host endothelial cells (68). It is therefore possible that binding to C4BP could promote pneumococcal adhesion as well. C4BP is also a ligand for the endocytic receptor CD91 (also known as the LDL receptor-related protein), and CD91 in association with heparan sulfate proteoglycan contributes to the cellular uptake of C4BP in vitro (69). Hence, it is tempting to speculate that C4BP binding could mediate pneumococcal invasion of host cells. Whether bacteria-bound C4BP could have a dual role in pneumococcal infection as suggested for bacteria-bound FH on mucosal surfaces promoting adherence and invasion and in the bloodstream promoting bacterial survival (66) remains to be investigated.

Our data show that C4BP binding seems to be restricted to certain serotypes and to certain clonal lineages such as ST124 of serotype 14, and this binding property may provide a virulence attribute to ST124 strains that are successfully spreading throughout the world. We are currently collecting a large number of strains isolated from either healthy carriers or patients with pneumonia or IPD from different parts of the world to obtain a global epidemiological overview of the importance of C4BP binding for immune evasion and invasive disease.

In conclusion, we have demonstrated for the first time that S. pneumoniae can recognize and bind complement inhibitor C4BP and identified a new allelic variant of the cell surface-expressed protein and major virulence factor PspC as a ligand for C4BP. This new allelic variant of PspC is expressed mostly by serotype 14 isolates commonly associated with invasive human disease. However, while we have identified a new function for PspC, a second ligand yet unidentified also promotes binding of C4BP on the bacterial surface. The identification of the second potential C4BP ligand is in progress.

Acknowledgments

The authors thank Prof. Mark Lipsitch for providing the isogenic capsular variants, Prof. Don Morrison for providing the competent sequence peptides and for providing advice on transformation techniques, Prof. Timothy J. Mitchell for providing the rabbit polyclonal anti-pneumolysin antiserum, Prof. Elaine Tumonanen for providing the rabbit polyclonal anti-PspC antiserum, and Prof. Björn Dahlbäck for the polyclonal rabbit anti-human C4BP and the murine monoclonal against CCP1 of C4BP (mAb104). We also thank Prof. Staffan Normark for critical reading of the manuscript. Christina Johansson, Gunnel Möllerberg, and Ingrid Andersson are greatly acknowledged for characterization of clinical pneumococcal isolates, and Helen Andersson and Raged El-Ali are acknowledged for technical assistance.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 B.A. was supported by grants from the Swedish Medical Research Council, Crafoord Foundation, the Swedish Society of Medicine, Lars Hiertas Memory Funds, Alfred Österlund Foundation, Längmanska Culture Foundation, and O. E. & Edda Johansson Scientific Foundation. A.M.B. received grants from the Swedish Medical Research Council and the Swedish Foundation for Strategic Research. B.H.N. was financed by the Swedish Medical Research Council, Torsten and Ragnar Söderbergs Foundation, the Swedish Royal Academy of Science, and by the European Union project Pneumococcal Resistance Epidemicity and Virulence—An International Study funded by Directorate General research with the Sixth Framework Program. S.M. was supported by grants from the Academy of Finland, the Sigrid Juselius Foundation, and the Helsinki University Hospital Funds (EVO).

  • ↵2 Address correspondence and reprint requests to Dr. Barbara Albiger, Department of Laboratory Medicine, Medical Microbiology, Malmö University Hospital, Lund University, Malmö, SE-205 02 Sweden. E-mail address: Barbara.Albiger{at}med.lu.se

  • ↵3 Abbreviations used in this paper: MAC, membrane attack complex; C4BP, C4b-binding protein; CCP, complement control protein; SCR, short consensus repeat; NHS, normal human serum; IPD, invasive pneumococcal disease; PspC, pneumococcal surface protein C; MLST, multilocus sequence typing; ST, sequence type; wt, wild type; CSP, competence stimulatory peptide; FH, factor H; FI, factor I; RT, room temperature; GVB, gelatin-veronal buffer; pC4BP, plasma-purified C4BP; sIgA, secretory IgA; PAF-R, platelet-activating factor receptor; pIgR, polymeric IgG receptor.

  • Received July 22, 2008.
  • Accepted April 10, 2009.

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