Title: Identification and Characterization of the C3 Binding Domain of the Staphylococcus aureus Extracellular Fibrinogen-binding Protein (Efb)
Abstract: The secreted Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) is a virulence factor that binds to both the complement component C3b and fibrinogen. Our laboratory previously reported that by binding to C3b, Efb inhibited complement activation and blocked opsonophagocytosis. We have now located the Efb binding domain in C3b to the C3d fragment and determined a disassociation constant (Kd) of 0.24 μm for the Efb-C3d binding using intrinsic fluorescence quenching assays. Using truncated, recombinant forms of Efb, we also demonstrate that the C3b binding region of Efb is located within the C terminus, in contrast to the fibrinogen binding domains that are located at the N-terminal end of the protein. Enzyme-linked immunosorbent assay-type binding assays demonstrated that recombinant Efb could bind to both C3b and fibrinogen simultaneously, forming a trimolecular complex and that the C-terminal region of Efb could inhibit complement activity in vitro. In addition, secondary structure analysis using circular dichroism spectroscopy revealed that the C-terminal, C3b binding region of Efb is composed primarily of α-helices, suggesting that this domain of Efb represents a novel type of C3b-binding protein. The secreted Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) is a virulence factor that binds to both the complement component C3b and fibrinogen. Our laboratory previously reported that by binding to C3b, Efb inhibited complement activation and blocked opsonophagocytosis. We have now located the Efb binding domain in C3b to the C3d fragment and determined a disassociation constant (Kd) of 0.24 μm for the Efb-C3d binding using intrinsic fluorescence quenching assays. Using truncated, recombinant forms of Efb, we also demonstrate that the C3b binding region of Efb is located within the C terminus, in contrast to the fibrinogen binding domains that are located at the N-terminal end of the protein. Enzyme-linked immunosorbent assay-type binding assays demonstrated that recombinant Efb could bind to both C3b and fibrinogen simultaneously, forming a trimolecular complex and that the C-terminal region of Efb could inhibit complement activity in vitro. In addition, secondary structure analysis using circular dichroism spectroscopy revealed that the C-terminal, C3b binding region of Efb is composed primarily of α-helices, suggesting that this domain of Efb represents a novel type of C3b-binding protein. Staphylococcus aureus is an important human pathogen that causes a wide range of infections and is the most common bacterium associated with septic arthritis. The disease often results in severe and persistent joint damage, and mortality rates from this type of infection are high (1Lowy F.D. N. Engl. J. Med. 1998; 339: 520-532Crossref PubMed Scopus (4638) Google Scholar, 2Kissane J.M. Connor D.H. Chandler F.W. Schwartz H.J. Manz H.J. Lack E.E. Pathology of Infectious Diseases. I. 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Malik Z. J. Clin. Microbiol. 1985; 22: 828-834Crossref PubMed Google Scholar, 54Na'was T. Hawwari A. Hendrix E. Hebden J. Edelman R. Martin M. Campbell W. Naso R. Fattom A.I. J. Clin. Microbiol. 1998; 36: 414-420Crossref PubMed Google Scholar), suggesting that other complement-inhibitory strategies are likely. A recent report from our laboratory demonstrated that the S. aureus protein Efb binds to C3b and inhibits both complement-mediated lysis and opsonophagocytosis via a mechanism that inhibits C3b deposition onto activator surfaces (17Lee L.Y. Höök M. Haviland D. Wetsel R.A. Yonter E.O. Syribeys P. Vernachio J. Brown E.L. J. Infect. Dis. 2004; 190: 571-579Crossref PubMed Scopus (109) Google Scholar). Efb is a secreted, constitutively expressed protein that also binds fibrinogen. In addition, the efb gene was present in all S. aureus isolates examined but not identified in any other staphylococcal species (21Bodén Wastfelt M.K. Flock J.I. J. Clin. Microbiol. 1995; 33: 2347-2352Crossref PubMed Google Scholar). Investigations into the roles of Efb in vivo have resulted in various hypotheses describing functions for Efb and include roles for Efb in potentiating S. aureus survival by delaying wound healing and by inhibiting platelet aggregation via its interactions with fibrinogen and the platelet receptor GPIIb/IIIa (23Palma M. Shannon O. Quezada H.C. Berg A. Flock J.I. J. Biol. Chem. 2001; 276: 31691-31697Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 55Heilmann C. Herrmann M. Kehrel B.E. Peters G. J. Infect. Dis. 2002; 186: 32-39Crossref PubMed Scopus (41) Google Scholar). Furthermore, these studies suggest that the fibrinogen binding domains were located at the N terminus of Efb and consist of two 22-amino acid repeats with homology to the fibrinogen binding domains of coagulase from S. aureus (20Bodén M.K. Flock J.I. Mol. Microbiol. 1994; 12: 599-606Crossref PubMed Scopus (89) Google Scholar). A third fibrinogen binding domain was also reported to reside in the C-terminal end of the protein; however, the fibrinogen binding activity of this site was less well defined since binding was dependent on whether fibrinogen was soluble or plate-bound (22Palma M. Wade D. Flock M. Flock J.I. J. Biol. Chem. 1998; 273: 13177-13181Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 57Shannon O. Flock J.I. Thromb. Haemostasis. 2004; 91: 779-789Crossref PubMed Google Scholar, 58Wade D. Palma M. Lofving-Arvholm I. Sallberg M. Silberring J. Flock J.I. Biochem. Biophys. Res. Commun. 1998; 248: 690-695Crossref PubMed Scopus (13) Google Scholar). Data presented in this report suggest that only the N-terminal half of Efb contains fibrinogen binding activity and that this domain is not involved in C3b binding. To define the C3b binding region(s), we engineered four recombinant forms of Efb that spanned the N- and C-terminal regions, respectively, and examined them in various binding assays for C3b and fibrinogen. Data presented in this report describe the C3 binding region in Efb to be located in the C terminus and that Efb binds specifically to the C3d fragment of C3 with high affinity. Furthermore, secondary structure analysis of recombinant Efb (rEfb) using circular dichroism spectroscopy suggested that the C3 binding region is primarily composed of α-helices and may represent a novel C3 binding motif. Cloning of and Expression of the Efb Truncations from S. aureus Strain Newman—The efb gene and efb truncations were amplified by PCR using S. aureus strain Newman DNA as a template. The following oligonucleotide primers were used for efb, efb104, efb120, and efb165, respectively (IDT Inc., Coralville, IA): 5′-CGC GGA TCC CCA AGA GAA AAG AAA CCA GTG AGT A-3′ (forward primer) and 5′-AAC TGC AGA GTT TTA TTT AAC TAA TCC TTG-3′ (reverse primer); 5′-CCAGCAGCGAAAACTGATGCAA-3′ (forward primer) and 5′-AAGTTTTATTTAACTAATCCTTG-3′ (reverse primer); 5′-CGC GGA TCC CCA AGA GAA AAG AAA CCA GTG AGT A-3′ (forward primer) and 5′-AAC TGC AGT TAT TCT CTC ACA AGA TTT TGA GCT TG-3′ (reverse primer); 5′-CCA GCA GCG AAA ACT GAT GCA ACT-3′ (forward primer) and 5′-AAC TGC AGA GTT TTA TTT AAC TAA TCC TTG-3′ (reverse primer). The resulting PCR products were subsequently cloned using the TA expression kit into the pCRT7/NT-TOPO expression vector (Invitrogen) and designated pCRT7/NT-rEfb, pCRT7/NT-rEfb104, pCRT7/NT-rEfb120, and pCRT7/NT-rEfb165. Nucleotide sequencing of efb, efb104, efb120, and efb165 was performed by automated sequencing (Molecular Genetics Core Facility, University of Texas-Houston Medical School). The recombinant proteins rEfb (17Lee L.Y. Höök M. Haviland D. Wetsel R.A. Yonter E.O. Syribeys P. Vernachio J. Brown E.L. J. Infect. Dis. 2004; 190: 571-579Crossref PubMed Scopus (109) Google Scholar), rEfb104, rEfb120, and rEfb165 were expressed as recombinant N-terminal His-tagged proteins that allowed for purification using metal ion-chelating chromatography as described previously (12Lee L.Y. Miyamoto Y.J. McIntyre B.W. Höök M. McCrea K.W. McDevitt D. Brown E.L. J. Clin. Investig. 2002; 110: 1461-1471Crossref PubMed Scopus (96) Google Scholar, 59Guo B.P. Brown E.L. Dorward D.W. Rosenberg L.C. Höök M. Mol. Microbiol. 1998; 30: 711-723Crossref PubMed Scopus (220) Google Scholar). Proteins were expressed and purified as previously described (59Guo B.P. Brown E.L. Dorward D.W. Rosenberg L.C. Höök M. Mol. Microbiol. 1998; 30: 711-723Crossref PubMed Scopus (220) Google Scholar, 60Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Protein concentrations were determined by UV spectroscopy, and proteins were stored at –20 °C until use. Western Blot Analysis—Recombinant proteins C3b, iC3b, C3c, or C3d (Advanced Research Technologies, San Diego, CA) (4 μg each) were subjected to SDS-PAGE and examined by staining with 0.05% Coomassie Brilliant Blue or electrotransferred onto a 0.45-μm Immobilon-P™ PVDF (polyvinylidene fluoride) membrane (Millipore, Bedford, MA) as described previously (17Lee L.Y. Höök M. Haviland D. Wetsel R.A. Yonter E.O. Syribeys P. Vernachio J. Brown E.L. J. Infect. Dis. 2004; 190: 571-579Crossref PubMed Scopus (109) Google Scholar). Membranes subjected to Western blot analysis were blocked overnight at 4 °C in 5% nonfat dry milk in TBST (0.15 m NaCl, 20 mm Tris-HCl, 0.05% Tween 20 (Sigma-Aldrich), pH 7.4) and probed accordingly and then developed with 10 ml of 1-Step™ nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution (Pierce). All incubations were performed in 15 ml of 1% TBST for 1 h with shaking at room temperature, and membranes were washed in TBST between all steps. Labeling with digoxigenin was performed as described previously according to the manufacturer's instructions (17Lee L.Y. Höök M. Haviland D. Wetsel R.A. Yonter E.O. Syribeys P. Vernachio J. Brown E.L. J. Infect. Dis. 2004; 190: 571-579Crossref PubMed Scopus (109) Google Scholar). ELISA-type Binding Assays—Immulon-1B microtiter plate wells (Dynatech Laboratories, Chantilly, VA) were coated with either 0.25 μg of C3b or fibrinogen in 100 μl of PBS overnight at 4 °C. The plates were washed and blocked with 200 μl of Super Block (Pierce) for 1 h. Recombinant proteins (0–500 nm in 100 μl final volume/well) were added to the wells and incubated for 1 h. In the next step 100 μl of anti-His antibodies (Amersham Biosciences) (1:5000) were added and incubated for 1 h followed by 100 μl of goat anti-mouse alkaline phosphatase (AP)-conjugated antibodies (1:5000). Alternatively, for digoxigenin-labeled proteins, 100 μl of AP-conjugated anti-digoxigenin antibodies ((Fab fragment) Roche Diagnostics) (1:5000) were added. Next, 100 μlof a 1 mg/ml Sigma 104 phosphatase substrate (Sigma) dissolved in 1 m diethanolamine, 0.5 mm MgCl2, pH 9.8, was added, and the plates were allowed to develop for 1 h. Plates were read at 405 nm using a microplate reader (Molecular Devices, Menlo Park, CA). Plates were washed between all steps with PBS, 0.05% Tween 20, and all incubations took place at 37 °C. All dilutions were made using Super Block unless otherwise specified. Inhibition ELISA—Immulon-1B microtiter plate wells (Dynatech Laboratories) were coated with either 0.25 μg of C3b or C3d (Advanced Research Technologies) in 50 μl of PBS overnight at 4 °C. The plates were washed and blocked with 200 μl of Super Block (Pierce) for 1 h. Increasing concentrations of either rEfb or the control protein decorin-binding protein A (DbpA) from B. burgdorferi (0–500 nm in 50 μl final volume/well) were added to C3b- or C3d-coated wells and incubated for 1 h followed by the addition of 50 μl of human Factor H (Advanced Research Technologies) (1 or 5 μg) and allowed to incubate for an additional hour. A 1:1000 dilution of goat-anti human Factor H was subsequently added (100 μl) and incubated for 1 h followed by 100 μlof rabbit anti-goat alkaline phosphatase (AP)-conjugated antibodies (1: 5000). Plates were washed between all steps with PBS, 0.05% Tween 20 unless otherwise indicated, and all incubations took place at 37 °C. All dilutions were made using Super Block unless otherwise specified. Complement Activity Assays—The EZ Complement CH50 clinical diagnostic assay kit (Diamedix, Miami, FL) was used to evaluate the effects of the rEfb truncations on classical complement pathway activation and used as described by the manufacturer. Briefly, human serum (5 μl of complement reference serum) was incubated in the presence of 5 μg of each recombinant protein at a final volume of 20 μl at 37 °C for 1 h before a 1-h incubation at room temperature with antibody-coated sheep red blood cells (RBCs) (3 ml). After incubating, the RBCs were centrifuged (1800 rpm for 10 min), and the absorbance of the supernatants (150 μl) was measured at 405 nm using a microplate reader as described above to determine the percent lysis of each sample. The data are expressed as percent lysis of the standard reference serum, and the values were derived using the equation, absorbance of sample/absorbance of reference serum × CH50 value of reference (Diamedix) (17Lee L.Y. Höök M. Haviland D. Wetsel R.A. Yonter E.O. Syribeys P. Vernachio J. Brown E.L. J. Infect. Dis. 2004; 190: 571-579Crossref PubMed Scopus (109) Google Scholar). Circular Dichroism (CD)—rEfb, rEfb104, rEfb120, and rEfb165 were dialyzed twice in 2 liters of 10 mm potassium phosphate buffer, pH 7.4, and their respective concentrations were determined using UV spectroscopy. CD spectra were obtained using a Jasco J-720 spectropolarimeter (Jasco, Inc., Easton, MD) calibrated with 10 mm potassium phosphate buffer, pH 7.4, using a round 0.2-mm quartz cell. The CD spectra were collected at a scan speed of 20 nm/min at 1-nm intervals with a 1-s response and a bandwidth of 1 nm. CD data were collected across a wavelength range of 180–260 nm and repeated through 20 iterations. The rEfb CD spectra were normalized by subtracting the CD spectra of buffer only, presented as units of mean residue ellipticity θMRW, and calculated by using the equation, θMRW = θ × 100 × MRW/c × d, where θ is the ellipticity in millidegrees, MRW is the mean residue weight of the recombinant protein, c is the concentration of the protein in mg/ml, and d is the cell path length in centimeters. Prediction of the secondary structure was obtained by combining data derived from three deconvolution programs (mean and S.E. were determined): self-consistent (Selcon) method, neural network (NN), and CONTIN (Sofsec version 1.2, Softwood Co., Brooksfield, CT) as described previously (61Sreerama N. Woody R.W. Anal. Biochem. 1993; 209: 32-44Crossref PubMed Scopus (946) Google Scholar, 62Pancoska P. Blazek M. Keiderling T.A. Biochemistry. 1992; 31: 10250-10257Crossref PubMed Scopus (36) Google Scholar). Fluorescence Measurements—The intrinsic tryptophan fluorescence of 1.3 μm C3d in PBS was examined using a LS50B spectrofluorimeter (PerkinElmer Life Sciences) at 25 °C. The excitation wavelength was set at 295 nm (5-nm slit width) while monitoring emissions from 310–370 nm (4-nm slit width). Quenching of tryptophan fluorescence after the addition of rEfb (from 60 nm to 3.5 μm) was analyzed according to the modified plot of Stern-Volmer (63Eftink M.R. Ghiron C.A. Anal. Biochem. 1981; 114: 199-227Crossref PubMed Scopus (1625) Google Scholar) the F0/(F0 – F) ratio, where F0 and F are the fluorescence intensities at 335 nm in the absence or presence of rEfb, respectively. The F0/(F0 – F) ratio determined for each concentration of rEfb was plotted against the reciprocal of the rEfb concentrations, which yielded a straight line whose x intercept equaled the value of the ass