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Previous Article | Next Article 
Infection and Immunity, June 2002, p. 2869-2876, Vol. 70, No. 6
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.6.2869-2876.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Division of Infectious Disease, Children's Hospital and Regional Medical Center and University of Washington, Seattle, Washington
Received 26 November 2001/ Returned for modification 14 January 2002/ Accepted 11 February 2002
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ABSTRACT |
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INTRODUCTION |
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The process of colonization involves microbial and host receptor-ligand interactions. Many streptococci express proteins that bind specifically to proteins of the extracellular matrix (ECM) and/or serum. The ECM surrounds cells in connective tissue such as skin, bones, and cartilage. It is heterogeneous, and various ECM proteins, including fibronectin (Fn), fibrinogen, laminin, collagen, and integrins are utilized as adhesin receptors by several different pathogens (47). The most widely described interaction is the binding to Fn, which is a large dimeric glycoprotein present in the ECM in a fibrillar form. Fn contains repeats of a characteristic amino acid triplet sequence, arginine-glycine-aspartic acid [RGD]), which can function as the binding site for some bacteria (39). Fn is also one of the major adherence targets for group A streptococci (GAS) (35). Adhesins include protein F/SfbI (13, 40), serum opacity factor (18), and Fn binding protein FBP54 (10). Fn binding proteins, such as SfbI from Streptococcus pyogenes, have also been demonstrated to act as invasins (26). Furthermore, SfbI induces a protective immune response against S. pyogenes in the sera and lungs of mice after intranasal vaccination (12), emphasizing the significance of Fn binding for pathogenesis of streptococcal infections.
Unlike GAS, Streptococcus dysgalactiae, and Staphylococcus aureus, GBS do not bind Fn in its soluble form (43), suggesting that Fn binding of GBS is a low-avidity interaction that requires multiple GBS adhesins (29). So far the GBS surface structure(s) that mediates binding to Fn has not been identified.
To identify Fn adhesins of pathogenic GBS, we have employed a shotgun phage display approach to screen a GBS genomic library for sequences which encode peptides that bind to solid-phase Fn. Using this screen, we have identified gene fragments from GBS with homology to other gram-positive adhesins and virulence genes, confirming the feasibility of our approach. One of the isolated Fn binding clones revealed homology to the C5a peptidase (ScpB), a highly specific endopeptidase found in GAS (48), GBS (15), and group G streptococci (GGS) (8). It cleaves and inactivates C5a, a component of the human complement system, thereby contributing to the ability of GBS to evade phagocytosis (3).
Based on the identification of a C5a peptidase-related peptide, we hypothesized that this protein is bifunctional, serving as a protease and mediating binding to Fn. In this report we demonstrate that GBS C5a peptidase binds to immobilized Fn and serves as a bacterial adhesin.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. E. coli MC1061 was used as the host strain for shuttle vector pDC123.
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) with 10 µg of chloramphenicol (CHL) per ml (MC1061/pDC123) and 50 µg of XP (5-bromo-4-chloro-3-indolylphosphate) (Sigma) per ml for blue-white selection.
Construction of scpB mutant TOH97.
For allelic replacement mutagenesis of scpB, the broad-host-range vector pVE6007, which mediates CHL resistance and replicates at 30°C but not at 37°C, was used. A 2.5-kb fragment of scpB was amplified from chromosomal DNA of GBS strain 78-471 (7) and cloned directly into pT7Blue (Novagen). The resultant plasmid was digested with KpnI and HindIII, and the scpB-containing fragment was cloned into pVE6007 digested with the same enzymes. A unique BglII site in scpB was used to insert the 2.3-kb BamHI fragment of pCIV2 containing 
-kan2 (30). The omega-KAN cassette terminates translation of ScpB at Glu-154. The resulting construct, pTH14, was transformed into DH5 and grown on selective LB agar plates containing CHL (5 µg/ml) and KAN (40 µg/ml) at 30°C. Plasmid DNA prepared from E. coli DH5 was then transformed into GBS strain COH1, and transformants were selected by plating on THA plus CHL (10 µg/ml) and growing at 30°C. Allelic exchange mutagenesis was carried out as described by Yim and Rubens (50).
Complementation of scpB mutant TOH97. C5a peptidase was PCR amplified with the Long Template Expand PCR system (Boehringer Mannheim) by using the primers CBP6_C5a and CBP17_C5a. The PCR product contained the ribosomal binding site and the cell wall-anchoring LPTTND motif inserted into a T-vector derivative of pDC123 (5). The ligation was used to transform E. coli MC1061 and selected on solid medium containing 10 µg of CHL per ml and 50 µg of XP per ml to monitor alkaline phosphatase activity. The resultant complementation vector, pBEC101, was sequenced, and the integrity and orientation of the insert were confirmed. Three micrograms of pBEC101 was transformed into competent scpB mutant TOH97. Competent GBS were derived by the method of Framson et al. (11). Transformants, designated BEC97, were grown on THA containing 10 µg of CHL per ml and 1 mg of KAN per ml.
Phenotypic assays. The quantification of type III capsule expression in COH1 and the mutant TOH97 was analyzed as described by Chaffin et al. (6). Beta-hemolysin and CAMP factor analysis was performed as described by Nizet et al. (28).
Construction of phagemid library. Genomic DNA was isolated from Fn binding Streptococcus agalactiae strain A909 as described previously (19). The DNA was sonicated, and fragments of between 100 and 1,000 bp were isolated by preparative gel electrophoresis, treated with T4 DNA polymerase to generate blunt ends, and ligated into the SmaI site of the phagemid vector pG3H6. AMP-resistant transformants were harvested, grown in liquid culture (2x YT) at 37°C, and, in the logarithmic growth phase (optical density at 600 nm of 0.6), superinfected with 2.5 x 109 of PFU helper phage VCSM13. After 1 h of incubation at 37°C, the culture was pelleted and resuspended in 2x YT containing 100 µg of AMP per ml, 10 µg of tetracycline per ml, 50 µg of KAN per ml, and 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside). Induction of expression continued overnight at 30°C (phage rescue).
Phage particles were isolated from the supernatants of the expression cultures by polyethylene glycol precipitation. The phage titer was determined by reinfection of log-phase E. coli XL1-Blue cells and plating on LB agar containing 100 µg of AMP per ml as described by Scott and Smith (34).
Panning of the phagemid library. Seven wells of a 96-well microtiter plate were coated overnight at 4°C with highly purified human plasma Fn (Sigma) at a concentration of 5 µg/well in phosphate-buffered saline (PBS) (150 mM NaCl, 10 mM sodium phosphate, pH 7.2) or with 5% nonfat dry milk in PBS as a negative control. Each well was blocked with 200 µl of 5% nonfat dry milk per ml in PBS for 1 h at room temperature and washed with 200 µl of PBS plus 0.05% Tween. Fifty microliters of the streptococcal phage display library, containing 5 x 1010 phagemid particles, was added to each well and incubated for 2 to 3 h at room temperature. After intensive washing of the wells with PBS-0.05% Tween, the bound phage was used to directly infect cells of E. coli XL1-Blue in the microtiter plate. Therefore, 100 µl of log-phase E. coli XL1-Blue cells per well was added to the wells and incubated for 1 h at 37°C. Ten microliters from each well was used to determine the titer of the bound phage particles by 10-fold dilutions and plating on LB agar with 100 µg of AMP per ml. The remaining 90 µl/well was plated on LB agar plus AMP and used for an additional cycle of phage rescue and panning on Fn.
Sequencing of displayed inserts. Individual clones were isolated after each panning cycle, and the insert DNA was amplified by standard PCR. Primers used for amplifying the insert hybridized on the plasmid pG3H6 to a region at the 5' end of the insert (CBP1_pG3H6) and at the 3' end of the insert (CBP2_pG3H6). The obtained PCR products were purified with the Qiagen PCR purification kit, and the DNA was sequenced from both ends by using the Big Dye terminator sequencing kit (PE Biosystems, Foster City, Calif.) with the primers CBP3_pG3H6 and CBP4_pG3H6. Analysis of the DNA and protein sequences was done with the BLAST (Basic Local Alignment Search Tool) programs of the National Center for Biotechnology Information, National Institutes of Health (1), to compare the GBS sequences with those in the GenBank database.
Construction and purification of fusion proteins. The scpB gene and the scpB gene fragment (scpB-PDF) were amplified by PCR from chromosomal DNA of GBS strain COH1. Primers CBP19_C5a and CBP20_C5a contained BamHI and XhoI restriction sites for cloning of scpB into the expression vector pGEX-4T3. Primers CBP25_C5a and CBP26_C5a were used to amplify scpB-PDF and also contained BamHI and XhoI restriction sites. Amplified products were purified using the Qiagen PCR purification kit, digested with BamHI and XhoI, and ligated separately into the BamHI- and XhoI-digested expression vector pGEX-4T3. The ligated DNA was transformed into E. coli BL21(DE3). Recombinant clones were analyzed by restriction enzyme analysis. The rScpB used in this study contains amino acids (aa) 1 to 1090 of the protein and is missing the cell wall anchor. rScpB-PDF consists of the 112 aa (aa 116 to aa 227) found in the phage display screen. Production and purification of the two glutathione S-transferase (GST) fusion proteins from E. coli were performed according to the instructions of the manufacturer (Amersham Pharmacia), and they were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%) followed by Western blot as described by Laemmli (21). The recombinant fusion proteins were detected with a goat anti-GST antibody (Amersham Pharmacia) followed by a horseradish peroxidase (HRP)-conjugated rabbit anti-goat immunoglobulin G (heavy plus light chains) antibody (Pierce). GST protein alone was prepared as a control.
Binding of recombinant proteins to Fn. The binding activities of purified rScpB and rScpB-PDF to immobilized Fn were measured by enzyme-linked immunosorbent assay (ELISA). Fn (2 µg/ml) was applied at 4°C overnight to 96-well U-bottom microtiter plates. After blocking with 5% BSA for 2 h, purified rScpB, rScpB-PDF, or rGST at various concentrations was added to the wells and incubated at 37°C for 2 h, and the wells were washed with PBS. A goat anti-GST antibody was added and allowed to bind for 1 h at room temperature. The wells were incubated with a secondary HRP-conjugated rabbit anti-goat immunoglobulin G antibody for 1 h at room temperature. The plate was washed three times with PBS and developed by adding substrate solution (ortho-phenyldiamine) (Sigma) according to the instructions of the supplier. After 15 min the reaction was stopped by addition of 10% sulfuric acid, and the color development was measured at 490 nm with an ELISA plate reader (Dynatech MR5000).
Radiolabeling of bacteria and adherence assays. GBS were grown to log phase and labeled with L-[4,5-3H]leucine (Amersham Pharmacia) as described previously (49).
The adherence assays with radiolabeled GBS were performed as described by Tamura and Rubens (43) with the following modifications: Fn (10 µg/ml) was applied to U-bottom microtiter plates (Falcon) overnight, and the bacteria remained on the plates for 2 h at 4°C after a centrifugation step. All determinations were performed in triplicate. To calculate the percent adherence of bacteria to Fn, the output counts per minute minus background (adherence to uncoated wells) were divided by the input counts per minute minus the background and multiplied by 100. CFU per milliliter were determined for each bacterium by plating dilutions of the radiolabeled bacteria, and the ratio of CFU to counts per minute was determined to verify comparable uptake of label for the wild-type and mutant strains in each experiment.
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RESULTS |
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To ensure that the library displays all potential reading frames, a particular GBS sequence has to be present multiple times in different lengths and fused in different reading frames. Our library contained 4 x 106 individual clones with inserts ranging from 100 to 1.000 bp. Given that GBS has an estimated genome size of 2.2 Mb, the library represents the GBS genome at least 200 times.
Affinity selection for clones with Fn binding activity. To identify phage expressing Fn binding peptides, we affinity screened the phage library by panning on Fn-coated plates. Bound phage were subsequently enriched by several cycles of binding and elution; in each cycle 3.5 x 1011 phagemid particles were used to ensure that several copies of each specific phage in the library were present. The phage titer increased with each cycle of binding, which is indicative of a specific enrichment. Four cycles of panning on Fn-coated plates resulted in an approximately 1,000-fold enrichment of eluted phage (Fig. 1). As a negative control, the phage library was also panned against 5% nonfat dry milk in PBS; 1,000-fold less binding was observed with this control experiment (data not shown), confirming that enrichment of the library for Fn binding phage was specific.
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We further characterized the role of ScpB in Fn binding by using the type III strain COH1 rather than A909 (type Ia), from which the phage display library was created, since the adherence of COH1 to Fn had been fully characterized previously (43) and GBS strain COH1 was also shown to express ScpB (4). The DNA sequence of the COH1 scpB gene fragment is identical to the one from A909 (data not shown), and an insertion mutation in COH1 scpB had already been constructed (see below).
To generate rScpB and rScpB-PDF encoded by the DNA insert of the isolated phagemid clone, chromosomal COH1 DNA was amplified by PCR and cloned into the pGEX-4T3 expression vector. These plasmid constructs created GST fusion proteins allowing expression and affinity purification of each protein with GST-Sepharose. The expected molecular weight and purity of each recombinant protein were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the nature of the fusion proteins was confirmed by Western blotting using an anti-GST antibody (data not shown).
Increasing concentrations of the purified fusion proteins were incubated on ELISA plates containing 2 µg of immobilized Fn per ml, and the binding characteristics were quantified using anti-GST antibody (Fig. 3). Purified rGST proteins served as a negative control and bound poorly in comparison to the fusion proteins rScpB and rScpB-PDF. The recombinant proteins bound to Fn in a concentration-dependent manner, although adherence of rScpB-PDF was 50% lower than that of rScpB. These results confirmed the observations from the phage display library experiments, specifically, that the peptide spanning the region of ScpB from aa 116 to 227 contains an Fn binding domain. We next sought to confirm that ScpB could mediate adherence of GBS bacterial cells to immobilized Fn.
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-kan2 cassette, which was inserted within the scpB gene at Glu-154. This insertion truncates the amino-terminal end and terminates transcription distal to the -kan2 insertion site, including the region encoding the Fn binding peptide described above. Southern blot analysis with COH1 genomic DNA and PCR confirmed that the wild-type gene indeed had been replaced in TOH97 by scpB::-kan2 (data not shown).
The absence of C5a peptidase expression by TOH97 was verified by Western blot analysis of mutanolysin surface extracts with C5a peptidase antiserum (monoclonal antibody F1) (kindly provided by J. F. Bohnsack, University of Utah), confirming the interruption of the C5a peptidase reading frame (data not shown). The -kan2 cassette contains a transcriptional terminator preventing transcription of downstream genes. Sequence analysis using the Genetics Computer Group software package revealed a putative terminator at the 3' end of scpB. The lmb gene, which codes for the laminin binding protein, is positioned downstream of scpB and is under the control of its own promoter (36). Therefore, it was unlikely that insertion of the -kan2 cassette affects Lmb expression. Furthermore, serotype III strain COH1 has been shown not to bind to laminin (43).
Further phenotypic analysis of the mutation showed no effect on bacterial growth in Todd-Hewitt broth, RPMI plus 5% Casamino Acids, or plasma. Colony morphology and expression of ß-hemolysin, CAMP factor, hippuricase, hyaluronidase, and caseinase were indistinguishable from those of the wild type, as were the amounts of type III capsule (6).
Analysis of Fn binding of the mutant. To address whether C5a peptidase mediates Fn binding of GBS, we compared the binding of COH1 and TOH97 as described previously (43). All experiments were performed at 4°C to prevent growth of the bacteria and to reduce metabolic activity, since bacterial metabolic enzymes such as proteases can influence adherence.
In agreement with our previous results (43), the wild-type strain COH1 adheres to Fn at 30 to 50% of the input inoculum for Fn concentrations of 20 to 100 µg/ml (Fig. 4). Coating wells with Fn concentrations of >20 µg/ml did not increase the binding significantly, whereas adherence dropped to 5 to 10% at a concentration of 2 µg/ml. The ScpB- mutant TOH97 showed a 50% decrease in adherence compared to COH1. Both COH1 and TOH97 showed less than 0.01% adherence to either uncoated wells or wells coated with BSA, indicating that the adherence to immobilized Fn was specific. These data suggest that C5a peptidase contributed significantly to the adherence of GBS. However, since binding was not completely abolished, the bacteria may produce other Fn binding adhesins.
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DISCUSSION |
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Some of the host molecules that serve as substrates for bacterial binding have recently been identified. They include cytokeratin 8 (44), laminin (36), and solid-phase (but not soluble) Fn (43).
In this study, we have used genomic peptide libraries displayed on the surface of bacteriophage to isolate Fn binding proteins of GBS. Phage display has been successfully used to isolate and characterize surface proteins of other gram-positive organisms, such as S. aureus (17, 51), S. dysgalactiae (45), S. epidermidis (27), and S. equi (22). One advantage of phage display compared to other types of expression libraries is that the screening process is replaced by panning, which allows fast identification of peptides from a complex library that demonstrate binding to a desired molecule.
We constructed a phage display library of a type Ia strain of GBS and isolated those phages which contained GBS peptides that mediate binding to immobilized Fn (Fig. 1). Some of the isolated peptides shared homology to other gram-positive proteins which have been previously implicated in adherence and virulence (Table 2). However, this approach also picks unrelated molecules in addition to potentially relevant ones. One particular clone, though, showed high homology to the C5a peptidase of S. agalactiae. The scpB gene is 97% identical between GAS and GBS (7), and furthermore, a homolog has been identified in GGS (8). C5a peptidase is a serine protease which cleaves the complement component C5a between His-67 and Lys-68 in the polymorphonuclear leukocyte binding site, destroying its chemoattractant function. Many GBS express the surface-associated serine protease, although it is not functional in all strains (4). Due to its ubiquitous expression, Bohnsack et al. (4) suggested that C5a peptidase might have a second, unknown function. Stafslien and Cleary (37) hypothesized a potential role for C5a peptidase from GAS (ScpA) as an adhesin or invasin, based on its relatively large size and very limited substrate specificity. For example, the Hap serine protease in Haemophilus influenzae has been shown to mediate bacterial adherence to epithelial cells (14, 38), providing a precedent for a serine protease to exhibit more than one function.
Our in vitro binding studies with the recombinant C5a peptidase protein and the peptide fragment expressed as recombinant GST fusion proteins indicated that ScpB does bind to immobilized Fn. However, rScpB-PDF (peptide fragment) did not display the same high binding affinity for Fn as the whole rScpB (Fig. 3). This could be explained by a different protein conformation of the fragment when fused with GST compared to the whole protein. Alternatively, it is also possible that a second binding domain exists, which is absent in the fragment but confers higher affinity on the entire protein.
Using the C5a peptidase mutant TOH97, we confirmed the role of this protease in binding Fn. This nonpolar scpB mutation was shown previously to destroy peptidase activity against C5a (J. F. Bohnsack, personal communication). The location of the mutation within scpB specifically interrupted the predicted Fn binding domain. Here we show that this mutant binds Fn 50% less than the wild-type strain. However, binding in the presence of recombinant ScpB had no effect on Fn binding of the wild-type or mutant strain (data not shown). The mutation had no effect on other phenotypic characteristics known for GBS, including capsular polysaccharide. Complementation of the mutation in trans with the wild-type gene expressed on a plasmid restored Fn binding. Since the scpB mutation did not completely inhibit adherence to Fn, our results also suggest that other bacterial factors may be involved in Fn binding.
Our experiments with truncated C5a peptidase suggest that a peptide domain (aa 116 to 227) within ScpB mediated binding to Fn. This sequence is part of the catalytic domain of scpB, including two of the active residues, Asp-130 and His-193 (Fig. 2). Ser-512 was not found to be part of the Fn binding domain. Sequence analysis of the C5a peptidase revealed that the protease contains two amino acid triplet sequences Arg-Gly-Asp (RGD), one in the N-terminal half and one in the C-terminal half of the protein. This tripeptide has been shown to mediate cell adhesion to many proteins and was originally identified in Fn (32). However, our data show that neither of the RGD motifs is part of the binding peptide identified by phage display. This result is in agreement with a previous report by Tamura and Rubens (43) showing that peptides containing the sequence RGD do not inhibit the binding of GBS to Fn. Therefore, adherence of GBS C5a peptidase to Fn must be mediated by another mechanism which is independent of the RGD motif.
Fn is a large glycoprotein of eukaryotic extracellular matrices and plasma and has been shown to be the binding substrate for a variety of pathogenic bacteria, such as S. pyogenes (9) and S. aureus (20). Respiratory epithelial cells, such as A549, produce Fn on their surface (data not shown), and the lung is one of the main sites of GBS colonization. If Fn is important for the binding of GBS to epithelial cells, the ScpB- mutant TOH97 should display a reduced level of adherence to this cell line. In preliminary experiments TOH97 demonstrated reduced adherence to A549 cells compared to the wild type (data not shown).
The results of a study by Cheng et al. (6) show that recombinant ScpB binds Fn and may mediate invasion of A549 cells in part, confirming our results. Taken together, these observations indicate that GBS adherence to epithelial cells is mediated in part by the interaction of C5a peptidase and Fn.
Based on our observations, we speculate that ScpB may facilitate S. agalactiae persistence and colonization of epithelial surfaces, which is a prerequisite for subsequent disease. Hendrixson and St. Geme (14) have demonstrated that the Hap serine protease in H. influenzae promotes bacterial interaction with epithelial cells and possibly degrades host proteins. Our report and that of Cheng et al. (6) demonstrate a second example of a serine protease showing two functions: in this case, adherence to a host molecule and evasion of the host immune system. We are continuing to characterize the Fn binding domain of ScpB and pursuing the role of this protease in the pathogenesis of GBS infections.
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ACKNOWLEDGMENTS |
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We thank Patrick Cleary and Qi Cheng for helpful discussions of the results and Anne Clancy for critical review of the manuscript.
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FOOTNOTES |
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) and rScpB-PDF (
) fusion proteins and the control rGST protein (•) to immobilized Fn. Fn-coated microtiter plates were blocked with 5% BSA in PBS, and various concentrations of recombinant proteins were allowed to bind for 2 h. Detection of bound proteins was performed with an anti-GST antibody and a secondary HRP-conjugated anti-goat IgG antibody (see Materials and Methods). The plates were developed with ortho-phenyldiamine, and color development was analyzed by optical density (OD) at 490 nm. Points represent means of triplicates, and standard deviations are indicated. Recombinant proteins bound in a concentration-dependent manner.
). Microtiter plates were coated with four different concentrations of Fn, and adherence of radiolabeled bacteria was performed as described in Materials and Methods. Points represent means of triplicates, and standard deviations are indicated. pBEC101 expressed ScpB in the complementing strain BEC971 and restored the binding to immobilized Fn to at least wild-type levels.