Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-75007 Uppsala, Sweden1
Department of Infectious Diseases and Medical Microbiology, Lund University, Sölvegatan 23, SE-22362 Lund, Sweden2
Department of Microbiology, Pathology and Immunology, Huddinge University Hospital, Karolinska Institutet, SE-14186 Huddinge, Sweden3
Author for correspondence: Lars Frykberg. Tel: +46 18673299. Fax: +46 18673392. e-mail: Lars.Frykberg{at}mikrob.slu.se
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ABSTRACT |
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Keywords: receptin, virulence factor, phage display
Abbreviations: HSA, human serum albumin; HRP, horseradish peroxidase; RT, room temperature; vWf, von Willebrand factor; vWbp, von Willebrand factor binding protein
a The GenBank accession number for the sequence reported in this paper is AY032850.
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INTRODUCTION |
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The mature form of von Willebrand factor (vWf) is a large multifunctional glycoprotein, consisting of 2050 aa arranged in four different types of repeats (A through D). vWf exists as homodimers about 540 kDa in size, or multimers of different sizes up to 20000 kDa. Analysis by SDS-PAGE of plasma-derived vWf with reduced disulfide bonds reveals a predominant band with mobility corresponding to an apparent molecular mass of 225 kDa. The molecular mass of the vWf subunit, based on its chemical composition, is approximately 270 kDa. vWf is synthesized exclusively by endothelial cells and megakaryocytes (Ruggeri, 1999 ). The endothelial cells generate a plasma pool of vWf with a concentration of
10 µg ml-1 as well as an intracellularly stored supply of vWf in WeibelPalade bodies. Megakaryocytes are responsible for vWf stored within the
-granule of platelets. By supporting platelet adhesion and aggregation to exposed subendothelium in damaged blood vessels, vWf is an essential component in the maintenance of haemostasis, especially under conditions of rapid blood flow. vWf mediates platelet adhesion through two distinct platelet receptors: the glycoprotein (GP) Ib in the GP IbVIX complex and the GP IIbIIIa (also called integrin
IIbß3). Furthermore, vWf transports and stabilizes the coagulation factor VIII. vWf contains an Arg-Gly-Asp (RGD) motif also recognized by the endothelial integrin
Vß3 and vWf can in addition bind to various subendothelial components, such as collagens (type I, III and VI) and heparin-like glucosaminoglycans (Ruggeri, 1999
; Ruggeri & Ware, 1993
). Malfunctional vWf, or a reduced amount of this glycoprotein, leads to one of several types and subtypes of von Willebrand disease, which is the most common inherited bleeding disorder (Mohlke et al., 1999
).
In this investigation a shotgun phage-display library was made from chromosomal DNA of S. aureus strain Newman. The library was affinity selected (panned) against recombinant vWf, which resulted in the finding of a novel von Willebrand factor binding protein (vWbp).
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METHODS |
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Bacterial strains, growth conditions and helper phage.
Escherichia coli TG1 (Sambrook et al., 1989 ) was used for construction of the phage library and production of phage stocks. E. coli BL21(DE3) (Novagen) was used for expression of recombinant vWbp. E. coli was grown in LuriaBertani (LB) broth or on LA plates (LB with 1·5% agar) supplemented with 50 µg ampicillin ml-1 (LA-amp) when appropriate. Phage R408 (Promega) was used as helper phage for production of phage stocks. The S. aureus strains used were Newman, 8325-4, Wood 46 and five different human clinical isolates kindly provided by Dr B. Christensson, University of Lund, Sweden. Clinical isolates of Staphylococcus epidermidis were used as negative controls. S. aureus Newman
Eap, an isogenic mutant strain of S. aureus Newman in which the gene for staphylococcal extracellular adherence protein (Eap) has been deleted (J.-I. Flock, unpublished), was used for purification of vWbp. Staphylococci were grown in Tryptic Soya Broth (TSB, Oxoid).
Construction of an S. aureus shotgun phage-display library.
A shotgun phage-display library was constructed from S. aureus strain Newman DNA as described previously (Jacobsson & Frykberg, 1995 , 1996
). In short, DNA fragments of approximately 0·55 kb, obtained by sonication, were ligated into the pG8SAET phagemid vector (Jacobsson & Frykberg, 1999
). After transformation into E. coli TG1 and infection with helper phage R408, the final library consisted of 107 individual clones and had a titre of 1·5x109 c.f.u. ml-1.
Panning of the shotgun phage-display library.
Microwells (Maxisorp, Nunc) were coated with 50 µg vWf in 200 µl coating buffer (0·05 M NaHCO3, pH 9·5) and incubated at room temperature (RT) with shaking for 1 h. The wells were then washed three times with phosphate-buffered saline containing 0·05% Tween 20 (PBS-T). Two hundred microlitres of the phagemid library were then added to the vWf-coated wells, together with casein at a final concentration of 100 µg ml-1. Panning was carried out at RT with shaking for 4 h. After washing extensively with PBS-T, bound phages were eluted with 200 µl elution buffer (0·05 M sodium citrate, 0·15 M NaCl, pH 2·0) at RT for 2 min. After neutralization in a tube with 25 µl 2 M Tris/HCl, pH 8·7, 0·00150 µl volumes of the eluate were added to 25 µl stationary-phase E. coli TG1, together with LB broth to a final volume of 200 µl. The infection was allowed to proceed for 2030 min at RT before the suspension was spread on LA-amp plates for determination of the number of c.f.u. in the eluate. The plates were incubated overnight at 37 °C, then 150 colonies were transferred to two identical plates for screening and sequencing. The rest of the colonies were collected and infected with 10 µl helper phage R408 (1011 p.f.u. ml-1) for production of enriched phage stocks. The infected bacteria were mixed with 5 ml 0·5% soft agar, poured on an LA-amp plate and incubated at 37 °C overnight. The phages produced were recovered and the resulting phage stocks used for subsequent repannings, which were carried out as for the panning described above, except that repannings were accomplished in 2 h.
Screening and sequencing of phagemid clones.
After each round of panning, 150 colonies were picked in an identical pattern to two LA-amp plates, transferred to nitrocellulose filters (Schleicher & Schuell) and subsequently screened for expression of the phagemid expression tag (E-tag) with the anti-E-tag antibody. Phagemid DNA from positive clones was prepared with a Qiagen Miniprep kit (Qiagen) according to the manufacturers instructions. The inserted DNA fragments were sequenced using the DYEnamic ET Terminator Cycle Sequencing Premix Kit (Amersham Biosiences) and the samples analysed using the ABI 377 DNA Sequencer (Perkin Elmer) according to the manufacturers instructions. NTI Vector software (Informax) was used for handling the sequences obtained.
Cloning of the vwb gene.
The vwb gene was PCR-cloned from S. aureus strain Newman using Pwo DNA polymerase (Roche Diagnostics) and the primers 5'-GAATTCTCATATGATTCATGAAGAAGCC-3' (upstream) and 5'-GAATTCGCCATGCATTAATTATTTGCC-3'(downstream). The primers were designed based on S. aureus sequence data available at TIGR (http://www.tigr.org/). The resulting PCR product was cloned into pUC18 for subsequent sequencing.
Binding specificity of phagemid particles of NvWb32.
A phage stock of clone NvWb32 (Fig. 1) was prepared by infecting 500 µl of E. coli TG1 cells harbouring the phagemid with 10 µl helper phage R408 as above. After propagation in soft agar on an LA-amp plate, the phagemid particles were recovered as described above. The phage stock generated (2x1010 c.f.u. ml-1) was used in an experiment to analyse the binding specificity of the phagemid particles, and also in an inhibition experiment. In the binding specificity experiment 109 c.f.u. of the phage stock was panned against uncoated microwells and microwells coated with 2 µg of fibrinogen, fibronectin, vitronectin, vWf, IgG, HSA or casein. After 2 h of panning at RT the wells were washed, and the c.f.u. ml-1 of the eluate determined as described above. In the inhibition experiment 5x107 c.f.u. ml-1 of the phage stock was mixed with chicken antibodies, either unspecific or specific against vWbp, at a final concentration of 0·0850 µg ml-1. After 1 h pre-incubation at RT the mixtures were transferred to vWf-coated microtitre wells (1 µg vWf per well) and incubated for 2 h, followed by washing and determination of the c.f.u. ml-1 of the eluate as above.
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Inibition of the binding between vWf and rvWbp-mat using specific antibodies.
Microwells were coated for 1 h with 100 µl of a solution containing 1 µg rvWbp-mat ml-1 and coating buffer (0·05 M NaHCO3, pH 9·5) at RT with shaking. The wells were then washed three times with PBS-T, after which PBS with chicken antibodies, either unspecific or specific against vWbp, were added and incubated for 30 min, whereupon 125I-labelled vWf (IODO-BEADS Iodination Reagent Kit, Pierce) was added to the wells and the mixture was further incubated for 1 h. The final concentrations of the antibodies were 0·01650 µg ml-1. The wells were washed three times with PBS-T and bound 125I-vWf was detected with a gamma-radiation counter (Searle).
Purification and N-terminal sequencing of vWbp from S. aureus.
vWbp was purified from an S. aureus strain Newman Eap mutant (J.-I. Flock, unpublished). One hundred millilitres of an exponential-phase culture (OD600 3·0) was pelleted; the supernatant was sterile-filtered and subsequently passed through a HiTrap column with immobilized anti-vWbp antibodies. The eluate was collected in 1 ml fractions; the fractions were TCA-precipitated, the precipitates were dried and each fraction was resuspended in 10 µl water. The N-terminal sequence of the purified vWbp was determined by Edman sequencing by Bo Ek, Department of Plant Biology, Swedish University of Agricultural Sciences (SLU).
SDS-PAGE and Western blot analysis.
Protein samples were prepared for gel electrophoresis by mixing equal volumes of protein solution and 2x sample buffer [1x sample buffer is 62·5 mM Tris/HCl pH 6·8, 10% (v/v) glycerol, 2% SDS, 5% ß-mercaptoethanol and 0·01% bromophenol blue]. After boiling, the samples were analysed by SDS-PAGE using the PhastSystem (Amersham Biosciences) with PhastGel Gradient 415% or 825% gels and PhastGel SDS Buffer Strips. Proteins were blotted onto ECL nitrocellulose filters (Amersham Biosience). The presence of vWbp was detected either with chicken anti-vWbp antibodies and HRPanti-chicken antibodies or with vWf, goat anti-vWf antibodies and HRPanti-goat antibodies. Bound antibodies were detected with 4-chloro-1-naphthol (Sigma). 125I-labelled vWf was also used to detect vWbp, and bound 125I-vWf was visualized with Kodak BioMax MS film.
Purification of vWf from human serum.
Human serum (15 ml) was passed over a HiTrap column with immobilized rvWbp-part. The eluate was collected in 500 µl portions, which were TCA-precipitated and resuspended in 5 µl distilled water. vWf was detected with goat anti-vWf antibodies and HRP-labelled anti-goat antibodies in a Western blot experiment as described above.
Peptide sequencing by mass spectrometry.
Protein bands were excised from a Coomassie-blue-stained SDS-PAGE gel and cleaved with trypsin by in-gel digestion. Peptide analysis was performed by electrospray ionization mass spectrometry according to Wilm et al. (1996) on a Q-Tof mass spectrometer using the Masslynx software (Micromass, Manchester, UK). This was done by H
kan Larsson, Department of Plant Biology, Swedish University of Agricultural Sciences (SLU).
Detection of vwb in S. aureus.
Chromosomal DNA from eight different S. aureus strains and five S. epidermidis strains was prepared by using the DNeasy Tissue kit from Qiagen, supplemented with lysostaphin at a final concentration of 250 µg ml-1 in the cell lysis step. DNA was cleaved with EcoRI, separated on a 0·7% agarose gel and blotted to a nylon filter using the VaccuGene blotting system (Amersham Biosciences). After UV-fixation the filter was probed overnight at 65 °C with a 32P-labelled probe spanning the whole vwb gene. After washing, the filter was exposed to a Kodak BioMax MR film for 24 h at -70 °C.
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RESULTS AND DISCUSSION |
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Sequence analysis of vwb
The vwb gene is located between the clfA and emp genes. clfA encodes clumping factor A, the main fibrinogen-binding adhesin in S. aureus (McDevitt et al., 1994 ; Ni Eidhin et al., 1998
) and emp encodes an extracellular-matrix-protein-binding protein (Emp), a cell-surface protein with multiple binding activities (Hussein et al., 2001
). The clfA, vwb and emp genes are all in the same orientation according to the newly published complete genome sequence of two S. aureus strains, N315 and Mu50 (Kuroda et al., 2001
). The same gene organization is also seen in the different S. aureus sequencing projects in progress at TIGR (http://www.tigr.org/), University of Oklahoma (http://www.genome.ou.edu/) and the Sanger Centre (http://www.sanger.ac.uk/). The gene vwb encodes a previously uncharacterized protein now named von Willebrand factor binding protein (vWbp). Kuroda et al. (2001)
denote the gene product as a possible staphylocoagulase (function unknown). The amino acid sequences of the staphylocoagulase and vWbp from strain N315 shared 25% amino acid identity when a pairwise BLAST was performed at NCBI (http://www.ncbi.nlm.nih.gov/). Including the signal sequence, the deduced protein vWbp from strain Newman is composed of 508 aa starting with a leucine. The putative start codon TTG is preceded by a plausible ribosome-binding site (AGGAGA). Sequence analysis shows that the deduced mRNA transcript, after the translation stop codon, has a putative transcription terminator sequence with two stemloop structures followed by a stretch of six U residues. It is not likely that vwb is co-transcribed with clfA, since the deduced clfA mRNA sequence also contains a putative transcription termination sequence. The sequence of vwb obtained from S. aureus strain Newman was found to be identical to the sequence available from TIGR (S. aureus strain COL) and one of the sequences from the Sanger Centre (a hyper-virulent community-acquired methicillin-sensitive S. aureus strain), was also found to be completely identical in all but one nucleotide, leading to the substitution of an isoleucine by a leucine. The vwb sequences from the published, complete genomes of S. aureus strains N315 (methicillin-resistant) and Mu50 (methicillin-resistant and vancomycin-resistant) are identical to each other but only 80% identical to the vwb sequence from strain Newman. The second sequence from the Sanger Centre (the epidemic methicillin-resistant S. aureus strain EMRSA-16), shares about 80% sequence identity at the nucleotide level compared to strain Newman and strain N315. The vwb sequence from the University of Oklahoma (strain 8325) is identical to vwb from strain Newman, except for one missing nucleotide. Also in one vwb sequence from the Sanger Centre (strain EMRSA-16) one nucleotide is missing but at a different position. If correct, these frameshifts would lead to early disruptions of the vwb ORF but it seems more likely that the missing nucleotide in vwb in these two unfinished genomes is due to sequencing errors. Most of the differences between the vwb genes from the different strains are found in the first half of the gene. Very few differences are found in the C-terminal half of the deduced protein sequences (data not shown), which includes the vWf-binding domain.
The observed clustering of several genes encoding putative virulence factors is another interesting thing to consider, since many chromosomal virulence determinants are often associated in so-called virulence blocks (Hacker et al., 1997 ). The clfA gene is expressed in the exponential growth phase, but the expression is highest during postexponential growth (Ni Eidhin et al., 1998
; Wolz et al., 1996
). The conditions for expression of vwb and emp are not yet clarified at the mRNA level. At the protein level, Emp is detectable from mid-exponential growth and the concentration gradually increases up to stationary phase (Hussein et al., 2001
). Preliminary data indicate that vWbp is expressed already early in the exponential phase, becomes more abundant during the exponential growth and decreases in the stationary growth phase (data not shown).
Characterization of vWbp and its interaction with vWf
vWbp has a functional secretory signal sequence, with positively charged lysine residues in the N-terminal region, a central hydrophobic region and a more polar C-terminal region containing a signal peptidase cleavage sequence with alanines at the -3 and -1 positions. However, vWbp has no cell-wall-anchoring sequence typical of surface-bound proteins in Gram-positive bacteria. Thus, the protein should be found in a soluble form outside the bacteria. To further investigate vWbp, the part of the vwb gene encoding amino acids 124392 of the mature vWbp was expressed in E. coli and the recombinant protein was purified (rvWbp-part). Chickens were subsequently immunized with rvWbp-part and the resulting anti-vWbp antibodies were used to detect vWbp in the concentrated culture supernatant of S. aureus strain Newman. In a Western blot, the anti-vWbp antibodies specifically recognized a protein of 66 kDa. A protein of the same size was also detected by using vWf and anti-vWf-antibodies or by 125I-labelled vWf directly (Fig. 2
).
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ACKNOWLEDGEMENTS |
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Received 18 February 2002;
accepted 5 March 2002.