- Email: [email protected]
A von Willebrand factor-binding protein from Staphylococcus lugdunensis
FEMS Microbiology Letters 234 (2004) 155–161 www.fems-microbiology.org
A von Willebrand factor-binding protein from Staphylococcus lugdunensis Wiebensj€ Ljungh b, Martin Nilsson a, Joakim Bjerketorp a, Asa o a, Asa Lars Frykberg a, Bengt Guss a,* b
a Department of Microbiology, Swedish University of Agricultural Sciences, SE75007 Uppsala, Sweden Department of Medical Microbiology, Dermatology and Infection, Lund University, S€olvegatan 23, SE223 62 Lund, Sweden
Received 29 January 2004; received in revised form 11 March 2004; accepted 12 March 2004 First published online 23 March 2004
Abstract In the present study, a phage display library covering the genome of Staphylococcus lugdunensis, was affinity-selected against von Willebrand factor (vWf). This led to the identification of a gene, vwbl, encoding a putative cell surface protein of 2060 amino acids, denoted vWbl. The deduced protein has an overall organisation typical of staphylococcal cell surface proteins, with an N-terminal signal peptide, and a C-terminal cell wall sorting signal. The vWf-binding part is located in repetitive domains and antibodies against vWbl or vWf can inhibit the binding. Southern blot analysis showed that vwbl was present in the 12 S. lugdunensis strains tested. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Coagulase-negative staphylococci; Staphylococcus lugdunensis; Phage display; von Willebrand factor; Adhesin; LPXTG motif
1. Introduction Staphylococcus lugdunensis is a coagulase-negative staphylococcal species (CoNS) that has been reported to cause serious human infections such as endocarditis, septicemia and osteomyelitis as well as less severe infections, e.g. skin infections [1–3]. In a mouse abscess model, S. lugdunensis was shown to posses a high pathogenic potential relative to some other CoNS [4]. Further, the infections caused by S. lugdunensis show more resemblance to infections typical for Staphylococcus aureus, rather than CoNS. S. lugdunensis binds several different plasma and extracellular matrix proteins and most strains bind fibrinogen, collagens, vitronectin and laminin [5]. The ability to interact with and bind to host proteins is assumed to aid staphylococci in the initial attachment to the host tissue and to *
Corresponding author. Tel.: +46-0-1867-3205; fax: +46-0-18673392. E-mail address: [email protected] (B. Guss).
evade the immune defence [6,7]. Concerning bacterial proteins binding to von Willebrand factor (vWf), there are only a few reports. The binding of S. aureus to vWf was first reported in 1997 [8] and later it was shown that Protein A mediates the adherence of S. aureus to vWf [9]. In addition, a secreted S. aureus protein (vWbp) that binds vWf has recently been identified using phage display [10]. vWf is a large glycoprotein found in plasma (10 lg ml1 ) and in storage granules of platelets and endothelial cells. vWf has a dual and essential role in hemostasis. It binds to the subendothelium, primarily to exposed collagen at sites of vascular damage, and promotes platelet adhesion as well as aggregation. It is also a carrier and stabiliser of factor VIII, which is a cofactor that accelerates the clotting cascade [11]. In this study, a phage display library was made from genomic S. lugdunensis DNA. The library was subsequently affinity-selected (panned) against vWf, which revealed the existence of a large protein, with repetitive vWf-binding domains.
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.03.024
156
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161
2. Materials and methods
2.4. Cloning of vwbl
2.1. Bacterial strains, growth conditions and plasmids
To isolate the complete gene encoding the putative vWf-binding protein, Southern blot analysis of genomic DNA from strain 2342 was performed using the insert of phagemid clone SlvW2 (Fig. 1(A)) as a probe. Subsequently, 4 kb EcoRI-fragments was ligated into pUC18. Ampicillin-resistant transformants were screened by standard colony-hybridisation methods using the same probe as above. Sequence analysis of one selected clone (pEcoRIvwbl) revealed that the cloned fragment contained the 30 -end of the gene but lacked the 50 -end. Thus, to isolate the remaining portion of the gene, an additional Southern blot analysis was conducted, using a probe comprising a fragment from the 50 -end of the EcoRI-insert. Based on the result from this experiment a 3.2 kb HincII-fragment was ligated into the SmaI-site of pUC18 and the sequence of one selected clone was determined.
The 12 clinical S. lugdunensis isolates used in this study [5] were grown on tryptone soya agar or in tryptone soya broth (Oxoid). Escherichia coli strains were grown in Luria–Bertani (LB)-broth or on LA plates (LB-broth supplemented with 1.5% agar). Ampicillin (100 lg ml1 ) was added when appropriate (LA-amp). All bacterial incubations were done at 37 °C. E. coli strains ER2566 (New England Biolabs) and TG1 were used for protein production and phage display, respectively. The phagemid vector pG8SAET was used to construct the phage display library [12]. For additional cloning the plasmid pUC18 was used. 2.2. Proteins and reagents Human vitronectin was a kind gift from Ph.D. D.-Q. Li, Lund, Sweden and human thrombospondin was a kind gift from Prof. J. Lawler, Boston. Recombinant human von Willebrand factor (rvWf) was a kind gift from Prof. F. Dorner, Vienna. The rvWf has been shown to have an activity comparable to human plasmaderived vWf [13]. Polyclonal antibodies against human vWf were from Kordia (Leiden, The Netherlands), and human IgG was from Kabi Vitrum (Stockholm, Sweden). Casein, human serum albumin (HSA) and human fibronectin were from Sigma, and human fibrinogen was from IMCO Corp. Ltd. (Stockholm, Sweden). The fibrinogen-binding part (amino acids 52–646) of protein Fbe [14] of Staphylococcus epidermidis was expressed in E. coli following the same general scheme as described below under Section 2.5. 2.3. Construction of a phagemid library and panning A phage display library was constructed from S. lugdunensis strain 2342 essentially as described by Jacobsson et al. [15]. In short, fragmented genomic DNA of 0.1–3.5 kb in size was inserted into the pG8SAET phagemid vector. After electroporation into E. coli TG1 cells, the library consisted of 2 108 individual clones, the insertion rate was 90%, and the average insert size 0.5 kb. After infection with helper phage R408 (Promega), the phagemid titre was 1 1010 colony forming units (cfu) ml1 . A microwell (Maxisorp, Nunc) was coated for 2 h at room temperature (RT) with 200 ll of rvWf at a concentration of 20 lg ml1 in 0.05 M NaHCO3 (pH 9.7). The well was blocked with PBST (140 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 1.4 mM KH2 PO4 [pH 7.4] + 0.05% Tween 20) containing 1 mg ml1 casein, for 1 h at RT. The affinity-selection (panning) procedure was done as described in detail elsewhere [15].
2.5. Recombinant proteins and production of antibodies The region of vwbl, corresponding to the amino acids (aa) 1247–1503 of vWbl (Wbl3R), was PCR-amplified, using the forward primer vWblF (50 -TATATAC CATGGCAACAATTCCAGATCGCG), the reverse primer vWblR (50 -TTTATACCCGGGTTCTGACTG GATACGTTCATAC) and DNA from clone pEcoRIvwbl as template. The underlined sequences correspond to complementary sequences of vwbl. The fragment obtained, was digested with NcoI and SmaI and ligated into the corresponding sites in the expression vector pTYB4 of the IMPACT-system (New England Biolabs). The ligated DNA was electrotransformed into E. coli ER2566. Plasmids harbouring inserts were isolated from transformants and verified by DNA sequencing. The Wbl3R protein was expressed and purified following the manufacturer’s instructions. Polyclonal antibodies (IgY) against Wbl3R were raised in chicken by AgriSera (V€ann€as, Sweden) and affinity-purified on a Wbl3R-coupled HiTrap-column (Amersham Biosciences). The affinity-purified antibodies were conjugated with horseradish peroxidase (HRP) according to the method of Wilson and Nakane [16]. The minimal vWf-binding domain of vWbp (26 aa) of S. aureus was expressed using the same general scheme as outlined above. The forward primer vWbp26F (50 -AA TTATCCATGGGCACATCACCGACTACATATAC) and the reverse primer vWbp26R (50 -TATACCCG GGAATTTGTTGCTGAGTTTGAC) were used to PCR-amplify the relevant fragment using DNA from S. aureus strain Newman as template. 2.6. ELISA Microwells were coated with different proteins at a concentration of 0.25 lg ml1 in 0.05 M NaHCO3 (pH
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161
157
A
SlvW8 1608-1650 R5
R6
1303
48
S
2060
R
A R1
R2
W R3
SlvW1 1413-1440 SlvW2 1392-1460 SlvW3 1408-1451 SlvW4 1393-1450 SlvW5 1395-1436 SlvW6 1411-1451 SlvW7 1409-1444 B R1
1303-IPNDAPQETPVALEVTRYVDSEGNEVQETEEGTHDAPGIIADKWQYTGQTAAENGITTHVYQRIQSE
R2
1370-IPNEAPQETPVALEVTCYVDSEGNEVQETEEGTHDAPGIIGDKWQYTGQTTTEDGITTHIYQRIQSE
R3
1437-IPNEAPQETPVALEVTRYVDSEGNEVQETEEGTHQPPSIIGDKWQYTGQTTTADGITTYVYERIQSE
R4
1504-IPNEAPKETPIQLEVTRYVDGEGNEVQETEEGTHHAPGIIGDKWQYTGQTTTESGITTHVYERIQSE
R5
1571-IPNEAPQETPVQLEVTRYVNSEGNEVQETEEGTHQPPGIIGDKWQYTGQTTTADGITTYVYERIQSE
R6
1638-IPNEAPKETPVQLEVTRYVDTDGNEVQETEEGTHQPPGIIGDKWQYTGRVTEKDGITTYVYERIQSA
R7
1705-IPNEAPQETPVQLEVTRYVDITGNEVQETEEGTHQPRYIIGDKWRYSGVTVTENGITKHVYERIQSK
R8
1772-VPNDAPQETPVQLEVTRYVDPEGNEIQETTEGKHQPPGIIGDRWQYTGKVTEKDGIITYVYERIQSE
R9
1839-IPNNPPQETPVELEVTRYVDGEGNEVQETTEGKHQPPSIIGDRWQYTGKVTEKDGITTYVYERIQSK .**..*.***..**** **. .***.***.**.*... **.*.*.*.* ... .**....*.****
R10 1906-VPNDAPRVDIDELKITIYVDTNGREIVPSRKGQLPPEQFIGQDWQYTGHKIEKDGITTYIYKKVENA .**..*. .
.*..* **. .*.*.
. .*
.
.*.. *.*.*
. .**....*.....
Fig. 1. (A) Schematic presentation of vWbl aligned against translated inserts from rvWf-binding phagemid clones. The different regions on vWbl are indicated as S (signal peptide), A (non-repetitive region), R (repeat-region) and W (wall-associated region). The clones SlvW1–SlvW7, originate from panning experiments where phagemid particles were eluted by lowering the pH. The clone SlvW8 originates from a panning when bound phage was recovered by adding E. coli TG1 cells directly to the well. (B) Alignment of the ten repeats from vWbl. Since R10 is considerably more diverged than the other repeats, it is separately aligned to more clearly demonstrate the high similarity between the other repeats. Amino acids perfectly conserved in all repeats are indicated with an asterisk and functionally conserved amino acids with a dot. The minimal binding domain of 24 aa is marked with a box. The nucleotide sequence of the vwbl gene has the GenBank Accession No. AY530288.
9.7) for 1.5 h at RT. Wells were washed with PBST, and blocked with PBST containing 1 mg ml1 casein for 1 h at RT. After washing, Wbl3R (40 lg ml1 ) was added with or without the various inhibitors, as described in the figure legends for the individual experiment, and incubated at 4 °C overnight. Wells were washed and peroxidase-conjugated anti-Wbl3R antibodies were added to the wells (2 lg ml1 ), which were incubated for 2 h at RT. The wells were washed again and bound protein was detected using 3,30 ,5,50 -tetramethylbenzidine. The reaction was stopped by the addition of 1 M H2 SO4 and the absorbance read at 450 nm. 2.7. Detection of vwbl-like genes in strains of S. lugdunensis Genomic DNA was prepared from strains of S. lugdunensis using a DNeasy Tissue kit (Qiagen) with the modification that an addition of lysostaphin at 200 lg ml1 was used to lyse the cells. The presence of
vwbl or a gene similar to vwbl was detected by Southern blot analysis using a PCR-generated fragment encompassing the 2 kb repeat-region, of vwbl, which were labelled with 32 P by nick-translation using the Ready-To-Go DNA Labelling kit (Amersham Biosciences). Southern blot analysis was performed as described in [17]. 2.8. DNA sequencing and analysis of sequences The nucleotide sequence of vwbl was determined with the Dyenamic ET Terminator Cycle Sequencing Premix kit (Amersham Biosciences) and a model 377 Perkin– Elmer DNA sequencer, using a primer walking strategy. Specific synthetic primers were from Invitrogen. The computer program PCGENE (Intelligenetics Inc., Mountain View, CA, USA), were used to analyse the sequence data. Similarity searches at the nucleotide and amino acid level were performed using BLAST (http:// www.ncbi.nlm.nih.gov/BLAST/).
158
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161
3. Results and discussion 3.1. Isolation of phagemid clones expressing vWf-binding polypeptides To identify interactions between S. lugdunensis and host proteins, a phage display library was constructed in the vector pG8SAET using genomic DNA from S. lugdunensis strain 2342. The library was panned against rvWf, and after two rounds of panning a specific enrichment was obtained. In the second panning 200 times more phage particles bound to rvWf compared to the control protein (casein; data not shown). The nucleotide sequence from ten phagemid inserts, showed that the majority overlapped the same region of the genome. Several independent panning experiments against rvWf were conducted, all resulting in isolation of phagemids containing different inserts derived from the same region, suggesting that a novel vWf-binding protein had been identified. 3.2. Cloning and sequencing of vwbl The sequence of the complete gene was obtained from two separate clones, which revealed an open reading frame of 6180 nucleotides starting with a TTG codon. The open reading frame is preceded by a typical ribosomal binding site (AGGAGA), situated a few nucleotides upstream of the start codon. The coding sequence is followed by a transcriptional terminating sequence consisting of an inverted repeat and a T-rich region. The gene, termed vwbl, encodes a putative protein of 2060 aa (Fig. 1(A)), named vWbl (von Willebrand factor-binding protein of S. lugdunensis). In the N-terminal portion of the protein, a signal peptide is predicted with the most likely cleavage site located between aa 47 and 48 [18]. The signal peptide also contains the motif YSIRKG/S, demonstrated to play a role in the efficient secretion of proteins equipped with this motif [19]. Based on the proposed signal peptide, the mature vWbl consists of 2013 aa with a predicted molecular mass of 226 kDa. Following the signal peptide there is a region, termed A, consisting of 1255 aa (Fig. 1(A)). The A-region has no apparent similarity to other proteins but it harbours the interesting motif, Arg-Gly-Asp (RGD), at position 1134–1136, a motif found in many integrin-binding proteins, e.g. fibronectin, laminin and vWf [20], as well as in cell surface proteins of several pathogens [21,22]. Although, to our knowledge, there has been no reports describing staphylococcal binding to integrins through an RGD-dependent mechanism, it is tempting to speculate that vWbl could mediate direct adherence of S. lugdunensis cells to eukaryotic cells. A repeat (R)-region consisting of ten repeats, termed R1-R10, where each repeat comprises 67 aa, follows the A-region. An alignment of the ten repeats shows high similarity
between them (Fig. 1(B)). Further, the repeats show homology to the repeats of the muramidase-released protein from Streptococcus suis. This protein is a cell wall anchored protein, associated with virulence of S. suis [23,24]. Apart from this homology, vWbl do not show any significant similarity to any known protein at the amino acid level. The C-terminal part of vWbl harbours several characteristic features found in cell surface proteins of Gram-positive bacteria. An LPXTGmotif (aa 2026–2030), known to be involved in anchoring surface proteins covalently to the cell wall [25], is followed by a membrane-spanning region and a stretch of charged amino acid residues. 3.3. The vWf-binding domains in vWbl Despite three independent panning experiments, only seven different clones were found (SlvW1–SlvW7) (Fig. 1(A)), many of which were isolated several times. Alignment of the inserts revealed that all contained the C-terminal end of the R2-repeat, from which a ‘‘minimal’’ binding domain consisting of only 24 aa, could be deduced (Fig. 1(B)). No clone contained a complete R2domain, nor were any of the other repetitive domains found under these panning conditions. This result is puzzling, since nine of the ten different repeats are very similar (Fig. 1(B)). The library size, the insertion frequency, and the size of the inserted fragments, should ensure that all domains are represented in the library. In another successful panning experiment using the same library against another ligand, the inserts of the selected clones encoded polypeptides 300–450 aa in length (unpublished results). However, the clones described in this paper were shorter than expected, the longest clone encoded 68 aa and the shortest 27 aa. A possible reason for this could be that phagemid particles displaying other repeats, or several repeats, remain bound to vWf after pH-elution, and thereby are never recovered. In an additional experiment, the panning procedure was modified and instead of eluting the bound phage by lowering the pH, E. coli cells were added directly to the well. This allowed bound phage to infect the cells, which subsequently were plated on LA-amp plates. This resulted in isolation of phagemid particles comprising parts of the R5- and R6-repeats (SlvW8 in Fig. 1(A)) in addition to clones containing the R2-repeat. Thus, most likely the other R-domains also mediate binding to vWf, but are not found with the elution conditions used in this study. 3.4. The binding to vWf is specific and can be inhibited by Wbl3R To investigate the binding between vWbl and rvWf, a phage stock, from clone SlvW5 (Fig. 1A), was generated. The phage stock was separately panned against
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161
rvWf, fibrinogen, fibronectin, IgG, vitronectin, HSA, thrombospondin and uncoated microwells. Approximately 103 more phagemid particles bound to rvWf than to any other protein in the assay (data not shown). In addition, the phage stock was used in two inhibition assays. In the first, recombinant protein, WblR3, comprising the C-terminal end of the A-region and repeats R1–R3 of vWbl, was incubated in rvWf-coated microwells prior to addition of the phage stock. As shown in Fig. 2(A), the binding between rvWf and the phage particles was inhibited by approximately 95% when WblR3 was present, while HSA had no effect. In the second inhibition assay, antibodies against Wbl3R was stepwise diluted and incubated with the SlvW5 phage stock before transfer to rvWf-coated wells. Fig. 2(B), shows that the specific antibodies inhibited phage
A
90000 80000 70000
Cfu ml
-1
60000 PBS Wbl3R HSA
50000 40000 30000 20000 10000 0
B 1,0E+09
Cfu ml-1
1,0E+08 PBS S
Anti-Wbl3R IgY
1,0E+07
Pre-immune IgY
1,0E+06
159
particles from binding to rvWf in a dose-dependent manner, whereas pre-immune antibodies had no effect. 3.5. Characterisation of the vWbl–vWf-binding To validate the specific binding between Wbl3R and rvWf, various ELISAs were performed. In one ELISA, rvWf was immobilised in microwells and Wbl3R was added together with rvWf, fibrinogen or fibronectin. The binding was inhibited by soluble rvWf in a dose-dependent manner (Fig. 3A), thus demonstrating that both immobilised and soluble rvWf are recognised by Wbl3R. Fibrinogen or fibronectin had no effect on the binding. In another ELISA, Wbl3R were incubated together with polyclonal anti-human-vWf antibodies or unspecific antibodies, in rvWf-coated microwells (Fig. 3(B)). The results showed that addition of unspecific antibodies at a high concentration had no effect, whereas the same concentration of anti-vWf antibodies lowered the binding with almost 90%. We have recently described a secreted vWf-binding protein, vWbp, encoded by S. aureus [9]. The deduced ‘‘minimal’’ binding domain of vWbp is 26 aa, and a recombinant peptide comprising these 26 aa (vWbp26) inhibited the interaction between vWbp and rvWf in a dose-dependent manner (Bjerketorp, unpublished results). As shown in Fig. 3(C), the binding of Wbl3R to rvWf was inhibited in the presence of vWbp26, suggesting that vWbl and vWbp bind to the same region of vWf. This was somewhat surprising since both binding polypeptides are small, and show no sequence similarities. Besides, vWf is a large protein, with many potential binding sites. Nevertheless, not only have these bacterial species evolved proteins that bind vWf, but these proteins also bind to the same site in vWf. It should be noted that vWbl is cell surface anchored whereas vWbp is secreted. This suggests that the binding of vWbl to vWf, not only mediates S. ludgunensis adherence to vWf, but may also affect some of the biological functions served by vWf.
1,0E+05 PBS
1:500 1:100
1:20
1:4
Dilution
Fig. 2. Inhibition of the binding between phagemid (SlvW5) particles and immobilised rvWf. (A) Microwells coated with rvWf were separately incubated for 1 h with PBS, or PBS supplemented with Wbl3R, or HSA, both at the concentration of 2 mg ml1 . One tenth of the volume was then exchanged for 5 ll of phagemid particles (5 105 cfu), and incubation continued for 2 h. (B) Phagemid particles (5 109 cfu ml1 ) were incubated with anti-Wbl3R IgY or pre-immune IgY and transferred to rvWf-coated microwells. The anti-Wbl3R IgY was diluted in steps of five. In both (A) and (B), the wells were washed and bound phagemid particles were eluted by lowering the pH. Aliquots were used to infect E. coli TG1 cells, which were plated on LA-amp plates. The result is shown as cfu ml1 eluate. In (A), each value is the means of totally four infections from two separate wells. In (B), bars represent mean values of duplicate experiments. Standard deviations are indicated.
3.6. Clinical isolates of S. lugdunensis possess vwbl-like genes The distribution of the vwbl gene, among clinical isolates of S. lugdunensis, was investigated using Southern blot analysis. Genomic DNA from strain 2342 and 11 other clinical strains were digested with EcoRI and probed in a Southern blot analysis with a labelled PCRfragment, covering the ten repeats of vwbl. All strains possessed a fragment that reacted with this probe (Fig. 4). Restriction endonuclease analysis of genomic DNA from S. lugdunensis has shown that different isolates of this species are very homogenous [26]. Interestingly, as indicated by Southern blot analysis, there was a clear difference in size of the hybridising EcoRI-fragments, between
160
A
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161 0.4
A450
0.3 PBS vWf Fg
0.2
Fn 0.1
0 PBS S
1
5
25
Protein concentratio concentration (µg ml-1)
B 0.3
A450
PBS
anti-vWf IgG
0.2
Fig. 4. The presence of the R-region of vwbl in S. lugdunensis isolates as demonstrated by Southern blot analysis. The genomic DNA of the different staphylococci was digested with EcoRI. Lane 1, S. aureus strain Newman (negative control); lanes 2–13 represent different clinical isolates of S. lugdunensis (2, G5-87; 3, G2-89; 4, G16-89; 5, G6-87; 10, 2342; 11, 49/90; 12, 49/91; 13, 6, G58-88; 7, G66-88; 8, G3A; 9, SA; A251 [5]). A nucleotide size marker is indicated.
0.1
0 PBS
1:6250
1:1250
1:250
Dilution
C 0.3
0.2 A450
PBS
vWbp Fbe
vWf-binding protein with features characteristic for staphylococcal cell surface proteins. The distribution of this gene seems to be widespread among the clinical strains, but the importance of the protein vWbl in the context of infections remains to be elucidated. Under conditions corresponding to arterial blood flow, vWf is the bridging molecule required for platelet adherence to exposed subendothelium and platelet aggregation [27,28]. Similarly, vWbl and its interaction with vWf might serve a critical role in the bacterial attachment to minor vascular lesions, and thereby in the colonisation preceding infections, e.g. endocarditis, caused by S. lugdunensis.
0.1
Acknowledgements 0 PBS S
1
5
25
Protein concentratio concentration (µg ml-1)
Fig. 3. Inhibition of the binding between WblR3 and rvWf detected with ELISA. Microwells were coated with rvWf and the binding of Wbl3R was inhibited with various proteins: (A) rvWf (vWf), fibrinogen (Fg), or fibronectin (Fn); (B) specific antibodies against human vWf (anti-vWf) or unspecific antibodies (IgG), the antibodies were diluted in steps of five, the highest concentration was 10 lg ml1 ; (C) vWbp26 (vWbp), or Fbe. Bound Wbl3R was detected with HRPlabelled anti-Wbl3R antibodies. Bars represent the mean values of duplicate experiments. Standard deviations are indicated.
tested strains (Fig. 4). In addition, PCR-amplification revealed heterogeneity between the strains in the length of the R-region (data not shown). In conclusion, we have demonstrated that S. lugdunensis strains possess a gene, vwbl, coding for a
This investigation was supported by grants from the Swedish Medical Research Council (K2000-16X-1305802B and 06X-11229), the Swedish Foundation for Strategic Research, Infection & Vaccinology Research Program (24/98) and Biostapro AB.
References [1] Etienne, J., Pangon, B., Leport, C., Wolff, M., Clair, B., Perronne, C., Brun, Y. and Bure, A. (1989) Staphylococcus lugdunensis endocarditis. Lancet 1, 390. [2] Fleurette, J., Bes, M., Brun, Y., Freney, J., Forey, F., Coulet, M., Reverdy, M.E. and Etienne, J. (1989) Clinical isolates of Staphylococcus lugdunensis and S. schleiferi : bacteriological characteristics and susceptibility to antimicrobial agents. Res. Microbiol. 140, 107–118. [3] Herchline, T.E. and Ayers, L.W. (1991) Occurrence of Staphylococcus lugdunensis in consecutive clinical cultures and relationship of isolation to infection. J. Clin. Microbiol. 29, 419–421.
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161 [4] Lambe Jr., D.W., Ferguson, K.P., Keplinger, J.L., Gemmell, C.G. and Kalbfleisch, J.H. (1990) Patogenicity of Staphylococcus ludgunensis, Staphylococcus schleiferi, and three other coagulasenegative staphylococci in a mouse model and possible virulence factors. Can. J. Microbiol. 36, 455–463. (1993) Serum and [5] Paulsson, M., Petersson, A.C. and Ljungh, A tissue protein binding and cell surface properties of Staphylococcus lugdunensis. J. Med. Microbiol. 38, 96–102. [6] Flock, J.-I. (1999) Extracellular-matrix-binding proteins as targets for the prevention of Staphylococcus aureus infections. Mol. Med. Today 5, 532–537. € k, M. (1998) Surface protein adhesins of [7] Foster, T.J. and H€ oo Staphylococcus aureus. Trends Microbiol. 6, 484–488. [8] Herrmann, M., Hartleib, J., Kehrel, B., Montgomery, R.R., Sixma, J.J. and Peters, G. (1997) Interaction of von Willebrand factor with Staphylococcus aureus. J. Infect. Dis. 176, 984–991. [9] Hartleib, J., Kohler, N., Dickinson, R.B., Chhatwal, G.S., Sixma, J.J., Hartford, O.M., Foster, T.J., Peters, G., Kehrel, B.E. and Herrmann, M. (2000) Protein A is the von Willebrand factor binding protein on Staphylococcus aureus. Blood 15, 2149– 2156. Flock, J.-I., Jacobsson, K. [10] Bjerketorp, J., Nilsson, M., Ljungh, A, and Frykberg, L. (2002) A novel von Willebrand factor binding protein expressed by Staphylococcus aureus. Microbiology 148, 2037–2044. [11] Vischer, U.M. and de Moerloose, P. (1999) von Willebrand factor: from cell biology to the clinical management of von Willebrand disease. Crit. Rev. Oncol. Hematol. 30, 93–109. [12] Jacobsson, K. and Frykberg, L. (1999) Gene VIII-based, phage display vectors for selection against complex mixtures of ligands. In: Expression Genetics: Accelerated and High-Throughput Methods (McClelland, M. and Pardee, A., Eds.), pp. 225–238. BioTechniques Books, Natick, MA. [13] Fischer, B.E., Schlokat, U., Reiter, M., Mundt, W. and Dorner, F. (1997) Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale. Cell. Mol. Life Sci. 53, 943950. [14] Nilsson, M., Frykberg, L., Flock, J.-I., Pei, L., Lindberg, M. and Guss, B. (1998) A fibrinogen-binding protein of Staphylococcus epidermidis. Infect. Immun. 66, 2666–2673. [15] Jacobsson, K., Rosander, A., Bjerketorp, J. and Frykberg, L. (2003) Shotgun phage display – selection for bacterial receptins or other exported proteins. Biol. Proced. Online 5, 123–135.
161
[16] Wilson, M.B. and Nakane, P.K. (1978) Recent developments in periodate method of conjugating horseradish peroxidase (HRPOD) to antibodies. In: Immunofluorescence and related staining techniques (Holubar, K. and Wick, G., Eds.), pp. 215– 224. Elsevier North Holland Biomedical Press, Amsterdam. [17] Sambrook, J. and Russel, D. (2001) Molecular Cloning: A Laboratory Manual, third ed. Cold Spring Harbor Laboratory Press, NY. [18] Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6. [19] Bae, T. and Schneewind, O. (2003) The YSIRK-G/S motif of staphylococcal protein A and its role in efficiency of signal peptide processing. J. Bacteriol. 185, 2910–2919. [20] Ruoslahti, E. and Pierschbacher, M.D. (1986) Arg-Gly-Asp: a versatile cell recognition signal. Cell 44, 517–518. [21] Isberg, R.R. and Tran Van Nhieu, G. (1994) Binding and internalisation of microorganisms by integrin receptors. Trends Microbiol. 2, 10–14. [22] Ruoslahti, E. and Pierschbacher, M.D. (1987) New perspectives in cell adhesion: RGD and integrins. Science 238, 491–497. [23] Vecht, U., Wisselink, H.J., Jellema, M.L. and Smith, H.E. (1991) Identification of two proteins associated with virulence of Streptococcus suis type 2. Infect. Immun. 59, 3156–3162. [24] Smith, H.E., Vecht, U., Gielkens, A.L. and Smits, M.A. (1992) Cloning and nucleotide sequence of the gene encoding the 136kilodalton surface protein (Muramidase-released protein) of Streptococcus suis type 2. Infect. Immun. 60, 2361–2367. [25] Navarre, W.W. and Schneewind, O. (1994) Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in gram-positive bacteria. Mol. Microbiol. 14, 115–121. [26] Etienne, J., Poitevin-Later, F., Renaud, F. and Fleurette, J. (1990) Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains. FEMS Microbiol. Lett. 55, 93–97. [27] Sakariassen, K.S., Bolhuis, P.A. and Sixma, J.J. (1979) Human blood platelet adhesion to artery subendothelium is mediated by factor VIII-Von Willebrand factor bound to the subendothelium. Nature 279, 636–638. [28] Ikeda, Y., Handa, M., Kawano, K., Kamata, T., Murata, M., Araki, Y., Anbo, H., Kawai, Y., Watanabe, K., Itagaki, I., Sakai, K. and Ruggeri, Z.M. (1991) The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J. Clin. Invest. 87, 1234–1240.
A von Willebrand factor-binding protein from Staphylococcus lugdunensis Wiebensj€ Ljungh b, Martin Nilsson a, Joakim Bjerketorp a, Asa o a, Asa Lars Frykberg a, Bengt Guss a,* b
a Department of Microbiology, Swedish University of Agricultural Sciences, SE75007 Uppsala, Sweden Department of Medical Microbiology, Dermatology and Infection, Lund University, S€olvegatan 23, SE223 62 Lund, Sweden
Received 29 January 2004; received in revised form 11 March 2004; accepted 12 March 2004 First published online 23 March 2004
Abstract In the present study, a phage display library covering the genome of Staphylococcus lugdunensis, was affinity-selected against von Willebrand factor (vWf). This led to the identification of a gene, vwbl, encoding a putative cell surface protein of 2060 amino acids, denoted vWbl. The deduced protein has an overall organisation typical of staphylococcal cell surface proteins, with an N-terminal signal peptide, and a C-terminal cell wall sorting signal. The vWf-binding part is located in repetitive domains and antibodies against vWbl or vWf can inhibit the binding. Southern blot analysis showed that vwbl was present in the 12 S. lugdunensis strains tested. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Coagulase-negative staphylococci; Staphylococcus lugdunensis; Phage display; von Willebrand factor; Adhesin; LPXTG motif
1. Introduction Staphylococcus lugdunensis is a coagulase-negative staphylococcal species (CoNS) that has been reported to cause serious human infections such as endocarditis, septicemia and osteomyelitis as well as less severe infections, e.g. skin infections [1–3]. In a mouse abscess model, S. lugdunensis was shown to posses a high pathogenic potential relative to some other CoNS [4]. Further, the infections caused by S. lugdunensis show more resemblance to infections typical for Staphylococcus aureus, rather than CoNS. S. lugdunensis binds several different plasma and extracellular matrix proteins and most strains bind fibrinogen, collagens, vitronectin and laminin [5]. The ability to interact with and bind to host proteins is assumed to aid staphylococci in the initial attachment to the host tissue and to *
Corresponding author. Tel.: +46-0-1867-3205; fax: +46-0-18673392. E-mail address: [email protected] (B. Guss).
evade the immune defence [6,7]. Concerning bacterial proteins binding to von Willebrand factor (vWf), there are only a few reports. The binding of S. aureus to vWf was first reported in 1997 [8] and later it was shown that Protein A mediates the adherence of S. aureus to vWf [9]. In addition, a secreted S. aureus protein (vWbp) that binds vWf has recently been identified using phage display [10]. vWf is a large glycoprotein found in plasma (10 lg ml1 ) and in storage granules of platelets and endothelial cells. vWf has a dual and essential role in hemostasis. It binds to the subendothelium, primarily to exposed collagen at sites of vascular damage, and promotes platelet adhesion as well as aggregation. It is also a carrier and stabiliser of factor VIII, which is a cofactor that accelerates the clotting cascade [11]. In this study, a phage display library was made from genomic S. lugdunensis DNA. The library was subsequently affinity-selected (panned) against vWf, which revealed the existence of a large protein, with repetitive vWf-binding domains.
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.03.024
156
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161
2. Materials and methods
2.4. Cloning of vwbl
2.1. Bacterial strains, growth conditions and plasmids
To isolate the complete gene encoding the putative vWf-binding protein, Southern blot analysis of genomic DNA from strain 2342 was performed using the insert of phagemid clone SlvW2 (Fig. 1(A)) as a probe. Subsequently, 4 kb EcoRI-fragments was ligated into pUC18. Ampicillin-resistant transformants were screened by standard colony-hybridisation methods using the same probe as above. Sequence analysis of one selected clone (pEcoRIvwbl) revealed that the cloned fragment contained the 30 -end of the gene but lacked the 50 -end. Thus, to isolate the remaining portion of the gene, an additional Southern blot analysis was conducted, using a probe comprising a fragment from the 50 -end of the EcoRI-insert. Based on the result from this experiment a 3.2 kb HincII-fragment was ligated into the SmaI-site of pUC18 and the sequence of one selected clone was determined.
The 12 clinical S. lugdunensis isolates used in this study [5] were grown on tryptone soya agar or in tryptone soya broth (Oxoid). Escherichia coli strains were grown in Luria–Bertani (LB)-broth or on LA plates (LB-broth supplemented with 1.5% agar). Ampicillin (100 lg ml1 ) was added when appropriate (LA-amp). All bacterial incubations were done at 37 °C. E. coli strains ER2566 (New England Biolabs) and TG1 were used for protein production and phage display, respectively. The phagemid vector pG8SAET was used to construct the phage display library [12]. For additional cloning the plasmid pUC18 was used. 2.2. Proteins and reagents Human vitronectin was a kind gift from Ph.D. D.-Q. Li, Lund, Sweden and human thrombospondin was a kind gift from Prof. J. Lawler, Boston. Recombinant human von Willebrand factor (rvWf) was a kind gift from Prof. F. Dorner, Vienna. The rvWf has been shown to have an activity comparable to human plasmaderived vWf [13]. Polyclonal antibodies against human vWf were from Kordia (Leiden, The Netherlands), and human IgG was from Kabi Vitrum (Stockholm, Sweden). Casein, human serum albumin (HSA) and human fibronectin were from Sigma, and human fibrinogen was from IMCO Corp. Ltd. (Stockholm, Sweden). The fibrinogen-binding part (amino acids 52–646) of protein Fbe [14] of Staphylococcus epidermidis was expressed in E. coli following the same general scheme as described below under Section 2.5. 2.3. Construction of a phagemid library and panning A phage display library was constructed from S. lugdunensis strain 2342 essentially as described by Jacobsson et al. [15]. In short, fragmented genomic DNA of 0.1–3.5 kb in size was inserted into the pG8SAET phagemid vector. After electroporation into E. coli TG1 cells, the library consisted of 2 108 individual clones, the insertion rate was 90%, and the average insert size 0.5 kb. After infection with helper phage R408 (Promega), the phagemid titre was 1 1010 colony forming units (cfu) ml1 . A microwell (Maxisorp, Nunc) was coated for 2 h at room temperature (RT) with 200 ll of rvWf at a concentration of 20 lg ml1 in 0.05 M NaHCO3 (pH 9.7). The well was blocked with PBST (140 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 1.4 mM KH2 PO4 [pH 7.4] + 0.05% Tween 20) containing 1 mg ml1 casein, for 1 h at RT. The affinity-selection (panning) procedure was done as described in detail elsewhere [15].
2.5. Recombinant proteins and production of antibodies The region of vwbl, corresponding to the amino acids (aa) 1247–1503 of vWbl (Wbl3R), was PCR-amplified, using the forward primer vWblF (50 -TATATAC CATGGCAACAATTCCAGATCGCG), the reverse primer vWblR (50 -TTTATACCCGGGTTCTGACTG GATACGTTCATAC) and DNA from clone pEcoRIvwbl as template. The underlined sequences correspond to complementary sequences of vwbl. The fragment obtained, was digested with NcoI and SmaI and ligated into the corresponding sites in the expression vector pTYB4 of the IMPACT-system (New England Biolabs). The ligated DNA was electrotransformed into E. coli ER2566. Plasmids harbouring inserts were isolated from transformants and verified by DNA sequencing. The Wbl3R protein was expressed and purified following the manufacturer’s instructions. Polyclonal antibodies (IgY) against Wbl3R were raised in chicken by AgriSera (V€ann€as, Sweden) and affinity-purified on a Wbl3R-coupled HiTrap-column (Amersham Biosciences). The affinity-purified antibodies were conjugated with horseradish peroxidase (HRP) according to the method of Wilson and Nakane [16]. The minimal vWf-binding domain of vWbp (26 aa) of S. aureus was expressed using the same general scheme as outlined above. The forward primer vWbp26F (50 -AA TTATCCATGGGCACATCACCGACTACATATAC) and the reverse primer vWbp26R (50 -TATACCCG GGAATTTGTTGCTGAGTTTGAC) were used to PCR-amplify the relevant fragment using DNA from S. aureus strain Newman as template. 2.6. ELISA Microwells were coated with different proteins at a concentration of 0.25 lg ml1 in 0.05 M NaHCO3 (pH
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161
157
A
SlvW8 1608-1650 R5
R6
1303
48
S
2060
R
A R1
R2
W R3
SlvW1 1413-1440 SlvW2 1392-1460 SlvW3 1408-1451 SlvW4 1393-1450 SlvW5 1395-1436 SlvW6 1411-1451 SlvW7 1409-1444 B R1
1303-IPNDAPQETPVALEVTRYVDSEGNEVQETEEGTHDAPGIIADKWQYTGQTAAENGITTHVYQRIQSE
R2
1370-IPNEAPQETPVALEVTCYVDSEGNEVQETEEGTHDAPGIIGDKWQYTGQTTTEDGITTHIYQRIQSE
R3
1437-IPNEAPQETPVALEVTRYVDSEGNEVQETEEGTHQPPSIIGDKWQYTGQTTTADGITTYVYERIQSE
R4
1504-IPNEAPKETPIQLEVTRYVDGEGNEVQETEEGTHHAPGIIGDKWQYTGQTTTESGITTHVYERIQSE
R5
1571-IPNEAPQETPVQLEVTRYVNSEGNEVQETEEGTHQPPGIIGDKWQYTGQTTTADGITTYVYERIQSE
R6
1638-IPNEAPKETPVQLEVTRYVDTDGNEVQETEEGTHQPPGIIGDKWQYTGRVTEKDGITTYVYERIQSA
R7
1705-IPNEAPQETPVQLEVTRYVDITGNEVQETEEGTHQPRYIIGDKWRYSGVTVTENGITKHVYERIQSK
R8
1772-VPNDAPQETPVQLEVTRYVDPEGNEIQETTEGKHQPPGIIGDRWQYTGKVTEKDGIITYVYERIQSE
R9
1839-IPNNPPQETPVELEVTRYVDGEGNEVQETTEGKHQPPSIIGDRWQYTGKVTEKDGITTYVYERIQSK .**..*.***..**** **. .***.***.**.*... **.*.*.*.* ... .**....*.****
R10 1906-VPNDAPRVDIDELKITIYVDTNGREIVPSRKGQLPPEQFIGQDWQYTGHKIEKDGITTYIYKKVENA .**..*. .
.*..* **. .*.*.
. .*
.
.*.. *.*.*
. .**....*.....
Fig. 1. (A) Schematic presentation of vWbl aligned against translated inserts from rvWf-binding phagemid clones. The different regions on vWbl are indicated as S (signal peptide), A (non-repetitive region), R (repeat-region) and W (wall-associated region). The clones SlvW1–SlvW7, originate from panning experiments where phagemid particles were eluted by lowering the pH. The clone SlvW8 originates from a panning when bound phage was recovered by adding E. coli TG1 cells directly to the well. (B) Alignment of the ten repeats from vWbl. Since R10 is considerably more diverged than the other repeats, it is separately aligned to more clearly demonstrate the high similarity between the other repeats. Amino acids perfectly conserved in all repeats are indicated with an asterisk and functionally conserved amino acids with a dot. The minimal binding domain of 24 aa is marked with a box. The nucleotide sequence of the vwbl gene has the GenBank Accession No. AY530288.
9.7) for 1.5 h at RT. Wells were washed with PBST, and blocked with PBST containing 1 mg ml1 casein for 1 h at RT. After washing, Wbl3R (40 lg ml1 ) was added with or without the various inhibitors, as described in the figure legends for the individual experiment, and incubated at 4 °C overnight. Wells were washed and peroxidase-conjugated anti-Wbl3R antibodies were added to the wells (2 lg ml1 ), which were incubated for 2 h at RT. The wells were washed again and bound protein was detected using 3,30 ,5,50 -tetramethylbenzidine. The reaction was stopped by the addition of 1 M H2 SO4 and the absorbance read at 450 nm. 2.7. Detection of vwbl-like genes in strains of S. lugdunensis Genomic DNA was prepared from strains of S. lugdunensis using a DNeasy Tissue kit (Qiagen) with the modification that an addition of lysostaphin at 200 lg ml1 was used to lyse the cells. The presence of
vwbl or a gene similar to vwbl was detected by Southern blot analysis using a PCR-generated fragment encompassing the 2 kb repeat-region, of vwbl, which were labelled with 32 P by nick-translation using the Ready-To-Go DNA Labelling kit (Amersham Biosciences). Southern blot analysis was performed as described in [17]. 2.8. DNA sequencing and analysis of sequences The nucleotide sequence of vwbl was determined with the Dyenamic ET Terminator Cycle Sequencing Premix kit (Amersham Biosciences) and a model 377 Perkin– Elmer DNA sequencer, using a primer walking strategy. Specific synthetic primers were from Invitrogen. The computer program PCGENE (Intelligenetics Inc., Mountain View, CA, USA), were used to analyse the sequence data. Similarity searches at the nucleotide and amino acid level were performed using BLAST (http:// www.ncbi.nlm.nih.gov/BLAST/).
158
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161
3. Results and discussion 3.1. Isolation of phagemid clones expressing vWf-binding polypeptides To identify interactions between S. lugdunensis and host proteins, a phage display library was constructed in the vector pG8SAET using genomic DNA from S. lugdunensis strain 2342. The library was panned against rvWf, and after two rounds of panning a specific enrichment was obtained. In the second panning 200 times more phage particles bound to rvWf compared to the control protein (casein; data not shown). The nucleotide sequence from ten phagemid inserts, showed that the majority overlapped the same region of the genome. Several independent panning experiments against rvWf were conducted, all resulting in isolation of phagemids containing different inserts derived from the same region, suggesting that a novel vWf-binding protein had been identified. 3.2. Cloning and sequencing of vwbl The sequence of the complete gene was obtained from two separate clones, which revealed an open reading frame of 6180 nucleotides starting with a TTG codon. The open reading frame is preceded by a typical ribosomal binding site (AGGAGA), situated a few nucleotides upstream of the start codon. The coding sequence is followed by a transcriptional terminating sequence consisting of an inverted repeat and a T-rich region. The gene, termed vwbl, encodes a putative protein of 2060 aa (Fig. 1(A)), named vWbl (von Willebrand factor-binding protein of S. lugdunensis). In the N-terminal portion of the protein, a signal peptide is predicted with the most likely cleavage site located between aa 47 and 48 [18]. The signal peptide also contains the motif YSIRKG/S, demonstrated to play a role in the efficient secretion of proteins equipped with this motif [19]. Based on the proposed signal peptide, the mature vWbl consists of 2013 aa with a predicted molecular mass of 226 kDa. Following the signal peptide there is a region, termed A, consisting of 1255 aa (Fig. 1(A)). The A-region has no apparent similarity to other proteins but it harbours the interesting motif, Arg-Gly-Asp (RGD), at position 1134–1136, a motif found in many integrin-binding proteins, e.g. fibronectin, laminin and vWf [20], as well as in cell surface proteins of several pathogens [21,22]. Although, to our knowledge, there has been no reports describing staphylococcal binding to integrins through an RGD-dependent mechanism, it is tempting to speculate that vWbl could mediate direct adherence of S. lugdunensis cells to eukaryotic cells. A repeat (R)-region consisting of ten repeats, termed R1-R10, where each repeat comprises 67 aa, follows the A-region. An alignment of the ten repeats shows high similarity
between them (Fig. 1(B)). Further, the repeats show homology to the repeats of the muramidase-released protein from Streptococcus suis. This protein is a cell wall anchored protein, associated with virulence of S. suis [23,24]. Apart from this homology, vWbl do not show any significant similarity to any known protein at the amino acid level. The C-terminal part of vWbl harbours several characteristic features found in cell surface proteins of Gram-positive bacteria. An LPXTGmotif (aa 2026–2030), known to be involved in anchoring surface proteins covalently to the cell wall [25], is followed by a membrane-spanning region and a stretch of charged amino acid residues. 3.3. The vWf-binding domains in vWbl Despite three independent panning experiments, only seven different clones were found (SlvW1–SlvW7) (Fig. 1(A)), many of which were isolated several times. Alignment of the inserts revealed that all contained the C-terminal end of the R2-repeat, from which a ‘‘minimal’’ binding domain consisting of only 24 aa, could be deduced (Fig. 1(B)). No clone contained a complete R2domain, nor were any of the other repetitive domains found under these panning conditions. This result is puzzling, since nine of the ten different repeats are very similar (Fig. 1(B)). The library size, the insertion frequency, and the size of the inserted fragments, should ensure that all domains are represented in the library. In another successful panning experiment using the same library against another ligand, the inserts of the selected clones encoded polypeptides 300–450 aa in length (unpublished results). However, the clones described in this paper were shorter than expected, the longest clone encoded 68 aa and the shortest 27 aa. A possible reason for this could be that phagemid particles displaying other repeats, or several repeats, remain bound to vWf after pH-elution, and thereby are never recovered. In an additional experiment, the panning procedure was modified and instead of eluting the bound phage by lowering the pH, E. coli cells were added directly to the well. This allowed bound phage to infect the cells, which subsequently were plated on LA-amp plates. This resulted in isolation of phagemid particles comprising parts of the R5- and R6-repeats (SlvW8 in Fig. 1(A)) in addition to clones containing the R2-repeat. Thus, most likely the other R-domains also mediate binding to vWf, but are not found with the elution conditions used in this study. 3.4. The binding to vWf is specific and can be inhibited by Wbl3R To investigate the binding between vWbl and rvWf, a phage stock, from clone SlvW5 (Fig. 1A), was generated. The phage stock was separately panned against
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161
rvWf, fibrinogen, fibronectin, IgG, vitronectin, HSA, thrombospondin and uncoated microwells. Approximately 103 more phagemid particles bound to rvWf than to any other protein in the assay (data not shown). In addition, the phage stock was used in two inhibition assays. In the first, recombinant protein, WblR3, comprising the C-terminal end of the A-region and repeats R1–R3 of vWbl, was incubated in rvWf-coated microwells prior to addition of the phage stock. As shown in Fig. 2(A), the binding between rvWf and the phage particles was inhibited by approximately 95% when WblR3 was present, while HSA had no effect. In the second inhibition assay, antibodies against Wbl3R was stepwise diluted and incubated with the SlvW5 phage stock before transfer to rvWf-coated wells. Fig. 2(B), shows that the specific antibodies inhibited phage
A
90000 80000 70000
Cfu ml
-1
60000 PBS Wbl3R HSA
50000 40000 30000 20000 10000 0
B 1,0E+09
Cfu ml-1
1,0E+08 PBS S
Anti-Wbl3R IgY
1,0E+07
Pre-immune IgY
1,0E+06
159
particles from binding to rvWf in a dose-dependent manner, whereas pre-immune antibodies had no effect. 3.5. Characterisation of the vWbl–vWf-binding To validate the specific binding between Wbl3R and rvWf, various ELISAs were performed. In one ELISA, rvWf was immobilised in microwells and Wbl3R was added together with rvWf, fibrinogen or fibronectin. The binding was inhibited by soluble rvWf in a dose-dependent manner (Fig. 3A), thus demonstrating that both immobilised and soluble rvWf are recognised by Wbl3R. Fibrinogen or fibronectin had no effect on the binding. In another ELISA, Wbl3R were incubated together with polyclonal anti-human-vWf antibodies or unspecific antibodies, in rvWf-coated microwells (Fig. 3(B)). The results showed that addition of unspecific antibodies at a high concentration had no effect, whereas the same concentration of anti-vWf antibodies lowered the binding with almost 90%. We have recently described a secreted vWf-binding protein, vWbp, encoded by S. aureus [9]. The deduced ‘‘minimal’’ binding domain of vWbp is 26 aa, and a recombinant peptide comprising these 26 aa (vWbp26) inhibited the interaction between vWbp and rvWf in a dose-dependent manner (Bjerketorp, unpublished results). As shown in Fig. 3(C), the binding of Wbl3R to rvWf was inhibited in the presence of vWbp26, suggesting that vWbl and vWbp bind to the same region of vWf. This was somewhat surprising since both binding polypeptides are small, and show no sequence similarities. Besides, vWf is a large protein, with many potential binding sites. Nevertheless, not only have these bacterial species evolved proteins that bind vWf, but these proteins also bind to the same site in vWf. It should be noted that vWbl is cell surface anchored whereas vWbp is secreted. This suggests that the binding of vWbl to vWf, not only mediates S. ludgunensis adherence to vWf, but may also affect some of the biological functions served by vWf.
1,0E+05 PBS
1:500 1:100
1:20
1:4
Dilution
Fig. 2. Inhibition of the binding between phagemid (SlvW5) particles and immobilised rvWf. (A) Microwells coated with rvWf were separately incubated for 1 h with PBS, or PBS supplemented with Wbl3R, or HSA, both at the concentration of 2 mg ml1 . One tenth of the volume was then exchanged for 5 ll of phagemid particles (5 105 cfu), and incubation continued for 2 h. (B) Phagemid particles (5 109 cfu ml1 ) were incubated with anti-Wbl3R IgY or pre-immune IgY and transferred to rvWf-coated microwells. The anti-Wbl3R IgY was diluted in steps of five. In both (A) and (B), the wells were washed and bound phagemid particles were eluted by lowering the pH. Aliquots were used to infect E. coli TG1 cells, which were plated on LA-amp plates. The result is shown as cfu ml1 eluate. In (A), each value is the means of totally four infections from two separate wells. In (B), bars represent mean values of duplicate experiments. Standard deviations are indicated.
3.6. Clinical isolates of S. lugdunensis possess vwbl-like genes The distribution of the vwbl gene, among clinical isolates of S. lugdunensis, was investigated using Southern blot analysis. Genomic DNA from strain 2342 and 11 other clinical strains were digested with EcoRI and probed in a Southern blot analysis with a labelled PCRfragment, covering the ten repeats of vwbl. All strains possessed a fragment that reacted with this probe (Fig. 4). Restriction endonuclease analysis of genomic DNA from S. lugdunensis has shown that different isolates of this species are very homogenous [26]. Interestingly, as indicated by Southern blot analysis, there was a clear difference in size of the hybridising EcoRI-fragments, between
160
A
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161 0.4
A450
0.3 PBS vWf Fg
0.2
Fn 0.1
0 PBS S
1
5
25
Protein concentratio concentration (µg ml-1)
B 0.3
A450
PBS
anti-vWf IgG
0.2
Fig. 4. The presence of the R-region of vwbl in S. lugdunensis isolates as demonstrated by Southern blot analysis. The genomic DNA of the different staphylococci was digested with EcoRI. Lane 1, S. aureus strain Newman (negative control); lanes 2–13 represent different clinical isolates of S. lugdunensis (2, G5-87; 3, G2-89; 4, G16-89; 5, G6-87; 10, 2342; 11, 49/90; 12, 49/91; 13, 6, G58-88; 7, G66-88; 8, G3A; 9, SA; A251 [5]). A nucleotide size marker is indicated.
0.1
0 PBS
1:6250
1:1250
1:250
Dilution
C 0.3
0.2 A450
PBS
vWbp Fbe
vWf-binding protein with features characteristic for staphylococcal cell surface proteins. The distribution of this gene seems to be widespread among the clinical strains, but the importance of the protein vWbl in the context of infections remains to be elucidated. Under conditions corresponding to arterial blood flow, vWf is the bridging molecule required for platelet adherence to exposed subendothelium and platelet aggregation [27,28]. Similarly, vWbl and its interaction with vWf might serve a critical role in the bacterial attachment to minor vascular lesions, and thereby in the colonisation preceding infections, e.g. endocarditis, caused by S. lugdunensis.
0.1
Acknowledgements 0 PBS S
1
5
25
Protein concentratio concentration (µg ml-1)
Fig. 3. Inhibition of the binding between WblR3 and rvWf detected with ELISA. Microwells were coated with rvWf and the binding of Wbl3R was inhibited with various proteins: (A) rvWf (vWf), fibrinogen (Fg), or fibronectin (Fn); (B) specific antibodies against human vWf (anti-vWf) or unspecific antibodies (IgG), the antibodies were diluted in steps of five, the highest concentration was 10 lg ml1 ; (C) vWbp26 (vWbp), or Fbe. Bound Wbl3R was detected with HRPlabelled anti-Wbl3R antibodies. Bars represent the mean values of duplicate experiments. Standard deviations are indicated.
tested strains (Fig. 4). In addition, PCR-amplification revealed heterogeneity between the strains in the length of the R-region (data not shown). In conclusion, we have demonstrated that S. lugdunensis strains possess a gene, vwbl, coding for a
This investigation was supported by grants from the Swedish Medical Research Council (K2000-16X-1305802B and 06X-11229), the Swedish Foundation for Strategic Research, Infection & Vaccinology Research Program (24/98) and Biostapro AB.
References [1] Etienne, J., Pangon, B., Leport, C., Wolff, M., Clair, B., Perronne, C., Brun, Y. and Bure, A. (1989) Staphylococcus lugdunensis endocarditis. Lancet 1, 390. [2] Fleurette, J., Bes, M., Brun, Y., Freney, J., Forey, F., Coulet, M., Reverdy, M.E. and Etienne, J. (1989) Clinical isolates of Staphylococcus lugdunensis and S. schleiferi : bacteriological characteristics and susceptibility to antimicrobial agents. Res. Microbiol. 140, 107–118. [3] Herchline, T.E. and Ayers, L.W. (1991) Occurrence of Staphylococcus lugdunensis in consecutive clinical cultures and relationship of isolation to infection. J. Clin. Microbiol. 29, 419–421.
M. Nilsson et al. / FEMS Microbiology Letters 234 (2004) 155–161 [4] Lambe Jr., D.W., Ferguson, K.P., Keplinger, J.L., Gemmell, C.G. and Kalbfleisch, J.H. (1990) Patogenicity of Staphylococcus ludgunensis, Staphylococcus schleiferi, and three other coagulasenegative staphylococci in a mouse model and possible virulence factors. Can. J. Microbiol. 36, 455–463. (1993) Serum and [5] Paulsson, M., Petersson, A.C. and Ljungh, A tissue protein binding and cell surface properties of Staphylococcus lugdunensis. J. Med. Microbiol. 38, 96–102. [6] Flock, J.-I. (1999) Extracellular-matrix-binding proteins as targets for the prevention of Staphylococcus aureus infections. Mol. Med. Today 5, 532–537. € k, M. (1998) Surface protein adhesins of [7] Foster, T.J. and H€ oo Staphylococcus aureus. Trends Microbiol. 6, 484–488. [8] Herrmann, M., Hartleib, J., Kehrel, B., Montgomery, R.R., Sixma, J.J. and Peters, G. (1997) Interaction of von Willebrand factor with Staphylococcus aureus. J. Infect. Dis. 176, 984–991. [9] Hartleib, J., Kohler, N., Dickinson, R.B., Chhatwal, G.S., Sixma, J.J., Hartford, O.M., Foster, T.J., Peters, G., Kehrel, B.E. and Herrmann, M. (2000) Protein A is the von Willebrand factor binding protein on Staphylococcus aureus. Blood 15, 2149– 2156. Flock, J.-I., Jacobsson, K. [10] Bjerketorp, J., Nilsson, M., Ljungh, A, and Frykberg, L. (2002) A novel von Willebrand factor binding protein expressed by Staphylococcus aureus. Microbiology 148, 2037–2044. [11] Vischer, U.M. and de Moerloose, P. (1999) von Willebrand factor: from cell biology to the clinical management of von Willebrand disease. Crit. Rev. Oncol. Hematol. 30, 93–109. [12] Jacobsson, K. and Frykberg, L. (1999) Gene VIII-based, phage display vectors for selection against complex mixtures of ligands. In: Expression Genetics: Accelerated and High-Throughput Methods (McClelland, M. and Pardee, A., Eds.), pp. 225–238. BioTechniques Books, Natick, MA. [13] Fischer, B.E., Schlokat, U., Reiter, M., Mundt, W. and Dorner, F. (1997) Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale. Cell. Mol. Life Sci. 53, 943950. [14] Nilsson, M., Frykberg, L., Flock, J.-I., Pei, L., Lindberg, M. and Guss, B. (1998) A fibrinogen-binding protein of Staphylococcus epidermidis. Infect. Immun. 66, 2666–2673. [15] Jacobsson, K., Rosander, A., Bjerketorp, J. and Frykberg, L. (2003) Shotgun phage display – selection for bacterial receptins or other exported proteins. Biol. Proced. Online 5, 123–135.
161
[16] Wilson, M.B. and Nakane, P.K. (1978) Recent developments in periodate method of conjugating horseradish peroxidase (HRPOD) to antibodies. In: Immunofluorescence and related staining techniques (Holubar, K. and Wick, G., Eds.), pp. 215– 224. Elsevier North Holland Biomedical Press, Amsterdam. [17] Sambrook, J. and Russel, D. (2001) Molecular Cloning: A Laboratory Manual, third ed. Cold Spring Harbor Laboratory Press, NY. [18] Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6. [19] Bae, T. and Schneewind, O. (2003) The YSIRK-G/S motif of staphylococcal protein A and its role in efficiency of signal peptide processing. J. Bacteriol. 185, 2910–2919. [20] Ruoslahti, E. and Pierschbacher, M.D. (1986) Arg-Gly-Asp: a versatile cell recognition signal. Cell 44, 517–518. [21] Isberg, R.R. and Tran Van Nhieu, G. (1994) Binding and internalisation of microorganisms by integrin receptors. Trends Microbiol. 2, 10–14. [22] Ruoslahti, E. and Pierschbacher, M.D. (1987) New perspectives in cell adhesion: RGD and integrins. Science 238, 491–497. [23] Vecht, U., Wisselink, H.J., Jellema, M.L. and Smith, H.E. (1991) Identification of two proteins associated with virulence of Streptococcus suis type 2. Infect. Immun. 59, 3156–3162. [24] Smith, H.E., Vecht, U., Gielkens, A.L. and Smits, M.A. (1992) Cloning and nucleotide sequence of the gene encoding the 136kilodalton surface protein (Muramidase-released protein) of Streptococcus suis type 2. Infect. Immun. 60, 2361–2367. [25] Navarre, W.W. and Schneewind, O. (1994) Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in gram-positive bacteria. Mol. Microbiol. 14, 115–121. [26] Etienne, J., Poitevin-Later, F., Renaud, F. and Fleurette, J. (1990) Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains. FEMS Microbiol. Lett. 55, 93–97. [27] Sakariassen, K.S., Bolhuis, P.A. and Sixma, J.J. (1979) Human blood platelet adhesion to artery subendothelium is mediated by factor VIII-Von Willebrand factor bound to the subendothelium. Nature 279, 636–638. [28] Ikeda, Y., Handa, M., Kawano, K., Kamata, T., Murata, M., Araki, Y., Anbo, H., Kawai, Y., Watanabe, K., Itagaki, I., Sakai, K. and Ruggeri, Z.M. (1991) The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J. Clin. Invest. 87, 1234–1240.