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• W2162585536 abstract "The opportunistic human pathogenStaphylococcus epidermidis is the major cause of nosocomial biomaterial infections. S. epidermidis has the ability to attach to indwelling materials coated with extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and collagen. To identify the proteins necessary for S. epidermidisattachment to collagen, we screened an expression library using digoxigenin-labeled collagen as well as two monoclonal antibodies generated against the Staphylococcus aureuscollagen-adhesin, Cna, as probes. These monoclonal antibodies recognize collagen binding epitopes on the surface of S. aureus andS. epidermidis cells. Using this approach, we identified GehD, the extracellular lipase originally found in S. epidermidis 9, as a collagen-binding protein. Despite the monoclonal antibody cross-reactivity, the GehD amino acid sequence and predicted structure are radically different from those of Cna. The mature GehD circular dichroism spectra differs from that of Cna but strongly resembles that of a mammalian cell-surface collagen binding receptor, known as the α1 integrin I domain, suggesting that they have similar secondary structures. The GehD protein is translated as a preproenzyme, secreted, and post-translationally processed into mature lipase. GehD does not have the conserved LPXTG C-terminal motif present in cell wall-anchored proteins, but it can be detected in lysostaphin cell wall extracts. A recombinant version of mature GehD binds to collagens type I, II, and IV adsorbed onto microtiter plates in a dose-dependent saturable manner. Recombinant, mature GehD protein and anti-GehD antibodies can inhibit the attachment of S. epidermidis to immobilized collagen. These results provide evidence that GehD may be a bi-functional molecule, acting not only as a lipase but also as a cell surface-associated collagen adhesin. The opportunistic human pathogenStaphylococcus epidermidis is the major cause of nosocomial biomaterial infections. S. epidermidis has the ability to attach to indwelling materials coated with extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and collagen. To identify the proteins necessary for S. epidermidisattachment to collagen, we screened an expression library using digoxigenin-labeled collagen as well as two monoclonal antibodies generated against the Staphylococcus aureuscollagen-adhesin, Cna, as probes. These monoclonal antibodies recognize collagen binding epitopes on the surface of S. aureus andS. epidermidis cells. Using this approach, we identified GehD, the extracellular lipase originally found in S. epidermidis 9, as a collagen-binding protein. Despite the monoclonal antibody cross-reactivity, the GehD amino acid sequence and predicted structure are radically different from those of Cna. The mature GehD circular dichroism spectra differs from that of Cna but strongly resembles that of a mammalian cell-surface collagen binding receptor, known as the α1 integrin I domain, suggesting that they have similar secondary structures. The GehD protein is translated as a preproenzyme, secreted, and post-translationally processed into mature lipase. GehD does not have the conserved LPXTG C-terminal motif present in cell wall-anchored proteins, but it can be detected in lysostaphin cell wall extracts. A recombinant version of mature GehD binds to collagens type I, II, and IV adsorbed onto microtiter plates in a dose-dependent saturable manner. Recombinant, mature GehD protein and anti-GehD antibodies can inhibit the attachment of S. epidermidis to immobilized collagen. These results provide evidence that GehD may be a bi-functional molecule, acting not only as a lipase but also as a cell surface-associated collagen adhesin. microbial surface components recognizing adhesive matrix molecules phosphate-buffered saline monoclonal antibody (Ab) bovine serum albumin enzyme-linked immunosorbent assay Staphylococcus epidermidis is now recognized as an important nosocomial pathogen. In the past 20 years it has emerged as a frequent cause of infections associated with indwelling devices such as catheters, artificial heart valves, and orthopedic implants (1Garrett D.O. Jochimsen E. Murfitt K. Hill B. McAllister S. Nelson P. Spera R.V. Sall R.K. Tenover F.C. Johnston J. Zimmer B. Jarvis W.R. Infect. Control. Hosp. Epidemiol. 1999; 20: 167-170Crossref PubMed Scopus (96) Google Scholar). In certain populations such as low birth weight infants and immuno-compromised patients S. epidermidis can be a prominent source of morbidity and mortality (2de Silva G.D.I. Justice A. Wilkinson A.R. Buttery J. Herbert M. Day N.P.J. Peacock S.J. Clin. Infect. Dis. 2001; 33: 1520-1528Crossref PubMed Scopus (28) Google Scholar). The molecular mechanisms of pathogenesis of S. epidermidisdisease are not well understood, but as with most infections, bacterial adherence to host surfaces is recognized as the first crucial step in the infection process and a prerequisite for colonization. A two-step process of S. epidermidis adherence is often described in which the first step is bacterial attachment to the biomaterial, and the second step includes microbial proliferation, intercellular adhesion, and biofilm formation. Almost all S. epidermidisstrains are able to attach to native abiotic surfaces (3Hogt A.H. Dankert J. Feijen J. J. Biomed. Mater. Res. 1986; 20: 533-545Crossref PubMed Scopus (135) Google Scholar, 4Pascual A. Fleer A. Westerdaal N.A. Verhoef J. Eur. J. Clin. Microbiol. 1986; 5: 518-522Crossref PubMed Scopus (117) Google Scholar, 5Tojo M. Yamashita N. Goldmann D.A. Pier G.B. J. Infect Dis. 1988; 157: 713-722Crossref PubMed Scopus (222) Google Scholar, 6Muller E. Hubner J. Gutierrez N. Takeda S. Goldmann D.A. Pier G.B. Infect. Immun. 1993; 61: 551-558Crossref PubMed Google Scholar). However, any foreign material implanted into the human body is quickly coated with various plasma proteins such as fibrinogen, fibronectin, and vitronectin (7Herrmann M. Vaudaux P., D. Pittet R.A. Lew P.D. Schumacher- Perdreau F. Peters G. Waldvogel F.A. J. Infect. Dis. 1988; 158: 693-701Crossref PubMed Scopus (429) Google Scholar, 8Yu J. Montelius M.N. Paulsson M. Gouda I. Larm O. Montelius L. Ljungh A. Biomaterials. 1994; 15: 805-814Crossref PubMed Scopus (36) Google Scholar), and Staphylococcus aureus, which is also a common cause of biomaterial centered infections, appears to adhere to this protein coat via adhesins of the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)1 type. Analysis of the adherence behavior of S. epidermidissuggests that this organism also expresses MSCRAMMs. In fact, a gene encoding a fibrinogen binding MSCRAMM (sdrG, also calledfbe) was cloned and sequenced from S. epidermidis(9Nilsson M. Frykberg L. Flock J.I. Lindberg M. Guss B. Infect. Immun. 1998; 66: 2666-2673Crossref PubMed Google Scholar). SdrG, a 119-kDa MSCRAMM, has a structural organization similar to the clumping factor (ClfA) from S. aureus and specifically recognizes the N-terminal region of the fibrinogen Bβ chain (10Davis S.L. Gurusiddappa S. McCrea K.W. Perkins S. Höök M. J. Biol. Chem. 2001; 276: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In addition, the autolysin AtlE, necessary for S. epidermidisattachment to polystyrene, was shown to specifically bind to biotin-labeled vitronectin (11Heilmann C. Hussain M. Peters G. Gotz F. Mol. Microbiol. 1997; 24: 1013-1024Crossref PubMed Scopus (574) Google Scholar). These data indicate that S. epidermidis, similarly to S. aureus, may express specific MSCRAMMs that mediate cell attachment to host protein-conditioned surfaces. In the present communication, we report that the GehD (12Longshaw C.M. Farrell A.M. Wright J.D. Holland K.T. Microbiology. 2000; 146: 1419-1427Crossref PubMed Scopus (59) Google Scholar) lipase binds to collagen type I, II, and IV and may mediate the adherence ofS. epidermidis cells to immobilized collagens. We identified GehD probing a S. epidermidis expression library with labeled collagen type I and monoclonal antibodies generated against theS. aureus collagen-binding protein, Cna. Staphylococcal lipases have been implicated as possible virulence factors in localized infections such as absesses (13Hedström S.A. Acta Pathol. Microbiol. Scand. Sect. B Microbiol. 1975; 83: 285-292Google Scholar, 14Hedström S.A. Nilsson-Ehle P. Acta Pathol. Microbiol. Scand. Sect. B Microbiol. 1983; 91: 169-173Google Scholar, 15Rollof J. Hedström S.A. Nilsson-Ehle P. Acta Pathol. Mocrobiol. Immunol. Scand. Sect. B Microbiol. 1987; 95: 109-113PubMed Google Scholar), and there is evidence that they are highly expressed during infection in a murine model (16Lowe A.M. Beattie D.T. Deresiewicz R.L. Mol. Microbiol. 1998; 27: 967-976Crossref PubMed Scopus (164) Google Scholar). The contribution of these enzymes to virulence is not clearly understood, although lipases may be important for the colonization and persistence of organisms on the skin (17Gribbon E.M. Cunliffe W.J. Holland K.T. J. Gen. Microbiol. 1993; 139: 1745-1751Crossref PubMed Scopus (113) Google Scholar). Another class of proteins that function as collagen binding adhesion receptors are the mammalian integrins. These proteins mediate the attachment of eukaryotic cells to the extracellular matrix. The integrins are transmembrane αβ heterodimeric proteins that mediate cell-cell and cell-matrix interactions of mammalian cells. In this extensive family of proteins, α1β1 and α2β1 are the primary collagen binding integrins. Within the α subunit of the collagen binding integrins, the ligand binding region is called I domain (33Lu C. Shimaoka M. Ferzly M. Oxvig C. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 98: 2387-2392Crossref Scopus (119) Google Scholar). Our data predict that mature GehD may adopt a structure that resembles that of the integrin α1 I-domain. The data described here show that the GehD lipase binds to collagens and may promote S. epidermidis attachment to immobilized collagens. Our data indicate that the GehD lipase may be a bifunctional molecule, acting as a glycerol ester hydrolase and a collagen adhesin. S. epidermidis strains 146, 9491, 12228, 14852, and 14990 were obtained from the ATCC collection. S. epidermidis 9, 2J24 (gehC::ermC), and KIC82 (gehD::ermC) were created by Christopher M. Longshaw (12Longshaw C.M. Farrell A.M. Wright J.D. Holland K.T. Microbiology. 2000; 146: 1419-1427Crossref PubMed Scopus (59) Google Scholar). S. aureus Cowan 1 spa::tet R strain was generously donated by T. Foster (University of Dublin, Ireland). All strains were grown in brain heart infusion or tryptic soy broth media (Difco) at 37 °C overnight. For the monoclonal antibody reactivity assays, bacteria were harvested and re-suspended in phosphate-buffered saline (PBS), pH 7.4 (140 mm NaCl, 270 μm KCl, 430 μm Na2HPO4, 147 μmKH2PO4) 0.02% sodium azide, washed, and adjusted to a cell density of 1010 cells/ml using a standard curve relating the A 600 to the cell number determined by counting cells in a Petroff-Hausser chamber. The cells were then heat-killed at 88 °C for 10 min. For all other assays, overnight cultures were diluted 1:1000 into fresh tryptic soy broth media, and the resultant culture was incubated until it reached logarithmic growth phase (A 6000.3–0.6). Bacteria were then harvested by centrifugation and used in attachment or Western assays. A S. epidermidis 9491 λZAP Express (Stratagene) expression library was constructed as follows. S. epidermidis 9491 chromosomal DNA was partially digested with MboI, and the fragments corresponding to 3–11 kilobases were isolated and purified. The purified fragments were ligated to the ZAP Express® (Stratagene) vector, predigested withBamHI, and dephosphorylated with CIAP (calf intestinal alkaline phosphatase). The resultant ligation product was packaged into phage particles using the Gigapack III Gold (Stratagene)-packaging extract. The obtained library was amplified and screened using the Escherichia coli XL1-Blue MRF′ strain. Clones of interest were excised from the λ ZAP Express® phage using the ExAssist® helper phage to generate the pBK-CMV phagemid vector packaged as filamentous phage particles. The filamentous phage stock was used to infect the E. coli XLOLR strain. The resultant colonies carrying the excised pBK-CMV phagemid vector were used for subsequent subcloning and dideoxy sequencing of the cloned inserts. A DNA fragment encoding the mature domain of the GehD lipase was PCR-amplified from S. epidermidis 9491 genomic DNA. The oligonucleotides primers 5′-TTT GAA TTC TGC GCA AGC TCA ATA TAA and 5′-TTT GCG GCC GCT ATC GCT ACT TAC GTG TAA were used to amplify the fragment designated as mature GehD. Constructs generated by PCR were cloned into the pETBlue-2 System using the E. coli NovaBlue strain as a cloning host and the E. coli Tuner (DE3) pLacI strain as the expression host. Large scale expression and preparation of recombinant proteins were as described previously using HiTrap nickel-chelating chromatography (10Davis S.L. Gurusiddappa S. McCrea K.W. Perkins S. Höök M. J. Biol. Chem. 2001; 276: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Protein concentrations were determined from the absorbance at 280 nm as measured on a Beckman Du-70 UV-visible spectrophotometer. The molar extinction coefficient of the proteins was calculated using the method of Pace et al. (18Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3471) Google Scholar). Purified collagen I (Vitrogen®, Cohesion, Palo Alto CA) was labeled with digoxigenin-3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxy-succinimide ester (digoxigenin) (Roche Molecular Biochemicals) according to the manufacturer's instructions. To label recombinant proteins with biotin, 7.5 mg of sulfosuccinimidyl-6-(biotinamido) hexanoate (NHS-LC-biotin; Pierce) was dissolved in 100 μl of dimethyl sulfoxide (Me2SO) and combined with 0.5 mg of recombinant protein in PBS. The total reaction (1 ml volume) was incubated in an end-over-end rotator at room temperature for 2 h then dialyzed against PBS and stored at 4 °C. Digoxigenin-labeled collagen or mAbs 11H11 and 1F6 (19Visai L., Xu, Y. Casolini F. Rindi S. Höök M. Speziale P. J. Biol. Chem. 2000; 275: 39837-39845Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) were used to screen the S. epidermidis 9491 λZAP Express (Stratagene) expression library. The library was plated using standard methods according to the vector manufacturer's instructions (Stratagene). After blocking additional protein binding sites on the filter lifts with a solution containing 3% (w/v) bovine serum albumin (BSA) in TBST (0.15 m NaCl, 20 mmTris-HCl, 0.05% (v/v) Tween 20, pH 7.4), digoxigenin-labeled collagen (0.5 μg/ml in TBST) was incubated with the filters. The bound digoxigenin-labeled collagen was incubated with anti-digoxigenin Fab conjugated to alkaline phosphatase (1:5000 in TBST, Roche Molecular Biochemicals). When mAbs 11H11 or 1F6 (1:500 in TBST) were used as probes, goat anti-mouse antibodies (1:4000 in TBST) conjugated to alkaline phosphatase (Bio-Rad) were used as secondary antibodies. Clones expressing collagen-binding proteins were identified by developing the membranes with 5-bromo-4-chlor-3-indoyl phosphatep-toluidine salt and p-nitro blue tetrazolium chloride (Bio-Rad). To test the reactivity of the mAbs generated against bacterial surface proteins, microtiter wells (Dasit, Milan, Italy) were coated overnight at 4 °C with 2 μg of human fibronectin in 100 μl of 50 mmsodium carbonate, pH 9.5, to provide a surface for bacterial attachment. The wells were washed five times with 10 mmsodium phosphate buffer, pH 7.4, containing 0.13 m NaCl and 0.1% (v/v) Tween 20 (PBST), and additional protein binding sites were blocked with a solution of 2% (w/v) BSA in PBS. Suspensions of 1 × 108 cells of S. epidermidis or S. aureus Cowan 1 spa::tet R) whole cells were added and incubated for 2 h at room temperature followed by 5 washes with PBS to remove unbound cells. Solutions of 2 μg of each monoclonal antibody in 100 μl of 2% (w/v) BSA in PBS were added, incubated for 2 h at room temperature, washed extensively with PBST, and detected with a 1:500 dilution of peroxidase-conjugated rabbit anti-mouse IgG (Dako, Gostrup, Denmark). The conjugated enzyme was incubated with o-phenylenediamine dihydrochloride (Sigma) as a substrate, and the color development absorbance was monitored at 492 nm using a microplate reader (Bio-Rad). To test protein-protein interactions, microtiter plates (Immulon 4, Dynex Technologies, Chantilly, VA) were coated with 1 μg of type I collagen in 100 μl of PBS/well overnight at 4 °C. Wells were then washed 3 times with PBS and blocked with 1% (w/v) bovine serum albumin in PBS for 1 h before the addition of varying concentrations of the biotinylated recombinant protein. After incubation at room temperature for 2 h with gentle shaking, the wells were extensively washed with PBS containing 0.05% (v/v) Tween 20 (PBST). Streptavidin-alkaline phosphatase conjugate (Roche Molecular Biochemicals) was diluted 10,000-fold with blocking buffer and added to the wells. After incubation at room temperature for 45 min, the wells were washed with PBST. For color development, 100 μl of 1.3m diethanolamine, pH 9.8, containing 1 mg/mlp-nitrophenyl phosphate (Sigma) was added to the wells. Absorbance at 405 nm (A 405 nm) was measured using a Thermomax microplate reader (Molecular Devices Corp., Menlo Park, CA) after 1 h of incubation at room temperature. Experiments were performed in triplicate and repeated with independently prepared protein preparations. Binding to BSA-coated wells was considered as background level and subtracted from binding to collagen. Data were presented as the mean value ± S.E. ofA 405 nm from a representative experiment (n = 3). The effect of antibodies as inhibitors of proteins binding to collagen was examined as described above except that biotinylated proteins were mixed with antibodies at varying ratios and added to the wells. The secondary structural composition of recombinant proteins was examined by CD spectroscopy. Far UV CD data were collected using a Jasco J720 spectropolarimeter calibrated with a 0.1% (w/v) d-10-camphorsulfonic acid solution using a bandwidth of 1 nm and integrated for 4 s at 0.2-nm intervals. All sample concentrations were less than 30 μm in 20 mm Tris-HCl buffer, pH 7.4. Spectra were recorded at ambient temperatures in 0.2-mm path length cuvettes. Thirty scans were averaged for each spectrum, the contribution from the buffer was subtracted, and quantitation of secondary structural elements was performed by deconvolution software provided by University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School (Piscataway, NJ) and D. Greenwood (Softwood Co., Brooksfield, CT). These deconvolution programs (SELCON and VARSLC1) are derived from databases of known protein structures. Analyses were performed using the BIAcore 1000 system (BIAcore AB, Uppsala, Sweden) as described previously (20Xu Y. Gurusidappa S. Rich R.L. Owens R.T. Keene D.R. Mayne R. Hook A. Hook M. J. Biol. Chem. 2000; 275: 38981-38989Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). The Cna protein was tested in HBS (10 mm HEPES, 150 mm NaCl, pH 7.4). The α1 I domain was tested in HBS containing 5 mmβ-mercaptoethanol, and mature GehD was tested in both HBS and glycine buffer (50 mm glycine, pH 7.4). Data from the equilibrium portion of the sensorgrams were used for analysis and calculation of the K D and n. Purified mature GehD was dialyzed in PBS, pH 7.4, before being sent to Rockland Immunochemicals, Inc. (Gilbertsville, PA) for immunization in rabbits and production of polyclonal antisera. IgGs were purified from both immune and preimmune serum by chromatography using protein A-Sepharose (Sigma). Microtiter plates (Immulon 4, Dynex Technologies, Chantilly, VA) were coated with 1 μg of type I collagen in 100 μl of PBS/well overnight at 4 °C. Wells were then washed 3 times with PBS and then blocked with 1% (w/v) bovine serum albumin in PBS for 1 h before the addition of bacteria. Early log-phase S. epidermidis cultures (A 600 of 0.5) were added, and the plates were incubated for 2 h at room temperature. After gentle washes, adherent cells were fixed with 100 μl of 25% (v/v) aqueous formaldehyde and incubated at room temperature for at least 30 min. The plates were then washed gently, stained with crystal violet, then washed again and read on an ELISA plate reader at 590 nm. To study inhibition of collagen binding by IgGs, S. epidermidis suspensions were preincubated with serial dilutions of purified IgGs in PBS for 2 h at room temperature. The cell suspensions were then transferred to ELISA plates coated with 1 μg of collagen/well, and their ability to attach to collagen was tested as described above. For whole-cell SDS-PAGE (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207530) Google Scholar), 2 × 107 S. epidermidis (previously treated with lysostaphin) cells or E. coli cells were boiled in 2% (w/v) SDS for 3–5 min under reducing conditions and subjected to electrophoresis through a 10% acrylamide gel at 150 V for 45 min. The separated proteins were stained with Coomassie Brilliant Blue. For Western ligand blot assays, whole cell lysates, or purified proteins were transferred from the polyacrylamide gel onto a nitrocellulose membrane in a semi-dry electroblot system (Bio-Rad). Additional binding sites on the membrane were blocked by incubating in 2% (w/v) BSA in TBST for 2 h at room temperature or overnight at 4 °C followed by three 10-min washes in TBST. The membrane was then incubated at room temperature with 0.5 μg of digoxigenin-labeled collagen/ml TBST for 1 h, washed, and incubated with 1:5000 anti-digoxigenin Fab alkaline-phosphatase conjugate (Roche Molecular Biochemicals) in TBST for 1 h. The membrane was washed, and collagen-binding proteins were visualized with 150 μg of 5-bromo-4-chlor-3-indoyl phosphate p-toluidine salt/ml and 300 μg of p-nitro blue tetrazolium chloride/ml (Bio-Rad) in carbonate bicarbonate buffer (14 mmNa2CO3, 36 mm NaHCO3, 5 mm MgCl2·6H2O, pH 9.8). The clinical isolate S. epidermidis 9491 was chosen as a prototype strain in our search for new MSCRAMMs. We tested its ability to adhere to immobilized bovine collagen type I, human fibrinogen, and human fibronectin. Each protein was immobilized in microtiter wells, and the bacteria attached to the wells were detected using crystal violet. The results presented in Fig.1 show that S. epidermidis9491 has the ability to attach to collagen, fibrinogen and fibronectin. Although previous studies have shown that S. epidermidisattachment to human fibrinogen is mediated by proteins such as Fbe and SdrG (9Nilsson M. Frykberg L. Flock J.I. Lindberg M. Guss B. Infect. Immun. 1998; 66: 2666-2673Crossref PubMed Google Scholar, 10Davis S.L. Gurusiddappa S. McCrea K.W. Perkins S. Höök M. J. Biol. Chem. 2001; 276: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), the bacterial components that mediate attachment to collagen or fibronectin were up to this point not identified. A panel of 22 monoclonal antibodies was previously generated against theS. aureus MSCRAMM Cna-(151–318) (19Visai L., Xu, Y. Casolini F. Rindi S. Höök M. Speziale P. J. Biol. Chem. 2000; 275: 39837-39845Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). We explored the possibility that at least some of these mAbs would cross-react with collagen-binding proteins on S. epidermidis by examining a panel of strains (S. epidermidis 146, 9491, 12228, 14852, and 14990). Two monoclonals, 11H11 and 1F6, cross-reacted with whole cells of all the S. epidermidis strains tested. Both of these antibodies were raised against the ligand binding central region of Cna-(151–318). Furthermore, these antibodies were shown to inhibit collagen binding to Cna and recognize conformationally dependent epitopes, presumably located in the ligand binding site of Cna-(151–318) (19Visai L., Xu, Y. Casolini F. Rindi S. Höök M. Speziale P. J. Biol. Chem. 2000; 275: 39837-39845Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). As expected, all of the anti-Cna mAbs bind toS. aureus Cowan 1 cells. The S. epidermidisstrains are recognized only by two antibodies. These results suggest that S. epidermidis exposes on its surface proteins that form epitopes similar to those present on Cna and that these proteins are recognized by 1F6 and 11H11. We constructed an expression library ligating MboI partially digested, size-selected genomic DNA from S. epidermidis 9491 to BamHI-digested λZAP Express II® vector. Using mAbs 1F6 and 11H11 as well as digoxigenin-labeled collagen, we screened ∼690,000 plaques. We isolated three clones that reacted with each mAb and labeled collagen. DNA sequencing of the excised phagemids revealed that 2 of the clones were identical, and the third had an additional 36 bp of upstream sequence. Further sequence analysis revealed that the cloned DNA immediately downstream of the T7lac sequence from the phagemid is 97% identical to the previously identified S. epidermidis second lipase gene, gehD (12Longshaw C.M. Farrell A.M. Wright J.D. Holland K.T. Microbiology. 2000; 146: 1419-1427Crossref PubMed Scopus (59) Google Scholar) (Fig.2 A). Previous studies of GehD and other staphylococcal lipases have shown that they are transcribed and translocated as 650–700-amino acid precursors that are processed post-translationally to extracellular mature lipases of about 360 amino acids with a size of ∼45 kDa (12Longshaw C.M. Farrell A.M. Wright J.D. Holland K.T. Microbiology. 2000; 146: 1419-1427Crossref PubMed Scopus (59) Google Scholar). To simulate the native protein in the mature form, we used the PCR to construct recombinant mature GehD (Fig. 2 A). The PCR product encoding mature GehD was cloned into the expression vector pETBlue-2 (Novagen). The protein was expressed as a C-terminal polyhistidine (His tag) fusion and purified by nickel-chelating chromatography. Mature GehD appears as a single polypeptide at ∼45 kDa when analyzed by SDS-PAGE (Fig. 2 B, lane 2). Amino acid sequence comparisons did not reveal any significant similarities between the linear amino acid sequences of Cna and mature GehD. Furthermore, the CD spectra of mature GehD shown in Fig.3 A is very different from that of Cna (Fig. 3 C). Deconvolution of the mature GehD data using the SELCON and VARSLC1 programs revealed that the predicted overall secondary structure of mature GehD consists of ∼26.5% α-helix, 20.6% β-sheet, and 52.9% coil. This secondary structure composition differs markedly from that of the reported crystal structure of Cna-(151–318): 8% α-helix, 53% β-sheet, and 39% coil (29Fagan P.K. Reinscheid D. Gottschalk B. Chhatwal G.S. Infect. Immun. 2001; 69: 4851-4857Crossref PubMed Scopus (33) Google Scholar). In contrast, the mature GehD CD spectra strongly resembles that of a mammalian cell-surface collagen binding receptor known as the α1 integrin I domain (Fig. 3 B). The secondary structure composition of this domain is 33.2% α-helix, 20.7% β-sheet, and 46.1% coil, which is comparable with that of mature GehD. The collagen binding activity of the recombinant, mature GehD was analyzed by Western ligand blot. Purified protein was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with digoxigenin-labeled collagen, mAbs 11H11 (anti-Cna), or 7E8 (anti-His tag) (Fig. 2 B, lane 4 and 5, respectively). In this assay, the recombinant, mature GehD binds collagen and both antibodies. It should be noted that a second lower mass polypeptide is detected in lanes 4 and 5. This is a contaminating polypeptide that is recognized by the secondary anti-mouse antibody used to detect mAbs11H11 and anti-His. The collagen binding activity of the recombinant, biotin-labeled mature GehD was also assessed by a solid phase, ELISA-type assay. Mature GehD bound in a concentration-dependent, saturable manner to collagens I, II, and IV coated on microtiter wells (Fig.4), whereas the binding to wells coated with albumin was minimal. From the ELISA-type assay we estimated that half-maximum binding occurred at about 0.25 μm mature GehD. In addition, we examined the ability of unlabeled mature GehD to inhibit the binding of biotinylated mature GehD to immobilized collagen. Unlabeled GehD could inhibit the binding of the labeled protein to immobilized collagen, whereas a fibrinogen binding recombinant protein from S. epidermidis (SdrG) had no inhibitory effect (data not shown). This suggests that both biotin-labeled and unlabeled mature GehD bind with similar affinity to immobilized collagen. We also tried to characterize the binding of mature GehD to collagen by surface plasmon resonance. In this assay, soluble recombinant mature GehD is run over a sensory chip coated with type I collagen. Using a Hepes-based buffer system, we could calculate a K Dof 4 μm for the interaction. Using a glycine buffer we recorded equilibrium data and calculated a K D of 3 μm and 1 binding site for mature GehD per collagen monomer. However, not only was the interaction of mature GehD with collagen dependent on the buffer system used, but the collagen binding activity declined as the purified mature GehD was stored for long periods of time. Clearly, these are aspects of the mature GehD binding to collagen that we do not understand at the present, and theK D values reported above must be taken with caution. We used a microtiter well attachment assay to study the adherence of S. epidermidis to collagen. Two independent, identical clones of S. epidermidis carrying a deletion of the g" @default.
• W2162585536 created "2016-06-24" @default.
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• W2162585536 date "2002-11-01" @default.
• W2162585536 modified "2023-10-18" @default.
• W2162585536 title "Is the GehD Lipase from Staphylococcus epidermidis a Collagen Binding Adhesin?" @default.
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