Ace Is a Collagen-binding MSCRAMM from Enterococcus faecalis*

Identification of E. faecalis Ace in a Microbial Genome Data Base

The amino acid sequence comprising the minimal collagen-binding region (residues 151–318) of the S. aureuscollagen adhesin, Cna (4, 5), was used to search for homologous sequences in the Microbial Genome Database at the National Center for Biotechnology Information. The BLAST (14) search resulted in the discovery of a novel putative gene sequence from E. faecalisof significant homology. The complete open reading frame comprising this sequence was subsequently obtained from The Institute for Genomic Research (TIGR) web site.

Structural Modeling Studies

The 335-amino acid sequence of Ace A domain was sent to the ExPASy SWISS-MODEL Automated Protein Modeling Server and modeled using the Cna 151–318 molecule structure (1AMX.pdb, Genbank accession no. M81736) as a template. A sequence alignment and model of the Ace A domain was returned by the ExPASy server composed of 145 amino acid residues based on the structure of Cna 151–318. Even when the 335-amino acid sequence of Ace A domain was sent to the SWISS-MODEL server without specific instructions to model the sequence on the Cna 151–318 structure, the Cna 151–318 structure file was chosen as a template automatically, as determined by a BLAST P(N) search of known protein structure sequences in the ExPDB modified Protein Data Bank data base. Manipulation of the Ace A domain model and Cna 151–318 (1AMX.pdb) was accomplished using the Swiss-PDB Viewer 3.0 software package available at the ExPASy web site, and images were rastered using the software package Persistence of View Ray-tracer (POV-Ray 3.0) (15-17). Temperature factors for the Ace model were higher in the loop regions, especially in regions where there are gaps in the sequence alignment. The root mean square deviation calculated between the two structures was 0.63 Å for the Cα atoms (142 total) and 0.61 Å for the analysis of all backbone atoms (426 total). These values for the root mean square deviation of the backbone atoms suggested a high level of accuracy for the model.

Bacterial Strains and Culture Conditions

Unless otherwise noted, chemicals and reagents were molecular-biology grade from Sigma or U.S. Biochemical Corp. Based on our previous report (12), theE. faecalis strains were grown in BHI medium (Difco) overnight at 46 °C. The two E. faecalis strains used for the Western blot analysis are designated EF1 (originally described by Caparon and Scott (18)) and EF2 (a clinical E. faecalisisolate obtained from University of Alabama at Birmingham), respectively. Strain OG1RFΔGel is a gelatinase mutant of strain OG1RF (12, 19) and was grown in BHI supplemented with 2 mg/ml kanamycin.S. aureus strain Phillips, a clinical isolate from an osteomyelitis case (11), was grown in tryptic soy broth (Difco) at 37 °C.

Cloning and Construction of Expression Plasmids

The nucleotide sequence encoding the Ace A or A+B domains (Fig. 1a) was obtained by PCR using a thermocycler (Perkin-Elmer Cetus 480) and chromosomal DNA (20) from E. faecalis strain EF1 as the template. Primers (USB Life Technologies) were designed to amplify nucleotides 94 (5′-GCAGGATCCGAATTGAGCAAAAGTTCAATC-3′) to 1101 (5′-GCAGTCGACTCAGTCTGTCTTTTCACTTGTTTC-3′) of the A domain and nucleotides 94 (5′-GCAGGATCCGAATTGAGCAAAAGTTCAATC-3′) to 1750 (5′-GCAGTCGACTCATGGCTGTTTTTTCTCAGTTGTAG-3′) of the A+B domain sequence as determined from the nucleotide information obtained from TIGR. The resulting gene fragments were subcloned into pQE-30 (Qiagen Inc., Chatsworth, CA), transformed into Eschericia colistrain JM101 and analyzed by automated DNA sequencing (University of Texas Medical School, Houston, TX). Construction of the Cna plasmids that yield the recombinant proteins in Fig. 1 d has been described previously (5).

Expression and Purification of Recombinant Proteins

Recombinant Ace A domain with a His tag at its N terminus was produced by inoculating 1-liter cultures of Luria broth (supplemented with 100 μg/ml ampicillin) with 40 ml of an overnight culture of the A domain expression construct described above. Following 2.5 h of growth at 37 °C, isopropyl-β-d-thiogalactoside was added to a final concentration of 0.2 mm to induce protein expression and the cultures were allowed to grow for another 3 h. Bacteria were harvested by centrifugation, the supernatant decanted, and the cell pellets resuspended in PBS before being stored at −80 °C. The suspension was later thawed in an ambient-temperature water bath for 30 min and the cells lysed using a French press. Insoluble cell debris was removed by centrifugation at 28,000 × g for 20 min, followed by filtration through a 0.45-μm membrane. Recombinant Ace A domain was then initially purified using metal-chelating chromatography. Bacterial lysates were applied to a 5-ml Ni²⁺-charged HiTrap chelating column (Amersham Pharmacia Biotech) and bound protein eluted with a 200-ml linear gradient of 0–200 mm imidazole in 4 mm Tris-HCl, 100 mm NaCl, pH 7.9, at a flow rate of 5 ml/min. Fractions corresponding to recombinant Ace A domain, as determined by SDS-PAGE, were pooled and dialyzed against 25 mm Tris-HCl, pH 8.0, before further purification by ion-exchange chromatography. Dialyzed protein was applied to a 5-ml HiTrap Q column (Amersham Pharmacia Biotech) and bound protein eluted with a 200-ml linear gradient of 0–0.5 m NaCl in 25 mm Tris-HCl, pH 8.0, at a flow rate of 5 ml/min. Fractions containing purified Ace A domain were identified by SDS-PAGE and estimated to be >90% pure. Production and isolation of recombinant Cna proteins was performed as described previously (5).

Preparation of Ace A Domain Polyclonal Antibodies

Purified Ace A domain was dialyzed against 10 mmNa₂HPO₄, 150 mm NaCl, pH 7.4 (PBS), before being sent to HTI Bio Products (La Jolla, CA) for immunization in rabbits and production of polyclonal antisera. For some experiments, IgGs were purified from both immune and pre-immune serum by chromatography using Protein A-Sepharose (Sigma).

Western Blot Analysis

Mutanolysin surface extracts (21) were prepared from E. faecalis strains EF1 and EF2 grown at 46 °C and analyzed by Western blot analysis. The presence of Ace was detected following incubation with anti-Ace A domain polyclonal antiserum, followed by goat anti-rabbit IgG horseradish peroxidase, and development in the presence of 4-chloronaphthol and H₂O₂.

Bacterial Adherence Assays

Enzyme-linked immunosorbent assay plates were coated with 5 μg of type I collagen in 100 μl of PBS/well overnight at 4 °C. Wells were then washed three times with PBS and then blocked with 1% bovine serum albumin in PBS for 1 h before the addition of bacteria. Bacteria (E. faecalis grown at 46 °C, S. aureus at 37 °C) were harvested from liquid cultures and diluted to a concentration having an absorbance of 1.0 at 600 nm in PBS (approximately 5 × 10⁸bacteria/ml) before being labeled with FITC (22). 100 μl of labeled bacteria were added per well, and the plates were incubated at 37 °C for 1 h. The total fluorescence (F _total) per well was measured after a 1-h incubation using a Fluoroskan II fluorescence reader (Labsystems, Beverly, MA), with λ_ex = 485 nm and λ_em = 535 nm. The wells were washed with PBS three times to remove unbound bacteria and the remaining fluorescence (F _test) measured. Adherence was calculated as follows: adherence =F _test/F _total. For the data shown in Fig. 6, adherence of labeled cells in the absence of antibodies was normalized to 100%. Bovine serum albumin-coated wells were used as negative controls. For inhibition assays, FITC-labeled bacteria were first incubated with anti-Ace A domain IgG for 1 h at 37 °C before addition of the mixture to the collagen-coated wells.

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Figure 6

Inhibition of E. faecalis strain OG1RFΔGel (a) andS. aureus strain Phillips (b) binding to type I collagen by anti-Ace A domain IgG. FITC-labeled bacteria were preincubated with anti-Ace A domain IgG (●) or pre-immune IgG (○) before addition to wells coated with type I collagen. Values are expressed as the percentage of adherence obtained in the absence of antibody and represent the mean ± standard deviation of quadruplicate measurements.

Absorption Spectroscopy

Absorption measurements were taken at ambient temperature (23 ± 2 °C) on a Beckman DU-70 UV-visible spectrophotometer using a 1.0-cm path length cuvette. All spectra were corrected for background noise. Molar extinction coefficients of each protein were calculated using values of Paceet al. (23) for the extinction coefficients of the individual residues.

Circular Dichroism Spectroscopy

Far-UV CD data were collected on a Jasco J720 spectropolarimeter calibrated withd-10-camphorsulfonic acid, employing a bandpass of 1 nm and integrated for 4 s at 0.2-nm intervals. All samples were less than 15 μm in 0.1 mmNa₂HPO₄, 1 mm NaCl, pH 7.0. Spectra were recorded at ambient temperature in cylindrical 0.5-mm path length cuvettes. Twenty scans were averaged for each spectrum, and the contribution from buffer was subtracted. Quantitation of secondary structural components was performed as described in Ref. 8. The validity of these results was confirmed by comparison with the results obtained from x-ray crystallographic data for Cna 151–318; the breakdown of secondary structural components is nearly identical for the solid- and solution-phase structures (Table I).

Surface Plasmon Resonance Spectroscopy

Analyses were performed using the BIAcore 1000 system. Bovine type I collagen predissolved in 0.1 m HCl (Collagen Corp., Fremont, CA) was immobilized on a CM5 sensor chip as described previously (5). Recombinant proteins in 150 mm NaCl, 50 mmHEPES, 0.005% P-20 surfactant, pH 7.4, were flowed over multiple flow cells containing different amounts of immobilized collagen. The slowest flow rate (1 μl/min) specified for the instrument was employed. Even at this rate, however, the association and dissociation of the recombinant Ace A domain protein with the collagen-coated surface were too rapid to be quantitated. Specific binding response data were obtained by subtracting the response obtained using a flow cell that was not coated with collagen. Analytical conditions were as described previously (24, 25). No mass transport effects were observed in these measurements.

The data for the construction of the Scatchard plots were obtained from the equilibrium portion of the surface plasmon resonance spectroscopy (SPR) sensorgrams (e.g. the response at approximately 900 s in the Ace sensorgram of Fig. 4 a). Values for the collagen-bound protein, ν_bound, and concentration of unbound protein, [P] _free, were calculated from Equations1-3. Equation 1 Equation 2 Equation 3

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Figure 4

a, representative profiles of the relative SPR responses for the binding of 20 μmrecombinant E. faecalis EF1 Ace A domain (——) andS. aureus Cna A domain residues 151–318 (- - -) to immobilized type I collagen. In the analyses shown here, the association occurs from 55 to 960 s and the dissociation begins at 960 s. Both profiles have been corrected for the response of protein over a flow cell containing no collagen. b, Scatchard plot of 1–70 μm E. faecalis EF1 Ace A domain binding to 2436 RU immobilized type I collagen as measured by SPR. The analysis was repeated with varying MSCRAMM concentrations and amounts of immobilized collagen. No SPR signal was detected for Ace A domain concentrations of less than 1 μm. From three measurements, K_D = 48 ± 7 μm; n = 5.3 ± 0.3.

In Equation 1, R is the SPR response, m is the molecular mass, P is the protein, C is collagen; in Equation 2, [P]_o is the concentration of total protein, vol _flowcell is the volume of sample in the flow cell, and area _flowcell is the surface area of the flow cell. Plotting ν_bound/[P]_free versusν_bound yields the plot shown in Fig. 4 b. The negative reciprocal of the slope yields the dissociation constant,K_D , and the x axis intercept is equivalent to the number of sites, n, in collagen at which the MSCRAMM protein binds.

E. faecalis Ace Is a Mosaic Protein Having Critical Sequence Homology with S. aureus Cna

In an attempt to identify novel collagen binding proteins, we searched microbial genome data bases for amino acid sequences which have significant similarity to that of Cna 151–318 (previously referred to as Cna M19; Refs. 4-8), the central region of the A domain of the S. aureus collagen-binding MSCRAMM (Fig. 1 c). A significant match was recorded in the E. faecalis genome data base.2 The complete sequence of the gene encoding this protein, which has been given the working name Ace (adhesin of collagen from E. faecalis), was obtained from TIGR and was present on contig gef 6285.3 Translation of the nucleotide sequence revealed a 74-kDa protein, which has a structural organization very similar to that of MSCRAMMs from other Gram-positive bacteria (Fig. 1 a). A possible signal sequence involving the first 31 amino acids is followed by a 335-amino acid-long A domain. The B domain is composed of 4.5 tandemly repeated 47-residue units of >90% identity. The C-terminal region is composed of a putative cell wall-associated domain rich in proline residues and contains the cell wall-anchoring LPXTG consensus sequence (26). An 18-amino acid hydrophobic transmembrane region followed by a short cytoplasmic tail represents the C-terminal end of the protein.

PCR primers were designed to amplify the nucleotide sequence encoding the A or A+B domains of ACE from E. faecalis strain EF1. The resulting PCR fragment for the A domain corresponded to the same size fragment encoded by strain V583 (27) in the TIGR sequence. However, the PCR fragment for the A+B domain construct was approximately 300 base pairs smaller than expected. DNA sequence analysis revealed that theace gene from strain EF1 contained only 2.5 B domain repeat units, whereas 4.5 B domain repeat units were present in the sequence of strain V583. With the exception of having two fewer B domain repeat units, the DNA sequence of ace from strain EF1 was greater than 95% identical to that of strain V583.

A central region (residues 174–319) in the A domain of E. faecalis Ace (from either strain EF1 or V583) has a high degree of sequence similarity to residues 151–318 of the S. aureusCna protein. Within this span of amino acids, 27% of the residues are identical to residues in Cna 151–318 and an additional 29% are similar (Fig. 1 e). Significant similarity (46%) continues throughout the A domain of Ace and the corresponding region of the Cna A domain; outside the A domains, however, there is no obvious sequence homology between Ace and Cna.

Structural Models Suggest a Similar Folding Motif for S. aureus Cna 151–318 and E. faecalis Ace 174–319

Modeling of Ace 174–319 onto the structure determined for Cna 151–318 gave the structure shown in Fig. 2 a. To obtain the best sequence alignment, three one-residue gaps and one three-residue gap were introduced into the sequence of Ace 174–319 and a two-residue gap was introduced into the sequence of Cna 151–318. It is noteworthy that the polypeptide region in Ace covered by residues 174–319 is predicted to fold in a “jellyroll” as Cna 151–318 does, even though a substantial number of the amino acids involved are different as shown in Fig. 2 a, where the residues conserved between the two proteins are presented in cyan segments and the residues unique to Ace 174–319 are presented in gray. The more substantial residue differences are located in loops connecting the β-strands. When the polypeptides of Ace 174–319 and Cna 151–318 are overlaid (Fig. 2 b), the β-strands are almost identical and the most notable folding differences are observed in the loops.

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Figure 2

a, ribbon diagram of E. faecalis Ace residues 174–319 mapped onto Cna 151–318 x-ray structure, with cyan segments denoting regions of sequence identity or similarity and gray denoting regions lacking homology. b, ribbon diagrams of E. faecalis Ace A domain residues 174–319 (green) overlaid with Cna 151–318 (red). c, space-filled model of E. faecalis Ace A domain residues 174–319 mapped onto Cna 151–318 structure. In panel c, residues within the putative collagen-binding trench that are conserved in S. aureus Cna 151–318 and E. faecalis Ace 174–319 are depicted in blue, trench residues that are not conserved are depicted in green, and the onecyan residue is a Thr in Cna 151–318 and a Val in Ace. These structures harbor the introduced gaps described under “Results.”

A trench present on the surface of the Cna 151–318 structure has been identified as the collagen-binding site and can accommodate a collagen-like triple-helical peptide (7). The structure predicted for Ace 174–319 contains a trench in the same orientation, as highlighted in Fig. 2 c. Approximately half of the Cna trench-lining residues are conserved in Ace. Of the conserved putative Ace trench residues shown in blue in Fig. 2 c, four (Tyr-180, Arg-193, Phe-195, and Asn-197) were shown to be critical for collagen binding in Cna (9). Another residue (Lys-237) known to be critical for collagen binding in Cna 151–318 is not conserved in Ace and is one of the residues shown in green (Fig. 2 c). These modeling studies, based on the known collagen-binding MSCRAMM, Cna, suggested that Ace: 1) can act as a collagen adhesin and 2) possesses a trench-shaped binding site.

A Recombinant Form of Ace A Domain Has a β-Sheet Structure and Binds Collagen

A recombinant form of the Ace A domain was expressed as a fusion protein with a N-terminal His tag. This protein was soluble and could be purified by chromatography on a Ni²⁺-charged IDA-Sepharose column and an anion-exchange column. Analysis of the protein by CD spectroscopy gave a spectrum with a maximum at 195 nm and a minimum at 217 nm (Fig.3). This spectrum was qualitatively similar to that of the intact A domain and residues 151–318 of Cna. Deconvolution of the spectra revealed very similar compositions of secondary structure for each of the three proteins dominated by β-sheet structures and with a small α-helical component (TableI).

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Figure 3

Far-UV CD spectra of recombinant proteins mimicking the E. faecalis EF1 Ace A domain (——) and the S. aureus Cna A domain, full-length (– – –) and residues 151–318 (- - -). Secondary structure compositions are reported in Table I. Mean residue weight ellipticity is reported in degrees · cm²/dmol.

We used SPR to analyze the predicted collagen-binding activity of Ace. The sensorgrams in Fig. 4 ashow that recombinant Ace A domain and Cna 151–318 both bind to type I collagen immobilized on a BIAcore sensor chip. However, the kinetics of the two interactions were dramatically different. The on and off rates of the Ace/collagen interaction were far too rapid to be determined from these measurements, whereas the association and dissociation rates of the binding of Cna 151–318 to collagen were slower and measurable (24, 25).

Scatchard analysis of SPR equilibrium binding data from increasing concentrations of Ace flowed over immobilized collagen yielded a linear plot (Fig. 4 b), indicating five copies of a single class of Ace A domain binding sites exist in type I collagen. The calculated dissociation constant (48 μm) indicated a relatively weak affinity. In contrast, our earlier analyses of the binding of Cna 151–318 and intact Cna A domain to type I or type II collagen yielded a concave upward Scatchard plot, indicating the presence of several classes of Cna binding sites in these collagens (24,25).4

Ace Is a Collagen-binding MSCRAMM

Analyses of theace gene sequence revealed many elements, including the cell wall-anchoring motif characteristic of cell wall-associated surface proteins from Gram-positive bacteria. This raised the question: is Ace a functional collagen-binding MSCRAMM present on the surface of enterococci? We have previously demonstrated that most strains ofE. faecalis can adhere to a collagen substrate after growth at 46 °C, indicating the presence of collagen-binding MSCRAMMs on the bacterial surface (12). Western blot analyses of proteins released from E. faecalis strain EF1 and EF2 grown at 46 °C by digestion with mutanolysin demonstrated the presence of two major bands reacting with antibodies raised against the Ace A domain, whereas pre-immune sera did not react with any protein (Fig.5). The larger band migrated at approximately 80 kDa and most likely represented the full-length Ace protein, whereas the smaller band may represent a proteolytically processed form of the protein. Ace from strain EF1 has an expected molecular mass of only 60 kDa. The difference between this mass and the apparent molecular mass observed in Fig. 5 may be due to the acidic nature (pI = 4.3) of the Ace protein.

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Figure 5

Western blot analysis of E. faecalis surface extracts. E. faecalisstrains EF1 (lanes 1 and 3) and EF2 (lanes 2 and 4) surface extracts were prepared by mutanolysin digestion and detected using anti-Ace A domain IgG (lanes 1 and 2) or pre-immune IgG (lanes 3 and 4). Prestained molecular mass standards are shown on the left.

Antibodies to Ace A domain were tested for their ability to inhibit the adherence of enterococcal cells to a substrate of type I collagen. As shown in Fig. 6a, as little as 1 μg/ml anti-Ace IgG almost completely inhibited bacterial adherence to immobilized collagen, whereas there was no effect of the pre-immune IgG over the range of concentrations examined. Neither immune or pre-immune IgG types had any effect on S. aureus strain Phillips adherence to type I collagen, indicating that anti-Ace A domain antibodies did not interfere with the binding of Cna to collagen (Fig.6 b). Taken together, these results demonstrate that Ace is present on the surface of E. faecalis cells and acts as a collagen adhesin.