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,
Departments of
*
Microbiology,
Surgery, and
Medicine, University of Mississippi Medical Center, Jackson, MS 39216;
Department of Microbial Pathogenesis, German Research Centre for Biotechnology, Braunschweig, Germany; and
¶ Department of Biochemistry, University of Texas Health Science Center, Tyler, TX 75708
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-helical domain of PspC, the binding of FH to PspC is not inhibited by sIgA. ELISA, surface plasmon resonance, and flow cytometry indicate that the two host proteins do not compete for binding with PspC and likely do not share the same binding sites. We confirmed by Western analysis that the binding sites are separate using recombinant PspC proteins. These PspC variants bind FH yet fail to bind sIgA. Thus, we conclude that FH and sIgA can bind concurrently to the -helical region of PspC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PspC, also known as CbpA and SpsA (2, 3), belongs to the family of choline-binding proteins that are present on the surface of S. pneumoniae. These proteins noncovalently bind to the phosphorylcholine on the cell wall teichoic acid and the membrane-bound lipoteichoic acid (4). PspC is similar in structure to PspA, another pneumococcal surface protein (5, 6). It consists of an N-terminal -helical domain followed by a proline-rich domain. The C-terminal half of PspC contains the choline-binding domain. A variant of PspC, Hic, contains the LPXTG motif that anchors the protein covalently to the bacterial surface (7).
The importance of PspC in adherence and colonization of S. pneumoniae to epithelial cells of nasal passages and lungs in mice is well established (8, 2). Pneumococcal strains with mutations in PspC are unable to colonize the mucosal surface or infect the lungs. The adherence properties of PspC may be due to its ability to bind glycoconjugates such as sialic acid and lactotetraoses, as well as C3 on activated epithelial cells of the host (2, 9).
PspC can also specifically interact with the secretory component (SC)4 or the SC portion of polymeric Ig receptor (pIgR) and secretory IgA (sIgA) (3, 10). This binding is species specific because it binds only to the human SC of IgA (11). The SC binding site has been mapped to a hexapeptide motif (YRNYPT) in the -helical domain of PspC that is conserved among many pneumococcal strains (11). Because sIgA plays several important functions including prevention of microbial adherence, opsonization for macrophages and neutrophils, neutralization of toxins, and Ab-dependent cellular toxicity, interaction of SC with PspC may have important biological consequences.
Factor H (FH) is a 150-kDa single-chain glycoprotein that like other plasma proteins is synthesized in the liver. It is found in the plasma at a concentration of 
400–500 mg/L (12). The protein is composed of 20 short consensus repeat (SCR) domains each containing 60 aa residues that fold into 
strands and sheets (13, 14). FH protects host cells and tissues from damage upon activation of the alternative pathway of complement by rapidly inactivating bound C3b on the host cell surface (12, 15). Specifically, FH controls activation of the alternative pathway by serving as a cofactor for cleavage of C3b by Factor I, dissociating the C3bBb complex, or by competing with factor B for binding to C3b (12, 14).
We have previously demonstrated that FH binds to pneumococci of various capsular types (16). In this study, we have localized the binding site for PspC to a region within SCR 6–10 of FH. To better understand the multifunctional role of PspC in the virulence and pathogenesis of S. pneumoniae in the host, we have further examined the interaction of PspC with FH and sIgA. Our results from in vitro studies indicate that sIgA does not compete with FH for PspC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pneumococcal strains used in this study for flow cytometry were cultured as previously described (16). Briefly, the bacteria were harvested at late mid-log phase by centrifugation and suspended in PBS (pH 7.2). The bacterial concentration (1 x 108 CFU/ml) was estimated by spectrophotometer (OD600) and confirmed by viable counts on blood agar plates. For ligand dot blot analysis, Escherichia coli Y1090 was grown in Luria-Bertani medium and used to prepare a control lysate as described previously (16).
Purified FH and sIgA
Purification of human FH from serum and sIgA from colostrum were described previously (3, 16). Both proteins were biotinylated using the EZ-Link sulfo-NHS-LC-biotinylation kit (Pierce, Rockford, Il). The concentrations of biotinylated FH and sIgA were 1.0 mg/ml, and biotinylation of proteins was confirmed by quantitative ELISA. Human sIgA was purified from colostrum as described previously (3). The proteins were dialyzed overnight at 4°C with three changes of PBS (pH 7.2).
Flow cytometry
The interaction of sIgA with PspC on live pneumococcus was examined by incubating bacteria with 100 µl of biotinylated sIgA (30 µg/ml) for 1 h at 37°C. The bacteria were washed three times with PBS and after centrifugation suspended in 100 µl of streptavidin (1.0 µg/ml) conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, OR) for 30 min at room temperature. The cells were washed as before, suspended in 2 ml of PBS, and analyzed by FACScan cytometer (BD Biosciences, Franklin Lakes, NJ).
To examine the binding of FH on the pneumococcus in the presence of sIgA, bacteria were incubated with a 200-µl mixture of FH (30 µg/ml) and biotinylated sIgA (30 µg/ml) for 1 h at 37°C. After washing and centrifugation steps, sIgA was detected by streptavidin conjugated to Alexa Fluor 488 as described above. FH was detected using murine monoclonal anti-human FH IgG1 (10 µg/ml; Quidel, Santa Clara, CA) and a Zenon R-PE mouse IgG1 Labeling Kit (Molecular Probes). The binding of FH and sIgA on pneumococci was analyzed as described above.
Cloning and expression
Recombinant PspC proteins used in this study are described in Table I. SpsA-SH2 (aa 38 to 283) from type 1 strain ATCC 33400 was expressed by the plasmid pQSH2. SpsA-SH2198–203 was derived by site-directed mutagenesis using pQSH2 as a template. It contained amino acid substitutions GGINED for YRNYPT at positions 198–203 of SpsA (S.H., unpublished data). Both proteins were expressed in E. coli M15[pREP4] (Qiagen, Valencia, CA) as described previously (11). Expression was induced by the addition of 1 mM isopropyl--D-thiogalactopyranoside to exponentially growing E. coli cells. PspC-LXS234 (aa 1–445) from type 2 strain D39 was expressed using the pET20b expression vector (Novagen, Madison, WI) as described previously (5). All recombinant PspC proteins contained His tags, which facilitated their purification by affinity chromatography (Pierce). The purity of the proteins was confirmed by SDS-PAGE using 4–20% Tris-glycine precast gels (BioWhittaker Molecular Applications, Rockland, ME). Concentrations were determined by Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA).
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Western blot analyses were performed as previously described (16). Purified PspC fragments were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membranes were incubated with biotinylated FH, biotinylated sIgA, or polyclonal anti-PspC Ab (5). Bound Abs and FH were detected using streptavidin-conjugated HRP and a chemiluminescent substrate (Pierce).
Ligand dot blot
The procedure used was similar to the one described for interaction of FH with M protein (17). Briefly, 5 µg of PspC-LXS234, albumin, and E. coli lysate (16) were dried onto a nitrocellulose membrane (Millipore, Bedford, MA) at 37°C for 30 min. After blocking in 1% BSA-PBS overnight at 4°C, the membranes were incubated with 0.3 µg of full length FH or recombinant FH representing specific deletions of the 20 SCR domains that were cloned and expressed as previously described (18) for 1 h at room temperature. The membrane was further incubated with goat anti-FH IgG Ab (Quidel) followed by rabbit anti-goat biotinylated IgG Ab (Southern Biotechnology Associates, Birmingham, AL) before detection with streptavidin-conjugated HRP. Bound FH was visualized using a chemiluminescent substrate (Pierce).
Hemolysis assay
The inhibition of the alternative pathway mediated by FH was examined by a hemolysis assay (7, 19). C2-deficient serum (1/12 dilution; Advanced Research Technology, San Diego, CA) was used as a source of complement and rabbit RBC (5 x 108; Advanced Research Technology) were used as target cells. A dilution of serum was used that resulted in 80% lysis of target cells after incubation at 37°C for 40 min. Subsequently, C2-deficient serum with or without recombinant purified PspC (10.4 µM), FH (6.3 µM), or a mixture of PspC and FH (2:1 molar ratio) in 200 µl of Veronal-buffered saline with 13.5 mM EGTA, Mg2+, and 0.1% gelatin was preincubated for 5 min at 37°C. The rabbit RBC (50 µl) were added to the incubation mixture. The reaction was stopped with 750 µl of cold Veronal-buffered saline, 10 mM EDTA at 5-, 10-, 20-, and 40-min intervals. After centrifugation at 3000 rpm for 5 min, the amount of hemoglobin released in the supernatant was measured with a Spectronic Genesys TM 5 spectrophotometer (Spectronic Instruments, Rochester, NY) at 412 nm.
ELISA
The plates were coated with 100 µl of PspC-LXS234 (5.0 µg/ml) for 3.5 h at 37°C. After washing with PBS-0.05% Tween 20 and blocking with 1% BSA, serial dilutions of sIgA (initial concentration, 25 µg/ml) were added to the plates. This was followed by addition of 50 µl of biotinylated FH (10 µg/ml) to the wells and incubation of the plates overnight at 4°C. The plates were then washed and incubated with streptavidin-alkaline phosphatase for 2 h at 37°C. After the final wash, the plates were incubated with substrate before reading at 405 nm. The experiment was performed in duplicate and repeated three times. As positive controls, exogenous PspC and unlabeled FH were used to inhibit binding of biotinylated FH to PspC-coated wells.
Surface plasmon resonance analysis
Surface plasmon resonance measurements were obtained on a Biacore X instrument and analyzed with BIAevaluation 3.0 software (Biacore, Uppsala, Sweden). PspC-LXS234 in PBS, pH 7.5, was covalently immobilized by amine coupling on research grade carboxylated dextran CM5 chips (Biacore) per manufacturer’s instructions. Binding was performed in HBS-P running buffer (Biacore), and 0.02% sodium azide at a flow rate of 1 µl/min. Factor H (100 µl; 1 mg/ml) in PBS was injected until signal reached a maximum and no further increase in resonance U was observed. Immediately, 100 µl of 1 mg/ml sIgA in PBS were injected. Binding to flow cell 2, which lacked coupled protein, was used as background. The surface was regenerated between experiments with 30 µl of 200 mM EDTA (pH 3.6), 0.02% sodium azide.
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Results |
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The reactivity of biotinylated sIgA with exponentially growing S. pneumoniae was tested by flow cytometry. S. pneumoniae cells were incubated with biotinylated sIgA, suspended in Alexa Fluor-conjugated streptavidin, and examined with a FACScan cytometer. Although TRE 108 cells (PspC–) did not bind sIgA (Fig. 1A), change in the mean fluorescence intensity was observed upon incubation of wild-type D39 cells with biotinylated sIgA (Fig. 1B). We also examined binding of biotinylated sIgA to cell lysates prepared from different pneumococcal capsular serotypes by Western blot analysis (data not shown). sIgA bound to all pneumococcal strains tested except the PspC– strain, TRE 108. Also, both the migration of bands and intensity of binding upon incubation with sIgA varied among the different lysates as expected, given known variation in PspC size among pneumococcal strains. These results were consistent with data published by Hammerschmidt et al. (3).
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We examined the binding of FH to purified recombinant PspC from D39 in the presence of sIgA by competitive inhibition analysis using ELISA (Fig. 2). sIgA did not inhibit binding of biotinylated FH to recombinant PspC adsorbed on microtiter plates. Likewise, FH did not inhibit the binding of biotinylated sIgA to PspC (data not shown). In contrast, binding of biotinylated FH to immobilized PspC was significantly reduced upon addition of PspC or unlabeled FH in solution. These results suggest that sIgA does not compete with FH for binding sites on PspC.
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The interaction of purified PspC-LXS234 with FH and sIgA was measured by surface plasmon resonance technology. In these experiments, PspC-LXS234 was covalently coupled to one flow cell of the sensor chip with the second uncoupled flow cell serving as a control for binding. We first examined the binding of only FH or sIgA to PspC-LXS234. We then determined the ability of sIgA to interact with PspC in the presence of bound FH. The off rate of sIgA was faster than the off rate of FH (data not shown). Thus, after FH binding on PspC-LXS234 reached steady state, sIgA was allowed to bind to the immobilized PspC-LXS234. PspC-sIgA binding also reached saturation (Fig. 3). The net increase in resonance observed upon binding of sIgA in the presence of FH was equal to the increase in resonance observed in the absence of FH (Fig. 3, inset), indicating that the PspC-FH interaction does not inhibit the PspC-sIgA interaction. These results further support the observation that the two proteins do not compete for binding to PspC as shown by ELISA.
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The binding of FH on live pneumococcal cells in the presence of sIgA was examined by flow cytometry. Bacterial cells were incubated with a mixture of sIgA and FH at a 1:1 ratio. Results of cells staining with either only FH (4.97% events), sIgA (21.90% events), or both FH and sIgA (19% dual-positive events) are shown in Fig. 4. We can thus conclude that FH and sIgA can bind concurrently on live pneumococcal cells.
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The binding of FH and sIgA to various recombinant PspC proteins was analyzed by Western Blot. PspC-LXS234 and SpsA-SH2 are wild-type recombinants derived from two different strains of S. pneumoniae. The protein SpsA-SH2198–203 has amino acid substitutions GGINED for the sIgA binding motif YRNYPT. Both FH and sIgA reacted efficiently with PspC-LXS234 and SpsA-SH2 (Fig. 5). As previously shown, SH2198–203 failed to bind sIgA (11). However, SpsA-SH2198–203 reacted efficiently with FH. This datum further validates that the binding site for sIgA is distinct from that for FH.
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We mapped the PspC-binding site on FH by a ligand dot blotting assay (17). PspC was immobilized on a nitrocellulose membrane and incubated with either full length FH or recombinant truncated proteins with specific deletions in the SCR domains. BSA and E. coli lysate were used as negative controls. FH and all recombinant proteins containing SCR 1–10 bound to purified PspC (Table II). The FH deletion mutant protein lacking SCR 6–10 (rFH 
6–10) failed to bind PspC. The results demonstrate that the PspC-binding site for FH is located inclusively between the SCR 6 and SCR 10 domains of this complement-regulatory protein.
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The functional activity of FH in the presence of PspC was assessed by hemolysis assay using C2-deficient serum and rabbit RBC as source of complement and target, respectively. Previously, this assay served as an indirect measure of complement activity based on the amount of lysis of the rabbit RBC upon activation of the complement in the serum. The results are shown in Fig. 6. Preliminary experiments were performed to determine the concentration of C2-deficient serum needed for 80% hemolysis of rabbit RBC after 40 min. Incubation of rabbit RBC with C2-deficient serum alone and in combination with purified PspC resulted in 75.0 ± 7.64% and 69.3 ± 8.9% hemolysis, respectively. However, lysis was greatly inhibited on introduction of FH to the reaction mixture (only 15 ± 0.58%). Moreover, FH activity was maintained in the presence of PspC as indicated by the inhibition of hemolysis of rabbit RBC (12.0 ± 0.58%) upon addition of a preincubated mixture of purified FH and PspC to the C2-deficient serum.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helical domain. Because the D39 mutant TRE 108 does not express PspC but still expresses PspA, its inability to bind FH suggests that the FH-binding site must be in the -helical domain of PspC which has limited homology with PspA. Previously, the binding of sIgA/SC has been mapped to a hexapeptide motif also in the -helical region of PspC (11). In the present study, we determined whether sIgA and FH can concurrently bind to the -helical domain of PspC.
We first confirmed the binding of biotinylated sIgA to PspC in cell lysates from several different pneumococcal strains commonly used in our laboratory. The interaction of sIgA with exponentially growing D39 and other pneumococcal strains was further examined by flow cytometry. As with FH, differences in sIgA binding were observed among the various pneumococcal isolates. This may be explained by the polymorphism of PspC among the different pneumococcal strains. Recently, the polymorphic pspC locus of 43 pneumococcal strains was characterized by DNA sequencing of PCR fragments (28). The majority (86%) of strains tested contained a single copy, although some strains (14%) had two tandem copies of the PspC gene. The DNA sequence of PspC varied in each of the 43 strains of pneumococci. The PspC proteins were placed in 11 different groups according to variations in the nucleotide sequence. The C-terminal end of the PspC variants contained either the choline-binding domain or the LPXTG motif. This ability to express and use two different anchorage domains may be unique to PspC of S. pneumoniae.
The molecular mass of PspC is in the range of 59–105 kDa based on its migration properties on SDS-PAGE (2). According to sequence analysis, variations in PspC size are strain dependent and have been attributed to the -helical domain (5, 28). Although sequence variability exists among different pneumococcal strains, the sIgA binding site has been mapped to a conserved hexapeptide, YRNYPT, in PspC of ATCC 33400 strain between aa 198 and 203 (11).
The biological significance of PspC interaction with the SC portion of sIgA or pIgR has been examined in several studies. It has been shown that binding to the ectodomain of pIgR, known as SC of sIgA, to PspC has resulted in increased pneumococcal adherence to mammalian cells and their transmigration from the apical to the basolateral surface of epithelial cells (10). This suggests a mechanism by which pneumococci can travel from one site of infection to another in the host. Reduced pneumococcal bacteremia and delayed sepsis in pIgR knockout mice provided further evidence that PspC-SC/sIgA interaction is important in the pathogenesis of S. pneumoniae (10). It has also been shown that glycans of the secretory component are important in the prevention of infections on the epithelial surface by directly binding to adhesion molecules of Helicobacter pylori, E. coli, and Clostridium difficile (29, 30, 31, 32). However, Zhang et al. (10) observed that the binding of sIgA to PspC is independent of glycans given that deglycosylation did not affect reactivity of the SC with PspC.
In this investigation, we also examined the functional significance of PspC-FH interaction by first mapping the PspC-binding sites on FH. The PspC-binding site was localized to SCR domains 6–10 of FH using recombinant truncated FH proteins. Other studies mapped the binding site on FH to be within SCR 8–15 for PspC and SCR 8–11 for the PspC variant, Hic of S. pneumoniae (33, 34). Although there are three C3b binding sites on FH, previous studies have also shown that C3b does not inhibit binding of FH to PspC (33). In addition, complement-regulatory activity due to C3b binding is directly attributed to only the first four SCR domains of FH (35). Thus, our findings that the binding of FH to PspC is within SCR 6–10 imply that the active site is still available to bind C3b and inhibit complement activation. Our data from the hemolysis assay provide evidence that FH is active in the presence of PspC. The regulatory activity of FH was also maintained after binding to Hic of type 3 pneumococci, YadA of Y. enterocolitica, and CRASP1 of B. burgdorferi (7, 20, 26). These proteins can control C3b degradation on or near the bacterial surface upon attachment of FH, because the binding site to the bacteria (SCR 5–7) and active site for C3b inhibition (SCR 1–4) are in separate regions within the molecule.
Several independent investigations have established the binding of sIgA and FH to PspC of S. pneumoniae. To our knowledge, this is the first study examining the interaction of FH to PspC in the presence of sIgA. We have shown by ELISA that sIgA does not compete with FH for binding to immobilized PspC. This was further confirmed by directly measuring the interaction of PspC with FH and sIgA using Biacore technology and observing both host proteins binding on live pneumococcal cells by flow cytometry. The efficient binding of both FH and sIgA to PspC at the same time suggests presence of two distinct binding sites within the -helical region of the molecule. This is supported by the ability of FH to bind efficiently to recombinant PspC proteins that failed to bind sIgA by Western analysis. These observations, although interesting, are not unique to S. pneumoniae. The Sir protein of Streptococcus pyogenes is able to bind both Ig and C4BP at separate sites within the N-terminal fragment of the molecule (36). Likewise, protein of group B Streptococcus binds sIgA at the N terminus, whereas the C terminus of this protein is involved in FH binding (37).
The interaction of PspC with FH may be an important mechanism by which pneumococci control C3b deposition on their surface and escape opsonophagocytosis. Alternatively, the interaction of PspC with the SC of IgA or pIgR may be critical for the pneumococci to translocate from the nasopharynx and spread to the normally sterile parts of the respiratory tract such as the lungs or the bloodstream during infection. The lack of competition between FH and sIgA for binding to PspC is interesting and requires further studies in the future to better understand the regulation of FH and sIgA binding to PspC as well its role in the virulence and pathogenesis of S. pneumoniae in humans.
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Acknowledgments |
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Footnotes |
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2 Current address: Research Center for Infectious Diseases, University of Wurzburg, Germany.
3 Address correspondence and reprint requests to Dr. Larry S. McDaniel, Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216. E-mail address: LMcDaniel{at}microbio.umsmed.edu
4 Abbreviations used in this paper: SC, secretory component; FH, factor H; pIgR, polymeric Ig receptor; sIgA, secretory IgA; SCR, short consensus repeat.
Received for publication October 6, 2003. Accepted for publication March 17, 2004.
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References |
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protein has separate binding sites for human factor H and IgA-Fc. J. Biol. Chem. 277:12642.This article has been cited by other articles:
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 J. V. McDowell, M. E. Harlin, E. A. Rogers, and R. T. Marconi Putative Coiled-Coil Structural Elements of the BBA68 Protein of Lyme Disease Spirochetes Are Required for Formation of Its Factor H Binding Site J. Bacteriol., February 15, 2005; 187(4): 1317 - 1323. [Abstract] [Full Text] [PDF]
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), and FH (
). Each experiment is done in triplicate.
