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Staphylococcus aureus continues to be a major pathogen, and adherence of S. aureus cells to host molecules is a prerequisite for tissue colonization and overt disease. In addition to the wall-bound MSCRAMM protein superfamily (for a review, see reference 15) conferring adhesion (4), cellular invasion (2, 17), and virulence (for a review, see reference 6), other adhesive S. aureus proteins are cell secreted and in part rebind to bacterial surface structures via mechanisms not yet clearly defined. Three of these molecules, i.e., coagulase (16), Efb (the extracellular fibrinogen [Fg] binding protein) (14), and a 60-kDa protein from S. aureus strain Newman (3) subsequently termed Eap (for extracellular adherence protein) (13) have been found to bind Fg. The size and function of Eap suggest close similarity with a group of 60- to 72-kDa proteins binding to a large spectrum of host molecules, including fibronectin (Fn), vitronectin (Vn), thrombospondin, bone sialoprotein, and collagen (11). The 72-kDa protein of strain FDA 574 has been cloned and sequenced and due to its subdomain homology with the major histocompatibility complex class II  chain has been termed Map (for major histocompatibility complex class II analog protein) (10). Similarity of Eap and Map has been further suggested upon comparison of the respective N-terminal sequences and amino acid compositions (13). While these studies suggest the presence and close similarity of Eap and Map analogs in different S. aureus laboratory strains, the degree of molecular homology has not yet been corroborated and furthermore, the presence of Eap and Map analogous sequences in clinical S. aureus and Staphylococcus epidermidis isolates has not yet been determined. Therefore, this study aimed to determine the prevalence of Eap and Map analogs in various clinical and laboratory strains, to further analyze their molecular nature, and to investigate the function of recombinant gene products.

Bacterial strains. Nine laboratory strains of S. aureusNewman D2C (ATCC 25904), Cowan 1 (ATCC 12598), SA113 (9), 8325-4 (12), 6850 (1), ATCC 12600, DSM 20044, DSM 10231, and Wood 46 (ATCC 10832)as well as 240 methicillin-susceptible S. aureus isolates derived from clinical specimens were tested in this study. These isolates were obtained either from blood (n = 100) or from the anterior nares (n = 140) during a German multicenter study (18). Only one isolate per patient was tested. In addition, two reference strains of S. epidermidis (ATCC 35984 and DSM 20044) as well as eight clinical S. epidermidis isolates from patients in the University Hospital of Muenster, Muenster, Germany were included. Isolates were identified at the species level as S. aureus or S. epidermidis using standard microbiological methods including commercial phenotypic testing kits (Api Staph ID 32; BioMerieux, Marcy-l'Etoile, France).

Phenotypic characterization of Map and Eap analogs. Bacterial cell surface proteins were selectively extracted by heating bacteria in 2% sodium dodecyl sulfate (SDS) in 100 mM Tris-HCl, pH 6.8, without significant release of intracellular contents or cells lysis (8). Briefly, bacteria were grown overnight, pelleted, resuspended in 2% SDS (Sigma-Aldrich, Deisenhofen, Germany) in 125 mM Tris-HCl (pH 7.0), heated at 95°C (3 min), and then centrifuged (10,000 × g, 5 min). The supernatant was dialyzed against distilled water to remove SDS and stored at 20°C. Human Fn (Chemicon, Temecula, Calif.), human Fg (Calbiochem, San Diego, Calif.), and Vn (purified according to the method presented in reference 19) were labeled with biotin (Boehringer, Mannheim, Germany) according to the instructions of the supplier. For Western ligand analysis, the cell surface extracts of S. aureus strains Newman, Cowan 1, Wood 46, 8325-4, and SA113 separated by SDS-polyacrylamide gel electrophoresis (PAGE) were blotted electrophoretically onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany), membranes were blocked with 3% bovine serum albumin, and proteins were probed with biotinylated Vn and Fn. A protein running between markers of 70 and 86 kDa was found in extracts of strain Wood 46, while in extracts of strains Newman, 8325-4, Cowan, and SA113, a protein close to the marker of 70 kDa was detected after incubation with Fn and Vn (Fig. 1). The 70-kDa protein of strain Newman (presumably corresponding to previously published Eap) was detected in remarkably large amounts and confirmed to be a Map analog based upon sequence comparison of the 18-amino-acid N-terminal sequence (AAKPL DKSSS SLHHG YSK; 89% identity to Map from S. aureus FDA 574 [EMBL accession no. U20503], 95.6% identity to a partially [121 amino acids] sequenced 70-kDa outer membrane protein precursor from S. aureus ATCC 25923 [EMBL accession no. X13404], and 74% identity to a 70-kDa outer surface binding protein [p70] from S. aureus Wood 46 [EMBL accession no. Y10419]).

View larger version (50K): [in this window] [in a new window]   FIG. 1.   Western ligand blot analysis with biotinylated Fn and Vn of S. aureus cell surface proteins. Cell surface proteins were extracted by boiling whole cells in 100 mM Tris-HCl, pH 6.8, containing 2% SDS. After separation by SDS-PAGE, proteins were blotted onto a nitrocellulose membrane and probed with biotin-labeled Fn (A) and Vn (B), and bands were visualized in a color reaction using avidin alkaline phosphatase. From left to right, lanes contain Newman, Wood 46, 8325-4, Cowan 1, and SA113. Upper arrow in each panel, Eap of Wood 46; lower arrow in each panel, Eap of the other strains tested.

Incidence and polymorphism of map analogs in clinical staphylococcal isolates. For isolation of genomic DNA, staphylococci from 0.5 ml of an overnight brain heart infusion culture were pelleted by centrifugation at 5,000 × g for 20 min, resuspended in 180 µl of TE buffer (20 mM Tris chloride, 2 mM EDTA [pH 8.0]) with 7.5 µl of recombinant lysostaphin (5 mg/ml; Sigma-Aldrich) by vortex mixing, and incubated at 37°C for 30 min. DNA was subsequently extracted with a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Nucleic acid samples were eluted with distilled water and adjusted to a final concentration of 1 µg/ml according to A₂₆₀ values. The PCR amplification was performed in a thermal cycler with a hot bonnet (iCycler; Bio-Rad, Munich, Germany). The PCR mixture consisted of 1 µg of DNA; 10 mM Tris-HCl, pH 8.3; 10 mM KCl; 3 mM MgCl₂; a 1 µM concentration of the primer; a 200 µM concentration (each) of dATP, dCTP, dGTP, and dTTP; 5 U of Taq DNA polymerase (QBIOgene, Heidelberg, Germany); and double-distilled water added to a final volume of 50 µl. A total of 30 PCR cycles were run under the following conditions: DNA denaturation at 95°C for 1 min (5 min for the first cycle), primer annealing at 50°C for 1 min, and DNA extension at 72°C for 2 min. After the final cycle, the reaction was terminated by keeping it at 72°C for 10 min. Ten microliters of the product was analyzed on 1% ethidium bromide-stained agarose gel.

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Agarose (1%) gel stained with ethidium bromide showing the PCR product amplified with the eap and map primers P2 and P3 from genomic DNA of S. aureus as described in Methods and Materials. Lanes: Mr, DNA molecular size marker (combined 100-bp and 1-kb DNA ladder [New England Biolabs]); 1, S. epidermidis DSM 20044; 2, S. aureus clinical isolate 7 (1.8 kb); 3, S. aureus Newman (2.0 kb); 4, S. aureus Wood 46 corresponding to group III (2.4 kb).

Sequencing of eap-N, eap-7, and eap-W. Plasmid DNA was prepared using the Qiagen plasmid Mini kit and used as a template for sequencing on an ABI 310 genetic analyzer. Sequencing was started with the M13 reverse and the T7 standard primers of the vector upstream and downstream of insert DNA, respectively, and continued by primer walking, with subsequent primers being synthesized according to obtained sequence information. Nucleotide and deduced protein sequences were analyzed with the program of the European Molecular Biology Laboratory (EMBL) outstation European Bioinformatic Institute (EBI) (Cambridge, United Kingdom, http://www.ebi.ac.uk/). We have determined the complete nucleotide sequences encoding Eap from strain Newman 2DC (eap-N), from clinical isolate 7 (eap-7), and from strain Wood 46 (eap-W). In these sequences, the putative translation start codon ATG is preceded by a putative ribosomal binding sequence characteristic of gram-positive bacteria. The first 30 amino acids have features typical of a bacterial signal peptide. Sequence data are summarized in Table 2. The deduced amino acids sequence of Eap-W and the deposited sequence of mature peptide of p70 are almost identical (99%), confirming the sequencing results of strain Wood 46 from two different laboratories. The deposited sequence of p70 is lacking a signal peptide sequence, and the eap-W sequence now provides the complete sequence information on Eap of Wood 46. The deposited nucleotide sequence of strain Newman (Kreikemeyer et al., EMBL accession no. AJ223806 [for brevity, in this report, it is designated map-N]) is lacking a part of the 5' sequence, yet on the protein level this sequence is 98% identical to the complete Eap-N sequence presented here.

View larger version (53K): [in this window] [in a new window]   FIG. 3.   Alignment of the deduced amino acid sequences of Eap-N, Eap-7, and Eap-W with published sequences of Map FDA574, p70, and Map-N. Alignment was determined with the analysis program, multiple sequence alignments 2CLUSTAL W (version 1.74), available at http://www2.ebi.ac.uk. Asterisks indicate consensus amino acids; dots indicate gaps introduced to maximize alignment. References and EBL data bank accession numbers are detailed in text.

View larger version (65K): [in this window] [in a new window]   FIG. 4.   Alignment of the deduced amino acid sequences of map FDA574, Eap-7, Eap-N, and Eap-W. Alignment was performed after deleting one A residue at nucleotide 1792 in eap-N (referred to as Eap-N_8A) and 2112 in eap-W (Eap-W_8A) with the sequence analysis program as described in Fig. 3.

Expression and functional analysis of recombinant eap eap-N, eap-7, and eap-W (lacking the signal peptide) were amplified by PCR with upper primer P3 for eap-N and eap-7 or upper primer P4 for eap-W and lower primer P2. PCR products were ligated into the QIAexpress pQE30 vector (Qiagen). The ligation mixture was then transformed in E. coli M-15. Six-His-tagged Map fusion proteins were expressed and purified according to the protocol provided by the manufacturer (Qiagen). The expression of His-tagged recombinant Eap using vector pQE30 in E. coli M15 allows single-step purification using Ni-nitrilotriacetic acid affinity resin. SDS-PAGE of recombinant Eap (Eap-W, 74 kDa; Eap-N, 65 kDa [in some expression and purification experiments, 74-kDa proteins were observed] [Fig. 5]; Eap-7, 65 kDa) revealed proteins of similar size compared to the Eap analogs from the respective S. aureus strains extracted with 2% SDS. The recombinant proteins also showed binding to biotin-labeled Fn, Fg, and Vn in Western ligand blots (Fig. 5). Control blots without ligands but with alkaline phosphatase-conjugated avidin did not reveal a signal.

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Western ligand blot analysis of recombinant proteins Eap-7 (lanes a), Eap-N (lanes b), and Eap-W (lanes c) with Fn, Fg, and Vn. Recombinant proteins were affinity purified as described in the text. After separation by SDS-PAGE, proteins were blotted onto nitrocellulose membrane and probed with biotin-labeled ligands, and spots were visualized by alkaline phosphatase reaction. The values on the left are molecular masses in kilodaltons. Note that in this experiment migration of Eap-N corresponds to a larger protein size than that of Eap-N in Fig. 1 (see also text).

Expression and localization of Eap analogs in clinical staphylococcal isolates. To confirm the expression of eap in isolates with a positive PCR for eap, Western immunoblots were probed with anti-Eap antibodies developed against recombinant Eap-N. Polyclonal antibodies were raised in two rabbits according to the method presented in reference 7 by injecting 50 µg of Ni-nitrilotriacetic acid-purified Eap-N and two consecutive booster injections. Naturally occurring antistaphylococcal antibodies were immunodepleted by mixing serum with 100 volumes of 2% SDS extract from a clinical isolate which does not contain eap as determined by the PCR analysis method and SDS-PAGE. SDS-PAGE-separated S. aureus surface proteins extracted with 2% SDS-containing buffer by heating at 95°C for 3 min were blotted onto a nitrocellulose membrane and probed with antiserum (dilution, 1:10,000), and blots were developed using alkaline phosphatase-conjugated goat anti-rabbit immunoglobulins (dilution 1:3,000; Dako A/S, Glostrup, Denmark). Of 20 clinical isolates tested, Eap expression was observed in 14 isolates belonging to groups I and II as determined by PCR analysis. Variation in the amount of Eap expression was observed, with maximum protein production by strain Newman (Fig. 6).

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Western immunoblot analysis of SDS surface protein extracts from three S. aureus isolates with anti-Eap antibodies. Lane 1, S. aureus Newman; lane 2, S. aureus isolate 7; lane 3, S. aureus Wood 46.

Nucleotide sequence accession numbers. The complete nucleotide sequences encoding Eap from strain Newman 2DC (eap-N), from clinical isolate 7 (eap-7), and from strain Wood 46 (eap-W) are deposited in the EMBL database under the following accession numbers: eap-N, AJ290973; eap-7, AJ243790; eap-W, AJ245439.