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Research Article

Identification and characterization of a novel autolysin (Aae) with adhesive properties from Staphylococcus epidermidis

Christine Heilmann, Günther Thumm, Gursharan S. Chhatwal, Jörg Hartleib, Andreas Uekötter, Georg Peters

  • 1Institute of Medical Microbiology, University of Münster, Domagkstr. 10, D-48149 Münster, Germany
  • 2Mikrobielle Genetik, University of Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany
  • 3Division of Microbiology, GBF-National Research Center for Biotechnology, Spielmannstr. 7, D-38106 Braunschweig, Germany
  • Correspondence
    Christine Heilmann
    heilmac{at}uni-muenster.de

    • Microbiology 2003; 149(10):2769–2778 · https://doi.org/10.1099/mic.0.26527-0

    View at publisher PubMed

    Abstract

    Staphylococcus epidermidis biofilm formation on polymer surfaces is considered a major pathogenicity factor in foreign-body-associated infections. Previously, the 148 kDa autolysin AtlE from S. epidermidis, which is involved in the initial attachment of the cells to polymer surfaces and also binds to the extracellular matrix protein vitronectin, was characterized. Here, the characterization of a novel autolysin/adhesin (Aae) in S. epidermidis is described. Aae was identified as a 35 kDa surface-associated protein that has bacteriolytic activity and binds vitronectin. Its N-terminal amino acid sequence was determined and the respective gene, aae, was cloned. DNA-sequence analysis revealed that aae encodes a deduced protein of 324 amino acids with a predicted molecular mass of 35 kDa. Aae contains three repetitive sequences in its N-terminal portion. These repeats comprise features of a putative peptidoglycan binding domain (LysM domain) found in a number of enzymes involved in cell-wall metabolism and also in some adhesins. Expression of aae by Escherichia coli and subsequent analysis revealed that Aae possesses bacteriolytic activity and adhesive properties. The interaction of Aae with fibrinogen, fibronectin and vitronectin was found to be dose-dependent and saturable and to occur with high affinity, by using the real-time Biomolecular Interaction Analysis (BIA). Aae binds to the Aα- and Bβ-chains of fibrinogen and to the 29 kDa N-terminal fragment of fibronectin. In conclusion, Aae is a surface-associated protein with bacteriolytic and adhesive properties representing a new member of the staphylococcal autolysin/adhesins potentially involved in colonization.

    • The EMBL/GenBank/DDBJ accession number for the aae DNA sequence reported in this article is AJ250905.

    INTRODUCTION

    Staphylococcus epidermidis is the major cause of infections associated with implanted medical devices, such as intravascular catheters, cerebrospinal fluid shunts, prosthetic heart valves, cardiac pacemakers and orthopaedic devices (Baddour et al., 1990; George et al., 1979; Inman et al., 1984; Karchmer et al., 1983; Kristinsson, 1989; Peters et al., 1982, 1984). The pathogenesis of foreign-body-associated infections is characterized by the ability of S. epidermidis to form a multilayered biofilm on the polymer surface. Biofilm formation is a two-step process that involves initial attachment of the bacteria to the polymer surface and subsequent proliferation of the bacteria leading to accumulation into multilayered cell clusters. The accumulation phase or intercellular adhesion is at least partly mediated by a polysaccharide adhesin (PIA) (Fey et al., 1999; Heilmann et al., 1996b; Mack et al., 1996). The direct interaction between bacteria and polymer surfaces plays a crucial role in the early stages of the adherence process in vitro and probably also in vivo. Additional factors are important in later stages of adherence in vivo, as implanted material rapidly becomes coated with host extracellular matrix and plasma proteins, such as fibronectin (Fn), fibrinogen (Fg), vitronectin (Vn), thrombospondin, collagen, von Willebrand factor, laminin and elastin (Cottonaro et al., 1981; Dickinson & Bisno, 1989; Kochwa et al., 1977). These host factors deposited on the implanted material could serve as specific receptors for colonizing bacteria (Chhatwal et al., 1987; Hartleib et al., 2000; Herrmann et al., 1988, 1991, 1997; Lopes et al., 1985). Accordingly, surface-bound Fn significantly promotes adherence of clinical isolates of coagulase-negative staphylococci. In contrast, adherence to immobilized Fg varies between S. epidermidis strains (Herrmann et al., 1988; Vaudaux et al., 1989). Recently, S. epidermidis genes encoding proteins that are involved in Fg-binding (fbe) (Nilsson et al., 1998; Pei & Flock, 2001) or Fn-binding (embp) (Williams et al., 2002) have been identified by using the phage display technique.

    We found that the surface-associated autolysin AtlE from S. epidermidis is involved in initial attachment of the cells to a polymer surface and thus in biofilm formation (Heilmann et al., 1996a, 1997). AtlE also binds to Vn, suggesting not only a role in colonizing polymer surfaces, but also in colonizing host-factor-coated material and host tissue. Thus, AtlE is a multifunctional, surface-associated protein having both enzymic (60 kDa amidase and 52 kDa glucosaminidase) and adhesive functions (Heilmann et al., 1997). Recent results demonstrated the importance of AtlE in S. epidermidis pathogenicity: the atlE mutant strain was significantly less virulent than the wild-type in an intravascular catheter-associated infection model in rats (Rupp et al., 2001). The staphylococcal autolysins Aas from Staphylococcus saprophyticus (Hell et al., 1998) and AtlC from Staphylococcus caprae (Allignet et al., 2002), which are homologous to AtlE and to Atl from Staphylococcus aureus (Foster, 1995; Oshida et al., 1995), respectively, have been found to bind to Fn. Aas also binds to sheep erythrocytes leading to haemagglutination (Hell et al., 1998). These results led to the proposal of a new class of staphylococcal adhesins – the autolysin/adhesins. Here, we describe the molecular characterization of Aae, a novel autolysin/adhesin from S. epidermidis having both bacteriolytic and adhesive properties.

    METHODS

    Bacterial strains, plasmids and media.

    The biofilm-forming clinical strain S. epidermidis O-47 that was used to isolate chromosomal DNA for amplification of the aae gene has been described previously (Heilmann et al., 1996a). For cloning of aae′, the vector pCR2.1 (Invitrogen) and Escherichia coli INVαF′ [F′ endA1 recA1 hsdR17 (

    Figure image not available in archive
    ) supE44 thi-1 gyrA96 relA1 ϕ80lacZΔM15 Δ(lacZYA–argF)U169 λ−] (Invitrogen) as the cloning host were used. For the expression of hexahistidine (His6)-tagged fusion proteins, the Qiaexpress vector pQE30 (Qiagen) and E. coli TG1 [supE hsdΔ5 thi Δ(lac–proAB) F′ (traD36 proAB+ lacIq lacZΔM15)] as the cloning host were used. The staphylococci were routinely cultivated in trypticase soy broth (TSB); trypticase soy agar contained 1·4 % agar. E. coli strains were grown in Luria–Bertani (LB) medium. LB agar contained 50 μg ampicillin ml−1 for selection of plasmids.

    DNA manipulations, transformation, PCR and DNA sequencing.

    DNA manipulations and transformation of E. coli were performed according to standard procedures (Sambrook et al., 1989). Plasmid DNA was isolated using the Qiagen Plasmid Kit according to the manufacturer's instructions (Qiagen). Chromosomal DNA from S. epidermidis was isolated according to the procedure of Marmur (1961). PCR was carried out with Taq DNA polymerase (Boehringer Mannheim) in accordance with the supplier's protocol. All primers were synthesized by MWG-Biotech. Primers CH62 (upper; 5′-GAG GAG GAT TTT AAA GTG C-3′) and CH69 (lower, internal; 5′-AAC ATG ACC ATA GTA ACC-3′) were used to amplify the aae′ gene from the chromosomal DNA of S. epidermidis O-47. For construction of His6–Aae and His6–Aae′ fusion proteins, primers CH95 (upper; 5′-CAG GGA TCC GCA ACA ACG CAT ACA GTA AAA AGT GG-3′; BamHI restriction site is underlined) and CH97 (lower; 5′-GTC CTG CAG CTT AAT GAA TAA ATT TGT AAT TTC TCA C-3′; PstI restriction site is underlined) were used for the amplification of aae, and primers CH95 (upper, see above) and CH96 (lower, internal; 5′-GTC CTG CAG AAC ATG ACC ATA GTA ACC-3′; PstI restriction site is underlined) were used for the amplification of aae′.

    For cloning the PCR-amplified aae′ gene into the vector pCR2.1, the TA cloning kit (Invitrogen) was used according to the manufacturer's instructions. DNA sequences of both strands of aae′ were determined by the dideoxy chain-termination method using the cycle sequencing protocol with the GeneAmp PCR system 2400 (Perkin Elmer) on an ABI Prism 310 Genetic analyser (Perkin Elmer) and by MWG-Biotech using a Li-COR DNA sequencer. To determine the whole DNA sequence of aae, sequencing of the S. epidermidis O-47 chromosomal DNA (Peschel et al., 1999) was done on a Li-COR DNA sequencer model 4000 L using primers CH107 (5′-GGGACAATGCATCAGCTGCTGATGG-3′), CH108 (5′-CATATTTCCAG GAGCTGCACTCCAG-3′) and CH109 (5′-TGTGCAATTGAGCCTACTGTAGG-3′).

    The DNA and deduced protein sequences were analysed using the program pc/gene (IntelliGenetics). The protein sequences were compared with those of known proteins using the programs blastp (Altschul et al., 1997) and fasta (Pearson & Lipman, 1988). The alignment was done using the program clustal w at the European Bioinformatics Institute ().

    Construction and purification of the His6–Aae and His6–Aae′ fusion proteins.

    For the construction of the His6-tagged fusion proteins, the above-mentioned primers were used to amplify the aae and aae′ genes without the nucleotide sequences encoding the signal peptides from genomic DNA of S. epidermidis O-47 introducing a BamHI site at the 5′ end and a PstI site at the 3′ end. The PCR-amplified fragments were restricted with BamHI and PstI and ligated with the vector pQE30, linearized by BamHI and PstI, so that the aae and aae′ genes are in-frame with the His codons. The respective His6-tagged fusion proteins were purified under denaturing conditions (8 M urea) using the Ni-NTA Spin Kit (Qiagen) according to the supplier's protocol. The yield of fusion proteins was in the range of 100–140 μg per 10 ml culture volume as determined by the Pierce BCA Protein Assay, which was performed according to the manufacturer's instructions (Pierce). For the use in Biacore experiments and ligand affinity blot analysis, the protein solutions were dialysed against 0·1 M phosphate buffer (pH 7·0) to remove the urea.

    Protein isolation, SDS-PAGE, ligand affinity blot analysis and N-terminal protein sequencing.

    Surface-associated proteins of S. epidermidis O-47 and mut1 were prepared from cultures that were grown overnight in brain–heart infusion (BHI) medium at 37 °C. The cells were harvested by centrifugation and the cell pellet was resuspended in 1 volume of Laemmli sample buffer and then heated for 5 min at 95 °C. Crude cell lysates of E. coli (pQaae) and E. coli (pQaae′) were prepared from non-induced and induced (addition of 1 mM IPTG and continued growth of 5 h) cultures. Additionally, as a negative control, a crude cell lysate was prepared from an induced culture of E. coli (pQE30). After centrifugation, 10 μl of the supernatant containing surface-associated staphylococcal proteins, 10 μl of the cell lysates from E. coli or 2–4 μl of purified proteins were subjected to SDS-PAGE (10 % separation gel and 4·5 % stacking gel). Proteins were stained with Coomassie brilliant blue R250 (0·1 %).

    To determine the binding domains of Fg and Fn to Aae, ligand affinity blot analysis was performed. For this, the disulfide bonds between the Fg chains (Aα-, Bβ- and γ-chains) were reduced by heating Fg (Calbiochem) in sample buffer (Bio-Rad) for 10 min at 95 °C. Human Fn (Roche) was protease-digested as described by McGavin et al. (1991). Briefly, Fn was diluted to 1 mg ml−1 in 25 mM Tris/HCl (pH 7·6), 50 mM NaCl, 2·5 mM CaCl2 and 0·5 mM EDTA and then digested with 5 μg thermolysin ml−1 (Calbiochem) for each 1 mg of Fn (2 h at room temperature). The reaction was stopped by the addition of EDTA to a final concentration of 5 mM. The Fg chains (5 μg per lane) or Fn fragments (5 μg per lane) were then separated by SDS-PAGE (10 or 12 % separation gel and 4·5 % stacking gel) and electrotransferred to a nitrocellulose membrane (Schleicher and Schuell) using a semi-dry-blot apparatus (Bio-Rad). Afterwards, the membranes were blocked in Tris-buffered saline/0·05 % Tween 20 (TBST)/5 % BSA overnight and washed three times with TBST. The membranes were then incubated with 40 μg purified Aae ml−1 in TBST/1 % BSA for 2–3 h at room temperature. As a negative control, incubation was performed in TBST/1 % BSA without a ligand or with 40 μg His6–dihydrofolate reductase ml−1 [purified from a culture of E. coli harbouring the control plasmid pQE40 (Qiagen)] in TBST/1 % BSA. Bound protein was detected by incubation with Ni-NTA–alkaline phosphatase conjugate (Qiagen) diluted 1 : 5000 in TBST/1 % BSA (1·5 h) and subsequent colour reaction (Bio-Rad).

    For N-terminal sequencing of Aae, surface proteins were separated by SDS-PAGE as described above and blotted onto a PVDF membrane (Millipore). After staining with Coomassie blue, the 35 kDa protein of strain mut1 was cut out and the N-terminal amino acid sequence was determined on an ABI 494-A automated sequencer (Applied Biosystems).

    Detection of bacteriolytic activity.

    The bacteriolytic activity of cell-surface-associated proteins was determined as described previously (Heilmann et al., 1997). Briefly, surface-associated proteins were isolated as described above and separated by SDS-PAGE on a polyacrylamide gel containing heat-inactivated Staphylococcus carnosus (0·05 %), S. epidermidis (0·05 %) or Micrococcus luteus cells (0·05 %) as a substrate in the separation gel (10 %). After electrophoresis, gels were washed for 30 min in distilled water and then incubated overnight in 0·1 M phosphate buffer (pH 7·0) at 37 °C. Bands with bacteriolytic activity were observed as clear zones in the opaque gel. After photography against a dark background, the clear zones appeared as dark bands.

    Real-time Biomolecular Interaction Analysis (BIA) for quantification of molecular interactions.

    Experiments were performed on a BIAcore 2000 instrument according to the general procedures recommended by the manufacturer of the instrument. Aae or Aae′ were immobilized on the sensor chip C1 to analyse their interaction with the extracellular matrix proteins Fg (Sigma), Fn (Chemicon) and Vn [purified from blood according to the protocol of Hayashi (1993); Aae′ only]. For immobilization, Aae or Aae′ was covalently coupled to the sensor chip surface C1 via primary amine groups. To prepare the surface of the sensor chip, a freshly prepared 0·1 M glycine/NaOH (pH 12)/0·3 % Triton X-100 solution at a flow rate of 20 μl min−1 was injected (2×30 μl). Afterwards, to activate the sensor chip, a mixture of 50 μl of 0·1 M N-hydroxysuccinimide/N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide (amine coupling kit; Biacore AB) was injected at a flow rate of 5 μl min−1. Each of the proteins Aae and Aae′ (∼50 μg ml−1) in 10 mM sodium acetate buffer (pH 5·0) was immobilized on the surface. Remaining N-hydroxysuccinimide-ester groups on the sensor chip surface were then blocked by injection of 55 μl of 1 M ethanolamine (pH 8·5) (amine coupling kit; Biacore AB). Non-specific binding of the extracellular matrix proteins to the sensor surface was avoided by coating the surface with 0·2 mg BSA ml−1 in HBS-buffer (10 mM HEPES, 150 mM NaCl, 3·4 mM EDTA, 0·005 % Surfactant pH 7·4) at flow rates of 30 μl min−1. HBS-buffer was also used as the running buffer. After Aae or Aae′ were immobilized on the C1 sensor chip surface (120 pg Aae per 0·26 mm2 and 130 pg Aae′ per 0·26 mm2), extracellular matrix proteins in increasing concentrations were injected over the sensor surface for the kinetic studies at a flow rate of 30 μl min−1. Binding of extracellular matrix proteins was monitored and presented in an overlay-plot of the sensorgrams (a plot of resonance unit versus time). Kinetic data were determined at 25 °C. For regeneration of the sensor chip surface after each injection, 10 mM NaOH was used resulting in a return to baseline. Kinetic data were analysed using BIA-evaluation software (version 3.1) from Biacore AB as described by Karlsson et al. (1991).

    RESULTS

    Identification of the Aae in S. epidermidis and determination of its N-terminal amino acid sequence

    A single band of 35 kDa with bacteriolytic activity was identified in the AtlE-negative mutant of S. epidermidis (mut1) using SDS-PAGE with heat-killed S. carnosus cells as the substrate in the separation gel (Heilmann et al., 1997). Western-ligand assays using biotin-labelled Vn revealed that a protein with the same molecular mass bound Vn (not shown). Therefore, the 35 kDa protein was termed Aae (autolysin/adhesin from S. epidermidis). To clone aae, the 35 kDa protein was isolated from mut1 and its N-terminal amino acid sequence was determined to be Ala-Thr-Thr-His-Thr-Val-Lys-Ser-Gly-Glu-Ser-Val-Trp.

    Cloning of the aae gene, nucleotide sequence analysis of aae and amino acid sequence analysis

    In the database of the unfinished genome of S. aureus COL from the Institute for Genomic Research (TIGR; ), an amino acid sequence that was 92 % identical to the N-terminal sequence of Aae was found. The respective ORF was analysed and primers were selected to amplify the respective gene from S. epidermidis O-47 genomic DNA. Only a 3′-internal primer that was used in the amplification reaction revealed a PCR product of 858 bp. The PCR-amplified DNA fragment was cloned into the vector pCR2.1 in E. coli, yielding the plasmid pCaae′. The nucleotide sequence of the truncated aae gene (aae′) was determined on both strands. The DNA sequence of the missing 3′ end of aae was determined by sequencing the chromosomal DNA of S. epidermidis O-47. aae consists of 972 nt encoding a deduced protein of 324 aa with a predicted molecular mass of 35 kDa. The truncated aae′ comprises an incomplete ORF of 843 nt encoding a deduced protein of 281 aa with a predicted molecular mass of 30 kDa. aae starts with the start codon GTG which is preceded by a putative ribosome-binding site. The predicted aae gene product is composed of 32·5 % hydrophobic, 9·6 % basic and 3·3 % acidic amino acids. The theoretical pI value of Aae is 9·86. Aae contains a putative signal peptide in the first 25 aa. The N-terminal amino acid sequence of Aae, determined by protein sequencing (see above), is predicted by the amino acid sequence deduced from the aae DNA sequence starting at Ala-26. According to the program hmmtop (), the mature protein is predicted to be located extracellularly. Sequence comparison of Aae with known proteins in databases revealed two domains. The N-terminal domain of Aae contains three direct repeated sequences. The repeats Re2 (62 aa, starting at Y-86) and Re3 (66 aa, starting at Y-149) are 60 % identical and the repeat Re1 (56 aa, starting at H-29) shares only 38 % identical amino acids with Re3 (Fig. 1⇓). The repeats are homologous to a consensus sequence that has been termed LysM domain (see below). The N-terminal domain of Aae is homologous to certain autolysins, i.e. 44 % identity to the cell-wall-associated 51 kDa endopeptidase LytF from Bacillus subtilis (Margot et al., 1999), 44 % identity to the putative 35 kDa endopeptidase LytE from B. subtilis (Margot et al., 1998), 38 % identity to the 70 kDa N-acetylmuramoyl-l-alanine amidase from Enterococcus faecalis (Beliveau et al., 1991) and 34 % identity to the 50 kDa invasion-associated protein P60 of Listeria species (Bubert et al., 1992). The C-terminal domain of Aae (starting at H-216) shows the highest similarity to the recently described, secretory, highly antigenic protein SsaA from S. epidermidis that is expressed in vivo (56 % identical amino acids) (Lang et al., 2000), to ORF1 from S. aureus (56 % identical amino acids; EMBL accession no. X97985) and to SceB from S. carnosus (55 % identical amino acids; EMBL accession no. U96107). SceB and probably also ORF1 are extracellular proteins. The functions of all three proteins remain unknown.

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    Fig. 1.

    Alignment of the deduced amino acid sequences of the repetitive sequences Re1, Re2 and Re3 of the autolysin/adhesin Aae from S. epidermidis O-47 (only the first 44 aa of the repeats are shown). The consensus sequence indicates the LysM domain that is proposed to have a general peptidoglycan-binding function. Letters in the second and third line of the consensus sequence indicate alternative amino acids. Highlighted letters indicate amino acids that match the consensus sequence.

    Construction, expression and purification of His6–Aae and His6–Aae′ fusion proteins in E. coli

    For expression of the aae and aae′ genes in E. coli, the PCR-amplified fragments were cloned into the BamHI and PstI sites of the expression vector pQE30 in E. coli TG1 resulting in an in-frame translation of the N-terminal His6-tag with the aae and aae′ genes. Representative clones expressing the aae and aae′ genes contained the plasmids pQaae and pQaae′, respectively. Subsequently, Aae and Aae′ were purified from E. coli cultures via their His6-tag using Ni-NTA affinity chromatography. SDS-PAGE of affinity-purified fusion proteins revealed a 35 kDa protein band (Aae) with E. coli (pQaae), which corresponded in size to the surface-associated Aae isolated from S. epidermidis O-47 and mut1 (Fig. 2⇓). The truncated 30 kDa Aae′ fusion protein purified from E. coli (pQaae′) was slightly smaller (Fig. 2⇓). The 35 and 30 kDa protein bands were absent in uninduced cultures from E. coli (pQaae) and E. coli (pQaae′), respectively, as well as in an induced culture of E. coli (pQE30) (not shown).

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    Fig. 2.

    SDS-PAGE (10 % separation gel) of His6-tagged Aae′ purified from E. coli (pQaae′) (lane 2), His6-tagged Aae purified from E. coli (pQaae) (lane 3), cell-surface-associated proteins of S. epidermidis O-47 (lane 4) and cell-surface-associated proteins of its AtlE-negative mutant mut1 (lane 5). The arrow indicates the 35 kDa Aae. The sizes of marker proteins (Bio-Rad prestained, lane 1) are indicated.

    Analysis of bacteriolytic activity of Aae and Aae′

    To analyse the bacteriolytic activities of Aae and Aae′, the His6-tagged proteins as well as cell-surface-associated proteins from S. epidermidis O-47 and mut1 were investigated using zymographic analysis: SDS-PAGE containing heat-inactivated S. carnosus, S. epidermidis or M. luteus cells as a substrate for lytic enzymes in the separation gel. Both Aae isolated from S. epidermidis O-47 and mut1 as well as the His6-tagged Aae purified from E. coli exhibited bacteriolytic activity when S. carnosus (Fig. 3⇓) or S. epidermidis cells (not shown) were used as a substrate. The truncated Aae′ purified from E. coli did not show bacteriolytic activity (Fig. 3⇓). No Aae-associated bacteriolytic activity was detected when M. luteus cells were used as a substrate in the separation gel (not shown).

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    Fig. 3.

    Zymographic analysis of the bacteriolytic activity of Aae. SDS-PAGE of His6-tagged Aae′ purified from E. coli (pQaae′) (lane 2), His6-tagged Aae purified from E. coli (pQaae) (lane 3), cell-surface-associated proteins of S. epidermidis O-47 (lane 4) and cell-surface-associated proteins of its AtlE-negative mutant mut1 (lane 5). The separation gel (10 %) contained heat-inactivated S. carnosus cells (0·05 %) as a substrate for bacteriolytic enzymes. Bacteriolytic activity is visible as a clearing zone after incubation in phosphate buffer. The arrow indicates Aae-associated bacteriolytic activity. The sizes of marker proteins (Bio-Rad prestained, lane 1) are indicated.

    Quantification of the Fg-, Fn- and Vn-binding activity of Aae and Aae′ as determined by real-time BIA

    BIA, based on optical detection of surface plasmon resonance, was performed to study the adhesive properties of the autolysin Aae and the truncated Aae′. In BIA, interactions are analysed between a ligand immobilized on a sensor chip and an analyte, which is in solution and flows over the surface of the chip. The use of this system allowed the study of the interaction of the autolysin (ligand) with the extracellular matrix protein Fg, Fn or Vn (analyte) without the need to label any of the proteins. After Aae or Aae′ was immobilized on the C1 chip, various concentrations of Fg (ranging from 27 to 328 nM), Fn (ranging from 23 to 227 nM) or Vn (ranging from 500 to 1300 nM) were sequentially injected over the immobilized proteins for the kinetic studies. The sensorgrams of the bindings of the matrix proteins to Aae and Aae′ were monitored as shown in Fig. 4⇓. The interactions of Aae and Aae′ with the extracellular matrix proteins were dose-dependent and saturable. In control experiments, Aae (not shown) and Aae′ did not bind to BSA (Fig. 4f⇓). From the quantitative data of the kinetics of these interactions, dissociation constants (Kd) were determined to be 4·0 nM for the interaction of Aae with Fg and 5·2 nM for the interaction of Aae with Fn. The Kd value was ascertained to be 3·5 nM for the binding of Aae′ to Fg and 13·9 nM for the binding of Aae′ to Fn. For the interaction of Aae′ with Vn, the Kd value was determined to be 149 nM.

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    Fig. 4.

    Determination of binding of Fg (a, c), Fn (b, d), Vn (e) and BSA (f) to immobilized Aae (a, b) and Aae′ (c, d, e, f) using the Biacore system. After Aae and Aae′ were immobilized on the C1 chip surface, extracellular matrix proteins were injected over the chip surface at a flow rate of 30 μl min−1. Binding of extracellular matrix proteins was monitored and presented in an overlay-plot of the sensorgrams [a plot of RU (resonance unit) versus time]. (a) Concentrations of Fg (from bottom to top): 27, 55, 82, 109, 137, 164, 192 and 328 nM. (b) Concentrations of Fn (from bottom to top): 23, 68, 114, 136, 159, 204 and 227 nM. (c) Concentrations of Fg (from bottom to top): 27, 55, 82, 137, 164, 192 and 328 nM. (d) Concentrations of Fn (from bottom to top): 23, 68, 114, 136, 159, 204 and 227 nM. (e) Concentrations of Vn (from bottom to top): 500, 600, 700, 800, 900, 1000, 1100, 1200 and 1300 nM. (f) Concentrations of BSA (from bottom to top): 0 nM, 100 nM, 500 nM, 1000 nM, 2000 nM, 5 μM and 10 μM.

    Identification of the Aae-binding domains of Fg and Fn by ligand affinity blot analysis

    Each Fg molecule consists of three pairs of non-identical polypeptide chains (AαBβγ) that are linked together in the central N-terminal domain by three interchain disulfide bridges (Doolittle, 1984). To identify the binding domains for Aae on Fg, the Aα- (67 kDa), Bβ- (55 kDa) and γ-chains (48 kDa) were separated by SDS-PAGE and analysed in a ligand affinity blot using His6–Aae as a probe followed by Ni-NTA–alkaline phosphatase conjugate. Fig. 5⇓ shows that Aae bound to the Bβ-chains and to the Aα-chains. In the negative controls, no significant binding of the Ni-NTA–alkaline phosphatase conjugate was detected. To identify the binding domain of Fn to Aae, human Fn was digested with the protease thermolysin, which produces a 29 kDa N-terminal Fn fragment and three larger polypeptide fragments with 40–45 kDa, 53–70 kDa and 120–140 kDa (Pankov & Yamada, 2002). In ligand affinity blot analysis, Aae was detected to bind to the 29 kDa Fn fragment (Fig. 6⇓). No significant binding was detected in the negative controls. However, the larger protein band that indicated binding of Aae represents the 37·5 kDa protease thermolysin that is present in the preparation as determined in control experiments (not shown).

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    Fig. 5.

    Determination of the binding domain of Fg to Aae by ligand affinity blot analysis. The Fg chains (Aα-, Bβ- and γ-chains) were separated by SDS-PAGE (10 % separation gel) (a, lane 2) and transferred to nitrocellulose filters that were probed with Aae (b, lane 3) or without a ligand (b, lane 2) followed by Ni-NTA–alkaline phosphatase conjugate. The sizes of marker proteins (MBI Fermentas prestained; a, b, lane 1) and the positions of the Aα-, Bβ- and γ-chains of Fg are indicated.

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    Fig. 6.

    Determination of the binding domain of Fn to Aae by ligand affinity blot analysis. Fn was proteolytically cleaved by thermolysin. Fn fragments were separated by SDS-PAGE (12 % separation gel) (a, lane 2) and transferred to nitrocellulose filters that were probed with Aae (b, lane 4), dihydrofolate reductase (b, lane 2) or without a ligand (b, lane 3) followed by Ni-NTA–alkaline phosphatase conjugate. The sizes of marker proteins (MBI Fermentas prestained; a, b, lane 1) are indicated. The arrow indicates the 29 kDa Fn fragment that interacted with Aae.

    DISCUSSION

    Bacterial autolysins are peptidoglycan hydrolases that play an important role in cell-wall turnover, cell division, cell separation and antibiotic-induced lysis of bacterial cells (Buist et al., 1995; Doyle et al., 1988; Höltje, 1996). Autolysins are also considered to be virulence factors. For instance, a number of autolysis-defective mutants exhibit less virulence in animal models than the corresponding wild-type strains, i.e. an autolysis-defective S. aureus mutant (Mani et al., 1994), a lytA-deletion mutant of Streptococcus (Str.) pneumoniae (Berry & Paton, 2000; Berry et al., 1989) and Listeria monocytogenes mutants that produce greatly reduced amounts of the lytic protein P60 (Kuhn & Goebel, 1989). The contribution of autolysins to virulence may be direct or indirect. The major autolysin LytA of Str. pneumoniae is thought to play an indirect role in the pathogenic process by mediating the release of pneumolysin and other inflammatory substances from the cytoplasm or the cell wall (Canvin et al., 1995; Diaz et al., 1992). However, recently it was suggested that the release of pneumolysin is independent of the activity of LytA (Balachandran et al., 2001). Some autolysins were reported to be involved in adherence suggesting a more direct role in the pathogenesis, i.e. the invasion-associated protein P60 has been proposed to be involved in the adherence (and invasion) of L. monocytogenes to mouse fibroblasts (Bubert et al., 1992; Kuhn & Goebel, 1989). Another autolysin from L. monocytogenes, Ami, also seems to function in the adherence to eukaryotic cells (Milohanic et al., 2000). During characterization of the multifunctional autolysin/adhesin AtlE from S. epidermidis (Heilmann et al., 1997), we identified another autolysin, which was distinct from AtlE. Because this 35 kDa autolysin was also found to bind Vn, it was named Aae (autolysin/adhesin from S. epidermidis). After determination of the N-terminal amino acid sequence of Aae, we cloned the corresponding gene, aae, as well as a 3′ deletion derivative, aae′, from S. epidermidis O-47.

    Aae has three repetitive sequences in its N-terminal portion which are highly homologous to a consensus sequence that is found in a variety of cell-wall-degrading enzymes (Fig. 1⇑). This consensus sequence has been termed LysM domain for Lysin motif and is thought to have a general peptidoglycan binding function (Pfam accession number of the LysM domain is Pf01476, available at and at ) (Bateman et al., 1999; Joris et al., 1992; Ponting et al., 1999). However, the precise binding partners and sites have not yet been determined. Lytic enzymes harbouring the LysM domain include the muramidase-2 of Enterococcus hirae (6 repeats), E. faecalis autolysin (4 repeats) and B. subtilis Φ PZA lysozyme (2 repeats); in contrast to Aae, these lysins contain the repeats in their C-terminal portions (Joris et al., 1992). The LysM domain has also been found in proteins which are not involved in cell-wall metabolism, but have been associated with adherence, such as the immunoglobulin binding protein A of S. aureus (one copy of the LysM domain located near the C terminus), the S. aureus elastin-binding protein EbpS (one copy of the LysM domain located in the C-terminal portion) (Downer et al., 2002) and intimin, which is an outer-membrane protein required for attachment of enterohaemorrhagic and enteropathogenic Escherichia coli to mammalian cells (one copy of the LysM domain located near the N terminus) (Bateman & Bycroft, 2000). The peptidoglycan-binding function of the LysM domain in Aae might be responsible for its surface-associated location, because Aae does not have a C-terminal anchor region with the LPXTG motif that is typical for surface proteins of Gram-positive bacteria (Schneewind et al., 1993). Other staphylococcal proteins that lack this anchor region and are surface-associated by a partially characterized or unknown mechanism include the homologous autolysins Atl, AtlE, Aas and AtlC (Allignet et al., 2002; Foster, 1995; Heilmann et al., 1997; Hell et al., 1998; Oshida et al., 1995), the elastin-binding protein EbpS, which is an integral membrane protein (Downer et al., 2002), the major histocompatibility complex class II analogous protein Map (Eap) (Flock & Flock, 2001; Hussain et al., 2002) and the extracellular matrix protein-binding protein Emp (Hussain et al., 2001).

    In ligand affinity blot analysis, Aae and Aae′ bound digoxigenin-labelled Fg, Fn and Vn (not shown). Using BIA, the interactions of Aae and Aae′ with Fg, Fn and Vn (established for Aae′ only) were found to be dose-dependent and saturable and binding occurred with high affinity. Ligand affinity blot analysis demonstrated that Aae binds to the Aα-chains as well as to the Bβ-chains of Fg. Likewise, the S. aureus clumping factor B (ClfB) binds to the Aα- and the Bβ-chains of Fg (Ni Eidhin et al., 1998). In contrast, the S. aureus clumping factor A (ClfA) binds to the γ-chain. Other known Fg-binding proteins include the extracellular Fg-binding protein Efb from S. aureus that binds to the Aα-chains (Palma et al., 2001) and Fbe from S. epidermidis that binds to the Bβ-chains (Pei et al., 1999). The domain of Fn that Aae interacts with is located within the 29 kDa N-terminal fragment of Fn that is produced after proteolytic cleavage. This domain contains five type 1 repeats and is a known target for binding of other fibronectin-binding proteins from S. aureus and Str. pyogenes (Schwarz-Linek et al., 2003).

    Aae isolated from S. epidermidis wild-type and mut1 as well as Aae purified from E. coli has bacteriolytic activity in contrast to the truncated Aae′. This indicated that the missing C-terminal part (43 aa) in Aae′ is required for bacteriolytic activity. In BIA, the binding of Aae′ to Fg and Fn is comparable to the binding of Aae. This suggested that the missing C-terminal part in Aae′ is not required for its adhesive properties. Therefore, the bacteriolytic and adhesive domains in Aae appear to be distinct.

    Muramidases and glucosaminidases cleave the glycosidic linkages of the peptidoglycan backbone; amidases cleave the amide bond between N-acetylmuramic acid and the l-alanine that forms part of the peptide cross-bridge. The 60 kDa amidase and the 52 kDa glucosaminidase that are cleavage products of the AtlE precursor molecule exhibit bacteriolytic activity against S. carnosus as well as against M. luteus cells; however, the glucosaminidase is much less active against staphylococcal cells than against M. luteus cells (Heilmann et al., 1997). Aae exhibits lytic activity against S. carnosus and S. epidermidis cells, but not against M. luteus cells. Therefore, it probably does not have amidase or glucosaminidase activity. Further analyses are warranted to identify the enzymic activity of Aae.

    Aae is an autolysin/adhesin with multiple binding functions. This is becoming a common theme among virulence factors. Other proteins with multiple binding functions include the well characterized Fn-binding protein FnBPA from S. aureus, which has been reported to possess a second binding function to Fg (Wann et al., 2000), Map (Eap) (Hussain et al., 2002; Palma et al., 1999) and the extracellular matrix protein-binding protein Emp (Hussain et al., 2001).

    The concept of multifunctional proteins exhibiting enzymic and adhesive properties has not only been found with autolysin/adhesins, but also with other surface-associated proteins, i.e. the serum opacity factor (SOF) which binds Fn and acts as an apolipoproteinase leading to serum opacity (Kreikemeyer et al., 1999; Rakonjac et al., 1995) and a glycerolaldehyde-3-phosphate dehydrogenase with multiple binding activities (Pancholi & Fischetti, 1993).

    In conclusion, we here report the molecular characterization of a new member of the staphylococcal autolysin/adhesins, Aae, which has bacteriolytic and adhesive properties and might be involved in colonization of host-factor-coated material or host tissue by S. epidermidis. Further analyses are necessary to identify its precise enzymic activity. Moreover, a site-directed aae deletion mutant will be constructed and analysed in animal models to determine the role that the autolysin/adhesin plays in S. epidermidis pathogenicity.

    Acknowledgments

    We thank B. Specht (Inventus BioTec, Münster, Germany) for performing the BIA and S. Weber and U. Hörling for excellent technical assistance. Sequence data for identification and cloning of the aae gene were obtained from the Institute for Genomic Research (). The work was partially supported by the German National Research Foundation for the Specialized Research Center no. 293, Münster, Germany.

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