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Infection and Immunity, June 2002, p. 2933-2940, Vol. 70, No. 6
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.6.2933-2940.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Insertional Inactivation of eap in Staphylococcus aureus Strain Newman Confers Reduced Staphylococcal Binding to Fibroblasts

Muzaffar Hussain,1* Axana Haggar,2 Christine Heilmann,1 Georg Peters,1 Jan-Ingmar Flock,2 and Mathias Herrmann3

Institute of Medical Microbiology, University of Muenster Hospital, D-48129 Muenster,1 Institute of Microbiology and Hygiene, University of Saarland Hospital, D-66421 Homburg/Saar, Germany,3 Department of Microbiology, Pathology and Immunology and Karolinska Institutet, Huddinge University Hospital, F82, S-141 86 Huddinge, Sweden2

Received 15 November 2001/ Returned for modification 14 January 2002/ Accepted 1 March 2002


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ABSTRACT
 
To initiate invasive infection, Staphylococcus aureus must adhere to host substrates, such as the extracellular matrix or eukaryotic cells, by virtue of different surface proteins (adhesins). Recently, we identified a 60-kDa cell-secreted extracellular adherence protein (Eap) of S. aureus strain Newman with broad-spectrum binding characteristics (M. Palma, A. Haggar, and J. I. Flock, J. Bacteriol. 181:2840-2845, 1999), and we have molecularly confirmed Eap to be an analogue of the previously identified major histocompatibility complex class II analog protein (Map) (M. Hussain, K. Becker, C. von Eiff, G. Peter, and M. Herrmann, Clin. Diagn. Lab. Immunol. 8:1281-1286, 2001). Previous analyses of the Eap/Map function performed with purified protein did not allow dissection of its precise role in the complex situation of the staphylococcal whole cell presenting several secreted and wall-bound adhesins. Therefore, the role of Eap was investigated by constructing a stable eap::ermB deletion in strain Newman and by complementation of the mutant. Patterns of extracted cell surface proteins analyzed both by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western ligand assays with various adhesive matrix molecules clearly confirmed the absence of Eap in the mutant. However, binding and adhesion tests using whole staphylococcal cells demonstrated that both the parent and mutant strains bound equally well to fibronectin- and fibrinogen-coated surfaces, possibly due to their recognition by other staphylococcal adhesins. Furthermore, Eap mediated staphylococcal agglutination of both wild-type and mutant cells. In contrast, the mutant adhered to a significantly lesser extent to cultured fibroblasts (P < 0.001) than did the wild type, while adherence was restorable upon complementation. Furthermore, adherence to both epithelial cells (P < 0.05) and fibroblasts (not significant) could be blocked with antibodies against Eap, whereas preimmune serum was not active. In conclusion, Eap may contribute to pathogenicity by promoting adhesion of whole staphylococcal cells to complex eukaryotic substrates.


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INTRODUCTION
 
Staphylococcus aureus continues to be a major cause of human disease, accounting for superficial skin infections as well as for serious invasive infections, such as endocarditis, osteomyelitis, and septic shock (22). Adherence of S. aureus to components of host tissues is an important first step in colonization and subsequent infection (10, 35) and is mediated by specific interactions between adhesins on the bacterial cell surface and host cell receptors (6). In addition to the well-characterized bacterial surface-located proteins (28) conferring adhesion to various extracellular matrix proteins (6) and invasion of eukaryotic cells (1, 31, 32), other adhesive S. aureus proteins are secreted. Three of these molecules, i.e., coagulase (29), the extracellular fibrinogen (Fg)-binding protein Efb (26), and the extracellular adherence protein Eap (24), have been shown to bind Fg. Eap of S. aureus Newman has been cloned and sequenced (14) and has previously been shown to bind to additional plasma proteins, including fibronectin (Fn) and prothrombin (Pt) (24). Eap can form oligomers, and by rebinding to the staphylococcal cell surface, it mediates bacterial agglutination. It also enhances binding to epithelial cells and fibroblasts by its dual affinity for eukaryotic components and the S. aureus surface (24). While these observations have been made with purified Eap, further precision in describing the role of Eap, as well as of the related molecule Map (for major histocompatibility complex class II analogous protein [19]), in intact S. aureus cells has been hampered by the lack of availability of defined Eap-negative mutants. Thus, we have constructed an eap-deficient mutant by allelic replacement, and here we report the genotypic and phenotypic characteristics of the mutant compared to the parent strain.


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MATERIALS AND METHODS
 
Bacterial strains and media. S. aureus Newman (kindly provided by T. Foster, Dublin, Ireland) was used to generate the {Delta}eap mutant. Recombinant plasmids cloned in Escherichia coli were passaged in a restriction-negative S. aureus strain, SA113 (16), before electroporation to S. aureus Newman. Staphylococcus carnosus TM300 (11) was used as an intermediate host in the construction of a complemented strain. The following strains of E. coli were used as cloning hosts: E. coli DH5{alpha}, E. coli TG1, and E. coli SCS 110 (Stratagene, La Jolla, Calif.).

For cultivation of S. aureus, tryptic soy broth and agar (Difco, Detroit, Mich.), brain heart broth and agar (Merck, Darmstadt, Germany), Mueller-Hinton broth and agar (Mast, Merseyside, United Kingdom), and Luria-Bertani (LB) broth and agar (Difco) were used. For cultivation of E. coli, LB broth and agar were used.

Solubilization of staphylococcal cell surface proteins. Cells were grown in 5 ml of brain heart infusion for 18 h and then centrifuged at 6,000 x g for 5 min. The pellet was resuspended in 500 µl of 2% sodium dodecyl sulfate (SDS) (Sigma, St. Louis, Mo.), heated at 95°C for 5 min, and then centrifuged at 10,000 x g for 5 min. The liquid supernatant was dialyzed against distilled water and stored at -20°C.

Western ligand blot analysis. Proteins separated by standard SDS-polyacrylamide gel electrophoresis (PAGE) were electrophoretically transferred (Trans-blot SD; Bio-Rad, Munich, Germany) onto a nitrocellulose membrane (Schleicher & Schüll, Dassel, Germany) blocked with 3% bovine serum albumin (BSA). Fn (Chemicon, Temecula, Calif.), Fg (Calbiochem, San Diego, Calif.), vitronectin (Vn) (prepared as previously published [15, 37]), and collagen (Cn) (type I; product no. C7774; Sigma) were separately labeled with biotin (Boehringer Mannheim GmbH, Mannheim, Germany), used to probe blotted proteins on nitrocellulose membranes, and subsequently detected in an avidin-alkaline phosphatase reaction (Bio-Rad). Crude staphylococcal supernatants were subjected to Western immunoblotting. Probing was done with sheep serum against Eap, followed by rabbit anti-sheep horseradish peroxidase-conjugated antibodies.

DNA manipulations and transformations. Manipulations were performed according to standard procedures (30). S. aureus cells were lysed with lysostaphin (20 U/ml; Ambicin L, recombinant; Applied Micro Inc., New York, N.Y.), and chromosomal DNA was prepared using the QIAmp blood kit (Qiagen, Hilden, Germany). Plasmid DNA was prepared using the Qiagen plasmid kit. DNA fragments were isolated from agarose gels using the QIAquick gel extraction kit (Qiagen). Selection for resistance to antibiotics in E. coli or S. aureus was performed with ampicillin (100 µg/ml; Sigma), erythromycin (10 µg/ml; Serva, Heidelberg, Germany), and chloramphenicol (10 µg/ml; Serva).

Construction of an Eap-deficient mutant. (i) Amplification of the eap gene. eap from chromosomal DNA of S. aureus strain Newman was amplified by PCR. Primer sequences derived from the map gene (19) were as follows: primer PI, 5' CTC GGA TCC ATG AAA TTT AAG TCA TTG ATT ACA ACA ACA TTA GCA TTA GG 3' (upper primer, including nucleotides 71 to 110; the BamHI restriction site is underlined), and primer PII, 5' CTC GGT ACC TTA AAA TTT AAT TTC AAT GTC TAC TTT TTT AAT GTC 3' (lower primer, including nucleotides 2107 to 2140; the KpnI restriction site is underlined). The PCR mixture contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl, 200 µM (each) deoxynucleoside triphosphates, 100 picomoles of each primer, 2.5 U of Ampli Taq DNA polymerase, and 1 µg of template DNA. The PCR was carried out in an Omni Gene Thermocycler (Hybrid, Heidelberg, Germany), and temperature cycling consisted of an initial denaturing at 96°C for 4 min followed by 30 cycles, each cycle consisting of denaturing at 96°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 3 min. A 10% volume of the amplified product was analyzed in a 1% agarose gel.

(ii) Cloning of eap. The eap PCR product and the cloning vector pUC 18 were separately restricted with BamHI and KpnI, and restriction enzymes were removed using the Qiagen PCR purification kit. The restricted PCR product was then ligated into pUC 18. Freshly prepared competent cells of E. coli DH5{alpha} were transformed with the ligation mixture, and the transformed cells were plated on LB plates containing ampicillin, IPTG (isopropyl-ß-D-thiogalactopyranoside; Sigma), and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (Sigma). One representative plasmid containing the amplified PCR product of eap was designated pMH1.

(iii) Insertion of ermB. The plasmid pEC4 (composed of pUC19 and the staphylococcal transposon Tn551) (2) containing the erythromycin cassette ermB was restricted with HpaI and SmaI. A 1.4-kb fragment containing the erythromycin cassette was isolated using the QIAquick gel extraction kit and ligated with HpaI-linearized pMH1. The HpaI restriction site is located in the eap gene at nucleotide 426. The ligation mixture was transformed in E. coli SCS110 and then plated on LB plates containing ampicillin and erythromycin. One representative plasmid conferring resistance to both antibiotics was designated pMH2.

(iv) Construction of a shuttle vector containing eap::ermB. The eap::ermB fragment was isolated from pMH2 as a 3.4-kb fragment by restriction with SstI and XbaI and ligated with vector pBT9 carrying a temperature-sensitive replicon for staphylococci. Plasmid pBT9, composed of parts of pBR322 and pTV1ts (38), is a shuttle vector able to replicate in E. coli and staphylococci. E. coli TG1 was transformed with the ligation mixture and plated on LB plates containing ampicillin, erythromycin, and chloramphenicol. One representative plasmid conferring resistance to all three antibiotics was designated pMH3.

(v) Inactivation of eap. Plasmid pMH3 from E. coli was first propagated in the restriction-deficient S. aureus SA113. One representative plasmid was isolated and designated pMH4. Plasmid pMH4 was then transformed into S. aureus strain Newman by electroporation (21), and a transformant clone containing pMH4 was selected. For construction of an eap allelic-replacement mutant, the method described by Palma et al. (25) was used. S. aureus Newman(pMH4) was cultivated overnight in LB medium in the presence of erythromycin (10 µg/ml) and chloramphenicol (10 µg/ml) with shaking at 32°C. The overnight culture was reinoculated (1:20) into LB medium containing only erythromycin and grown at 43°C overnight, thus selecting for clones with plasmid integration by single recombination. The culture was reinoculated (1:20) in LB medium and grown at 43°C for 24 h without antibiotic presence, selecting for stable insertions by a second recombinational event, resulting in stable erythromycin resistance due to chromosomal integration of the eap gene and concomitant loss of the chloramphenicol resistance. Various dilutions from this culture were incubated at 43°C on LB plates containing erythromycin (10 µg/ml). Chloramphenicol-sensitive and erythromycin-resistant colonies were detected by replica plating onto plates containing chloramphenicol or erythromycin. A clone designated AH12 (eap::Eryr), sensitive to chloramphenicol and resistant to erythromycin, was taken for further analysis.

(vi) Confirmation of the eap::Eryr allele replacement. Genomic DNAs from S. aureus strain Newman and mutant AH12 (mAH12) were extracted using a Qiagen DNA purification kit. Forward primer PIV (5' CTC GGA TCC ATC ATA AAA AAG GAG TGA TAA TTT 3', including nucleotide sequence 43 to 66 of eap of S. aureus Newman [EMBL accession no. AJ132841]) and reverse primer PII were used. PCR amplification by 30 repeated cycles was performed with an annealing at 55°C and an elongation time of 3 min at 72°C. From S. aureus Newman and the eap mutant mAH12, amplification products of 2.1 and 3.4 kb, respectively, were expected. Plasmid pMH4 does not give any amplification, since the primer PIII sequence is upstream of the eap gene and not included in pMH4.

Complementation of S. aureus mAH12. A PCR product of eap from genomic DNA of S. aureus Newman, including the ribosomal binding site, was prepared using primers PIII (5' CTC GGA TCC AAG GAG TGA TAA TTT ATG AAA TTT AAG TC 3', including nucleotide sequence 52 to 80 of eap of S. aureus Newman [EMBL accession no. AJ132841]; the BamHI restriction site is underlined) and PII and ligated into pCX19. Plasmid pCXI9 is a derivative of the xylose-inducible expression vector pCXI5 (36). The ligation mixture was transformed into S. carnosus protoplasts (12). A representative plasmid containing eap as an insert was designated pCXEap, and accordingly, the transformed S. carnosus strain was designated TM300(pCXEap). The plasmid pCXEap was isolated from S. carnosus and transformed by electroporation into mAH12. Transformants were grown on tryptic soy agar plates (containing 10 µg of chloramphenicol and 10 µg of erythromycin per ml), and one representative clone expressing Eap upon xylose induction (as detailed above for expression of recombinant Eap) was designated mAH12(pCXEap).

Binding of Eap to staphylococci. 125I-labeled Eap (IODO-Gen, precoated tube; Pierce, Rockford, Ill.) (the radioiodination procedure was performed as described by the manufacturer) ranging from 0.02 to 1.4 µg was added to a 100-µl volume containing 7 x 106 CFU in phosphate-buffered saline (PBS). The mixtures were incubated with shaking at room temperature for 60 min, and the cells were collected by centrifugation at 10,000 x g for 10 min. The liquid supernatants were discarded, the pellets were washed, and the radioactivity associated with the pellets was measured in a gamma counter. The specific iodination was determined by measuring the 125I activity in a known amount of Eap.

Adherence of bacteria to immobilized Eap. Microtiter plates were coated overnight at room temperature with 100 µl of Eap (40 µg/ml). After being coated, the wells were blocked by addition of 100 µl of 2% BSA for 1 h at 37°C. After three washes with phosphate-buffered saline containing 0.05% Tween 20 (PBST), a 100-µl bacterial suspension (S. aureus Newman, mutant mAH12, Bacillus subtilis, E. coli, or Streptococcus mutans) containing 6 x 108 CFU in PBST was added, and after 2 h of incubation at 37°C, the wells were washed three times with PBST. The bacteria were fixed for 40 min at 56°C. After the bacteria were fixed, 100 µl of crystal violet (0.65% [wt/vol]) was added to the wells, and the plates were incubated for 45 min at 37°C. The wells were washed six times with PBST. After the wells were washed, 100 µl of citric buffer (pH 4) was added to the wells, and the plates were incubated for 10 min at 37°C. The number of bound bacteria, reflected by the amount of crystal violet present in the wells, was measured with a spectrophotometer at an absorbance at 540 nm. Background values due to binding of bacteria to the BSA or to the wells themselves were obtained by omitting Eap, and these values were subtracted.

Adherence to Fg- and Fn-coated surfaces. S. aureus strain Newman and the eap mutant mAH12 (eap::Eryr) were grown in LB medium overnight with shaking at 37°C. Comparisons of binding between Newman and the eap mutant mAH12 were performed by mixing the strains in 50:50 proportions, which were then added to microtiter wells coated with either Fg (20 µg/ml) or Fn (40 µg/ml) and subsequently blocked with 2% BSA for 1 h at 37°C. After adherence for 2 h at room temperature, the wells were washed three times with PBST, and adherent bacteria were detached from the wells by adding 100 µl of 10% trypsin. The bacteria were serially diluted and plated in triplicate on blood agar plates. At least 100 colonies were picked from the plates onto LB plates containing 4 µg of erythromycin/ml to determine the ratio between the two strains. The ratio was determined in nine separate experiments. The exact ratio between the two strains before adherence was determined in the same way. The clonal purity of colonies from the mixed incubation was checked for 20 colonies, which were found to be unmixed, i.e., interstrain aggregation does not occur during adherence and plating.

Agglutination of bacteria by Eap. S. aureus Newman and the eap mutant mAH12 were grown in LB medium. After overnight growth, the strains were washed and suspended in PBS. A 40-µl bacterial suspension containing 6 x 108 CFU was placed on glass slides together with Eap at the final concentrations shown in Table 1. Agglutination was visible within 15 to 20 min at room temperature and was scored as follows: -, no agglutination; +, weak agglutination; ++, medium agglutination; and +++, strong agglutination.


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  		TABLE 1. Agglutination of bacteria in the presence of various concentrations of Eap

Binding of bacteria to fibroblasts. Fibroblasts (human fetal lung cells) were cultured in Eagle's medium (Gibco BRL Life Technologies, Paisley, Scotland) supplemented with 10% fetal calf serum (HyClone, Logan, Utah), HEPES buffer, {alpha}-glutamine, penicillin (100 U/ml), and streptomycin (100 U/ml). The fibroblasts were seeded (8 x 104 cells/ml) in 24-well culture plates (Costar, Cambridge, Mass.) and incubated at 37°C under 5% CO2. For determination of binding, the following standard assay was used. Upon reaching confluence, cells were washed with standard medium (Eagle's medium without supplements) prior to the experiment. After the cells were washed, 900 µl of standard medium was added. The cells were inoculated with 100 µl of bacteria (50 µl from each strain) adjusted to contain 107 bacteria per well. After incubation for 2 h at 37°C under 5% CO2, the wells were washed three times with PBS; 200 µl of 10% trypsin was added to the wells to detach the cells from the plates, and 800 µl of sterile water was added to lyse the cells. The ratio between the two strains was determined as described above in nine separate experiments. For determination of the adherence of the complemented mutant mAH12(pCXEap) to fibroblasts, the strain was grown in LB medium with 10 µg of chloramphenicol/ml. After 2 h of incubation, xylose (0.5% final concentration) was added to induce expression of Eap. Wells with confluent fibroblast layers had to be incubated in this experiment with each of the strains separately, as reliable enumeration in the coincubation assay used for the mAH12 mutant was not possible due to the instability of the plasmid in the complemented mutant. Enumeration of the complemented strain was performed in the presence of chloramphenicol. To observe the effect of anti-Eap antibodies on adherence to fibroblasts, a 50-µl bacterial suspension (107 bacteria/ml) was preincubated for 30 min at 37°C with 50 µl of Eap antibodies (8 mg/ml). Control wells were inoculated with bacteria and preimmune immunoglobulin G (7 mg/ml). The immunoglobulin G was purified using protein G-Sepharose (Pharmacia, Uppsala, Sweden) following the procedure recommended by the manufacturer. The bacteria were then added to the fibroblasts grown to confluence in the wells. Adherence was determined as described above. Adherence to fibroblasts by strains Newman and mAH12 was also done in the presence of Eap: 50 µl of each strain was preincubated for 30 min at 37°C with 50 µl of Eap (80 µl/ml), and the bacteria were then added to the cells. Control wells were inoculated with bacteria preincubated without Eap.


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RESULTS
 
Construction of an Eap-deficient mutant. Staphylococcal surface proteins from strain Newman were extracted by 2% SDS, and a protein of 60 kDa corresponding to Eap was shown to be recognized in Western ligand assays by soluble Fg, Fn, Vn, and type I Cn. The N-terminal amino acid sequence of the protein was determined (AAKPL DKSSS SLHHG YSKVH VPY) and evaluated for homology with the SWISSPROT and TrEMBL databases (http://www2.ebi.ac.uk). A 100% homology to the 70-kDa outer membrane surface protein precursor from S. aureus ATCC 25923, a 78% homology to the major histocompatibility complex class II analogous protein from S. aureus FDA 574 (19), and a 74% homology to a 70-kDa outer surface protein from S. aureus Wood 46 (17) were revealed. These findings confirmed the presence of intact and functional Eap in S. aureus Newman, which was used in this study.

For generation of a genetically defined and stable {Delta}eap mutant, insertional deletion by allelic replacement was employed. map of S. aureus strain FDA 574 (19), comprised of a single open reading frame of 2,070 nucleotides, was used to synthesize two oligonucleotides for amplification of map-analogous eap from chromosomal DNA of S. aureus Newman by PCR. On an agarose gel, the PCR product appeared as a single band of ~2,100 nucleotides. An erythromycin cassette was inserted in the PCR product of eap and ligated into pBT9, a replication temperature-sensitive shuttle vector; propagated in S. aureus SA113; and then transformed into S. aureus Newman. The eap mutant was constructed by homologous recombination at the nonpermissive temperature with selection for erythromycin resistance and susceptibility to chloramphenicol.

Genetic characterization of mutant mAH12 (eap::Eryr). To address the possible role of Eap in the pathogenesis of staphylococcal infection, a stable and defined mutant was generated for further analysis. Figure 1 shows the map of eap as well as the positions of the primer pair PIV-PII and the expected sizes of the amplified PCR products. Insertion of the Eryr gene into eap was examined by PCR analysis using these primers as well as primers EryF (5' ATG AAC AAA AAT ATA AAA TAT TCT CAA AAC 3') and EryR (5' TTA TTT CCT CCC GTT AAA TAA TAG ATA AC 3'). These primers are designed from sequences of Tn551 (NCBI accession no. Y13600) and ermB (NCBI accession no. AF239773) of Streptococcus intermedius. The PCR amplification product of the parent strain Newman showed a band at a size corresponding to the expected size of 2.1 kb. As expected, the PCR product from the isogenic mutant Newman mAH12 revealed a 1.4-kb-larger band corresponding to the insertion of the erythromycin cassette, as shown in Fig. 1. This also confirms physical integration of eap::Eryr into the chromosome, since the PIV primer position is not included on the plasmid pMH4.



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  		FIG. 1. (A) Genetic map of eap showing primer positions for PCR. (B) PCR products amplified with primers PIV and PII or primers EryF and EryR from genomic DNA of strain Newman and mutant strain mAH12 and plasmid DNA of pMH4. Lanes 1 and 8 show 1-kb markers. WT, S. aureus Newman; mAH12, S. aureus mutant Newman AH12; SD, ribosome binding site.

Phenotypic characterization. Conventional microbiologic characterization, as well as biochemical comparison of the mutant with the parent strain, using three commercially available kits (ID 32 biochemical test kit and Staphyslide-test [Biomérieux, Marcy l'Etoile, France] and Pastorex Staph-Plus [Sanofi Diagnostic Pasteur, S.A., Marnes la Coquette, France]), revealed no differences between the eap mutant mAH12 and the parent strain (not shown). In Coomassie blue-stained SDS-PAGE gels, the protein band pattern of an SDS extract from the mutant was similar to that of the parent strain, except that a band of 60 kDa, corresponding to the size of Eap, was missing in the extract of the eap mutant (Fig. 2A). The Western affinity ligand blot analysis using biotinylated Fn, Fg, Vn, and Cn revealed the presence of Eap in the SDS extract of the parent strain but did not detect Eap in the SDS extract of the {Delta}eap deletion mutant (Fig. 2B). Control blots probed with avidin-alkaline phosphatase but without prior incubation with biotinylated ligands did not reveal band recognition (not shown). Western immunoblotting was performed using sheep serum directed against Eap. Eap could be detected in the culture supernatant of strain Newman; however, no Eap could be detected in supernatants from the eap mutant mAH12 (Fig. 2C). Additional analysis of Fg-binding proteins of strains Newman and mAH12 by affinity chromatography followed by ionic-exchange fast protein liquid chromatography and analysis of eluates by enzyme-linked immunosorbent assay and Western immunoblotting also failed to demonstrate Eap in mAH12 (not shown).



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  		FIG. 2. (A) Analysis of cell surface proteins extracted using 2% SDS and separated in an SDS-7.5% PAGE gel. WT, wild type. (B) Western ligand analyses of cell surface protein extracts. Nitrocellulose membranes with blotted proteins were probed with biotinylated Fn, Fg, Vn, or Cn, and protein-protein interaction was detected with avidin in an enzymatic color reaction. (C) Western immunoblot of Eap. Detection of Eap was done with sheep anti-Eap antibodies followed by anti-sheep conjugated antibodies. Lane 1, crude supernatant from strain Newman; lane 2, crude supernatant from strain mAH12 (eap::Eryr); lane 3, recombinant Eap. Protein A is seen in lanes 1 and 2.

Complementation of S. aureus mAH12. S. carnosus TM300 was complemented with eap and examined for expression of functional Eap. While Eap was not demonstrable in 2% SDS surface protein extracts of wild-type TM300 analyzed by SDS-PAGE, it was detected in extracts of xylose-induced TM300(pCXEap) cultures (not shown). Accordingly, S. aureus mAH12 complemented with eap was examined for expression of functional Eap (Fig. 3A). As expected, Eap recognizing Fg, Fn, Vn, and Cn in Western ligand blot analysis was found to be present in SDS extracts of mAH12(pCXEap) (Fig. 3B). Eap was detected in 2% SDS extracts of xylose-induced TM300(pCXEap) and mAH12(pCXEap) in Western immunoblots probed with anti-Eap antiserum (data not shown).



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  		FIG. 3. (A) Coomassie blue-stained SDS-PAGE gel of SDS extract from strain Newman (lane 1), mutant mAH12 (lane 2), and mAH12 complemented with eap (lane 3). (B) Western ligand blot analyses of cell surface proteins extracted from wild-type strain S. aureus Newman (W) and eap mutant mAH12 (C) complemented with eap. The arrow indicates Eap. The molecular mass standards are in kilodaltons.

Adherence of S. aureus Newman, mAH12, and mAH12(pCXEap). (i) Binding to soluble Eap. It was shown earlier that externally added Eap can rebind to S. aureus (24), either to endogenous surface-located Eap by an Eap-Eap interaction or to other components, one of which is a phosphatase (8). Radiolabeled Eap was added to strain Newman and to the eap mutant mAH12. Binding was found to be dose dependent, and no significant difference between the two strains could be seen, indicating that rebinding of Eap to S. aureus appears to be largely independent of endogenous Eap (Fig. 4).



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  		FIG. 4. Binding of radiolabeled Eap to Newman and mAH12 cells. The indicated amounts of labeled Eap were added to cells of strain Newman or mAH12. After the cells were washed, the amount of bound Eap was determined. Diamonds, Newman; squares, mAH12.

(ii) Binding of bacteria to Eap-coated surface. The comparable extents of binding of different strains to Eap were confirmed by coating microtiter plates with Eap (Fig. 5). Strains Newman and Newman mAH12 bound equally well to Eap. These results indicate that the binding of these strains appears to be largely independent of endogenous Eap, a finding further supported by the fact that other gram-positive bacterial species showed a similar extent of adhesion to Eap surfaces (Fig. 5).



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  		FIG. 5. Binding of bacterial strains to Eap immobilized on microtiter plate wells. Bacteria in suspensions were added to microtiter wells coated with Eap. After the plates were washed, the adherence of bacteria was assessed by crystal violet staining. Adherence is expressed as a percent in relation to that of strain Newman. The error bars show standard deviations; n = 9.

(iii) Adherence to immobilized Fn and Fg. In a coincubation assay, a 1:1 proportion of microorganisms from strains Newman and mutant mAH12 was added to Fg and Fn immobilized in microtiter wells. The bacteria were detached from the wells with trypsin, and the two strains were differentiated based on erythromycin resistance. After adherence of the bacteria to either Fg or Fn, the ratio between the strains remained the same as before adherence, indicating similar adherence properties (Fig. 6).



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  		FIG. 6. Relative binding of strains Newman and mAH12. The two strains were mixed in a 1:1 proportion and added to microtiter wells coated with Fg or Fn. After the plates were washed, the bound bacteria were released by trypsinization and plated on blood agar plates. Colonies were checked for erythromycin resistance by cultivation on erythromycin-containing plates to determine the proportion between the two strains. Hatched bars, proportion of strain Newman; open bars, proportion of mAH12. The error bars show standard deviations; n = 9.

(iv) Binding to fibroblasts. In an earlier study, it was shown that Eap externally added to fibroblasts and epithelial cells could enhance the binding of S. aureus Newman to such cells (24). A confluent layer of fibroblasts was inoculated with a mixture of S. aureus Newman and the mutant mAH12 and incubated for 2 h at room temperature. Bound bacteria were detached, and the ratio between the two strains was determined. After incubation with the fibroblasts, a significant difference in adherence (P < 0.001) of the wild type versus the mutant could be observed, with the wild type adhering to a greater extent (Fig. 7A). The complemented strain mAH12 (eap::Eryr) showed a significantly higher adherence (P < 0.01) versus the mutant strain mAH12 (Fig. 7B).



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  		FIG. 7. (A) Relative binding of strains Newman and mAH12 to confluent fibroblasts. The two strains were mixed in a 1:1 proportion and added to the fibroblasts. After adherence and washing, assessment of relative binding was done as for Fig. 6. The error bars show standard deviations; n = 9. ***, P < 0.001 (unpaired t test). (B) Binding of strain mAH12 (eap::Eryr) and strain mAH12(pCXEap) to confluent fibroblasts. The strains were added to fibroblasts separately. After adherence and washing, the percentage of bound bacteria was determined. The error bars show standard deviations; n = 12. **, P < 0.01 (unpaired t test).

(v) Adherence to fibroblasts in the presence of Eap. Addition of external Eap could significantly (P < 0.01) enhance the adherence of both strains Newman and mAH12 (Fig. 8).



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  		FIG. 8. Adherence of strains Newman and Newman mAH12 in the presence of externally added Eap. Confluent layers of fibroblasts were inoculated with either strain Newman or Newman mAH12 with or without Eap present and incubated for 2 h. The bacteria were detached, and a viable count was estimated. The error bars show standard deviations; n = 4. **, P < 0.01 (unpaired t test).

(vi) Adherence to fibroblasts in the presence of antibodies against Eap. For the adherence assay on fibroblasts or epithelial cells, strain Newman was pretreated with antibodies against Eap prior to addition to the cells. These antibodies significantly reduced the adherence of strain Newman to epithelial cells (P < 0.05) (Fig. 9A), while the adhesion to fibroblasts was also reduced, albeit not significantly due to a large variability of binding in the presence of preimmune serum (Fig. 9B).



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  		FIG. 9. Adherence of strain Newman in the presence of antibodies against Eap. Confluent layers of epithelial cells (A) or fibroblasts (B) were incubated with strain Newman and either with antibodies against Eap or with preimmune serum and incubated at 37°C for 2 h. The bacteria were detached, and viable counts were estimated. The error bars show standard deviations; n = 6. *, P < 0.05 (unpaired t test).

Aggregation assay. To elucidate the capacity of Eap to mediate the interaction between bacterial cells, we performed aggregation assays using purified Eap and washed S. aureus Newman cells or mAH12 cells. Although upon careful close examination, a low level of spontaneous agglutination could be demonstrated, agglutination could clearly be enhanced by the addition of increasing doses of Eap. At concentrations of 20 µg of Eap/ml and above, visible agglutination was found to be maximal (Table 1). Interestingly, no difference in agglutination between wild-type and mutant strains could be observed, suggesting that production of endogenous Eap is not necessary for the agglutination reaction.


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DISCUSSION
 
Here, we report the construction and characterization of a genetically defined S. aureus mutant with functional deletion of eap. Eap is an adhesive protein secreted by S. aureus Newman (24) with high homology with a number of proteins identified in various other S. aureus strains, such as Map (19), the partially sequenced 70-kDa protein precursor from S. aureus ATCC 25923 (EMBL accession no. X13404), and a 70-kDa outer surface binding protein (p70) from S. aureus Wood 46 (EMBL accession no. Y10419). These Eap analogs have been associated with adhesive interactions of S. aureus with a broad spectrum of host molecules, because in Western ligand blotting experiments they have been shown to bind bone sialo protein, Fn, Fg, Vn, thrombospondin (23), and osteopontin (19). Eap analogs not only possess broad-spectrum binding characteristics, in S. aureus surface protein extract analyses from strains such as FDA 574 (23) or Newman, but appear as the most prominent protein in SDS-PAGE. However, work with mutants deficient in fnbA and fnbB (13, 34), as well as with mutants deficient in clfA and clfB (5) or cna (27), clearly demonstrates that the latter adhesins are the major structures pivotal for functional binding of their respective matrix ligands.

In this respect, the successful construction of a {Delta}eap mutant for the first time allowed us to more precisely characterize the role of Eap. Since strain Newman expresses functional ClfA and ClfB (5), as well as FnbA and FnbB (20), it was not surprising to observe that mutant mAH12 interacted with immobilized Fg and Fn similarly to the wild type. Strain Newman, however, adheres less well to immobilized Fn (33), although it appears to express a functional Fn-binding protein(s) (20). While in these assays the contribution of Eap to staphylococcal adherence to Fn and Fg appeared to be minimal, its relative contribution may depend on the amount of Eap retained on the surface, a factor which varies with age and culture conditions (unpublished findings and reference 24). A mutant defective in both FnbA and FnbB (13) still shows residual binding to Fn (7), and it is possible that surface-associated Eap contributes to this binding.

Consequently, in intact staphylococcal cells, the interaction of Eap with extracellular matrix proteins may be masked by the interaction of other specific adhesins with their respective ligands. However, during binding of staphylococci to eukaryotic cells, additional molecules are thought to contribute to the interaction, such as integrins (18) or heat shock proteins (4), which in turn recognize either Fn (9) or Fg (3) or may bind directly to staphylococcal Fn-binding proteins (4). In a previous study, confirmed here, we could demonstrate that Eap increases attachment to fibroblasts and to epithelial cells, enhancing the adherence exerted by other staphylococcal binding functions (24). The enhancement effect exerted by Eap has been confirmed in this work for both the wild-type and the mAH12 strains. Here, we demonstrate that the {Delta}eap mutant adheres to cultivated fibroblasts to a significantly reduced extent compared to the wild type and that antibodies against Eap reduce this adherence. Binding of the mutant was restored upon complementation, making a polar effect of the mutation unlikely. This demonstrated effect of Eap could suggest that eukaryotic cellular receptors may specifically recognize staphylococcal Eap, but the presence of such receptors has not yet been experimentally proven.

Attachment to eukaryotic cells might be enhanced by the ability of the microorganisms to aggregate. Externally added Eap has been shown to aggregate staphylococci (24), and here we show that no discernible difference between the wild type and mutant could be detected with respect to binding of soluble Eap, adherence to immobilized Eap, or cell aggregation upon addition of external Eap. Therefore, the binding of Eap to staphylococci appears to occur upon interaction with components other than endogenous Eap (8).

In summary, characterization of the {Delta}eap mutant of S. aureus Newman presented here shows that Eap does not contribute significantly to adherence to Fn or Fg, but adherence to fibroblasts is significantly enhanced by Eap. The availability of this mutant will help to further elucidate the precise functional role of this multiply interactive, highly expressed staphylococcal molecule in additional in vivo analyses as well as in adequate animal models.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Collaborative Research Center 492, project B9; by a grant from the Swedish Medical Research Council (K2000-16X-12218-04B); and by Biostapro AB.

We are indebted to R. Brückner, Tübingen, Germany, for providing plasmids pEC4 and pBT9.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Microbiology, University of Muenster Hospital, Domagkstr. 10, D-48129 Muenster, Germany. Phone: 49-251-835 5357. Fax: 49-251-835 5350. E-mail: muzaffa{at}uni-muenster.de. Back

Editor: V. J. DiRita


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Infection and Immunity, June 2002, p. 2933-2940, Vol. 70, No. 6
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.6.2933-2940.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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