Ectodomains 3 and 4 of Human Polymeric Immunoglobulin Receptor (hpIgR) Mediate Invasion of Streptococcus pneumoniae into the Epithelium* □ S

Streptococcus pneumoniae binds to the ectodomain of the human polymeric Ig receptor (pIgR), also known as secretory component (SC), via a hexapeptide motif in the choline-binding protein SpsA. The SpsA-pIgR interaction mediates adherence and internalization of the human pathogen into epithelial cells. In this study the results of SpsA binding to human, mouse, and chimeric SC strongly supported the human specificity of this unique interaction and suggested that binding sites in the third and fourth Ig-like domain of human SC (D3 and D4, respectively) are involved in SpsA-pIgR complex formation. Binding of SpsA to SC-derived synthetic peptides indicated surface-located potential binding motifs in D3 and D4. Adherence and uptake of pneumococci or SpsA-coated

The polymeric immunoglobulin receptor (pIgR) 1 is synthesized as an integral membrane glycoprotein in the rough endoplasmic reticulum of mucosal epithelial cells and comprises a polymeric IgA (pIgA)-binding extracellular region, a 23-amino acid membrane-spanning region, and a cytoplasmic tail of 103 amino acids (1,2). The extracellular region of pIgR is divided into the leader peptide, and five Ig-like domains (D1 to D5) of 100 -110 amino acids each, out of which D1 represents the initial pIgA-binding domain (3). A sixth non-Ig-like domain connects the extracellular domains to the transmembrane region (1). The pIgR is crucial for the generation of secretory IgA (SIgA) because it mediates transport of pIgA to mucosal secretions (4,5). The pIgR-pIgA complex is endocytosed from the basolateral pole and translocated by vesicular transport to the apical surface of the epithelium. Release of SIgA and free secretory component (FSC, from unoccupied receptor) occurs by proteolytic cleavage of the pIgR near the plasma membrane (1,2,6,7).
SIgA represents the first line of specific immune defense on mucosal surfaces (8 -12) performing immune exclusion of antigens such as bacteria, viruses, parasites, and toxins (10,11,(13)(14)(15)(16). In addition to its role in pIgA transport, pIgR/SC provides increased stability and mucosal anchoring to secretory antibodies (17,18). Moreover, free or IgA-bound SC may compete with pathogens for adhesion sites on the apical surface of mucosal epithelial cells (19,20) and also act as a nonspecific microbial scavenger (21,22). Some pathogens have developed strategies to exploit these host defense functions of the pIgR, for their invasion into the epithelium. In infections by type 2 herpes simplex virus and Epstein-Barr virus, virus-specific pIgA antibodies act as a bridge that connects the pathogen with the pIgR-expressing epithelium thereby facilitating internalization of virus (23)(24)(25)(26). Recently, adherence of Streptococcus pneumoniae to nasopharyngeal epithelial cells was associated with pIgR-mediated translocation of the bacteria across the epithelial barrier (27). S. pneumoniae is a frequent resident of the upper and lower respiratory tract of humans, but pneumococci are also important human pathogens, causing relatively harmless local infections such as otitis media and sinusitis as well as life-threatening diseases such as pneumonia, septicemia, and meningitis (28,29). Pneumococcal dissemination starts at the respiratory tract after penetration of the epithelial barrier, and disease outcome depends on bacterial virulence and host defense factors. The surface-displayed choline-binding protein SpsA (also designated CbpA and PspC) of S. pneumoniae exhibits a unique interaction with FSC or the SC portion of pIgR and SIgA (27,30). This interaction may be of pathogenic significance because it provides a mechanism for adherence of pneumococci to nasopharyngeal pIgR-expressing cells. Apical recycling of the pIgR may allow bacterial invasion and subsequent translocation across human epithelial barriers (27). Whether this apical to basolateral translocation occurs by utilizing the pIgR-transcytosis machinery in reverse or by other mechanisms is not known.
We have previously shown that SC from mouse, rat, guinea pig, and rabbit does not interact with SpsA in vitro (31). Furthermore, a hexapeptide motif (YRNYPT), located in a highly conserved part of the N-terminal region of SpsA, was identified as the minimal pIgR/SC-binding motif by synthetic peptide technology. Systematic replacement analysis indicated a crucial role of the YPT sequence in this motif (31). In this study we demonstrate by the sensitive surface plasmon resonance (SPR) technique that pIgR/SC from mouse, rat, guinea pig, hamster, rabbit, dog, cow, and horse do not bind to SpsA. Furthermore, we used two complementary approaches to identify the SpsA binding site on human SC: first, chimeric domains swap molecules between human and mouse SC identified human D3 and D4 as required for the interaction with SpsA; second, synthetic peptide array technology identified several epitopes in human pIgR capable of interacting with SpsA. Putative binding sites were identified in extracellular domains D2 133-148 , D2/3 208 -225 , and D4 349 -375 . Crucial roles of the hexapeptide binding motif of SpsA and the identified SpsA binding sites in D3 and D4 in hpIgR-mediated adherence of SpsA coated latex beads and in pneumococcal invasion of epithelial cells was confirmed.
Protein Expression and Purification-Escherichia coli M15[pREP4] (Qiagen, Hilden, Germany) was used as host for recombinant pQE expression plasmids and cultured at 37°C on Luria-Bertani (LB) agar or grown on LB-agar containing 100 g ml Ϫ1 ampicillin. Expression and purification of His-tagged fusion proteins was performed as described previously (31). To obtain His-tagged fusion proteins SpsA SH2, SpsA SH12, SpsA SH3, SpsA SM1, and SpsA SH2 201 (31) of highest purity and to eliminate contaminations that might interfere in the BIAcore analysis, affinity purified His-tagged proteins were subjected to gel filtration chromatography using HiLoad 16/60 Superdex 75 according to the manufacturer's instructions (Amersham Biosciences). To obtain the bacterial adhesin for hpIgR/SC in its native form, SpsA from S. pneumoniae ATCC 33400 serotype 1 was purified by immunoaffinity chromatography. Protein A-purified polyclonal anti-SpsA IgG (10 mg) generated in rabbit against SpsA protein SH2 (31) was coupled to CNBractivated Sepharose 4B (Amersham Biosciences) according to the manufacturer's instructions. Fractionation and enrichment of solubilized pneumococcal cell wall proteins was performed as described previously (33). The cell wall protein fraction was applied to the coupled antiserum and SpsA was eluted with 0.1 M glycine, pH 2.4. Its purity was confirmed by SDS-PAGE followed by silver stain and its SC binding activity by immunoblot analysis. SIgA and FSC from humans and other species such as rats, mice, rabbits, guinea pigs, bovines, equine, canines, and hamsters were purified as described earlier from milk or bile (31).
Surface Plasmon Resonance-Association and dissociation reactions of FSC and SIgA were analyzed by surface plasmon resonance using a BIAcore TM optical biosensor 2000 (Amersham Biosciences). SpsA purified from S. pneumoniae and SpsA derivatives SH2, SH12, SM1, and SH2 201 were covalently immobilized on CM5 sensor chips as described previously (34). Briefly, SpsA proteins, each in a concentration of 1 mg ml Ϫ1 in 20 mM sodium acetate, pH 4.0, were coupled at 10 l min Ϫ1 onto N-hydroxysuccinimide (0.05 M), N-ethyl-NЈ-(diethylaminopropyl)carbodiimide (0.2 M)-activated sensor chips using a volume of 70 l with a flow rate of 10 l min Ϫ1 . Binding analysis was performed in a HBS BIAcore running buffer (10 mM HEPES, 150 mM NaCl, 1.4 mM EDTA, 0.05% Tween 20, pH 7.4) using a flow rate of 10 l ml Ϫ1 in all experiments. Regeneration of the affinity surface between sequential analyte injections was conducted twice with 20 l of 20-30 mM NaOH. The kinetics of FSC and SIgA binding to immobilized SpsA proteins was analyzed from raw data of the BIAcore sensorgrams using a linear kinetic model included in BIAevaluation software version 3.0. For every evaluation, a minimum of six data sets corresponding to FSC/SIgA binding reactions at concentrations between 0.19 and 500 nM were analyzed. Sensorgram data were fitted globally to the algorithms representing a simple 1:1 Langmuir kinetic.
Construction of Chimeric SC Molecules and Binding to SpsA-A pcDNA3.1-derived (Invitrogen) expression plasmid encoding human FSC has been described previously (35). A similar expression plasmid for murine FSC was made by amplifying the 6 extracellular domains of murine pIgR and inserting the coding region into pcDNA3.1his6. Domain swap chimeras indicated in Figs. 3 and 4 were made by PCR splice overlap extension and the chimeric cDNA was inserted into pcDNA3.1his6 (details provided upon request). Domain boundaries of the chimeric constructs were verified by DNA sequencing, and were as indicated in Piskurich et al. (2) and shown in Fig. 4. For recombinant expression of wild-type human and murine SC as well as all the chimeric and mutant SC variants, 293E cells, maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 2 mM L-glutamine, FIG. 1. Models of the SpsA proteins used for biochemical and in vitro analysis. SpsA SH2 is the mature NH 2terminal region of the choline-binding protein SpsA from S. pneumoniae serotype 1 (ATCC 33400) and SpsA SH12 represents the mature NH 2 -terminal region of SpsA of S. pneumoniae serotype 35A (NCTC 10319) (30) and the latter is identical to the mature NH 2 -terminal CbpA sequence of D39 and R6x (38). SM1 is an amino-terminal truncated SpsA derivative containing the binding site YRNYPT for pIgR. The SpsA derivative SH2 201 contains an individual Asp substitution at position 201 and is, therefore, unable to bind to SC in in vitro assays (31). SpsA SH3 was constructed by deletion of a HindIII DNA fragment of the SpsA SH2 encoding DNA cloned in expression vector pQE30 and does not contain the SC/pIgRbinding motif. and 50 g ml Ϫ1 gentamicin, were transiently transfected by FuGENE (Roche) according to the manufacturer's protocol. Supernatants were harvested 2, 4, and 6 days after the end of the transfection, pooled (30 ml in total), SC precipitated by adding 12 g of ammonium sulfate, and resuspended in 1 ml of phosphate-buffered saline (PBS) with 0.05% azide. Binding to SpsA fragments was analyzed by ELISA. Microtiter plates (Costar) were coated with 1.5 g ml Ϫ1 of SpsA fragments in 0.02 M ammonium acetate, pH 7.0, overnight and then blocked with 1% bovine serum albumin in PBS for 90 min. Dimeric IgA was coated at 3.0 g ml Ϫ1 . Titration experiments demonstrated that maximal OD was achieved when the SC concentration was between 0.5 and 1.0 g ml Ϫ1 . All SC variants were therefore diluted to ϳ2 g ml Ϫ1 in ELISA buffer (PBS with 0.5% bovine serum albumin, 0.05% Tween 20) and incubated for 90 min, and binding was determined by incubation with a mixture of rabbit antiserum to human SC (DAKO; diluted 1/3000) and murine SC (gift from Blaise Corthesy, diluted 1/10000), and revealed by alkaline phosphatase-conjugated goat anti-rabbit serum.
Analysis of Spot-synthesized hpIgR Peptides for SpsA Binding Activity-The leader sequence and extracellular domains of the hpIgR from amino acid 1 to 592 were divided into 200 overlapping peptide fragments, each consisting of 15 amino acids (see Supplementary Materials), with an offset of three amino acids (36). The peptides were synthesized as an array of spots on an aminophenylated cellulose membrane (AIMS Scientific Products GmbH, Braunschweig, Germany) as described previously (37), and are NH 2 -terminal acetylated and remain covalently attached to the membrane via their carboxyl terminus. Binding of SpsA to hpIgR-derived peptides was carried out by using a SpsA-derivative SH12 and anti-SpsA IgG followed by horseradish peroxidase-conjugated anti-rabbit IgG as described previously (31).
Pneumococcal Adherence and Invasion-Pneumococcal adherence to and invasion of Calu-3 and MDCK-hpIgR-expressing cells were performed in 24-well plates (Greiner, Germany) on glass coverslips (diameter, 12 mm). Confluent epithelial cells with a density of ϳ2 ϫ 10 5 cells were inoculated with 5 ϫ 10 6 to 1 ϫ 10 7 pneumococci and infections were carried out for 4 h in Dulbecco's minimal essential medium-HEPES at 37°C under 5% CO 2 . To remove non-adherent bacteria, the cells were rinsed several times with PBS. Synthetic peptides used in inhibition experiments were purified by preparative high-performance liquid chromatography (RP-8) with water/acetonitrile gradients containing 0.5% trifluoroacetic acid and characterized by amino acid analysis.
Immunofluorescence-Extra-and intracellular pneumococci were stained using a polyclonal anti-pneumococcal antiserum and secondary goat anti-rabbit IgG coupled to Alexa 488 (green) or Alexa 568 (red) (MoBiTec). Pneumococcal antiserum was generated in rabbit against heat-inactivated pneumococci (R6x and ATCC 11733) and it reacts equally well against different pneumococcal strains. Briefly, extracellular bacteria, adhering to epithelial cells, were incubated for 15 min with the polyclonal anti-pneumococcal antiserum, washed, and fixated in 3.7% paraformaldehyde. After washing coverslips were incubated with the Alexa 488 goat anti-rabbit Ig conjugate. The intracellularly located invasive pneumococci were stained after permeabilization of the cells with 0.1% Triton X-100 for 5 min, washed in PBS, followed by incubation with anti-pneumococcal antiserum and Alexa fluor 568 goat anti-rabbit Ig conjugate. Extracellular pneumococci appear yellow, whereas intracellular appear red. At least 50 cells per glass coverslip were scored for pneumococcal adherence and invasion. Each experiment was repeated at least five times and results were expressed as mean Ϯ S.D.
Transfection of Epithelial Cells-Transfection of MDCK cells with pcDNA3.1hpIgR and constructs pcDNA3.1hpIgRD2⌬ 133-148 , pcDNA3.1hpIgRD2/3⌬ 206 -229 , and pcDNA3.1hpIgRD4⌬ 349 -389 , respectively, were conducted using FuGENE 6 (Roche) as transfection reagent according to the instructions of the supplier. Briefly, 1 ϫ 10 4 MDCK cells were seeded on coverslips in medium without antibiotics. Serumand antibiotic-free medium was mixed with FuGENE reagent and 0.5 g of DNA was added (FuGENE:DNA in a ratio of 3:1). The solution was mixed gently and incubated for 30 min at room temperature. The complex mixture was added to the cells and the 24-well plate was swirled to ensure even dispersal. Cells were incubated at 37°C in 5% CO 2 for at least 2 days prior to incubation with the SpsA-coated latex beads. SpsA-coated latex beads were detected by anti-SpsA IgG followed by goat anti-rabbit IgG coupled to Alexa 568 and efficiency of transfection was visualized by immunofluorescence using rabbit anti-SC antiserum as primary antibody followed by Alexa fluor 488 goat anti-rabbit Ig conjugate (MoBiTec).
Attachment and Internalization of SpsA Derivatives Coupled to Latex Beads-SpsA derivatives (rSpsA SH12, rSpsA SH2, rSpsA SH 201 ) expressed as recombinant proteins were coated on 3-m polystyrene latex beads (Sigma) by incubating 10 8 beads with 5 g of the proteins in PBS for 12 h at 4°C. After washing with PBS, beads were resuspended in culture medium to a final concentration of 10 8 beads ml Ϫ1 . Efficiency of protein coating was verified by fluorescence-activated cell sorting (data not shown). Binding of 10 7 SpsA-coated beads was assayed to 2 ϫ 10 5 Calu-3 or MDCK cells. Cells were grown on a 12-mm glass coverslip and incubated with latex beads for 1 to 4 h incubation at 37°C under 5% CO 2 . After washing six times with adhered or internalized PBS, beads were visualized by light microscopy, immunofluorescence, or scanning electron microscopy.
Scanning Electron Microscopy-Following incubation with SpsAcoupled latex beads, Calu-3 or MDCK-hpIgR cells were fixed with a fixation solution containing 2% glutaraldehyde and 3% formaldehyde in cacodylate buffer for 1 h on ice and washed with cacodylate buffer. After washing several times in TE buffer, samples were dehydrated with a gradient series of acetone (10,30,50,70,90, and 100%) on ice, each step 15 min, followed by critical point drying with liquid CO 2 . Samples were examined in a Zeiss field emission scanning electron microscope DSM982 Gemini at an acceleration voltage of 5 kV using the Everhard Thornely SE-detector and the inlens-SE detector in a 50:50 ratio.
Statistical Analysis-The differences in adherence were analyzed by the Student's t test, unpaired, and statistically significant for p Ͻ 0.05 and p Ͻ 0.001, respectively.

Association and Dissociation Kinetics of SC/SIgA to SpsA
Proteins-The complex formation of SpsA-SC and SpsA-SIgA were analyzed using SPR. Kinetics of hFSC and SIgA binding to SpsA purified from S. pneumoniae and recombinant N-terminal SpsA derivatives SH2, SH12, SM1, and SpsA protein SH2 201 , containing an amino acid substitution at position 201 (Tyr 3 Asp) of the hexapeptide binding motif (Fig. 1), were measured. Representative sensorgrams are shown in Fig. 2. The observed rates of complex formation between native SpsA, SH2, SH12, and SM1 were dependent on the concentration of applied hFSC or SIgA (Fig. 2, A and B). SpsA with a single substitution of a critical amino acid in the hexapepetide motif did not bind hFSC or human SIgA as shown by a total lack of increase of resonance units (Fig. 2C). Assuming a simple onestep bimolecular association reaction and fitting, therefore, the data globally according to the 1:1 Langmuir kinetic model revealed similar association and dissociation rate constants for native SpsA-hFSC/SIgA (Table I). The equilibrium dissociation constants as defined by K D ϭ k off /k ass are in the nanomolar range (Table I) (Table I).
The hexapeptide motif YRNYPT of SpsA, which represents the minimal SC binding motif (31), is present once in SpsA derivative SH2 at position 198 -203 and twice in SpsA derivative SH12, at positions 163-168 and 325-330. Nevertheless, SPR revealed that the number of SC binding motifs does not affect rates of association and dissociation of the SpsA and SC interaction. Moreover, SPR analysis confirmed our previous findings that SC from mouse, rat, and SIgA from bovine, canine, equine, guinea pig, hamster, mouse, rabbit, and rat did not bind to SpsA as indicated by a total lack of increase of resonance units (data not shown).
Targeting SpsA Binding to pIgR Ectodomains 3 and 4 of Human-Mouse Chimeric SC Molecules-To identify the SpsAbinding domain of hpIgR, we used two complementary approaches. First, we utilized the species specificity of the SpsA-SC/pIgR interaction by investigating the interaction between SpsA and chimeric human-mouse SC molecules. A series of human-mouse and mouse-human chimeras with species boundaries after each Ig-like domain were constructed and binding to SpsA was investigated in an ELISA (Fig. 3). An alignment of the amino acid sequences of human and mouse SC, indicating the boundaries of the Ig-like domains, is shown in Fig. 4. All SC molecules that contained human D3 and D4 bound to SpsA (SH2), whereas SC molecules that lacked either of these domains were not able to bind. None of the chimeras bound to a truncated SpsA fragment were deleted in the hexapeptide binding motif SpsA (SH3) (Fig. 3A). As a positive control, binding of all the chimeric SC molecules to dimeric IgA was shown to occur with similar A 405 (ELISA) values at the concentrations tested for binding (Fig. 3, A and B). To test whether human D3 and/or D4 were sufficient to mediate SpsA binding we constructed chimeras where only these domains were of human origin and again assessed their binding to SpsA (Fig. 3B). The results showed that human D3 and D4 were both required and sufficient for binding to SpsA (Fig. 3B). The SpsA variant SH2 201 with a mutation in the hexapeptide motif failed to bind any of the chimeric SC molecules, whereas the SpsA variant with a duplication of the hexapeptide motif (SH12) showed similar binding specificities and strength as the variant with a single hexapeptide motif (SH2) (Fig. 3B).
Binding of SpsA to Spot Synthesized pIgR Peptides-Although mouse SC failed to interact with SpsA, interacting epitopes in the SC molecule could include both species-divergent and conserved regions. We therefore proceeded to analyze the SpsA-hpIgR interaction by using synthetic peptides representing sequences of hpIgR. The sequence of hpIgR, which covers the leader peptide and extracellular domains D1 to D5, was divided into 200 overlapping 15-mer peptides, having an offset of three amino acids (see Supplementary Materials). Results of the spot membrane overlay assay, probed with SpsA, demonstrated binding of SpsA to five spot peptides of D1 and two regions in D2 consisting each of five positive peptide spots (Figs. 4 and 5). The sequence in D1 was not investigated further because it overlaps the pIg binding site and would therefore not be accessible in SIgA. The sequences of the positively assayed peptides localized in D2 of hpIgR are at amino acid positions 133-159 (D2 133-159 ) and 160 -186 (D2 160 -186 ), respectively. In addition, binding was also observed to peptides that represent amino acid residues 208 -225 (D2/3 208 -225 ) and 265-282 (D3 265-282 ) in D2 and D3 of hpIgR, respectively (Figs. 4 and 5). The screening of the membrane-spotted peptides showed also a positive reaction of membrane-spotted synthetic peptides spanning amino acids 349 -375 of D4 (D4 349 -375 ) of hpIgR (Figs. 4 and 5). Computer-aided primary and secondary structural analysis (39,40,41) revealed that the region D2/3 208 -225 , which comprises the end of domain 2 and the beginning of domain 3, and the region D4 349 -375 are with the highest probability located on the surface of the pIgR and therefore should be accessible for the SpsA-pIgR interaction. The region span- Kinetic parameters of the interaction of SpsA derivatives coupled to the surface of CM5 sensor chips and FSC and SlgA, respectively. Listed are the association and dissociation rate constants (k a and k d ) and equilibrium constants (K A , association, and K D , dissociation). Data were fitted globally according to the algorithms representing a simple 1:1 Langmuir binding model included in the BlAevolution 3.0 software. R max represents the maximal response units and represents a unit for the binding of the analyte to the CM5-coupled ligand. The values of the 2 -statistical test (Chi2) indicated that the selected reaction scheme was a good mathematical representation of the FSC/SlgA binding sensorgram data. ning amino acids 138 -144 in D2 was also predicted to be surface located. In contrast, regions spanning amino acids 160 -186 of D2 and 265-282 of D3, respectively, were not predicted to be surface located. Impact of the SpsA Hexapeptide on Pneumococcal Adherence and Invasion-SpsA-mediated adherence to and invasion of mucosal epithelial cells occurs in a cell-specific manner and depends on expression of hpIgR (27,42). To investigate the role of the hexapeptide motif in SpsA and the roles of the proposed binding sites in hpIgR in pneumococcal adherence and invasion, in vitro infection assays were carried out with hpIgR-expressing lung epithelial cells, Calu-3, and with hpIgR-transfected MDCK cells (MDCK-hpIgR) (Fig.  6). Immunofluorescence microscopy of the cell culture infec-tions indicated adherence and invasion of encapsulated type 35A (Fig. 6) and type 2 and of unencapsulated R6x pneumococci (data not shown), whereas the corresponding SpsA knockout pneumococcal strain was deficient in both of these processes (Fig. 6). Infection of the non-transfected MDCK cells could be disregarded because of the low number of attached pneumococci (data not shown). To assess the role of SpsA and the hexapeptide binding motif in the infection process of S. pneumoniae, blocking experiments were conducted. Adherence and invasion of a representative Cpsϩ pneumococcal strain (type 35A) was substantially blocked by the NH 2 -terminal part of SpsA (SH2) and anti-SpsA antiserum (Fig. 6, A and B). In contrast, adherence to and invasion of pIgR-expressing cells was not affected by mutated SpsA (SH2 201 ) with a critical Tyr to Asp amino acid substitution at position 201, underscoring the important role of the hexapeptide motif in the pneumococcal-cell interaction (Fig.  6, A and B). Pneumococcal adherence and invasion was also inhibited in the presence of hSIgA, hFSC, and anti-FSC polyclonal serum (data not shown).
Role of the Ectodomains of pIgR on Adherence and Invasion of S. pneumoniae in Epithelial Cells-To investigate the impact of the putative binding sites D2 133-159 , D2/3 208 -225 , and D4 349 -375 , respectively, on pathogenesis, the adherence was also analyzed in the presence of chimeric SC derivatives or synthetic peptides. Recombinant human SC inhibited pneumococcal adherence similarly to native purified SC, whereas recombinant mouse SC did not. The key role of domain 4 was further underscored by the fact that the chimeric humanmouse SC Hd4 and particularly Hd3d4, in which only domain D4 or D3 and D4 are of human origin, were able to inhibit significantly the adherence of pneumococci to pIgR-expressing cells (Fig. 7A). Moreover, SC and SIgA from mice, rabbit, and rat did not affect pneumococcal adherence or infection confirming the species-specific interaction of SpsA with hpIgR (data not shown). Adherence to and invasion of A549 cells and HEp-2 cells, which do not express hpIgR, was not affected by the proteins and antibodies used in blocking experiments (data not shown). The synthetic peptides D2 peptide 136 -159 , FKTENAQ-KRKSLYKQIGLYPVLVI (amino acids 136 to 159 of D2), D2/3 peptide 206 -229 , DDSNSNKKNADLQVLKPEPELVYE (amino acids 206 to 229 of D2/3), and D3 peptide 258 -283 , GENCDV-VVNTLGKRAPAFEGRILLNP (amino acids 258 -283 of D3), which was used as control peptide, did not affect significantly adherence and invasion (Fig. 7B). In contrast, the synthetic peptide D4 peptide 349 -375 , VAVLCPYNRKESKSIKYWCLWE-GAQNG (amino acids 349 to 375 of D4), substantially affected attachment and invasion of pneumococci to pIgR-expressing cells (Fig. 7B). Taken together, these experiments revealed a crucial role for the SpsA hexapeptide motif and receptor domains D4 and D3 for SpsA-SC/pIgR complex formation and pneumococcal invasion.
Adherence of SpsA-coated Latex Beads to MDCK-hpIgR Cells-To verify that hpIgR expression and its interaction with the hexapeptide binding motif of the SpsA protein is sufficient to mediate adherence and invasion, SpsA derivatives SH2, SH2 201 , and SH12 were immobilized on 3-m latex beads and attachment and internalization of the beads was followed by light, fluorescence, and electron microscopy (Fig.  8). Latex beads coated with wild-type SpsA SH2 and SpsA SH12 bound to MDCK-hpIgR cells, whereas beads coated with mutated SpsA SH2 201 did not, confirming the critical role of the SpsA hexapeptide as the SC/pIgR-binding motif (Fig. 8A). Kinetic studies indicated that SpsA-coated beads already bound to hpIgR-expressing cells after 1 h, but that their internalization required more than 4 h of incubation (Fig. 8B). The uptake of SpsA-coated latex beads by MDCK cells that express hpIgR, indicated that the SpsA-hpIgR in-teraction promotes adherence and internalization of pneumococci.
Adherence of SpsA-coated Latex Beads to Epithelial Cells Expressing Mutated hpIgR-To confirm the crucial role of ectodomains 3 and 4 of hpIgR, binding of SpsA SH12 latex beads to transiently transfected MDCK cells was investi-  Fig. 4) in which the proposed binding sites were deleted. The results of the adherence studies definitively demonstrated that a deletion of amino acids 133-148 in D2 did not affect binding of SpsA to hpIgR (Fig. 8C). Moreover, deletion in D2 showed that mutated pIgR is translocated to the epithelial cell surface and suggested a correct folding of the protein. HpIgR variants with deletions in D3 or D4 were also expressed at the cell surface (Fig. 8C). However, individual deletions of the proposed binding sites in D3 and D4 resulted in complete loss of attachment of the SpsA latex particles, indicating the pivotal role of these binding sites for SpsA-hpIgR complex formation that finally is able to mediate attachment and invasion of pneumococci to hpIgR-expressing epithelial cells (Fig. 8C). DISCUSSION In this study, we identified the binding sites for the S. pneumoniae choline-binding adhesin, SpsA, in D3 and D4 of human pIgR/SC. Furthermore, we show that this interaction is specific for human pIgR/SC and required for invasion of pIgR-expressing epithelial cells by S. pneumoniae. The function of SpsA as a bacterial adhesin, which also promotes uptake of pneumococci via the pIgR has been demonstrated by both in vitro and in vivo studies (27,38). A pneumococcal cbpA (spsA) knockout strain exhibited a 100-fold lower efficiency to colonize the nasopharynx of rats (38). We have, however, previously shown that SC and SIgA from mouse, rat, rabbit, and guinea pig did not interact with SpsA (31). In the current study, we further substantiated the species specificity of the pIgR/SC-SpsA interaction by the sensitive SPR technique. The specificity for human pIgR-transfected but not rabbit pIgR-transfected MDCK cells for pneumococcal invasion was also demonstrated by Zhang et al. (27). In mice with deficiencies in the pIgR or in p62 yes (a protein-tyrosine kinase required for normal pIgR transcytosis (43)), the efficiency to colonize the nasal cavity, or to cause bacterial sepsis, respectively, was reduced compared with the control group of wild-type mice (27). It has been speculated that low affinity binding to rodent pIgR/SC under the detection limit of in vitro assays might still be sufficient to mediate in vivo binding and subsequent translocation (44). SC and SIgA derived from different animals and used in this study for SPR analysis clearly failed to bind to SpsA, and thus, emphasize the human species specificity of the unique interaction of human pIgR/SC with SpsA. Therefore, the results of this study fail to explain the results of another study with p62 yes knockout mice (27).
The intracellular transport pathways of pIgR across polarized epithelial cells have been studied in detail and provided new insights into the nature and regulation of endocytotic and transcytotic pathways (45). Whether pathogens that invade via the pIgR are delivered to identical compartments and endosomes that normally engage the internalization of the pIgR-dIgA from the basolateral surface, by co-opting the pIgR for ''reverse'' transcytosis, has yet to be determined. The pIgR/SCbinding domain in the pneumococcal adhesin was mapped precisely to a hexapeptide motif in the NH 2 -terminal and highly conserved part of the SpsA protein. Individual amino acid substitutions identified tyrosine 201, proline 202, and threonine 203 of S. pneumoniae type 1 (ATCC 33400) SpsA as crucial for the interaction with the SC (31). The hexapeptide motif Y(H/R)NYPT is present twice as part of a repetitive sequence in the NH 2 -terminal part of most of the SpsA proteins expressed among pneumococci of different serotype (46,47). We used an NH 2 -terminal SpsA protein variant with a single hexapeptide motif and a variant containing a duplication of this sequence, as ligands in the SPR studies and for interaction studies with human, mouse, and chimeric SC molecules. The results did not reveal significant differences in the kinetic constants or species specificity of the different SpsA variants. In fact, the dissociation constants were in the same range as estimated by Scatchard analysis (30). SPR indicated a simple one-step bimolecular association with the extracellular portion of pIgR and also with SIgA via the hexapeptide motif in SpsA as demonstrated by loss of binding to the mutated SpsA 201 protein. The impact of the hexapeptide motif on pneumococcal invasion in pIgRexpressing epithelial cells was confirmed by the inability of SpsA SH2 201 to block adherence and invasion of pneumococci to hpIgR-expressing cells. Identification of the binding site(s) in pIgR was first achieved by assaying binding of human-mouse chimeric SC molecules to SpsA and further by employing synthetic peptide array technology. These complementary ap- FIG. 8. Binding of SpsA-coated latex beads to MDCK-hpIgR cells. A, binding of latex beads coated with the mature NH 2 -terminal part of SpsA (SpsA SH2 or SpsA SH12) or mutated derivative SpsA SH2 201 was visualized by light microscopy. B, binding and uptake of SpsA-coated latex beads incubated with MDCK-hpIgR cells for 1 or 4 h WERE visualized by field emission-scanning electron microscopy. C, binding of SpsA(SH12)-coated latex beads to transiently transfected MDCK cells. Latex beads and pIgR-expressing MDCK cells were detected by two-color immunofluorescence as described under ''Experimental Procedures.'' Cells staining positively for the wild type hpIgR or hpIgR variants D2⌬ 133-148 , D2/3⌬ 206 -229 , and D4⌬ 349 -389 are indicated by arrows.
proaches identified critical binding sites in D3 and D4 of hpIgR, which were localized in surface displayed regions and therefore should be accessible for the interaction with SpsA. The results further pointed out that the SpsA-pIgR/SC interaction is independent of the glycosylation of the receptor, thereby confirming previous results (27). The impact of the binding sites in D3 and D4 on pathogenesis was illustrated through the construction of mutated hpIgR, in which binding sites in D3 and D4 were individually deleted and adherence of SpsA-coated latex beads was substantially diminished. Final evidence was provided in in vitro infection experiments, where the human-mouse chimeric SC molecules Hd3d4 and Hd4 as well as the synthetic peptide D4 peptide 349 -375 inhibited adherence of pneumococci to hpIgR-expressing cells. Taken together, these results indicated a key role of amino acids 349 -375 in D4 of hpIgR for binding of SpsA. However, the results suggest that also D3, but not D2, is involved in this interaction.
Although pIgR transport in retrograde, from apical to basal, is less efficient than the basal-to-apical transcytosis, pneumococci are translocated from the apical surface across nasopharyngeal epithelial cells via this specific interaction (27). A broader study suggested also that factors other than the pIgR and its pneumococcal adhesin might be involved in uptake of clinical isolates in pIgR-expressing cells. Polymeric Ig-mediated invasion has been proposed to be cell-and strain-specific (42), although the nonspecific attenuation of the capsule and the colony morphology, which might favor adherence or virulence (48), have not been considered. Furthermore, the respiratory mucosa is saturated with FSC and SIgA because of efficient processing of unloaded or pIgA-complexed pIgR at the apical surface. These secreted proteins, therefore, compete with the immobilized pIgR at the apical surface for binding to its adhesin SpsA and might, thus, inhibit attachment of pneumococci to the cells (30,44). In fact, this cell-specific uptake strongly depends on the balance of free and pIgA-complexed soluble SC with that of uncleaved membraneous pIgR on the epithelium. Viral infections and enhanced levels of cytokines including interferon-␥, interleukin-1, interleukin-4, and tumor necrosis factor, which cause an up-regulation of the pIgR (49 -53), might favor invasiveness of pneumococci. To gain more insight into the SpsA-pIgR-mediated pneumococcal invasion and epithelial translocation, the cell compartments involved in this specific process and morphological studies of the invaded epithelium have to be conducted.