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Infect Immun. Aug 2006; 74(8): 4615–4623.
PMCID: PMC1539602

SdrI, a Serine-Aspartate Repeat Protein Identified in Staphylococcus saprophyticus Strain 7108, Is a Collagen-Binding Protein


A gene encoding a serine-aspartate repeat protein of Staphylococcus saprophyticus, an important cause of urinary tract infections in young women, has been cloned and sequenced. In contrast to other SD repeat proteins, SdrI carries 21 additional N-terminal repeats with a consensus sequence of (P/A)ATKE(K/E)A(A/V)(T/I)(A/T/S)EE and has the longest SD(AD)(1-5) repetitive region (854 amino acids) described so far. This highly repetitive sequence contains only the amino acids serine, asparagine, and a distinctly greater amount of alanine (37%) than all other known SD repeat proteins (2.3 to 4.4%). In addition, it is a collagen-binding protein of S. saprophyticus and the second example in this organism of a surface protein carrying the LPXTG motif. We constructed an isogenic sdrI knockout mutant that showed decreased binding to immobilized collagen compared with wild-type S. saprophyticus strain 7108. Binding could be reconstituted by complementation. Collagen binding is specifically caused by SdrI, and the recently described UafA protein, the only LPXTG-containing protein in the genome sequence of the type strain, is not involved in this trait. Our experiments suggest that, as in other staphylococci, the presence of different LPXTG-anchored cell wall proteins is common in S. saprophyticus and support the notion that the presence of matrix-binding surface proteins is common in staphylococci.

Attachment of microorganisms to host tissues is commonly regarded as the crucial initial step in the pathogenesis of microbial infections. Staphylococcus saprophyticus, an important cause of urinary tract infections, binds fibronectin (7, 42), laminin (33), and collagen (33, 35) and hemagglutinates sheep erythrocytes (10, 16).

Two major surface proteins have been characterized, the S. saprophyticus surface-associated protein (Ssp), a 95-kDa protein (6, 35) which has recently been identified as a surface-associated lipase, and an autolysin (Aas) (15), a multifunctional protein responsible for agglutination of sheep erythrocytes, fibronectin binding, and autolysis. In addition, the genomic sequence of the type strain of S. saprophyticus has been published recently (20). The sequence contains only one protein that is predicted to be covalently linked to the cell wall via an LPXTG motif (20). This protein, UafA, seems to mediate attachment to a bladder carcinoma cell line and causes hemagglutination. However, it cannot be the sole factor responsible for attachment and/or pathogenesis since nonhemagglutinating strains may cause infections (10, 16) and still adhere to collagen (35). In addition, all other sequenced staphylococcal genomes contain more than one cell wall-anchored protein.

In staphylococci, binding to extracellular matrix (ECM) proteins is often mediated by surface receptors designated MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) (30) that interact with proteins of the ECM such as fibrinogen (5, 25, 27, 43), collagen (31, 41), fibronectin (17, 39), vitronectin (21), or elastin (29).

Many MSCRAMMS are organized as SD repeat (Sdr) proteins that are characterized by a common organization comprising an amino-terminal signal sequence, a functional domain often labeled the A region, the SD repeat region, a cell wall-spanning region, an LPXTG motif, and a hydrophobic membrane-spanning domain, followed by a series of positively charged residues (19, 22, 23). The LPXTG motif is the target of a transpeptidase that cleaves the motif between threonine and glycine residues and anchors the protein to the peptidoglycan of the cell wall (37).

Some Sdr proteins contain additional repeats, termed B repeat regions, located between the A regions and SD repeat regions (18).

In this report, we describe the cloning and characterization of an Sdr protein of S. saprophyticus, which is also a wall-anchored protein of this species. It binds collagen and represents a description of an Sdr protein of S. saprophyticus. In addition, it contains N-terminal repeats of unknown function.


Bacterial strains and plasmids.

The bacterial strains and plasmids used in this study are listed in Table Table1.1. S. saprophyticus strain 7108, a hemagglutinating, fibronectin-binding, andcollagen-binding clinical isolate, has been described previously (10, 15, 35). For cloning, a lambda ZAP Express library kindly provided by W. Hell was used (15). Escherichia coli XL1 Blue MRF′ (Stratagene, La Jolla, CA) was the host for the phage and the phagemids. E. coli XLOLR (Stratagene) was used for in vivo excision. E. coli DH5 was the host for expression experiments and the intermediate host during construction of the plasmids for allelic replacement (12). Shuttle plasmid pBT2 (4) contains the temperature-sensitive replicon of pE194, the chloramphenicol resistance determinant of pC194, and the multiple cloning site of pUC18. Plasmid pEC4 (4) was used as the ermB source. Plasmid pQE (QIAGEN, Hilden, Germany) was used for recombinant protein expression. Also, we used the pCRII TOPO vector (Invitrogen, Karlsruhe, Germany).

Strains and plasmids used in this study

Bacterial growth media and antibiotics.

E. coli strains harboring plasmids were grown in Luria broth or on L agar. S. saprophyticus strain 7108 was grown in peptone yeast extract broth, in tryptic soy broth (TSB; Oxoid, Wesel, Germany), or on agar plates (6). Bacteria were usually incubated at 37°C, but in some experiments 30°C and 42°C were also used. Concentrations of 100 μg/ml ampicillin, 25 μg/ml kanamycin, and 12.5 μg/ml tetracycline were used for selection of plasmids in E. coli. For selection of plasmids or chromosomal markers in S. saprophyticus, 10 μg/ml chloramphenicol and 5 μg/ml erythromycin were used.


For screening of the library, a polyclonal antiserum toward S. saprophyticus surface proteins was used (6). A polyclonal antiserum directed to the SdrI A region was generated by subcutaneous immunization of New Zealand White rabbits with the cloned and expressed SdrI A fragment (see below) by using an adjuvant (MPL + TDM + CWS adjuvant system; Sigma, Taufkirchen, Germany). The secondary antiserum was swine anti-rabbit serum (1:20,000) conjugated with alkaline phosphatase (DAKO, Hamburg, Germany) (2). The specificity of the antiserum for SdrI A was verified by inhibition experiments using soluble recombinant antigen to inhibit binding of the antibody to the immobilized antigen.

DNA techniques. (i) DNA manipulation.

Restriction, ligation, and transformation were performed by standard techniques as described elsewhere (36). All restriction enzymes were obtained from Roche. T4 ligase (Roche, Mannheim, Germany) was used for ligation.

(ii) DNA preparation.

Chromosomal DNA of S. saprophyticus strain 7108 was prepared from cleared lysates and purified by double cesium chloride density centrifugation as described previously (8).

(iii) Southern blot hybridization.

Southern blot hybridization was performed as previously described (36). Chromosomal DNA was digested with EcoRI, resolved on 0.8% Tris-borate-EDTA gels, and transferred onto positively charged nylon membranes (Roche). Probes were prepared with a PCR labeling kit (Roche). For construction of an ermB cassette, primers ermB4R (5′ TCTAGAACTAGTGGATCCC 3′) and ermB4/PstI (5′ tattgtCTGCAGCCGAGAGTGATTGGTCTT 3′, where the lowercase letters indicate nonhomologous parts of the primers with incorporated restriction sites) were used. For construction of the cat probe, primers CAT-forw. (5′ GTTACAGTAATATTGACT 3′) and CAT2-rev. (5′ CATAAACAATCCTGCATG 3′) were used. Hybridization and washing were carried out under stringent conditions. Hybridization was done with 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS)-50% formamide at 42°C, and washing was done three times for 20 min each time with 2× SSC-0.1% SDS at 68°C. For detection of hybridized DNA, a digoxigenin luminescence detection kit (Roche) was used. The membrane was subjected to autoradiography with Amersham Hyperfilm MP (Amersham, Freiburg, Germany).

(iv) DNA sequencing.

Both strands of the cloned DNA were automatically sequenced (LI-COR DNA Sequencer 4000; LI-COR, Bad Homburg, Germany). For initial sequencing, the standard pUC/M13 primers (labeled with IRD800; MWG, Ebersberg, Germany) were used. Extension of DNA sequences was achieved by primer walking.

(v) In vitro transposon mutagenesis.

Since the SD(AD)(1-5) repeats were highly conserved, they could not be sequenced by primer walking because it was impossible to find new sequencing primers. The GPS-1 genome priming system (New England Biolabs, GmbH, Frankfurt am Main, Germany) contains a Tn7 transposon-based in vitro system that uses TnsABC transposase to insert a transposon (Transprimer) randomly into the DNA target (40). Primers that bind to sequences of this transposon can then be used to sequence the target. The repeat region was excised from pBSA11 with PstI und HindIII to obtain a 3.2-kb DNA fragment which included the complete SD(AD)(1-5) repeats of the sdrI gene. This insert was ligated into pUC18. The resulting plasmid (pBSA12) was incubated with the transposon mixture. After transformation, clones with an inserted transposon cassette were detected by selection with ampicillin and kanamycin. Plasmids that contained the transposons were digested with restriction endonucleases, and clones with centrally inserted transposons were sequenced. Sequencing was performed with primers from both transposon ends (primer N and primer S) and with M13 primers. Sequencing was performed by Seqlab Laboratories (Göttingen, Germany) with a long sequencing gel.

(vi) Screening of the expression library.

The lambda ZAP Express library was screened with a polyclonal antibody directed toward S. saprophyticus surface proteins (6). Positive clones were converted into phagemids by coinfection of E. coli XLOLR and ExAssist helper phage. Phagemids were transformed back into E. coli XL1 Blue MRF′.

(vii) Construction of an insertion mutant by allelic replacement.

The sdrI gene was interrupted by insertion of the ermB resistance gene, and pBT2 (4) was used as the replacement vector. For construction of pBTSA2, the ermB cassette was amplified from pEC4 with primers ermB4R (5′ TCTAGAACTAGTGGATCCC 3′) and ermB4/PstI (5′ tattgtctgcagCTGCAGCCGAGAGTGATTGGTCTT 3′, where the lowercase letters indicate nonhomologous sequences with added restriction sites), which yielded a PCR product that had PstI sites on both sides. The insert of pBSA1 was excised from the phagemid with SalI and HindIII and ligated into temperature-sensitive replacement vector pBT2. This plasmid, pBTSA1, was digested with PstI and ligated with the erythromycin resistance cassette (ermB). This plasmid was designated pBTSA2. By this method, the sdrI gene was interrupted at bp 2002.

The pBTSA2 constructs were purified from E. coli DH5 by cesium chloride density ultracentrifugation and transformed into S. saprophyticus strain 7108 by protoplast transformation (15). Chloramphenicol- and erythromycin-resistant clones were grown in the presence of erythromycin (5 μg/ml at 30°C for 24 h) and used to inoculate 1,000 ml of prewarmed (42°C) broth containing erythromycin (5 μg/ml). After overnight incubation, appropriate dilutions were plated onto P agar containing erythromycin (5 μg/ml). Clones that grew on erythromycin but not on chloramphenicol had lost the plasmid and were checked for SdrI expression.

(viii) Complementation of the SdrI-negative mutant.

The sdrI gene with its own promoter was excised from pBSA11 with BamHI and HindIII and cloned into the pBT2 vector. The resulting plasmid (pBTSA11) was transformed into E. coli, purified from this strain, and introduced into the SdrI-negative mutant by protoplast transformation.

Protein techniques. (i) Expression and purification of recombinant SdrI A fragment.

The SdrI A domain was amplified by PCR with primer 95.1.29/BamHI and primer 95.1.33/HindIII and with chromosomal DNA from S. saprophyticus strain 7108 as the template. The fragment were expressed by induction with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Gibco-BRL, Karlsruhe, Germany) for 2 to 4 h in E. coli strain M15 (pRep4; QIAGEN). Purification of the six-His-tagged fusion protein was performed according to the manufacturer's (QIAGEN) guide.

(ii) PCR for sdrI and uafA.

To test whether the recently described uafA gene and/or the sdrI gene were present in the S. saprophyticus strains used, we used a PCR with specific primers S.sa_SSP0135_seq3 and S.sa_SSP0135_rev2 for uafA and 95.1.29/BamHI and 95.1.33/HindIII for sdrI (Table (Table2).2). As the template, we used genomic DNA prepared from overnight cultures with a QIAamp DNA kit (QIAGEN, Hilden, Germany). The PCR was carried out with Taq polymerase for 30 cycles at 95°C for 30 s, 52°C (for uafA) or 50°C (for sdrI) for 30 s, and 72°C for 1 min. In addition to strains 7108 and CCM 883, we also tested 123 clinical isolates of S. saprophyticus which originated from urinary tract infections.

Cloning and PCR primers used in this study

(iii) Binding of bacteria to immobilized collagen.

Microtiter plates (Immulon 4; Dynex Technologies, Chantilly, VA) were coated with 1 μg of collagen I (Vitrogen; Cohesion, Palo Alto, CA) in 100 μl of PBS (140 mM NaCl, 0.27 mM KCl, 0.43 mM Na2HPO4, 0.147 mM KH2PO4, 0.02% NaN3, pH 7.4) per well overnight at 4°C. Wells were then washed three times with PBS and blocked with 1% bovine serum albumin in PBS for 1 h before the addition of bacteria. Bacteria were grown in 25 ml of PY broth to the mid-logarithmic phase or to the stationary phase, pelleted, washed with PBS, and adjusted to an optical density at 600 nm of 6.0. Bacterial suspensions were added to the wells (100 μl of cells/well), and the plates were incubated for 2 h at room temperature. The bacterial suspensions were carefully aspirated, and the wells were washed twice with 200 μl of PBS. Adherent cells were fixed with 100 μl of 25% aqueous formaldehyde and incubated at room temperature for 30 min. Wells were gently washed two times with 200 μl of PBS, stained with carbol fuchsin for 5 min, washed again with PBS, and read with an enzyme-linked immunosorbent assay plate reader at 550 nm. Staphylococcus aureus Cowan I, which is known to express Cna (41), served as the positive control, and S. aureus RN 6390 (Cna negative) (11) was used as the negative control. We used S. saprophyticus CCM883, which does not bind to collagen (35), as an additional control.

(iv) Flow cytometry.

The expression of SdrI by S. saprophyticus cells was determined by flow cytometry with a FACScalibur flow cytometer (B-D Biosciences, San Jose, Calif.) equipped with an argon ion laser (488 nm). Overnight cultures (20 ml of TSB) were pelleted and resuspended in PBS. A dilution of bacteria (1:100 in PBS) was incubated with the antiserum against the SdrI A fragment (1:1,000 in PBS) for 1 h at 37°C. After being washed once with PBS, cells were incubated with fluorescein isothiocyanate-conjugated swine anti-rabbit antibody at a 1:20 dilution for 1 h at 37°C. The cells were pelleted and washed twice with PBS. The labeled cell suspensions were aspirated through the flow cytometer, and a fluorescence emission measurement was performed in which at least 20,000 events were collected and analyzed with the Cell Quest Pro software provided with the flow cytometer. In some experiments, the bacterial cells were preincubated (2 h) with different concentrations of collagen, SdrI A antiserum was added (diluted 1:1,000 in PBS), and the mixture was incubated (1 h, 37°C) and further processed as described above to show that the antiserum and collagen competed for the same receptor.

Computer analysis.

Database and homology searches were carried out by the EMBL services (34) and the NIH BLAST program (1).

SignalP (28, 44) was used for signal sequence prediction.

Nucleotide sequence accession number.

The DNA sequence reported in this article has been deposited in the GenBank nucleotide sequence database under accession no. AF402316.


Cloning of sdrI.

Several plaques that reacted with the antiserum were found, and the DNA of these clones was converted into phagemids. One of these clones was designated pBSA1. Sequencing of the DNA of clone pBSA1 showed an incomplete open reading frame that coded for a protein with similarities to Sdr proteins. For isolation of the complete gene encoding the SdrI protein of S. saprophyticus, inverse PCR was used as described previously (35). We religated XbaI-digested chromosomal DNA and used it as a template for inverse PCR with primers 95.1.21 and 95.1.22 (Table. (Table.2).2). The resulting PCR fragment of 3 kb was reamplified with primers 95.1.28 and 95.1.31, subcloned into the pCRII TOPO vector (Invitrogen), and then sequenced (Fig. (Fig.1).1). By this method, the N-terminal part of the sdrI gene was obtained.

FIG. 1.
SdrI nucleotide and amino acid sequences. The putative promoter region (−10 and −35) and the RBS (ribosome-binding site) are underlined with broken lines. The hypothetical signal sequence is underlined with a solid line. The conserved ...

Since the SD repeats were very long, we wanted to verify their length and exclude any cloning artifacts (e.g., due to slipped-strand mispairing). We used a primer C terminally and one N terminally of the repeats to amplify the DNA fragment from wild-type strains, the cloning donor, and the phagemid. In all cases, a size of approximately 2.6 kb for the SD(AD)(1-5) repeats was found. Complete sequencing of the SD(AD)(1-5) repeats by primer walking turned out to be impossible because the coding sequence was highly conserved (Fig. (Fig.1).1). To complete the sequence, we constructed clones with in vitro-generated transposon insertions at different positions in the SD(AD)(1-5) repeats and sequenced from primers binding within this transposon. Finally, we determined a size of 854 amino acids for the SD(AD)(1-5) repeats.

Sequence analysis of the SdrI protein.

The deduced amino acid sequence of the S. saprophyticus SdrI protein predicts a polypeptide of 1,893 amino acids (Fig. (Fig.1).1). The calculated molecular mass of the primary translation product is 195.06 kDa. The primary amino acid sequence organization of the deduced S. saprophyticus Sdr protein is similar to that of the Sdr protein family. Also, it shows features typical of cell surface proteins of gram-positive bacteria. These features include positively charged residues (with the amino acid sequence RRKNKNNEEK) at the extreme C terminus, preceded by a hydrophobic membrane-spanning region and an LPXTG motif (Fig. (Fig.1).1). SdrI possesses a signal sequence (54 amino acids) followed by 21 N-terminal repeats with a highly conserved 12-amino-acid-long sequence (Fig. (Fig.2).2). C terminally of the repeat sequence, an A region of 432 amino acids is found. The A region of SdrI contains the TYTFTDYVD motif (Fig. (Fig.11).

FIG. 2.
Alignment of the 21 N-terminal repeats. The 21 highly conserved N-terminal repeats of SdrI are shown. Conserved residues are in bold type with asterisks below. The sequences were aligned by using CLUSTAL W. Colons, conserved substitutions; periods, semi- ...

SdrI possesses two B repeat regions (Fig. (Fig.1).1). The repeats are composed of 110 and 119 amino acids. Finally, it has long, highly conserved SD(AD)(1-5) repeats with a size of 854 amino acids.

Construction of an sdrI-isogenic mutant of S. saprophyticus.

To investigate the function of SdrI, an sdrI knockout mutant was constructed. Plasmid construct pBTSA2, which contains the sdrI gene interrupted by an ermB cassette, was transformed into S. saprophyticus strain 7108, cells were cured of the plasmid by cultivation at 42°C, and insertion mutants were selected with erythromycin. Insertion of ermB and loss of cat were verified by Southern hybridization and PCR as shown in Fig. Fig.3c.3c. Also, the correct position of the insertion was verified by sequencing.

FIG. 3.
The mutant contains one ermB insertion only. Southern hybridization of the knockout mutant and wild-type strain 7108 with ermB and cat sequences (a and b). Lanes M contained molecular weight marker II (labeled with digoxigenin; Roche, Mannheim, Germany). ...

Loss of SdrI expression in the knockout mutant was verified by fluorescence-activated cell sorting with antiserum against the A region of SdrI. In contrast to the wild type, the knockout mutant showed no expression of the SdrI protein (Fig. (Fig.4).4). The reaction of the mutant with the specific antiserum was virtually identical to that of the wild type with preimmune rabbit serum (Fig. (Fig.44).

FIG. 4.
Fluorescence-activated cell sorter analysis of wild-type, mutant, and complemented strains. Wild-type S. saprophyticus strain 7108, the sdrI knockout mutant, and the complemented mutant were grown overnight in TSB. The bacteria were incubated with antiserum ...

S. saprophyticus sdrI mutant shows decreased binding to collagen.

Since binding to collagen has been described for some S. saprophyticus strains (33, 35), we analyzed the binding of strain 7108, the mutant, and the complemented strain to collagen-coated microtiter wells (Fig. (Fig.5).5). The binding of the mutant was drastically reduced compared with that of the wild-type strain. This difference was highly significant (P < 0.0001, t test). Reintroduction of the gene into the mutant reconstituted collagen binding (Fig. (Fig.5).5). The positive control (S. aureus Cowan I) (41) showed the expected binding to the matrix protein, whereas the negative control, S. aureus RN 6390 (11), did not bind to collagen-coated wells. In addition, type strain CCM 883, which does possess uafA but not sdrI (20), did not attach to collagen. Strain 7108 contained both uafA and sdrI, as analyzed by PCR (data not shown). Binding of strain 7108 to immobilized collagen could be inhibited by the antiserum to SdrI A (data not shown). In addition, binding of the SdrI A antiserum to the wild-type strain could be inhibited by preincubation with collagen (Fig. (Fig.66).

FIG. 5.
Adherence of S. saprophyticus wild-type strain 7108 and the SdrI mutant to immobilized collagen. Cell suspensions of both strains were added to microtiter wells coated with collagen and incubated for 3 h at 37°C. The adherent bacterial films were ...
FIG. 6.
Collagen inhibits binding of SdrI A antiserum to S. saprophyticus. Binding of anti-SdrI A antiserum to SdrI was inhibited by increasing concentrations of collagen. Reactions of the antiserum with the wild-type strain without collagen and with 1,500 μg/ml ...

Prevalence of the sdrI gene in S. saprophyticus.

Clinical isolates of S. saprophyticus (123 strains), strain 7108, and type strain CCM883 were tested by PCR for the presence of the sdrI and uafA gene sequences (primers 95.1.29/BamHI and 95.1.45/HindIII for sdrI and S.sa_SSP0135_seq3 and S.sa_SSP0135_rev2 for uafA). We found sdrI in 11% of the strains and uafA in 94%. Strain 7108 contained uafA and sdrI, whereas strain CCM 883 harbored solely uafA.


S. saprophyticus is an important cause of urinary tract infections, especially in young female outpatients. In earlier experiments, it has been shown that the urease of this organism acts as an important virulence factor (8, 9) by contributing to pathogenicity in the bladder. Also, hemagglutination of sheep erythrocytes (10, 16), adherence to uroepithelial cells (26), and binding to laminin (33), fibronectin (7, 9, 42), and collagen (33, 35) have been described for S. saprophyticus.

Two major surface proteins have been described, an autolysin (Aas), a multifunctional protein without an LPXTG motif that is responsible for cell wall turnover and fibronectin binding and may be involved in the agglutination of sheep erythrocytes (7, 9, 15), and the S. saprophyticus surface-associated protein (Ssp), a 95-kDa protein (6) that exhibits lipase activity (35). In contrast to GehD (3), the lipase of Staphylococcus epidermidis, Ssp, is not involved in collagen binding (35). Interestingly, the type strain of S. saprophyticus, CCM883 (the same as recently sequenced strain ATCC 15305), hemagglutinates sheep erythrocytes but does not bind to collagen (20, 35). Hemagglutination is apparently caused by UafA, the only protein with a cell wall anchor that is present in the genomic sequence of this strain (20). We therefore sought to identify an additional collagen-binding factor.

For cloning of the sdrI gene, we used a polyclonal antibody directed toward S. saprophyticus surface proteins to screen a lambda ZAP Express library of hemagglutinating and collagen-binding strain 7108 (15, 35). The inserts of the clones obtained by this screening showed an N-terminally incomplete open reading frame. We used inverse PCR to complete the N terminus. Sequencing showed that the reading frame of the sdrI gene of S. saprophyticus codes for a 1,893-amino-acid-long polypeptide with a deduced molecular mass of 195.06 kDa. Homology analysis suggested that the gene is a member of the Sdr surface protein family (19, 23). It contains motifs typical of gram-positive surface proteins (LPXTG, membrane-spanning region, charged C terminus), as well as an A region containing the TYTFTDYVD motif, two B repeats, and a highly repetitive sequence containing only the amino acids serine, asparagine, and alanine.

Sequencing of the SD(AD)(1-5) repeats was technically demanding and necessitated the use of in vitro transposon mutagenesis to insert Tn7 into the conserved repeats. The sequence of the repeat region was completed with primers from the vector and primers from the transposon. SdrI of S. saprophyticus contains one of the longest SD(AD)(1-5) amino acid sequences described so far, comprising 854 amino acid residues (18, 19, 23, 24).

In addition, the amino acid composition of this repeat sequence is different from that of other SD repeats. This region contains a very high percentage of alanine residues (37%). The presence of alanine has been described in other Sdr proteins but at lower percentages (2.3% for ClfA [14, 24, 25] and 4.7% for SdrF [23]). The second known cell wall-anchored protein of S. saprophyticus, UafA, also contains a C-terminal repeat region which is composed of conserved SESESL repeats spanning 1,451 amino acids (20).

The lengths of the SD repeat regions differ considerably among SD proteins, with only 56 for SdrG (23), 308 for ClfA (24, 25), 558 for SdrF (23), and 854 for SdrI. Although the function of the SD repeat region and of its variation is not understood, experiments with ClfA showing that at least 72 residues of the SD repeats are needed for efficient binding of S. aureus to fibrinogen (14) indicate that a minimal length of this region is required for correct surface presentation of ECM-binding domain A.

In contrast to the Sdr proteins identified so far, SdrI possesses 21 12-amino-acid-long repeats with a consensus sequence of (P/A)ATKE(K/E)A(A/V)(T/I)(A/T/S)EE located between the signal sequence and A region. Most interestingly, Aas, an autolysin of S. saprophyticus, also contains N-terminal repeats but with a distinctively different amino acid sequence (15); UafA, however, does not contain such repeats (20). To date, the function(s) of these repeats is unknown.

The 432-amino-acid A region of S. saprophyticus SdrI is located between the 21 N-terminal repeats and the B repeats. Comparisons revealed that the A region of SdrI is 55% identical to the A region of the SdrF protein of S. epidermidis (23). The TYTFTDYVD motif, whose function is unknown and which is present in all of the Sdr proteins described so far (19), is also found in the A region of SdrI (Fig. (Fig.11).

The two B repeat regions of SdrI are located between the A region and the SD(AD)(1-5) repeats. The repeats are composed of 110 and 119 amino acids, and each contains a Ca2+-binding EF-hand motif with a 12-amino-acid consensus sequence [DX(N/D)X(D/N)GXX(D/N/G)XX(E/D)] (18). Although it has been shown in vitro for SdrD that the EF hands are functional (18), their physiological role remains elusive. For Cna, the collagen-binding MSCRAMM of S. aureus (11, 13, 31, 41, 32), it was shown that binding of the ECM is not dependent on the presence of the B repeats (13, 32).

For further analysis of the function of S. saprophyticus SdrI, an SdrI knockout mutant of strain 7108 was constructed by inserting a single ermB cassette at amino acid position 667. In adherence assays with immobilized collagen, the mutant showed less binding than the wild type, which could be restored by introduction of a plasmid carrying sdrI. This result indicates that SdrI is responsible for collagen binding by S. saprophyticus. Binding of whole bacteria to immobilized collagen could be inhibited by the antiserum directed toward the A domain of SdrI, and binding of this antibody to S. saprophyticus could be inhibited by collagen. These results indicate that the SdrI A region is specifically involved in collagen binding. CCM883, which does express UafA and is SdrI negative, does not bind to collagen, which indicates that UafA is not involved in collagen binding. Our experiments with wild-type strain 7108 (sdrI and uafA positive) and its SdrI-negative mutant support this notion since the sdrI mutant did not bind to collagen. In E. coli, collagen binding is thought to play an important role in the pathogenesis of urinary tract infections (38), and it is tempting to speculate that it acts in a similar manner in S. saprophyticus. Sequences encoding SdrI were found at a comparatively low percentage (11%) only. It is not known if the protein is associated with increased pathogenicity or is another example of a redundantly encoded adhesin in staphylococci.

In this study, we characterized an Sdr protein of S. saprophyticus. In contrast to other SD repeat proteins, SdrI carries 21 additional N-terminal repeats and has the longest SD repetitive region described so far. In addition, it is the first characterized collagen-binding protein of S. saprophyticus and the second example of a surface protein of S. saprophyticus carrying the LPXTG motif.

Our experiments suggest that, similar to other staphylococci, binding to matrix proteins and adhesion are redundantly encoded in S. saprophyticus and support the notion that the presence of Sdr proteins is common in staphylococci.


Parts of this work were supported by the Deutsche Forschungsgemeinschaft (grants DFG Ga 352/5-1 and Ga 352/5-2) and by the Ruhr-Universität Bochum (FoRUM F2 11/00).

The excellent technical assistance of S. Ortmann and S. Friedrich is gratefully acknowledged.


Editor: V. J. DiRita


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