Crystal Structure of the Tp34 (TP0971) Lipoprotein of Treponema pallidum
2006; Elsevier BV; Volume: 282; Issue: 8 Linguagem: Inglês
10.1074/jbc.m610215200
ISSN1083-351X
AutoresRanjit K. Deka, Chad A. Brautigam, Farol L. Tomson, Sarah B. Lumpkins, D.R. Tomchick, Mischa Machius, Michael V. Norgard,
Tópico(s)HIV Research and Treatment
ResumoThe Tp34 (TP0971) membrane lipoprotein of Treponema pallidum, an obligate human pathogen and the agent of syphilis, was previously reported to have lactoferrin binding properties. Given the non-cultivatable nature of T. pallidum, a structure-to-function approach was pursued to clarify further potential relationships between the Tp34 structural and biochemical properties and its propensity to bind human lactoferrin. The crystal structure of a nonacylated, recombinant form of Tp34 (rTp34), solved to a resolution of 1.9Aå, revealed two metaloccupied binding sites within a dimer; the identity of the ion most likely was zinc. Residues from both of the monomers contributed to the interfacial metal-binding sites; a novel feature was that the δ-sulfur of methionine coordinated the zinc ion. Analytical ultracentrifugation showed that, in solution, rTp34 formed a metal-stabilized dimer and that rTp34 bound human lactoferrin with a stoichiometry of 2:1. Isothermal titration calorimetry further revealed that rTp34 bound human lactoferrin at high (submicromolar) affinity. Finally, membrane topology studies revealed that native Tp34 is not located on the outer surface (outer membrane) of T. pallidum but, rather, is periplasmic. How propensity of Tp34 to bind zinc and the iron-sequestering lactoferrin may relate overall to the biology of T. pallidum infection in humans is discussed. The Tp34 (TP0971) membrane lipoprotein of Treponema pallidum, an obligate human pathogen and the agent of syphilis, was previously reported to have lactoferrin binding properties. Given the non-cultivatable nature of T. pallidum, a structure-to-function approach was pursued to clarify further potential relationships between the Tp34 structural and biochemical properties and its propensity to bind human lactoferrin. The crystal structure of a nonacylated, recombinant form of Tp34 (rTp34), solved to a resolution of 1.9Aå, revealed two metaloccupied binding sites within a dimer; the identity of the ion most likely was zinc. Residues from both of the monomers contributed to the interfacial metal-binding sites; a novel feature was that the δ-sulfur of methionine coordinated the zinc ion. Analytical ultracentrifugation showed that, in solution, rTp34 formed a metal-stabilized dimer and that rTp34 bound human lactoferrin with a stoichiometry of 2:1. Isothermal titration calorimetry further revealed that rTp34 bound human lactoferrin at high (submicromolar) affinity. Finally, membrane topology studies revealed that native Tp34 is not located on the outer surface (outer membrane) of T. pallidum but, rather, is periplasmic. How propensity of Tp34 to bind zinc and the iron-sequestering lactoferrin may relate overall to the biology of T. pallidum infection in humans is discussed. Syphilis is a chronic, complex sexually transmitted disease of humans caused by the noncultivatable spirochete Treponema pallidum (1LaFond R.E. Lukehart S.A. Clin. Microbiol. Rev. 2006; 19: 29-49Crossref PubMed Scopus (338) Google Scholar, 2Peeling R.W. Hook E.W. J. Pathol. 2006; 208: 224-232Crossref PubMed Scopus (133) Google Scholar). Despite the availability of effective antimicrobials, syphilis remains a significant threat to global health and can facilitate the transmission of human immunodeficiency virus (3Gerbase A.C. Rowley J.T. Heymann D.H. Berkley S.F. Piot P. Sex. Transm. Infect. 1998; 74 (Suppl): S12-S16PubMed Google Scholar, 4Simms I. Fenton K.A. Ashton M. Turner K.M.E. Crawley-Boevey E.E. Gorton R. Thomas D.R.H. Lynch A. Winter A. Fisher M.J. Lighton L. Maguire H.C. Solomou M. Sex. Transm. Dis. 2005; 32: 220-226Crossref PubMed Scopus (178) Google Scholar, 5Beltrami J.F. Weinstock H.S. Berman S.M. Swint E.B. Fenton K.A. Morb. Mortal Wkly. Rep. 2006; 55: 269-273PubMed Google Scholar). After cutaneous inoculation, the bacteria become blood-borne and invade various organ systems, including the liver, heart, and nervous system (1LaFond R.E. Lukehart S.A. Clin. Microbiol. Rev. 2006; 19: 29-49Crossref PubMed Scopus (338) Google Scholar, 2Peeling R.W. Hook E.W. J. Pathol. 2006; 208: 224-232Crossref PubMed Scopus (133) Google Scholar). Humans are the only known reservoir for T. pallidum, and although syphilis is one of the oldest recognized sexually transmitted diseases, it remains very poorly understood (1LaFond R.E. Lukehart S.A. Clin. Microbiol. Rev. 2006; 19: 29-49Crossref PubMed Scopus (338) Google Scholar, 2Peeling R.W. Hook E.W. J. Pathol. 2006; 208: 224-232Crossref PubMed Scopus (133) Google Scholar). This largely is a consequence of the fact that T. pallidum cannot be cultivated continuously in vitro (1LaFond R.E. Lukehart S.A. Clin. Microbiol. Rev. 2006; 19: 29-49Crossref PubMed Scopus (338) Google Scholar), thereby hampering studies on treponemal virulence and syphilis pathogenesis. Approaches for discerning T. pallidum membrane biology, which could reveal key aspects of the enigmatic parasitic strategy of the bacterium, thus, have been substantially limited (6Cox D.L. Chang P. McDowall A. Radolf J.D. Infect. Immun. 1992; 60: 1076-1083Crossref PubMed Google Scholar, 7Radolf J.D. Trends Microbiol. 1994; 2: 307-311Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 8Radolf J.D. Mol. Microbiol. 1995; 16: 1067-1073Crossref PubMed Scopus (79) Google Scholar). In an attempt to elucidate molecules that could mediate interactions of T. pallidum with its human host, solubilization with the nonionic detergent Triton X-114 was employed to identify a group of integral membrane lipoproteins (9Radolf J.D. Chamberlain N.R. Clausell A. Norgard M.V. Infect. Immun. 1988; 56: 490-498Crossref PubMed Google Scholar, 10Chamberlain N.R. Brandt M.E. Erwin A.L. Radolf J.D. Norgard M.V. Infect. Immun. 1989; 57: 2872-2877Crossref PubMed Google Scholar). This approach identified at least five lipoproteins, most of them representing dominant immunogens of T. pallidum (10Chamberlain N.R. Brandt M.E. Erwin A.L. Radolf J.D. Norgard M.V. Infect. Immun. 1989; 57: 2872-2877Crossref PubMed Google Scholar). This finding constituted a major advance in the treponemal field, because there is increasing evidence that the lipoproteins serve functions vital to the membrane biology of T. pallidum (11Haake D.A. Microbiology. 2000; 146: 1491-1504Crossref PubMed Scopus (150) Google Scholar). In other bacteria, membrane lipoproteins have importance as virulence factors, modular components of ATP binding cassette (ABC) 3The abbreviations used are: ABC, ATP binding cassette; bLF, bovine lactoferrin; hLF, human lactoferrin; HSQC, heteronuclear single quantum coherence; hTF, human transferrin; ITC, isothermal titration calorimetry; LBP, ligand-binding protein; SeMet, selenomethionine; SV, sedimentation velocity; MOPS, 3-[N-morpholino]propanesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine; IL-2, interleukin-2; IL-2R, IL-2 receptor. transporters, enzymes, receptors, protective immune targets, and proinflammatory agonists that contribute significantly to innate immune responses (11Haake D.A. Microbiology. 2000; 146: 1491-1504Crossref PubMed Scopus (150) Google Scholar, 12Hayashi S. Wu H.C. J. Bioenerg. Biomembr. 1990; 22: 451-471Crossref PubMed Scopus (428) Google Scholar, 13Sutcliff I.C. Russell R.R.B. J. Bacteriol. 1995; 177: 1123-1128Crossref PubMed Google Scholar, 14Becker P.S. Akins D.R. Radolf J.D. Norgard M.V. Infect. Immun. 1994; 62: 1381-1391Crossref PubMed Google Scholar, 15Steere A.C. Sikand V.K. Meurice F. Parenti D.L. Fikrig E. Schoen R.T. Nowakowski J. Schmid C.H. Laukamp S. Buscarino C. Krause D.S. Group The Lyme Disease Vaccine Study N. Engl. J. Med. 1998; 339: 209-215Crossref PubMed Scopus (576) Google Scholar, 16Ochsner U.A. Vasil A.I. Johnson Z. Vasil M.L. J. Bacteriol. 1999; 181: 1099-1109Crossref PubMed Google Scholar, 17Radolf J.D. Arndt L.L. Akins D.R. Curetty L.L. Levi M.E. Shen Y. Davis L.S. Norgard M.V. J. Immunol. 1995; 154: 2866-2877Crossref PubMed Google Scholar, 18Norgard M.V. Arndt L.L. Akins D.R. Curetty L.L. Harrich D.A. Radolf J.D. Infect. Immun. 1996; 64: 3845-3852Crossref PubMed Google Scholar). Whereas early experiments revealed only five treponemal lipoproteins (10Chamberlain N.R. Brandt M.E. Erwin A.L. Radolf J.D. Norgard M.V. Infect. Immun. 1989; 57: 2872-2877Crossref PubMed Google Scholar), more recent analysis of T. pallidum genome sequence information has predicted at least 45 lipoprotein genes (19Setubal J.C. Reis M. Matsunaga J. Haake D.A. Microbiology. 2006; 152: 113-121Crossref PubMed Scopus (145) Google Scholar). Most of these predicted that open reading frames encode proteins with no known homologies to other structurally or functionally characterized bacterial proteins. In addition, the absence of techniques for in vitro cultivation and genetic manipulation of T. pallidum has greatly hindered the ability to characterize the functional aspects of T. pallidum membrane lipoproteins. As an alternative approach to investigating the peculiar membrane biology of T. pallidum, we have adopted a structure-to-function approach to formulate new testable hypotheses regarding the potential function(s) of a number of the treponemal lipoproteins. That a structural biology approach for analyzing T. pallidum lipoproteins is a fruitful avenue of investigation is exemplified in at least three recent studies on Tp47, Tp32, and PnrA (20Deka R.K. Machius M. Norgard M.V. Tomchick D.R. J. Biol. Chem. 2002; 277: 41857-41864Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 21Deka R.K. Neil L. Hagman K.E. Machius M. Tomchick D.R. Brautigam C.A. Norgard M.V. J. Biol. Chem. 2004; 279: 55644-55650Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 22Deka R.K. Brautigam C.A. Yang X.F. Blevins J.S. Machius M. Tomchick D.R. Norgard M.V. J. Biol. Chem. 2006; 281: 8072-8081Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The current structural study focuses on the Tp34 membrane lipoprotein (also known as Tp0971) of T. pallidum. Early studies by Staggs et al. (23Staggs T.M. Greer M.K. Baseman J.B. Holt S.C. Tryon V.V. Mol. Microbiol. 1994; 12: 613-619Crossref PubMed Scopus (20) Google Scholar) denoted a protein, most likely Tp34, as having lactoferrin binding properties. Lactoferrin is a mammalian iron-binding glycoprotein found on mucosal surfaces and within biological fluids, including milk, saliva, and seminal fluid (24Kanyshkova T.G. Buneva V.N. Nevinsky G.A. Biochemistry (Mosc). 2001; 66: 5-13Crossref Scopus (156) Google Scholar, 25Levay P.F. Viljoen M. Haematologica. 1995; 80: 252-267PubMed Google Scholar, 26Fernaud S. Evans R.W. Mol. Immunol. 2003; 40: 395-405Crossref PubMed Scopus (582) Google Scholar). Despite the preliminary functional characterization of Tp34, NCBI Clusters of Orthologous Groups data base includes Tp34 with five other bacterial proteins as COG3470, which is denoted as “uncharacterized proteins probably involved in high affinity Fe2+ transport.” However, the structural and functional aspects of this group of proteins remain undetermined. To investigate further the potential of Tp34 to serve as a ligand-binding protein (LBP), we determined the three-dimensional structure of Tp34 using x-ray crystallography. The relevance of our findings to the notion that Tp34 is involved in lactoferrin binding as well as the potential implications of this interaction for iron utilization by T. pallidum are discussed. Construction of His6-tagged rTp34—To produce a nonlipidated, recombinant version of Tp34 (rTp34) in Escherichia coli, a fragment encoding amino acid residues 2-185 (cloned without the post-translationally modified N-terminal Cys residue) was amplified by PCR from treponemal genomic DNA using primer pairs encoding the predicted 5′ and 3′ termini. The PCR products were ligated into pProExHTa vector (Invitrogen) using BamHI and PstI restriction sites such that the resultant construct encoded a fusion protein with a His6 tag at its N terminus; this His6 tag region is cleavable by tobacco etch virus protease. Ligation mixtures were transformed into E. coli XL1-Blue (Stratagene), and the sequences of plasmids isolated from colonies that tested positive by restriction digest were verified using standard DNA sequencing techniques. Expression and Purification of rTp34—E. coli XL1-Blue was grown at 37 °C in LB medium containing 100 μg/ml ampicillin until the cell density reached an A600 of 0.6. The culture was then induced for 4 h with 600 μm isopropyl 1-thio-β-d-galactopyranoside. Cells derived from 1 liter of culture were harvested by centrifugation and lysed at room temperature with gentle rocking for 30 min using 50 ml of B-PER II (Pierce). The resulting suspension was centrifuged at 25,000 × g for 15 min to remove cell debris. rTp34 was isolated from the supernatant by nickel-nitrilotriacetic acid-agarose (Qiagen) using a standard method (22Deka R.K. Brautigam C.A. Yang X.F. Blevins J.S. Machius M. Tomchick D.R. Norgard M.V. J. Biol. Chem. 2006; 281: 8072-8081Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The N-terminal His6 tag was removed via cleavage with tobacco etch virus protease (Invitrogen) at room temperature for 3 h, and then the protease was removed according to the manufacturer’s protocol. Removal of the fusion segment was verified by SDS-PAGE. rTp34 was concentrated and bufferexchanged with 20 mm Hepes, 20 mm NaCl, pH 7.5 (buffer A) using an Amicon ultracentrifuge filter device (Millipore) with a 10,000 molecular weight exclusion limit. The concentrated protein was applied to a HiLoad 16/60 Superdex 75 prep grade column and purified on anAökta fast performance liquid chromatography system (GE Healthcare) using buffer A. Subsequent to elution, peak fractions were analyzed by SDS-PAGE. At this stage, the protein was pure to apparent homogeneity (i.e. >95%). Fractions containing purified rTp34 were pooled and concentrated to 10 mg/ml in buffer A for protein crystallization. Protein concentration was determined spectrophotometrically using an absorption coefficient of 1.491 mg-1ml-1cm-1 at 280 nm (27Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar). To express protein for nuclear magnetic resonance (NMR) characterization, bacteria were grown in M9 minimal medium supplemented with 15NH4Cl as the sole nitrogen source. The resultant 15N-labeled protein was purified as described above. For the production of a selenomethionine (SeMet) variant of rTp34, the expression vector was transformed into the E. coli methionine auxotroph B834 cells (Novagen) and grown in the presence of M9 medium containing 5% LB supplemented with 125 mg/liter each of adenine, uracil, thymine, and guanosine, 2.5 mg/liter thiamine, 4 mg/liter d-biotin, 20 mm glucose, 2 mm MgSO4, 50 mg/liter each of 19 l amino acids (excluding methionine), and 50 mg/liter l-selenomethionine (Sigma). Labeled protein was prepared as described above with the addition of 15 mm β-mercaptoethanol to the purification buffers. NMR Spectroscopic Screening—Suitability of rTp34 for structure determination was evaluated by NMR screening of 15N-labeled protein (28Yee A. Chang X. Pineda-Lucena A. Wu B. Semesi A. Le B. Ramelot T. Lee G.M. Bhattacharyya S. Gutierrez P. Denisov A. Lee C-H. Cort J.R. Kozlov G. Liao J. Finak G. Chen L. Wishart D. Lee W. McIntosh L.P. Gehring K. Kennedy M.A. Edwards A.M. Arrowsmith C.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1825-1830Crossref PubMed Scopus (181) Google Scholar). All 1H,15N heteronuclear single quantum coherence (HSQC) spectra of labeled protein were acquired at 25 °C by using a Varian INOVA 500-MHz spectrometer with NMRPipe (29Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar) used for data processing and NMRView (30Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2686) Google Scholar) for analysis. Data were collected on 500 μm samples in 50 mm K3PO4, 50 mm NaCl, pH 7.5. Preparation of PnrA, Tp32, Human Milk Lactoferrin (hLF), Bovine Milk Lactoferrin (bLF), and Human Serum Transferrin (hTF)—The purification procedures for PnrA and Tp32 have been described previously (21Deka R.K. Neil L. Hagman K.E. Machius M. Tomchick D.R. Brautigam C.A. Norgard M.V. J. Biol. Chem. 2004; 279: 55644-55650Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 22Deka R.K. Brautigam C.A. Yang X.F. Blevins J.S. Machius M. Tomchick D.R. Norgard M.V. J. Biol. Chem. 2006; 281: 8072-8081Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). As a final step, both proteins were dialyzed against buffer B (20 mm Hepes, 100 mm NaCl, 2 mm octyl β-glucoside, pH 7.5). Lyophilized hLF, bLF, and hTF were obtained from Sigma and reconstituted in buffer A at protein concentrations of 10 mg/ml. Proteins were then dialyzed overnight into buffer B. ApohLF was prepared in the same buffer after exhaustive dialysis against 0.1 m citric acid (31Masson P.L. Heremans J.F. Eur. J. Biochem. 1968; 6: 579-584Crossref PubMed Scopus (133) Google Scholar). Crystallization, Data Collection, and Structure Determinations—Crystals were grown at 20 °C using the vapor diffusion method in hanging drop mode by mixing 4 μl of protein (10 mg/ml in buffer A) with 4 μl of reservoir solution (2.4 m ammonium sulfate, 0.1 m Bicine, pH 9.0) and equilibrating against 500 μl of reservoir solution. Crystals appeared within 24 h and grew to average dimensions of 0.2 × 0.6 × 0.1 mm in about 3 days. The crystals were cryo-protected in 2.8 m ammonium sulfate, 15% (v/v) ethylene glycol, 0.1 m Bicine, pH 9.0, and then flash-cooled in liquid propane. Crystals exhibited the symmetry of space group P212121 with cell dimensions of a = 34 Aå, b = 66 Aå, c = 151 Aå and contained two molecules per asymmetric unit and 41% solvent. Crystals for the selenomethionine variant of rTp34 (rTp34-SeMet) were produced under the same conditions. Diffraction data were collected at beamlines 19-ID and 19-BM (SBC-CAT) at the Advanced Photon Source (Argonne National Laboratory, Argonne, IL) and processed with HKL2000 (32Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). Crystals diffracted to a minimum Bragg spacing (dmin) of about 1.63 Aå. Phases for rTp34-SeMet were obtained from a single anomalous diffraction experiment using x-rays with an energy near the selenium K absorption edge. Using data to 2.5 Aå, 6 of 12 possible selenium sites were located and refined with the program CNS (33Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar), resulting in a figure of merit of 0.35. Phases were extended to 1.7 Aå and improved by density modification (CNS), resulting in a final overall figure of merit of 0.92. The resulting electron density map was of sufficient quality to automatically construct an initial model using the program ARP/wARP (34Perrakis A. Morris R.J. Lamzin V.S. Nat. Struct. Biol. 1996; 6: 458-463Crossref Scopus (2565) Google Scholar). Manual rebuilding was carried out with the program Coot (35Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar). Refinement was accomplished using the program REFMAC5 (36Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) from the CCP4 package (37Number Collaborative Computational Project Acta Crystallogr. D. Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The rTp34-SeMet model was used as a starting model for the refinement of native rTp34. The electron density revealed strong, spherical features near His-124 in each monomer, indicative of metal ions. These features were not present in rTp34-SeMet. An anomalous difference Fourier electron-density map showed peaks of about 6.4 and 4.7 σ that coincided with the features. Subsequently, a crystal of rTp34 was incubated with 10 mm zinc acetate (rTp34-Zn), and diffraction data were collected using x-rays with an energy near the zinc K absorption edge. The anomalous difference Fourier electron-density map revealed very strong peaks of about 32 and 33 σ at the previously observed positions. Zinc ions were, therefore, included in the model. An additional six zinc ions were located, but they had rather high B values and are, thus, minor sites. Although the nature of the metal ions in native rTp34 was not unambiguously clear, the peaks were interpreted as zinc ions at this stage. These metal-binding sites were not fully occupied as indicated by the comparatively weak electron density and high B factors. Furthermore, a second conformation appeared for some of the zinc-ligand residues, whereas these residues adopted only a single conformation in rTp34-Zn, where the main zinc sites were presumably fully occupied. As a control, a dataset was collected from a crystal of rTp34 that was grown in the presence of 1 mm EDTA (rTp34-EDTA). Density observed at the zinc sites was weak and was interpreted as water molecules. Final models had an Rwork and Rfree of 18.4 and 21.5% for rTp34-SeMet, 18.8 and 23.5% for native rTp34, 19.0 and 23.3% for rTp34-Zn, and 18.9 and 22.0% for rTp34-EDTA, respectively. About 31 N-terminal residues could not be located in all cases. Figure Generation—Figures that feature structural details of rTp34 were generated using PyMOL. They were rendered using POV-Ray. Isothermal Titration Calorimetry—The binding of rTp34 or other T. pallidum lipoproteins to various target proteins was analyzed by injecting a solution of each lipoprotein into a solution of target protein and monitoring the resultant heat changes upon binding using a VP-ITC titration microcalorimeter (MicroCal, Inc.). All proteins were exhaustively exchanged into buffer B before the titration experiment. Protein concentrations were obtained by measuring the absorbance of proteincontaining solutions spectrophotometrically followed by the calculation of the protein concentration using Beer’s Law (A = ɛcl, where A is absorbance, ɛ is the molar extinction coefficient, c is the protein concentration, and l is the path length). In the case of rTp34, hLF, and bLF, the extinction coefficients were experimentally determined following the method of Pace et al. (27Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar). For other proteins ɛ was calculated by the ProtParam tool available at the ExPASy Proteomics Server. The titration was carried out using 31 serial injections of 8 μl of lipoprotein at concentrations of 500-600 μm into a stirred reaction cell (1.4 ml) containing solutions of the target protein at ∼30 μm.To study the potential binding of hTF to rTp34, a solution of hTF (126 μm) was injected into a solution of rTp34 (13.6 μm) as described above. The heats of interaction resulting from the titration were monitored by the VP-ITC instrument until the target protein was saturated with the lipoprotein. Integration of the resultant thermogram yielded the binding isotherm, which was analyzed using the Microcal-ORIGIN software package to yield the association constants and the thermodynamic parameters associated with binding. Titrations using rTp34 as the injectant and hLF as the target protein were carried out in triplicate; others were performed only once. Analytical Ultracentrifugation—Sedimentation velocity experiments were performed in a Beckman XL-I analytical ultracentrifuge. For rTp34 sedimentation, samples of 390 μl of reference buffer C (20 mm Tris-Cl, 20 mm NaCl, pH 7.5) or rTp34 (19 μm) diluted in reference buffer were loaded into a dualsector charcoal-filled epon centerpiece. In experiments that contained divalent metal ions, they were included at a concentration of 3 mm unless otherwise noted. The samples were centrifuged at 50,000 rpm in an An60-Ti rotor, and sedimentation was monitored using either laser interferometry or absorbance spectrophotometry at a wavelength of 280 nm. Data were analyzed using the program SEDFIT, which generates a continuous c(s) distribution for the sedimenting species (38Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar). For experiments designed to monitor the binding of rTp34 to another protein, the same buffer was used. The volumes, rotor, centrifugation speed, and instrumentation were as described above. Three simultaneous sedimentation experiments were performed in these cases, with the solution contents being (i) rTp34 (15 μm) alone, (ii) target protein (5 μm) alone, and (iii) a mixture of rTp34 (7.5 μm) and the target protein (2.5 μm). For such experiments, two signals from the proteins were concurrently used to follow their sedimentation. For some experiments, the two signals were absorbance at 280 nm and refractive index detection by laser interferometry; for others, the signals were absorbances at 280 and at 250 nm. Such data were analyzed according to the multisignal ck(s) methodology of Balbo et al. (39Balbo A. Minor K.H. Velikovsky C.A. Mariuzza R.A. Peterson C.B. Schuck P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 81-86Crossref PubMed Scopus (112) Google Scholar), as implemented in the program SEDPHAT. Briefly, the sedimentation profiles obtained from experiments i and ii were used to calculate the unknown extinction coefficient of the protein at 250 nm or the unknown interferometric extinction coefficient. This is possible because the extinction coefficient of the proteins at 280 nm is known. Once the extinction coefficient at both signals is known, a characteristic signal increment ɛkλ of each protein component k at each signal λ can be defined that allows the spectral decomposition of the c(s) distribution into two ck(s) distributions, one for each of the protein components. Integration of a ck(s) peak yields the concentration of a given component in that peak. The concentrations of co-sedimenting proteins may, thus, be derived by analyzing the two ck(s) distributions; comparison of the two concentrations yields the stoichiometry of the proteins in that peak. The program SEDNTERP was used to estimate the partial specific volume of the proteins as well as the density and viscosity of the buffer solutions. All sedimentation coefficients quoted in this study have been corrected to values that would be observed under standard conditions (i.e. s20,w). Cultivation and Isolation of Treponemes—T. pallidum subspecies pallidum (Nichols strain) was maintained and passaged by intratesticular inoculation of adult male New Zealand White rabbits as previously described (40Robertson S.M. Kettman J.R. Miller J.N. Norgard M.V. Infect. Immun. 1982; 36: 1076-1085Crossref PubMed Google Scholar). Spirochetes were harvested in T. pallidum medium (41Cox D.L. Riley B. Chang P. Sayahatheri S. Tassell S. Hevelone J. Appl. Environ. Microbiol. 1990; 56: 3063-3072Crossref PubMed Google Scholar) and flushed with a reduced oxygen atmosphere of 3% O2, 5%CO2, and the balance of nitrogen. The medium consisted of Earle’s balanced salt solution, minimum essential medium (MEM) (Hyclone) amino acids, MEM nonessential amino acids, MEM vitamin, 2 mm l-glutamine, 550 μm d-mannitol, 320 μm l-histidine, 24 mm sodium bicarbonate, 25 mm MOPS, 900 μm sodium pyruvate, 14 mm d-(+)glucose, 650 μm dithiothreitol, and 20% (v/v) fetal bovine serum (Mediatech, Inc.) with a final pH of 7.5. Rabbit testicular debris was removed from treponemal suspensions by two successive rounds of slow-speed centrifugation (200 × g for 8 min). Spirochetes remaining suspended were enumerated by darkfield microscopy and diluted to a concentration of 108 ml-1 in T. pallidum medium. Indirect Immunofluorescence Detection of Antigens in T. pallidum—The agarose gel microdroplet method (42Cox D.L. Akins D.R. Porcella S.F. Norgard M.V. Radolf J.D. Mol. Microbiol. 1995; 15: 1151-1164Crossref PubMed Scopus (49) Google Scholar) was utilized to assess whether lipoproteins were exposed on the surface of T. pallidum. T. pallidum was encapsulated within agarose gel microdroplets as previously described (42Cox D.L. Akins D.R. Porcella S.F. Norgard M.V. Radolf J.D. Mol. Microbiol. 1995; 15: 1151-1164Crossref PubMed Scopus (49) Google Scholar). The encapsulation method stabilizes the fragile outer membrane of T. pallidum during antibody exposure and washing treatments. Mouse monoclonal anti-Tp47 (clone 11E3) (43Jones S.A. Marchitto K.S. Miller J.N. Norgard M.V. J. Exp. Med. 1984; 160: 1404-1420Crossref PubMed Scopus (27) Google Scholar) and mouse monoclonal anti-Tp34 (44Swancutt M.A. Radolf J.D. Norgard M.V. Infect. Immun. 1990; 58: 384-392Crossref PubMed Google Scholar) were diluted 1:20 and added directly to 1 ml of aliquots of microdroplets with or without 0.15% (v/v) Triton X-100. The microdroplets were incubated overnight at 4 °C. The microdroplets were collected by centrifugation at (500 × g, 5 min) and washed 5 times with fresh T. pallidum medium. Two μg of goat anti-mouse Alexa Fluor 488 (Molecular Probes) were added to the microdroplets for 2 h before being re-washed 5 times and viewed on glass slides with a Olympus BH2 microscope (darkfield lens and BP490 (green) filter (Olympus America Inc.)). Images were obtained using a SPOT Pursuit camera (Diagnostic Instruments, Inc.). Slides were prepared from each of 3 independent microdroplet preparations, and ∼100 bacteria were counted per slide. Any spirochete with a fluorescent signal, which was often punctated, was considered positive and compared with the total number of spirochetes observed via darkfield microscopy. SDS-PAGE and Immunoblotting—Samples
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