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Mediates Adhesion to Host Epithelial Cells andStreptococcus pneumoniaeProtein of

PspCInteracts via Two Contact Sites with the The Host Immune Regulator Factor H

Steffi Haelbich, Christine Skerka and Peter F. ZipfelSven Hammerschmidt, Vaibhav Agarwal, Anja Kunert,

http://www.jimmunol.org/content/178/9/5848doi: 10.4049/jimmunol.178.9.5848

2007; 178:5848-5858; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2007 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Host Immune Regulator Factor H Interacts via TwoContact Sites with the PspC Protein of Streptococcuspneumoniae and Mediates Adhesion to Host Epithelial Cells1

Sven Hammerschmidt,2* Vaibhav Agarwal,* Anja Kunert,† Steffi Haelbich,† Christine Skerka,†

and Peter F. Zipfel†‡

Pneumococcal surface protein C (PspC) of Streptococcus pneumoniae is a key virulence factor that mediates adhesion to hostcells and immune evasion of the host complement. PspC binds the host immune and complement regulator factor H, whichis composed of 20 short consensus repeats (SCR). This interaction contributes to pneumococcal virulence. In this study, weidentified within the factor H protein two separate PspC binding regions, which were localized to SCR8 –11 and SCR19 –20,by using recombinant factor H deletion constructs for Western blotting assays and surface plasmon resonance studies. Adetailed analysis of binding epitopes in these SCR by peptide spot arrays identified several linear binding regions within thesequences of SCR8 –11 and SCR19 –20. In addition, the factor H binding site was mapped within the pneumococcal PspCprotein to a 121-aa-long stretch positioned in the N terminus (residues 38 –158). Factor H attached to the surface of pneu-mococci via PspC significantly enhanced pneumococcal adherence to host epithelial and endothelial cells. This adhesion wasspecific and was blocked with a truncated N-terminal factor H-binding fragment of PspC. In conclusion, the acquisition offactor H by pneumococci via PspC occurs via two contact sites located in SCR8 –11 and SCR19 –20, and factor H attachedto the surface of the pneumococcus promotes adhesion to both host epithelial and endothelial cells. The Journal of Immu-nology, 2007, 178: 5848 –5858.

S treptococcus pneumoniae is a natural resident of the naso-pharyngeal cavity. In addition to colonization, pneumo-cocci cause severe local infections including otitis medium,

sinusitis, and life-threatening invasive diseases such as lobar pneu-monia, sepsis, and meningitis (1–4). The bacterium uses severalstrategies for colonization of the respiratory tract, transcytosisthrough host cells, and transmigration of the blood-brain-barrier.Several virulence factors of S. pneumoniae have been identifiedthat are involved in the progression of pneumococcal diseases (5–7). Pneumococcal surface protein C (PspC3; also named CbpA orSpsA) is a major pneumococcal virulence factor. To date, 11 dif-ferent subtypes of PspC proteins have been identified and, based

on their different anchorages in the bacterial cell wall, divided intotwo subgroups (8).

The classical PspC proteins (subtypes 1–6) are choline-bindingproteins and constitute subgroup 1. The C-terminal choline-bindingdomain attaches the classical PspC proteins noncovalently to a cellwall via an interaction with the phosphorylcholine of lipoteichoic andteichoic acids. Members of the second subgroup, representing atypicalor PspC-like proteins (subtypes 7–11) such as Hic (PspC11.4), areanchored in a sortase-dependent manner to the peptidoglycan of thecell wall by an LPXTG motif. The N-terminal regions of the firstPspC subgroup show a common structure and organization. All pro-teins have a leader peptide and an N-terminal domain that is followedby either one or two single repeated domains (termed R1 and R2) anda proline-rich sequence (8–11).

PspC mediates pneumococcal adherence by binding the extracel-lular Ig-like domain, also known as the secretory component (SC), ofthe polymeric Ig receptor (pIgR) (12, 13). The specific binding to thehuman Ig-like ectodomains D3 and D4 of the SC occurs throughhexapeptide motifs that are located in the direct repeats R1 and R2 ofPspC (11, 13–15). One R domain is sufficient for binding to the SC ofpIgR, to a free SC, or to a SC as part of secretory IgA (10, 11, 15).

In addition to its role as an adhesin, PspC also mediates immuneevasion by binding the host complement and the innate immune reg-ulators, factor H and C3 (16, 17). Apparently PspC uses two differentepitopes for binding the soluble host proteins factor H and SC (18).The factor H binding residues of the subgroup II PspC11.4 protein(Hic) were mapped to residues 29–269 (19). Interestingly, a region(38–149) of Hic shows considerable sequence homology with theN-terminal sequences of the subgroup I PspC proteins (8, 19).

Factor H is a fluid phase regulator of the alternative complementpathway and consists of 20 domains that are termed short consensusrepeats (SCR), each consisting of �60 aa. This plasma glycopro-tein is the key fluid phase regulator of the alternative complement

*University of Wuerzburg, Research Center for Infectious Diseases, Wuerzburg, Ger-many; †Department of Infection Biology, Leibniz Institute for Natural Product Re-search and Infection Biology, Hans Knoell Institute, Jena, Germany; and ‡FriedrichSchiller University, Jena, Germany

Received for publication November 13, 2006. Accepted for publication February20, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by the Deutsche Forschungsgemeinschaft (Sonder-forschungsbereich 479, Teilprojekt A7; to S.H.) and the Bundesministerium furBildung und Forschung (CAPNETZ C8; to S.H.). V.A. is a recipient of a GermanAcademic Exchange Service (DAAD) fellowship.2 Address correspondence and reprint requests to Dr. Sven Hammerschmidt at thecurrent address: Max von Pettenkofer Institute for Hygiene and Microbiology,Ludwig Maxmilians University Munich, Pettenkoferstrasse 9a, D-80336 Munich,Germany. E-mail address: hammerschmidt@mvp.uni.muenchen.de3 Abbreviations used in this paper: PspC, pneumococcal surface protein C; CPS,capsular polysaccharide; FHL-1, factor H-like protein 1; GMFI, geometric mean flu-orescence intensity; HBMEC, human brain-derived microvascular endothelial cell;pIgR, polymeric IgR; PVDF, polyvinylidene fluoride; SC, secretory component; SCR,short consensus repeat.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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pathway and acts as a cofactor in the factor I-mediated proteolysisof C3b. Proteolytic cleavage of C3b results in the formation of theinactive iC3b fragment, which remains covalently linked to thesurface (20).

The binding of factor H to the surface of pathogens has beenobserved for other streptococci such as Streptococcus agalactiaeand Streptococcus pyogenes and several other pathogenic mi-crobes including Borrelia burgdorferi, Candida albicans, Neisse-ria gonorrhoeae, and Neisseria meningitidis (21–28). Host-de-rived factor H attached to the surface of a pathogen inhibitscomplement activation and thus prevents complement-mediatedkilling. The lack of PspC in the pneumococci of serotype 2 strainD39 and the lack of Hic in serotype 3 strain A66 attenuated pneu-mococcal virulence during sepsis. Apparently this defect results indecreased uptake by polymorphonuclear leukocytes (29, 30). Inaddition, pneumococci deficient in PspC, but not wild-type pneu-mococci, are efficiently killed by microglia cells, which representthe resident phagocytes in the brain (31). Thus, PspC expressionseems to be important for colonization and for pneumococcal sur-vival in an immune-competent host (6).

Recently, it was shown that factor H binds to the cell surfaces ofhost cells via polyanionic cell surfaces such as proteoglycans,sialic acids, heparan sulfate chains, or glycosaminoglycans (32–34). Moreover, factor H and its splicing variant factor H-like pro-tein 1 (FHL-1), which consists of the first seven SCR, bind via anRGD sequence of SCR4 to host cells. Factor H binding interfereswith fibronectin binding, suggesting that both molecules use iden-tical cellular receptors (35). Similarly, human polymorphonuclearleukocytes bind to immobilized factor H via integrin CD11b/CD18, i.e., CR3 (36). In contrast, binding to human endothelialcells is mediated via the heparin/glycosaminoglycan-binding sitewithin SCR20 of factor H (37).

In part, inconsistent data were reported for the interaction ofPspC and Hic with factor H. The pneumococcal protein Hic, whichis preferentially produced by serotype 3 strains, binds to SCR8–11and SCR12–14 of factor H. In contrast, for the PspC of serotype 2strain D39, binding to SCR6–10 and SCR13–15 of factor H hasbeen reported (16, 38). The goal of this study was to evaluate theinteraction of complement regulator factor H with the bacterialPspC protein by localizing the binding sites within the host proteinas well as within the bacterial protein. In addition, the impact ofbacterial cell surface-bound factor H on pneumococcal adhesion tohost cells was demonstrated for the first time.

Materials and MethodsBacterial strains, culture conditions, and PspC proteinpurification

S. pneumoniae was cultured on blood agar plates (Oxoid) at 37°C and 5%CO2 or in Todd-Hewitt broth (Roth) supplemented with 0.5% yeast extractto a density of 5 � 108 CFU ml�1 (OD600 of �0.5). The wild-type pneu-

mococcal strains and isogenic mutants that do not produce PspC or strainsthat are deficient for the capsular polysaccharide (CPS) are listed in TableI. The PspC protein has a modular organization and is divided into differentsubtypes; therefore, the PspC nomenclature is also included in Table I.Isogenic mutants that do not express PspC were constructed for the non-encapsulated R6x (PspC3.1) and S. pneumoniae NCTC 10319 (PspC3.3),which is a low encapsulated strain and perfectly suitable for cell cultureinfection experiments as described earlier (13, 39). PspC-deficient mutantsof R6x and NCTC 10319 were generated by replacement of the pspCsequence with the erythromycin gene cassette. Briefly, the full-length pspCgene was amplified by PCR from the chromosomal DNA of S. pneumoniaeNCTC 10319 with the primers 5�-GGATCCTTGTTTGCATCAAAAAGCGAAAG-3� and 5�-AAGCTTGTTTAGTTTACCCATTCACCATTGGC-3�, which incorporated flanking BamHI and HindIII (underlined) restrictionsites. The amplified DNA was cloned into similarly digested pQE30 (Qia-gen). A SpeI digest of the inserted pspC fragment (nt 550-1080) was de-leted and the erythromycin gene cassette was blunt-end ligated with theplasmid. The integrity of the antibiotic gene cassette was verified by se-quence analysis using ABI Prism dye terminator cycle sequencing (Ap-plied Biosystems). Erythromycin (5 �g ml�1) was added to the growthmedium for the mutants. The transformation of pneumococci was per-formed as described previously (15).

Escherichia coli BL21 (DE3) (Stratagene), cultivated at 30°C on Luria-Bertani agar or broth containing 100 �g ml�1 of ampicillin, was used as thehost strain for expression of a His6-tagged fusion protein. The His6-taggedfusion proteins were purified by Ni2� affinity chromatography with theProtino Ni prepacked column kit according to the manufacturer’s instruc-tions (Macherey-Nagel). The His6-tagged PspC proteins used in this studyhave been described earlier (13, 15). Briefly, the recombinant His6-taggedPspC proteins represent PspC group 2.1, which is expressed by strainATCC 33400 (serotype 1), or PspC group 3.3, which is expressed by strainNCTC 10319 (see Fig. 3A).

Factor H, Abs, and human sera

Human factor H and the polyclonal anti-factor H Ab were purchased fromCalbiochem. Recombinant factor H deletion mutants representing SCR1–7,SCR8–20, SCR8–11, SCR11–15, and SCR15–20 were expressed in thebaculovirus expression system as described (40, 41). Purification of anti-PspC IgG, which was generated by immunization of a rabbit with PspC2.1(15), and polyclonal anti-pneumococcal IgG (13) was performed by proteinA-Sepharose 4B affinity chromatography. The inhibitory role was assayedfor the mAbs L20, E14, and C18, whose binding sites in the factor Hprotein have been mapped to SCR19 (L20) and SCR20 (E14 and C18) (37,42). Nonimmune human sera or plasma proteins were obtained fromhealthy individuals upon informed consent.

SDS-PAGE and binding of 125I-radiolabeled factor H

Whole cell lysates of pneumococci or His6-tagged PspC derivatives weresubjected to SDS-PAGE and transferred to a polyvinylidene fluoride(PVDF) membrane (Immobilon-P; Millipore) using a semidry blotting sys-tem (Bio-Rad). Factor H was radiolabeled with 125I by a standard chlora-mine-T method and binding assays with pneumococci were performed asdescribed (10) and quantitated in a counter (1600 TR; Packard Instrument).

For blot overlay assays with 125I-radiolabeled factor H, whole proteinlysates of pneumococci were separated by SDS-PAGE and transferred to aPVDF membrane. After blocking with 10% skim milk (Oxoid), the mem-brane was washed and incubated with 125I-radiolabeled factor H (300,000cpm ml�1) in 5 ml of PBS-Tween 20 (0.05%) for 4 h at room temperature.After extensive washing, bound factor H was detected by autoradiography.

Table I. Streptococcus pneumoniae strains used in this study

Strain Capsule Type Resistance PspC Subtype Reference

NCTC10319 35A PspC3.3 10NCTC10319�pspC 35A Erythromycin �pspC This studyR6x Nonencapsulated PspC3.1 55R6x�pspC Nonencapsulated Erythromycin �pspC This studyR800 Nonencapsulated ND 45ATCC11733 2 PspC3.2 45TIGR4 4 PspC3.4 8TIGR4�cps Nonencapsulated Kanamycin PspC3.4 56A66 3 PspC11.4 (Hic) 8, 45A66�cps Nonencapsulated PspC11.4 (Hic) 45

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Immunoblot and Western blot analysis

Identical amounts of His6-tagged PspC deletion mutants were spotted on aPVDF membrane using a Bio-Dot SF microfiltration apparatus (Bio-Rad).Immobilization of the proteins was confirmed by immunodetection usingpurified anti-PspC IgG together with a HRP-conjugated rabbit Ab. Thebinding of factor H (2 �g ml�1) to the various PspC deletion mutants wasvisualized using factor H antiserum (diluted 1/800; Calbiochem) in com-bination with a HRP-conjugated goat antiserum and detected by ECLchemiluminescence (GE Health Care).

PepSpot analysis

A library of 115 peptides representing SCR8–11 and a library of 64 pep-tides representing SCR19–20 of factor H were synthesized and spotted ona cellulose membrane (Intavis MultiPep System). Each peptide was 12aa in length and differed from the next peptide in two amino acid res-idues; thus the peptides have an overlap of 10 residues. Membraneswere incubated with recombinant PspC protein (PspC SH2; 10 �gml�1) as described (13, 37) and bound PspC protein was detected usingpolyclonal PspC IgG in combination with HRP-conjugated rabbit IgGand ECL.

Surface plasmon resonance assays

Protein-protein interactions were analyzed by a surface plasmon resonancetechnique using a Biacore 3000 instrument as described (13, 37). Briefly,factor H, factor H SCR8–20, and the recombinant PspC derivative SH2were dialyzed against 10 mM sodium acetate buffer (pH 4.0). All proteinswere coupled via a standard amine-coupling procedure to the flow cells ofa sensor chip (CM5, Biacore). Control flow cells were prepared in the sameway but without injecting the protein. The analytes including PspC pro-teins, factor H, or the recombinant truncated factor H derivatives SCR1–7,SCR8–20, SCR8–11, SCR11–15, and SCR15–20 were dialyzed againstrunning buffer (i.e., PBS (pH 7.4)). Binding of the analytes was analyzedafter separate injection into the flow cells coupled with the binding partnersand into a control cell using a flow rate of 10 �l ml�1 at 25°C in allexperiments. Each interaction was measured at least three times.

Flow cytometric analysis of factor H binding to pneumococci

Binding of factor H to viable pneumococci in competitive inhibition ex-periments was tested using flow cytometry. Bacteria were cultured inTodd-Hewitt broth supplemented with 0.5% yeast extract, and 5 � 107

bacteria in 100 �l of PBS were incubated in the absence or presence ofPspC proteins that were used as competitors. The suspensions were incu-bated for 30 min at 37°C and thereafter bacteria were washed three times.The binding of factor H to pneumococci was detected after incubation withthe factor H antiserum for 30 min at 37°C followed by FITC-conjugatedanti-goat Ig Ab (MoBiTec). Bacteria were washed and fluorescence wasanalyzed by flow cytometry using a FACSCalibur apparatus (BD Bio-sciences). The pneumococci were detected using log forward and log sidescatter dot plot, and a gating region was set to exclude debris and largeraggregates of bacteria. Ten thousand bacteria were analyzed for fluores-cence using log scale amplification. The geometric mean fluorescence in-tensity (GMFI) � percentage of labeled bacteria was recorded as a measurefor binding activity.

Cell lines and culture conditions

The cultivation of cell lines was performed as described (39, 43). Briefly,human A549 cells (lung alveolar epithelial cells type II pneumocytes;ATCC catalog no. CCL-185) were cultivated in DMEM supplemented with10% FCS, 5 mM glutamine, 100 U/ml penicillin, and streptomycin (allfrom PAA Laboratories) at 37°C in 5% CO2. Detroit 562, human naso-pharyngeal epithelial cells (ATCC catalog no. CCL 138) that express thepolymeric Ig receptor, were cultivated in RPMI 1640 (PAA Laboratories)supplemented with 10% FCS, 2 mM glutamine, and 1 mM sodium pyru-vate. Human brain-derived microvascular endothelial cells (HBMEC) werecultured in RPMI 1640 based medium with 10% FCS, 10% Nu-Serum IV(BD Biosciences), 1% nonessential amino acids, 1% MEM vitamins, 1 mMsodium pyruvate, 2 mM glutamine, 100 U/ml penicillin, and 0.1 mg/mlstreptomycin.

Pneumococcal adhesion assay

Epithelial cells and HBMEC were seeded at a density of 5 � 104 in plainmedium on either 24-well tissue culture plates (Greiner) and cultivated for48 h or placed on glass cover slips (12-mm diameter) when assayed byimmunofluorescence. Confluent monolayers were washed thoroughly andinfected with pneumococci for 3 h in 500 �l of DMEM-HEPES (PAA

Laboratories) supplemented with 1% FCS at 37°C using a multiplicity ofinfection of 40. The role of factor H for adherence was analyzed by incu-bating pneumococci for 10 min with factor H in a total volume of 100 �lDMEM-HEPES at 37°C before infection and the infection assay was con-ducted in a total volume of 500 �l after adding the preincubated bacteria.After infection, cells were washed three times with PBS to remove un-bound bacteria. Adherent bacteria were visualized following the fixation ofcells with 3.7% paraformaldehyde by immunofluorescence or by platingthe bacteria on blood agar after the detachment and lysis of cells with

FIGURE 1. Binding of factor H to pneumococci. A, Immunoblot anal-ysis of the binding of factor H to S. pneumoniae serotype 35A(NCTC10319), which were incubated in human serum and plasma, respec-tively. The samples were separated by SDS-PAGE, transferred to a PVDFmembrane, and analyzed with factor H antiserum. Lane 1, Plasma as con-trol (Ctrl); lanes 2 and 6, whole cell lysate of pneumococci (W) incubatedin PBS; lane 3, proteins eluted from the pneumococcal cell surface (E) bytreatment with 2 M NaCl after incubation in human plasma; lanes 4 and 8,whole cell lysate (W) after incubation of pneumococci with human plasmaor serum; lane 5, serum as control (Ctrl); lane 7, proteins eluted (E) fromthe pneumococcal cell surface after incubation in human serum; M, proteinmarker (New England Biolabs). B, Binding of 125I-radiolabeled factor H towild-type (WT) NCTC10319 and isogenic �pspA mutant. Bacterial lysateswere separated by SDS-PAGE, transferred to a PVDF membrane, and usedfor an overlay assay with 125I-labeled factor H. C, Binding of soluble125I-radiolabeled factor H to viable pneumococcal wild-type (WT) strainsNCTC10319 (Cps�) and R6x (Cps�) and their isogenic �pspC mutants.Both wild-type strains bound strongly factor H and binding was reduced inthe mutants that are deficient in PspC. D, Recruitment of factor H to en-capsulated and nonencapsulated pneumococci producing different PspCsubtypes. Binding of factor H (2 �g) was determined by flow cytometryand results were expressed as GMFI � percentage of FITC-labeled andgated bacteria. Representative data from independent experiments areshown.

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saponin (1%; w/v). The number of viable intracellular bacteria was quan-titated by an antibiotic assay as described (43).

Determination of adherent pneumococci by immunofluorescence

Pneumococci attached to the cells were stained with a polyclonal pneu-mococcal antiserum in combination with a secondary goat anti-rabbit IgGcoupled with Alexa Fluor 488 (green) or Alexa Fluor 568 (red) (MoBiTec)(43). After blocking nonspecific binding sites with 10% FCS, cells werethoroughly washed with PBS and then incubated for 30 min with the pneu-mococcal antiserum (1/100). Bound Abs were detected with an Alexa Fluor488-labeled goat anti-rabbit Ig conjugate (MoBiTec). The glass cover slipswere embedded “upside down” in Moviol, sealed with nail polish, andstored at 4°C. At least 50 cells were counted using a fluorescence micro-scope (Zeiss Axioskop) and a confocal laser scanning microscope and soft-ware (Zeiss) were used for image acquisition. Each experiment was re-peated at least three times and results were expressed as mean � SD.

Statistical analysis

Adherence data were expressed as the mean � SD. Differences in adher-ence were analyzed by the two-tailed unpaired Student’s t test. In all an-alyzes, p � 0.05 was considered statistically significant.

ResultsBinding of factor H to pneumococcal PspC protein

PspC3.3 expressing pneumococci (NCTC10319, serotype 35A)were analyzed for factor H binding. Pneumococci were incubatedeither in human plasma or serum. After incubation in human

plasma, the elute fraction and the extract prepared from plasma-treated pneumococci were separated by SDS-PAGE, and analyzedby Western blotting using a factor H antiserum. Factor H wasdetected in samples of eluted proteins and whole cell lysates (Fig.1A, lanes 3 and 4), thus demonstrating that factor H derived fromhuman plasma binds to the surface of pneumococci. The sameresults were observed when pneumococci were incubated in hu-man serum (Fig. 1A, lanes 7 and 8). FHL-1, which is also presentin serum, did not bind to pneumococci (data not shown).

Similarly, the binding of radiolabeled factor H was assayed.125I-radiolabeled factor H bound to the wild-type pneumococci ofstrain NCTC10319 but not to the pneumococci of the isogenicpspC knockout strain (Fig. 1B). In addition 125I-radiolabeled factorH bound to the wild-type strain but not to the isogenic pspC mutantstrains representing the encapsulated strain NCTC10319 or thenonencapsulated R6x. (Fig. 1C). These results indicate that PspCis the major and most likely the only surface protein of pneumo-cocci that binds the host regulator factor H.

PspC is a highly variable surface protein with a modular orga-nization (8, 10). To assess whether PspC variability affects thebinding of factor H, pneumococci producing different PspC sub-types, including the serotype 3 strain A66 that expresses Hic(PspC11.4), were used in binding experiments. As demonstratedby flow cytometric analysis (Fig. 1D), all of the pneumococcal

FIGURE 2. Binding of factor H to PspC proteinanalyzed by surface plasmon resonance. Binding offactor H to the PspC protein SH2 (PspC2.1) was an-alyzed by surface plasmon resonance. PspC deriva-tive SH2 was coated on the surface of the sensor chipand human factor H was applied as an analyte withthe indicated concentrations. Sensorgrams show con-centration-dependent rates of factor H binding toPspC. Co, Injection of PBS.

FIGURE 3. Schematic models of PspC deletionconstructs and factor H. A, Structures of PspC2.1 andPspC3.3 deletion constructs. The binding regionidentified for factor H derivatives SCR8 –20 andSCR8 –11 is shown in black and the hexapeptidebinding epitope (Y/R)RNYPT of the SC is indicatedin the R domains of PspC variants. PspC2.1 is thePspC protein produced by S. pneumoniae ATCC33400 (serotype 1) and PspC3.3 is produced by S.pneumoniae NCTC10319 (serotype 35A). The latteris identical to the PspC of pneumococcal strains D39and R6x which have been used in previous studies toinvestigate the PspC-factor H interaction (16). LP,Leader peptide; CBD, choline-binding domain; P,proline-rich sequence; R, R domain. B, Factor H andfactor H deletion constructs SCR8 –20 and SCR8 –11.

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strains analyzed recruited factor H to the bacterial cell surface andbinding was independent of the PspC subtype. Similarly as in bind-ing studies with secretory IgA and SC (10), factor H binding ef-ficiency increased significantly when nonencapsulated pneumo-coccal strains were used (Fig. 1D), indicating that the CPSinterferes with factor H binding.

Binding of factor H to PspC analyzed by surface plasmonresonance

Surface plasmon resonance was used to characterize the binding offactor H to the SH2 domain of PspC2.1. Factor H binding to theimmobilized PspC was dose dependent and increased with increas-ing concentrations of factor H (Fig. 2). The PspC derivativeSH2198–203, in which the critical amino acids for the interactionwith the secretory component were exchanged (Fig. 3A), showedcomparable binding (data not shown). Thus, factor H-PspC com-plex formation occurs independently of the SC-binding epitope ofthe PspC protein.

Mapping of the factor H-binding domain in PspC

To map the binding site within the bacterial PspC protein, deletionmutants of PspC2.1 and one of PspC3.3 were used (Fig. 3A). Thefactor H deletion mutants used in this study are shown in Fig. 3B.PspC2.1 and PspC3.3 derivatives were immobilized and the bindingof factor H SCR8–11 or SCR8–20 was assayed. Both factor H mu-tants did bind to the PspC2.1 derivatives SH2, SH2198–203, and SH3and to the PspC3.3 fragment SH12. No binding was detected toPspC2.1 deletion mutants SM1, SM2, SM5, SM6, and SM7 (Fig. 4A).These results indicate that the factor H binding site of PspC is locatedwithin the N terminus, i.e., amino acid residues 38–158.

Because the factor H SCR8–20 mutant did not bind to N-terminalPspC mutants that contain only the R domain, a truncated R domain,or a C-terminal extended R domain (Figs. 3A and 4A), it is concludedthat the SC-binding epitope within the R domain(s) of PspC2.1 aredispensable for factor H binding. These results are consistent with aprevious study showing that factor H binding is independent of the SC

FIGURE 4. Mapping of the factor H binding site(s)in the pneumococcal PspC proteins by ligand affinityassays. A, Various PspC2.1 and PspC3.3 deletion con-structs (compare lower right panel) were immobilizedonto a PVDF membrane using a Bio-Dot SF microfil-tration apparatus (Bio-Rad) and probed with the factorH deletion mutant SCR8–20 (upper left panel), factor HSCR8–11 (upper right panel), or anti-PspC antiserum(lower left panel). Binding of both factor H SCR8–11and factor H SCR8–20 was demonstrated to PspC de-rivatives SH12 (PspC3.3), SH2, SH2198–203, and SH3(all PspC2.1). PspC derivatives containing the func-tional SC-binding domain (R domain) or C-terminallyextended R domains (SM1, SM2, SM5, or SM6) do notbind. B, Binding of the indicated PspC2.1 derivatives toimmobilized factor H as analyzed by surface plasmonresonance. C, Binding of PspC2.1 fragments to factor HSCR8–20. PspC derivatives were used as analytes andbinding is shown for all PspC derivatives containing theaa 38–158 (SH3, SH2, SH2198–203) but not for SM1,which starts at aa 158 of PspC2.1. Co, Injection of PBS.

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binding motif YRNYPT and that factor H binding does not competewith binding of SC (18). These results were confirmed by using acomplementary approach. In surface plasmon resonance studiesthe PspC derivatives SH12, SH2, SH2198 –203, SH3, and SM1were used as analytes to measure the binding to immobilizedfactor H and factor H SCR8 –20. Similarly as in the ligand blotoverlay assays, the binding to factor H or SCR8 –20 was ob-served with PspC proteins that contained the 121-aa-long (po-sitions 38 –158) N-terminal domain (Fig. 4, B and C).

Factor H has two contact sites for pneumococcal PspC

Given that the factor H SCR8–11 deletion mutant binds to PspC,we were interested in identifying the essential and minimaldomains of factor H that are required for this interaction. First,surface plasmon resonance was used to verify and compare thebinding of the factor H deletion mutants SCR1–7, SCR8–11,SCR11–15, SCR8–20, and SCR15–20 to immobilized PspC. Thefactor H mutant SCR8–11 bound to PspC2.1 as indicated by the

FIGURE 5. Surface plasmon mea-surements of the PspC interactionwith the factor H mutant SCR8–20.PspC2.1 derivative SH2 was immobi-lized to the surface of the CM5 sensorchip. A, Binding of factor H and theindicated factor H deletion mutants,which were each used at a concentra-tion of 50 �g ml�1, were analyzed. B,Binding of factor H SCR8–20 wasanalyzed in the absence and in thepresence of a mAb C18, which bindsto a linear epitope within SCR20 (42).Binding of the factor H deletion con-struct SCR8–20 was significantly de-creased in the presence of the mAbC18. C, Heparin inhibited the bindingof factor H to PspC SH2 in a dose-dependent manner. Co, Injection ofonly PBS.

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association and dissociation profile. Factor H construct SCR8–20showed a more pronounced binding and construct SCR15–20showed a rather low level of binding (Fig. 5A). In contrast,SCR1–7 and SCR11–15 showed no binding (Fig. 5A). Theseresults are indicative of a second PspC-binding domain in factor H.To confirm the existence of a second binding site within the Cterminus of factor H, the binding of factor H SCR8–20 to PspCwas analyzed in the presence of three domain-mapped mAbs thatbind to the C-terminal SCR19 (mAb L20) or to overlapping do-mains within the SCR20 of factor H (mAbs E14 and C18). The twomAbs that bind to SCR20, but not mAb L20, blocked the interac-tion with SCR19–20. In the presence of the two mAbs E14 andC18, which bind to SCR20, the binding of the SCR8–20 constructwas reduced, whereas the mAb L20 had no effect on binding (Fig.5B and data not shown). This effect of the blocking Abs is inagreement with presence of a second binding site in the C terminusof factor H. Factor H contains heparin binding sites in the SCR9and the SCR20 of factor H (44). Heparin blocked the binding ofSCR8–20 to PspC in a dose-dependent manner (Fig. 5C). Appar-ently the SCR20 binding region acts in concert with the bindingdomain in SCR8–11. These data suggest that factor H uses twobinding domains for interaction with PspC2.1 that are locatedwithin SCR8–11 and SCR19–20.

Mapping of the linear PspC binding sites within SCR8–11 andSCR19–20 of factor H

Peptide spot analyses were used to identify linear PspC-bindingsequences within two factor H regions. Peptides with a length of12 aa spanning the region of the SCR8–11 and the SCR19–20 offactor H were generated and probed with the recombinant SH2fragment of PspC2.1. Several linear binding regions were identi-fied (Fig. 6). PspC binding was indicated to linear sequences inSCR9, i.e., to a 20-aa-long stretch (510–529) that included threepositively charged residues, a 28-aa-long stretch (582–609) withinSCR10 that included five charged residues, and a linear stretch of24 residues (656–679) with two positively charged residues withinSCR11 (Fig. 6A). In addition, two linear binding regions wereidentified in SCR20, i.e., 18 residues (1177–1194) with three pos-

itively charged residues and a 22-aa-long stretch (1195–1216) withfive positively charged residues (Fig. 6B). The peptide spot anal-yses identified two contact sites and minimal PspC-bindingepitopes in SCR8–11 and SCR19–20 of factor H. The results in-dicate a critical role for the two regions in the factor H protein inbinding to PspC.

Factor H facilitates adhesion of pneumococci to host cells

Factor H mediates complement control at the surface of pneumo-cocci. To identify additional functions for the attached host regu-lator, we assayed the role of factor H in the adhesion of pneumo-cocci to human cells. The adhesion of pneumococci (NCTC10319)preincubated with factor H to human epithelial cells was studied.Factor H increased the attachment of pneumococci to host cells ina dose-dependent manner. Apparently factor H mediated adher-ence is a general mechanism, as this effect was observed for sev-eral human cell lines including epithelial and endothelial cells (Fig.7, A and B). These data further confirm that factor H and the SC ofpIgR do not share the binding sites in the bacterial protein PspC,because the factor H effect was also demonstrated for Detroit 562cells, which produce the pIgR.

In addition, the internalization of S. pneumoniae (NCTC10319)to human cells was quantitated. The attached bacteria were killedby antibiotics and internalized bacteria were recovered. Factor Htreatment of the bacteria significantly increased the number of in-ternalized bacteria (Fig. 7C). However, the increase in uptake waslower as compared with the increase in adherence. These resultssuggest that bacteria-bound factor H plays a pivotal role in adhe-sion and influences internalization.

The CPS of pneumococci interferes with bacterial adherence tohost cells (45). To elucidate whether the CPS also affects the factorH-mediated adherence of pneumococci to host cells, the adherenceof the wild-type TIGR4 was compared with that of the CPS-defi-cient mutant TIGR4�cps. As observed earlier for other pneumo-coccal strains (45), removal of the CPS, in this case TIGR4�cps,significantly increased the number of host cell-attached bacteria as

FIGURE 6. Localization of the linear PspC bindingregions in factor H SCR8–11 and SCR19–20.SCR8–11 (A) and SCR19–20 (B) were divided into12-aa peptides with a 10-aa transition with the next pep-tide. The spot membrane of SCR8–11, which comprisesaa 446–685 of factor H, contains 115 synthetic pep-tides. SCR19–20 was divided into 64 peptides and com-prises aa 1104–1231 of factor H. Membranes were in-cubated with PspC derivative SH2 (PspC2.1) andbinding was detected with an anti-PspC antiserum.

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compared with the encapsulated wild-type TIGR4 (Fig. 7D). Sim-ilar to our results with NCTC10319, pretreatment of the nonen-capsulated TIGR4�cps with factor H increased adherence signif-icantly (Fig. 7D). In contrast, pretreatment of the wild-type TIGR4with factor H was less efficient. Adherence to host cells was onlyslightly enhanced and the absolute values of host cell-bound pneu-mococci remained relatively low (Fig. 7D). These data demon-strate that the factor H-binding protein PspC is at least partiallyconcealed below the CPS. In conclusion, the cell culture infectionassays demonstrated a significant role of bacteria-bound factor Hin pneumococcal adherence independent of the cell type.

Blocking of factor H-mediated pneumococcal adherence

To confirm a role of surface-attached factor H in pneumococcaladhesion via binding to the very N-terminal part of PspC, blockingexperiments were performed. First, the binding of factor H topneumococci was measured by flow cytometry in the presence ofthe SH3 domain of PspC or PspC SM1. Flow cytometric analysisindicated a dose-dependent binding of factor H to pneumococci(data not shown) and a competitive inhibition of factor H bindingto pneumococci by PspC protein SH3, which contains the factorH-binding epitope (Fig. 8A). In contrast, the PspC derivative SM1,which represents the SC-binding R domain of PspC and lacks thefactor H binding region, showed no inhibitory effect (Fig. 8A).

In cell culture infections the PspC derivative SH3 was assessedfor its ability to inhibit factor H-mediated adherence of pneumo-cocci. Host cells were infected with pneumococci that had beenpretreated with a mixture of factor H and the PspC derivative SH3.The results showed that the factor H-binding domain SH3 of PspCinhibited factor H-mediated pneumococcal adhesion to host cells(Fig. 8, B and C). In conclusion, inhibition experiments confirmedthe specific interaction of amino acid residues 38–158 of PspC forfactor H-mediated pneumococcal adherence, which is mediated bythe interaction of PspC with the SCR8–11 and the SCR19–20 offactor H.

DiscussionIn this study we characterize the interaction of the host comple-ment inhibitor factor H and the microbial PspC surface protein anddemonstrate that this interaction is relevant for the adherence ofthe human pathogenic S. pneumoniae to host cells. The microbialPspC protein has one major binding site for factor H. The bindingsite was localized to a 121-aa-long stretch in the N-terminal regionof PspC comprising amino aa 38–158 of PspC, which is orientatedto the outside of the pathogen. The host regulator factor H interactswith the pneumococcal PspC protein via two regions that werelocalized to SCR8–11 and SCR19–20. In our peptide spot arrays,PspC epitopes were mapped to three linear binding regions withinSCR8–11 and to two linear binding regions within SCR19–20.The recruited factor H controls complement activation at the bac-terial surface and we demonstrate that pneumococci exploit sur-face-attached host regulators for adhesion to host cells. In conclu-sion, the PspC interacts with factor H via two separate contactssites in the host factor H and this interaction is essential for im-mune evasion and host cell attachment of S. pneumoniae.

The PspC protein is a multifunctional bacterial protein of S.pneumoniae and binds at least the SC of pIgR and the immune

FIGURE 7. Pneumococcal surface-bound factor H mediates bacterialadhesion to host cells. A, Attachment of pneumococcal strain NCTC10319(Cps�, serotype 35) was counted by immunofluorescence microscopy afterinfection of the epithelial cells Detroit 562 and A549, respectively, or theendothelial cell line HBMEC. The infection assays were conducted with orwithout the preincubation of pneumococci with 3 �g of factor H. B, Im-munofluorescence microscopy of adherent pneumococci. C, Invasion andintracellular survival of S. pneumoniae NCTC10319 in host cells as deter-mined by the antibiotic protection assay. Results are shown as the foldincrease in the invasion of pneumococci that were pretreated with factor H

relative to untreated pneumococci. D, Factor H mediated adherence ofwild-type pneumococcal strain TIGR4 and its nonencapsulated mutantTIGR4�cps to Detroit 562 nasopharyngeal epithelial cells as determinedby immunofluorescence microscopy. �, p � 0.005; ��, p � 0.02; relativeto infections conducted in the absence of factor H.

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regulator factor H (5, 6). To date, 11 subtypes of the PspC proteinfamily have been identified that are attached via two different waysto the bacterial surface (8). Subgroup I PspC proteins have a cho-line-binding domain and subgroup II proteins, including Hic, areanchored in a sortase-dependent mechanism in the microbial pep-

tidoglycan backbone via the C-terminal LPXTG motif. All iden-tified members of the PspC protein family show sequence poly-morphism and sequence variability. The identified 121-aa-longfactor H binding region the PspC of subgroup I proteins showssequence identity percentages to the subgroup II protein Hic(PspC11.4) of 34% (PspC3.3), 62% (PspC2.1), and 100%(PspC3.4), respectively. Within the N-terminal region of Hic threeputative factor H-binding epitopes were identified (19, 46). Theidentified sequences are conserved in all sequenced variants ofPspC. Recently, a 12-aa factor H binding motif was identified inthe first 104 amino acids of PspC3.1 (47). However, a syntheticpeptide representing this 12-aa-long stretch did not inhibit thebinding of factor H to PspC3.1 (47). Similarly, a synthetichexapeptide that was identified as the minimal SC-binding epitopedid not inhibit the binding of SC to PspC (15). Thus, amino acidsadjacent to the binding epitope and also the structure of the bindingregion seem to be pivotal for the complex formations. The iden-tification of factor H- and SC-binding epitopes in different N-ter-minal domains of PspC is furthermore in accordance with datafrom Dave et al. (18), who demonstrated that factor H and secre-tory IgA bind independently to PspC. This suggests that factor Hand SC bind different regions in PspC. Taken together, experimen-tal evidence shows that the binding epitope(s) for factor H arelocated in the very N-terminal region of PspC, whereas thehexapeptide SC-binding site is located in the R domains of PspC.

In this study we have localized the binding region of PspC toSCR8–11 in the middle region of factor H. In addition, we haveidentified a second contact site for PspC in the SCR19–20 of factorH. The sensorgrams of the surface plasmon resonance studiesshow a stronger PspC binding of SCR8–20, which includes thetwo binding regions, as compared with SCR8–11, which has onlyone binding region. In a previous study PspC binding has beenmapped to SCR6–10 and it has been suggested that the SCR13–15contributes to the complex formation (18, 38). As for PspC, theSCR8–11 of factor H has been shown to be involved in the inter-action of factor H with the Hic protein of serotype 3 pneumococciand the Bac (� protein) of S. agalactiae (46). Hic has further beenshown to bind to a region outside of the middle region of factor H.This second binding site of Hic, which has a lower affinity com-pared with the SCR8–11 binding region, has been localized to theSCR12–14 of factor H (46). In conclusion, the strength of complexformation seems to be determined by an interaction of the differentPspC variants with two contact sites in factor H. The simultaneousinteractions with discontinuous peptide sequences in these twocontact sites might finally result in a high avidity binding of factorH to PspC. Independent of the exact attachment points, host factorH is attached to the bacterial surface in a way such that the com-plement regulatory region SCR1–4 is oriented toward the outside.

Peptide mapping analysis showed the binding of factor H todiscontinuous and partially homologous sequences of pneumococ-cal Hic and Bac of S. agalactiae. Therefore, it was suggested thatthe bacterial adherence molecules Hic and Bac are related (30).Similarly, the Hic and PspC proteins of subgroup I are structurallyrelated and the localized factor H binding regions of the two pro-teins show sequence homology. The homology of PspC, Hic, andBac and their recognition of peptide sequences in SCR8 –11implies a general and probably conserved strategy for factor Hacquisition to the streptococcal surface.

PspC and the other related bacterial proteins use a unique regionfor the attachment of the host immune regulator factor H. All threeproteins bind to the middle region of factor H i.e., SCR8–11. Pre-viously, a heparin-binding domain has been localized to the SCR9of factor H (44). As shown here, the interaction of PspC and factorH SCR8–20 is blocked by heparin, suggesting that the heparin

FIGURE 8. Blocking of factor H binding to pneumococci and factorH-mediated adherence to host cells by PspC. A, Competitive inhibitionexperiments. Factor H (2 �g) binding to pneumococci (NCTC10319) wasmeasured in the absence of exogenous added PspC proteins, in the presenceof PspC derivatives as indicated and also in the presence of His6-enolase(10 �g; open bar) of S. pneumoniae, which was used as control protein.The binding of factor H was determined by flow cytometry and the resultswere expressed as GMFI � percentage of FITC-labeled and gated bacteria.Representative data from independent experiments are shown. B, In cellculture blocking experiments PspC SH3 (2.5 �g) was used. Adherence ofS. pneumoniae in the presence of factor H was set to 100%. The resultsrevealed an inhibitory effect of PspC SH3 on factor H-mediated pneumo-coccal attachment to host cells. Results are from a representative experi-ment. Inhibition studies were performed at least three times with similarinhibition patterns. C, Immunofluorescence microscopy of pneumococciattached to host cells when PspC SH3 was used to block factor H-mediatedadherence.

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binding regions in SCR9 and SCR20 of factor H mediate binding.The peptide spot assays identified three linear regions in SCR9,SCR10, and CR11 that mediate binding and include a total of 10positively charged amino acid residues. In addition, two linearbinding regions with a total of eight positively charged residueswere identified in SCR20 (Fig. 6). The presence of positivelycharged residues in the identified linear regions of factor H is inagreement with the observed inhibitory effect of heparin.

The identified type of attachment of factor H to PspC of S.pneumoniae differs from that of other pathogens such as Candidaalbicans, Aspergillus fumigatus, S. pyogenes, and Borrelia species(20, 48). Theses pathogens use SCR6–7 and SCR19–20 or a com-bination of both domains for the surface attachment of factor H. Incontrast to these pathogenic microorganisms, pneumococci do notbind FHL-1 from human plasma and none of the PspC variantsinteract with FHL-1.

Pneumococci are versatile pathogens that use multiple surfaceproteins or the capsular polysaccharide to control complement ac-tivation at the surface. A central role for complement, and partic-ularly for the alternative pathway of complement, has been shownfor clearance of pneumococci in mouse models. Several pneumo-coccal proteins were identified that mediate complement control.These include PhpA (also named PhtB and BVH-11), the toxinpneumolysin, which mediates complement-mediated clearance(49), and the PspA protein, which, similarly as PspC, is a memberof the choline-binding protein family. The deletion of PspA atten-uates virulence and increases the complement receptor-mediatedclearance of pneumococci (50). It has been suggested that PspAfunctions as an inhibitor of C3b deposition by controlling factorB-mediated alternative complement pathway activation (51, 52).The recruitment of factor H to the surface of pneumococci effi-ciently prevents the activation of C3b and the complement-medi-ated opsonophagocytosis of pneumococci (46). The improved sur-vival of pneumococci expressing PspC or Hic in a systemic mouseinfection model and in microglial cells provides further evidencefor the importance and versatility of PspC in different host niches(29, 31). A recent study indicated that carriage isolates, whichproduce lesser amounts of CPS than the invasive isolates, recruitsignificantly more factor H than the systemic isolates (53). Simi-larly, our binding experiments indicate that the CPS interferes withthe recruitment of factor H.

The adhesion of pneumococci to mucosal epithelial cells is acentral step for the bacteria to enter mucosal niches of the host andsubsequently disseminate into tissues. PspC is the major adhesin inthe nasopharyngeal cavity of the host (6, 12). Factor H enhancespneumococcal binding to host epithelial and endothelial cells. Fac-tor H binding to pneumococci and factor H-mediated adherence ofpneumococci to host cells was inhibited by the PspC SH3 domainof PspC, which includes the factor H binding region. In contrast,the SC/pIgR-binding domain of PspC, which includes thehexapeptide SC binding motif, had no effect. However it is cur-rently unclear which receptors on the host cells are involved in thisinteraction. Factor H has been shown to interact with integrin re-ceptors and, when unfolded, a properly accessible RGD bindingsite in SCR4 interacts with specific surface receptors on host ep-ithelial cells as well as endothelial cells (35–37, 42).

The infection experiments show that factor H significantly in-creased pneumococcal attachment to host cells. This effect wasobserved for both endothelial and also for epithelial cells, includ-ing nasopharyngeal cells, lung epithelial cells, and human brain-derived endothelial cells (Fig. 7). Thus, factor H attached to thebacterial surface acts as a molecular bridge and mediates adher-ence to host cells, in particular when the amount of CPS is rela-tively low. A similar role has been reported for factor H and the

related FHL-1 protein for adhesion and invasion of Fba-expressingS. pyogenes (54).

In conclusion, factor H binding to pneumococci occurs via aninteraction of the N-terminal part of PspC with two contact sites infactor H. This complex formation on the pneumococcal cell sur-face plays dual roles in pneumococcal infections. On mucosal sur-faces, bacteria-bound factor H promotes adherence to host cells.Moreover, in invasive infections factor H binding to pneumococciimproves survival by inhibiting complement-mediated lysis of thebacteria.

AcknowledgmentWe thank Francesco Iannelli (University of Siena, Siena, Italy) for pro-viding us the TIGR4�cps mutant strain.

DisclosuresThe authors have no financial conflict of interest.

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