Selective Release of the Treponema pallidum Outer Membrane and Associated Polypeptides with Triton X-114

Transcription

1 JOURNAL OF BACTERIOLOGY, Dec. 1988, p /88/ $02.00/0 Copyright ( 1988, American Society for Microbiology Vol. 170, No. 12 Selective Release of the Treponema pallidum Outer Membrane and Associated Polypeptides with Triton X-114 THOMAS M. CUNNINGHAM,'t ELDON M. WALKER,' JAMES N. MILLER,' AND MICHAEL A. LOVETT' 2* Department of Microbiology and Immunology' and Division of Infectioius Diseases, Department of Medicine,* University of California at Los Angeles, Los Angeles' California Received 25 March 1988/Accepted 30 July 1988 The effects of the nonionic detergent Triton X-114 on the ultrastructure of Treponema pallidum subsp. pallidum are presented in this study. Treatment of Percoll-purified motile T. pallidum with a 1% concentration of Triton X-114 resulted in cell surface blebbing followed by lysis of blebs and a decrease in diameter from ,um to ,im. Examination of thin sections of untreated Percoll-purified T. pallidum showed integrity of outer and cytoplasmic membranes. In contrast, thin sections of Triton X-114-treated treponemes showed integrity of the cytoplasmic membrane but loss of the outer membrane. The cytoplasmic cylinders generated by detergent treatment retained their periplasmic flagella, as judged by electron microscopy and immunoblotting. Recently identified T. pallidum penicillin-binding proteins also remained associated with the cytoplasmic cylinders. Proteins released by Triton X-114 at 4 C were divided into aqueous and hydrophobic phases after incubation at 37 C. The hydrophobic phase had major polypeptide constituents of 57, 47, 38, 33-35, 23, 16, and 14 kilodaltons (kda) which were reactive with syphilitic serum. The 47-kDa polypeptide was reactive with a monoclonal antibody which has been previously shown to identify a surface-associated T. pallidum antigen. The aqueous phase contained the 190-kDa ordered ring molecule, 4D, which has been associated with the surface of the organism. Full release of the 47- and 190-kDa molecules was dependent on the presence of a reducing agent. These results indicate that 1% Triton X-114 selectively solubilizes the T. pallidum outer membrane and associated proteins of likely outer membrane location. Definition of the surface molecules of Treponema pallidum remains a major goal of research focused on relating the structure of this noncultivable pathogen to the pathogenesis of syphilis. Application of conventional methods for determining the structure of the gram-negative cell envelope have not yielded consistent results, despite the availability of virulent treponemes purified from rabbit host tissue debris (10). Several properties which distinguish T. pallidum from other groups of bacteria have been major factors in complicating this basic structural analysis. Unlike other bacteria, freshly extracted virulent T. pallidum does not readily bind specific antibodies to its surface (13, 14, 25, 28). The molecular basis of this relative antigenic inertness of the treponemal surface is unknown, but could reflect either coating with host tissue components (1) or an innate structural property of the treponemal surface. Only with 4 h or more of incubation in vitro does the organism become susceptible to complement-dependent serum bactericidal activity (2), raising the possibility that surface antigens become exposed only when the treponemal surface becomes altered on in vitro incubation (28). Although the antigenic inertness of the treponemal surface may explain in part how the organism can evade the host immune response in vivo, it has complicated identification of surface molecules by direct antibody binding. The antigenic inertness of the surface is paralleled by the finding that it has not been possible to identify surface proteins of T. pallidium by radioiodination (19) or radioimmunoprecipitation (28) techniques. Another factor in complicating the basic structural analysis of T. pallidum has been the difficulty encountered in consistently demonstrating an outer membrane by electron * Corresponding author. t Present address: Division of Bacterial Products, Food and Drug Administration, Bethesda, MD microscopy of thin sections. The absence of such a reliable ultrastructural means of judging cell envelope integrity has complicated attempts to develop treponemal cell fractionation protocols. In this report, using modified procedures for preparing thin sections of T. pallidum which consistently demonstrate outer membrane structure, we provide ultrastructural evidence that the nonionic detergent Triton X-114 (TX-114) removes the outer membrane of T. pallidum without altering the bilayer appearance of the cytoplasmic membrane. We describe the partitioning of the detergent-extracted polypeptides into the aqueous and hydrophobic phases formed by TX-114 at temperatures above its cloud point. We also show that the periplasmic flagella and recently described penicillin-binding proteins (6) of T. pallidum remain with the cytoplasmic cylinder after detergent treatment. (This work was originally presented at the University of California Los Angeles Symposium on Bacteria-Host Cell Interaction, at Park City, Utah, on 15 February 1987 and at the 87th Annual Meeting of the American Society for Microbiology, at Atlanta, Ga. [Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, D23, p. 75]. It has been submitted by T. M. Cunningham in partial fulfillment of the requirements for the Ph.D. degree from the University of California Los Angeles, 1987.) 5789 MATERIALS AND METHODS Bacteria. The Nichols strain of T. pallidum subsp. pallidum was cultivated by passage in New Zealand White rabbits injected intramuscularly with 10 mg of cortisone acetate (Merck Sharpe & Dohme, Rahway, N.J.) per kg of body weight. Rabbits were housed individually, maintained at 18 to 20 C, and given antibiotic-free food and water. Treponemes were extracted from infected testes in phosphate-buffered saline with 10% heat-inactivated normal rab-

2 5790 CUNNINGHAM ET AL. Percoll purified Treponema pallidum 4 degrecs C 1% Triton X degrces C Centrifugation "COLD PELLET" "TX-114 EXTRACTED" 37 degrees 30 min. 25 dcgrecs Centrifrugation "AQUEOUS PHASE" "TX-114 PHASE" "WARM PELLET" FIG. 1. Diagram of TX-114 extraction and phase partitioning procedure. Intact T. pallidum was extracted in 1% TX-114 at 4 C as described in Materials and Methods. Particulate material was removed by centrifugation at 4 C (cold pellet). After incubation of TX-114-extracted material at 37 C, centrifugation resulted in the formation of an aqueous phase, a TX-114 phase, and a pellet termed the warm pellet. bit serum (56 C for 30 min). After two low-speed centrifugations (400 x g for 7 min), the organisms were purified from host components by Percoll density gradient centrifugation as described previously (10). Antisera. Specific antiflagellar serum was a gift of David Blanco and was prepared against the periplasmic flagella isolated from T. pallidum or T. phagedenis biotype Reiter (3). Antibodies to the 19-kilodalton (kda) monomer of recombinant 4D were affinity purified on a recombinant 4D Reacti-gel 6X (Pierce Chemical Co., Rockford, Ill.) column as previously described (23). A monoclonal antibody reactive with a 47-kDa T. pallidum protein (15) was a gift of Michael V. Norgard, University of Texas Health Science Center, Dallas, Tex. Sera from patients with secondary syphilis have been described previously (9). TX-114 treatment of T. pallidum. The steps in TX-114 treatment of T. pallidum and in phase partitioning of TX- 114-extracted material are diagramed in Fig. 1. Percoll (Pharmacia Fine Chemicals, Piscataway, N.J.)-purified organisms were suspended at a concentration of 2.5 x 109/ml at 4 C in TX-114 extraction solution (1% TX-114, 10 mm Tris [ph 7.5], 5 mm EDTA). The suspension was placed on a rotating platform for 4 h at 4 C. In some cases T. pallidum was extracted as above but with the addition of 50 mm dithiothreitol (DTT). After extraction, the suspensions were centrifuged at 50,000 x g at 4 C for 1 h, resulting in a pellet, termed the cold pellet, and a supernatant, termed TX-114- extracted, which was processed as described below for phase partitioning. Phase partitioning with TX-114. The 20 C cloud point of TX-114 facilitates phase partitioning of detergent-solubilized proteins and recovery of proteins with hydrophobic character (5, 21). TX-114-extracted samples of T. pallidum were incubated at 37 C for 30 min in a water bath and then centrifuged at 25 C at 50,000 x g for 1 h to separate the phases. A lower TX-114 phase and an upper aqueous phase were formed; a pellet also resulted from the centrifugation and was termed the warm pellet, reflecting its solubility in TX-114 at 4 C but not at 25 or 37 C. The warm pellet was J. BACTERIOL. suspended directly in electrophoresis sample buffer for analysis. After the upper aqueous phase was removed, the lower TX-114 phase was decanted and washed twice with 20% of its volume in 10 mm Tris (ph 7.5)-5 mm EDTA to remove any aqueous material remaining at the detergent-aqueousphase interface. Each of the fractions was precipitated with 10 volumes of acetone on ice for 45 min. These fractions were centrifuged at 12,600 x g at 4 C for 30 min, and the pellets were air dried prior to sodium dodecyl sulfate SDSpolyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis and immunoblotting. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and immunoblotting were performed as previously described (9). Samples were suspended in final sample buffer (FSB; 62.5 mm Tris [ph 6.8], 2% [wt/vol] SDS, 5% [vol/vol] 2-mercaptoethanol, 20% [vol/vol] glycerol, 0.1% bromphenol blue dye) and placed in a 100 C boiling-water bath for 10 min before being loaded. After electrophoresis on an SDS-12.5% polyacrylamide gel, the proteins were stained with Coomassie blue or transferred to nitrocellulose membranes for immunological analysis with immune sera and 125I-protein A. Autoradiography was performed with Kodak XAR 5 X-ray film (Eastman Kodak Co., Rochester, N.Y.) and intensifying screens at -70 C. The amount of each sample used for SDS-polyacrylamide gel electrophoresis analysis was chosen to reflect the same molar proportion of the unfractionated organisms. Electron microscopy. Samples of T. pallidum (15 pj) were applied to Formvar (Ted Pella, Inc., Redding, Calif.)-coated, carbon-stabilized cooper grids (200 mesh) for 4 min at 23 C in a moist chamber. The grids were then dried by being blotted on filter paper. Further treatment of the treponemes was carried out by floating grids on drops of the following solutions in a moist chamber. The grids were exposed for 30 s, 1 min, 15 min, and 4 h to 1% TX-114 extraction buffer (1% TX-114, 10 mm Tris [ph 7.5], 1 mm EDTA) over an ice bath. Control grids were processed without TX-114 treatment. Following treatment, the grids were fixed in 1% glutaraldehyde in sodium cacodylate buffer (ph 7.2), washed by serial passage through 10 drops of phosphate-buffered saline (ph 7.2), treated for 15 s with an aqueous solution of 50,ug of bacitracin (Sigma Chemical Co., St. Louis, Mo.) per ml as a wetting agent, and negatively stained in 1% aqueous uranyl acetate. Excess stain was absorbed by blotting with filter paper, and the grids were allowed to air dry. The grids were observed with a 100 CX II electron microscope (JEOL U.S.A. Inc., Peabody, Mass.). In experiments to assess the ultrastructural effects of Percoll purification and TX-114 extraction, T. pallidum was extracted from infected rabbit testes in 50% serum-saline and centrifuged at 400 x g for 15 min to remove gross tissue debris. A portion of the fresh suspension containing 5.0 x 109 organisms was processed for thin sectioning as described below. The remainder of the suspension was purified by the Percoll procedure (10) and was divided into a control sample and a second sample for a 4-h treatment with 1% TX-114 before being fixed for thin sectioning. Samples were fixed in suspension with a final concentration of 1.25% paraformaldehyde, 2.0% glutaraldehyde, 0.03% picric acid, and 3.0 mm calcium chloride in M cacodylate buffer (ph 7.4) and then pelleted by centrifugation at 10,000 x g. They were postfixed in 1.0% osmium tetroxide in 0.1 M cacodylate buffer (ph 7.4) with 3.0 mm calcium chloride added, suspended in 2.0% lonagar (Oxoid Colab Laboratories, Inc., Chicago Heights, Ill.), cut into 1-mm3 blocks, and stained en bloc with 1.0% uranyl acetate

3 VOL. 170, 1988 I A p t.... s. T. PALLIDUM OUTER MEMBRANE 5791 B it 9 *@,l 10 U j.....; A,I -,,., r :: :.. S;R e.:, '". #j.-. 4: w"'w :::.4,-.,...:.r ok.1 Downloaded from Cif t : V... A 40 FIG. 2. Effects of TX-114 treatment on T. pallidum ultrastructure. Electron micrographs of T. pallidum before (A) and after treatment with TX-114 for 15 min (B) and 30 (D). TX-114-treated and untreated organisms were mixed on the same grid (C), demonstrating that differences in diameters were not artifacts of processing on separate grids for electron microscopy. Parallel striations in panel A are adherent periplasmic flagella (arrow). Unwound periplasmic flagella (arrow) attached to the cytoplasmic cylinder are shown in panel B. Bar, 1,um. in veronyl acetate buffer. The material was dehydrated in an acetone series and embedded in Spurr epoxy resin. Silver to gray sections were prepared with an ultramicrotome (Ivan Sorvall, Inc., Norwalk, Conn.), placed on Formvar-coated copper keyhole grids, and stained with 1.0% aqueous uranyl acetate and Reynold lead citrate before being observed with a 100 CX II electron microscope. RESULTS Release of the T. pallidum outer membrane by TX-114. The effect of TX-114 on the structure of freshly isolated, Percollpurified virulent T. pallidum was examined by whole-mount electron microscopy. Untreated control organisms exhibited no blebbing of the outer membrane, retained tightly adherent periplasmic flagella on their cytoplasmic cylinders, and had diameters ranging from 0.25 to 0.35 p.m (Fig. 2A). In contrast, 15-min TX-114 treatment unwound but did not release the periplasmic flagella from the morphologically intact cytoplasmic cylinders and reduced the diameters of the organisms to 0.1 to 0.15 jm (Fig. 2B); identical results were observed after a 4-h treatment with TX-114 (results not shown). The difference in diameter between freshly isolated and TX-114-treated T. pallidum was further demonstrated when detergent-treated and untreated organisms were placed on the same grid (Fig. 2C). Exposure of the organism to TX-114 for 30 s resulted in blebbing (Fig. 2D). Immediate blebbing, followed by lysis of blebs, was also observed during a similar time course when treponemes were treated in suspension with TX-114 and monitored by darkfield microscopy (results not shown). We examined thin sections of T. pallidum to directly determine the fate of the outer and cytoplasmic membranes after treatment with TX-114. Figure 3 shows representative C on December 4, 2018 by guest

4 5792 CUNNINGHAM ET AL. J. BACTERIOL. A ~~~~~~~~~~~7 *:: -'.T : t~ 4Af.Lt ~ ~ ~~ FIG. 3. Release of the T. pallidum outer membrane by TX-114. Transmission electron micrographs show thin sections of freshly extracted (A) and Percoll-purified (B) T. pallidum cells before TX-114 detergent treatment and Percoll-purified T. pallidum cells after TX-114 treatment (C). Outer (arrowhead) and cytoplasmic (arrow) membranes were present on organisms not treated with TX-114. Only the cytoplasmic membrane remained on organisms treated with TX-114. Bar, 1 p.m. thin sections indicating that a readily discernable outer membrane found on motile T. pallidum prior to Percoll purification (Fig. 3A) was unaltered by Percoll purification (Fig. 3B). TX-114 treatment of the Percoll-purified treponemes resulted in loss of outer membrane structure, while the appearance of the cytoplasmic membrane was unchanged (Fig. 3C). We therefore conclude that the decreased B 2 0 FIG. 4. Composition of fractions derived from TX-114 extraction and phase partitioning of T. pallidum. The figure shows a Coomassie blue-stained SDS-polyacrylamide gel of molecular mass markers (lane MKS) (shown in kilodaltons) and the following TX-114 fractions derived from extraction and phase partitioning (Fig. 1): whole T. pallidum (lane WHOLE); the cold pellet of TX-114-insoluble cytoplasmic cylinders (lane INSOL); the total set of TX-114-extracted polypeptides (lane SOL); the aqueous phase (lane AQ); the TX-114 phase (lane TX-114); and the warm pellet (lane WP). Each sample represents an approximately equal percentage of the total number of T. pallidum cells extracted with TX-114. diameter of TX-114-treated T. pallidum shown in Fig. 2 resulted from selective release of the outer membrane. Composition of fractions derived from TX-114 extraction and phase partitioning of T. pallidum. As described in Materials and Methods, Fig. 1 outlines the major steps in extraction of Percoll-purified T. pallidum with Triton X-114. Figure 4 is a Coomassie blue-stained gel, which shows the results of TX-114 fractionation and phase partitioning of T. pallidum proteins under reducing conditions. Most of the cellular proteins of the T. pallidum cytoplasmic cylinders remained in the insoluble cold pellet (Fig. 4, lane INSOL) after TX-114 treatment. However, polypeptides of 57, 47, 38, 23, 19, 16 (doublet), 14, and 12 kda were consistently TX-114 extracted (lane SOL). Smaller amounts of at least six polypeptides between 33 and 37 kda were also detected. After phase partitioning, the aqueous phase (lane AQ) contained polypeptides of 57, 19, 16, and 12 kda. The TX-114 phase contained polypeptides of 57, 47, 35, 33, 16, and 14 kda (lane TX-114). The 35-, 33-, 16-, and 14-kDa polypeptides which partitioned into TX-114 were enriched relative to their molar representation in the TX-114-extracted fraction (lane SOL). The warm pellet (lane WP) contained a portion of the 47-kDa polypeptide and all the 38- and 23-kDa polypeptides extracted by TX-114. Association of T. pallidum penicillin-binding proteins with the cytoplasmic cylinders after TX-114 extraction. T. pallidum penicillin-binding proteins were labeled with 500 nm and 5,uM [35S]benzylpenicillin as described previously (6). The labeled treponemes were extracted with TX-114 and fractionated as shown in Fig. 1. Figure 5 is an autoradiogram of an SDS-polyacrylamide gel on which samples of whole, unextracted treponemes (lanes WH), the insoluble cold

5 VOL. 170, nanomolar WH INSOL SOL kd _ w_ e8- wow 61-5 micromolar WH INSOL SOL ir, M_~,~f -ama m.a FIG. 5. Fate of T. pallidum penicillin-binding proteins in TX-114 fractionation. T. pallidum was incubated in two different concentrations of [35S]benzylpenicillin (left lanes, 500 nm; right lanes, 5,uM) to allow covalent binding of penicillin. Labeled organisms were then extracted with TX-114, yielding insoluble cold-pellet (lanes INSOL) and TX-114-extracted (lanes SOL) fractions (Fig. 1). Lanes WH represent whole T. pallidum. Molecular mass markers are shown in kilodaltons (kd) a- 31-mo 6 WH INSOL SOL AO TXSOL WP T. PALLIDUM OUTER MEMBRANE 5793 pellet (lanes INSOL), and the TX-114-extracted (lanes SOL) fractions were separated. The penicillin-binding proteins, as detected by using either concentration of labeled penicillin, remained with the cytoplasmic cylinder cold pellet. The finding is consistent with the electron-microscopic evidence presented above that TX-114 does not destroy the integrity of the cytoplasmic membrane of T. pallidum. Association of T. pallidum endoflagella with the cytoplasmic cylinder after TX-114 extraction. An immunoblot was prepared that contained samples of whole T. pallidum and the fractions derived from TX-114 extraction and phase partitioning. Figure 6 shows the results of probing the immunoblot with antiflagellar serum at a 1:200 dilution. Endoflagellar bands were detected only in whole T. pallidum (Fig. 6, lane WH) and in the insoluble cold pellet (lane INSOL), which containis the cytoplasmic cylinders. This result is consistent with the electron micrograph presented in Fig. 2B, which showed that the periplasmic endoflagella were not released from the cytoplasmic cylinders by TX-114. Fate of T. pallidum antigens in TX-114 extraction and phase partitioning. The immunoblot of the TX-114-extracted T. pallidum fractions used to detect endoflagella (Fig. 6) was treated with glycine hydrochloride (ph 2.5) to remove bound antibodies and reprobed with human syphilitic serum diluted 1:100. Figure 7 shows that antibodies in syphilitic serum bound to all the major polypeptide constituents of the TX-114-extracted phase, the aqueous phase, the TX-114 phase, and the warm pellet, identified in Fig. 4. In addition, several polypeptides, ranging in molecular mass from 33 to 35 kda, which partitioned into the TX-114 phase were very antigenic relative to their molar representation in this fraction. Although this molecular mass range overlaps that of T. pallidum endoflagella, it is clear that these 33- to 35-kDa polypeptide antigens are distinct from the endoflagellar antigens which were shown not to be extracted by TX-114 (Fig. 6). l WH INSOL SOL AG TXSOL WP *ssw ^ ^ FIG. 6. Fate of endoflagella in TX-114 extraction. An immunoblot of the following TX-114 fractions derived from extraction and phase partitioning (Fig. 1) was probed with monospecific antiendoflagellar antibodies. Lanes: WH, whole T. pallidum; INSOL, the cold pellet of cytoplasmic cylinders; SOL, TX-114-extracted polypeptides; AQ, aqueous phase; TXSOL, TX-114 phase; WP, warm pellet. Molecular mass markers are shown in kilodaltons. FIG. 7. Fate of T. pallidum antigens in TX-114 extraction and phase partitioning. The immunoblot described in the legend to Fig. 6 was treated with glycine hydrochloride as described in the text to remove bound antibody and then reprobed with human syphilitic serum. Lanest WH, whole T. pallidum; INSOL, the cold pellet of cytoplasmic cylinders; SOL, TX-114-extracted polypeptides; AQ, aqueous phase; TXSOL, TX-114 phase; WP, warm pellet. Molecular mass markers are shown in kilodaltons.

6 5794 CUNNINGHAM ET AL. 19, (Non-reduced) WH SOL INSOL TX SOL WP AO 47. U.. 35 (+50mmn DTT} WH SOL INSOL TXSOL WP AO FIG. 8. Fate of the 190- and 47-kDa antigens in TX-114 fractionation. An immunoblot of TX-114 fractions (derived as shown in Fig. 1) was probed with anti-47-kda monoclonal and anti-4d antibodies. The left panel shows fractions derived without addition of DTT; the right panel shows fractions derived from TX-114 extraction in the presence of 50 mm DTT. Samples: lanes WH, whole T. pallidum; lanes SOL, the TX-114-extracted fraction; lanes INSOL, the cold pellet; lanes TXSOL, the TX-114 phase; lanes WP, the warm pellet; lanes AQ, the aqueous phase. Molecular mass markers are shown in kilodaltons. Effect of disulfide bonding on the 190-kDa (4D) and 47-kDa surface antigens in TX-114 fractionation. Immunoblots were prepared that contained samples of T. pallidum extracted with TX-114 in the presence or absence of 50 mm DTT and fractionated by the phase partitioning procedure. Figure 8 shows the results when the immunoblots were probed with affinity-purified anti-4d antiserum (23) mixed with an anti- 47-kDa monoclonal antibody (15). The 4D antigen, detected as a 19-kDa monomer and 34-kDa dimer, remained with the insoluble cytoplasmic cylinders (Fig. 8, left panel, lane INSOL) in the absence of DTT. However, when TX-114 extraction was performed in the presence of DTT, 4D was fully extracted (right panel, lane SOL) and subsequently partitioned into the aqueous phase (right panel, lane AQ). The amount of the 4D antigen, which is less than that of other polypeptides extracted by TX-114 in these samples, is expected, given our previous calculation that there is about 2,ug of 4D per 109 T. pallidum cells (23). Although much of the 47-kDa protein was removed by TX-114 extraction in the absence of DTT (Fig. 8, left panel lanes SOL and INSOL), the addition of DTT to TX-114 resulted in its complete extraction (right panel, lanes SOL and INSOL). The partitioning of the 47-kDa antigen into the TX-114 phase and warm pellet was not affected by the presence of DTT. Extraction with TX-114 in the presence of DTT did not otherwise alter the polypeptide composition or antigenicity of the TX-114-extracted fraction or the integrity of the cytoplasmic membrane (data not shown). 0 DISCUSSION ft 40 T. pallidum is highly evolved for coexistence with its hosts. Perhaps reflecting this adaptation, certain surface properties of the virulent organism are distinct from those of other bacteria. It has been appreciated for three decades that freshly extracted intact treponemes are not reactive in J. BACTERIOL. serological tests (11, 17). It has also been shown by immunoelectron microscopy that the surface of the virulent organism is resistant to the binding of antitreponemal antibodies in the absence of complement (13, 14, 25). The time required for in vitro complement-dependent serum bactericidal reactions, 4 h or longer in the T. pallidum immobilization and in vitro-in vivo neutralization tests (2), is probably also indicative of this resistance to antibody binding. Further evidence demonstrating the unusual nature of the surface of T. pallidum has been provided by Stamm and co-workers (28). Their attempts to immunoprecipitate surface proteins identified periplasmic flagella as being the most susceptible to antibody binding; they also reported that the outer membrane could be removed by 0.04% SDS with the release only of periplasmic flagellar protein (28). Implicit in such observations is the suggestion that the outer membrane is relatively devoid of protein. Other possibilities to explain the antigenic inertness of the surface and its resistance to radioiodination include a model in which the surface is coated with host molecules lacking the substrates for radioiodination and/or a model of outer membrane architecture wherein epitopes of its composite proteins are only transiently exposed owing to the motility of the organism and membrane fluidity. In support of the idea that a fluid outer membrane allows at least transient surface exposure of subsurface structures, we have found that antibodies to T. pallidum endoflagella have treponemicidal activity (4). These unusual surface properties of living T. pallidum cells have prompted the use of the term surface associated, rather than the term outer membrane, to describe the cellular location of proteins demonstrated on the surface of the organism by immunoelectron microscopy under the conditions of the T. pallidum immobilization test (8, 25). In this study we have used modified methods for thinsection electron microscopy to readily and consistently demonstrate the T. pallidum outer membrane. Our finding of the integrity of the outer membrane after Percoll purification corroborates our earlier report on the usefulness of this procedure for purifying virulent treponemes from host tissue components (10). We have found that the nonionic detergent TX-114 removes the outer membrane without visually apparent damage to the lipid bilayer-type structure of the cytoplasmic membrane. Whole-mount electron microscopy of treponemes treated for 30 s with 1% TX-114 showed that removal of the outer membrane is preceded by an extensive blebbing process followed by lysis of blebs in the detergent solution. These results are in contrast to the well-described susceptibility of the cytoplasmic membrane of isolated Escherichia coli cell envelopes to nonionic detergent solubilization (27). In E. coli, covalent and strong noncovalent forces link outer membrane proteins to peptidoglycan and thus stabilize the association of the outer membrane with the cell; in contrast, Ipp and ompa mutants of E. coli exhibit blebbing (16). Thin sections of T. pallidum have demonstrated a layer whose appearance is thought to be consistent with that of peptidoglycan juxtaposed to the outer leaflet of the cytoplasmic membrane (12). The stability of the cytoplasmic membrane of T. pallidum following TX-114 treatment may be based on tight links to peptidoglycan or its equivalent, in a manner analagous to the association of the E. coli outer membrane with peptidoglycan, or on strong protein-protein interactions. If treponemal peptidoglycan were tightly bound to the spirochetal outer membrane, movement mediated by the periplasmic flagella might be impossible. This study has demonstrated the utility of the endoflagella and penicillin-binding proteins of T. pallidum in cell fraction-

7 VOL. 170, 1988 ation studies. Penicillin-binding proteins known to be anchored in the cytoplasmic membranes of many bacterial species (29) have been identified recently in T. pallidum (6). Our finding that T. pallidum penicillin-binding proteins remain associated with the cytoplasmic cylinder following TX-114 treatment provides additional evidence that TX-114 treatment does not solubilize the T. pallidum cytoplasmic membrane. We recognize the possibility that some cytoplasmic membrane proteins are sensitive to TX-114 solubilization, but it seems improbable that there could be extensive solubilization of cytoplasmic membrane protein without concomitant disruption of phospholipid bilayer appearance. Schnaitman has shown that under conditions where Triton X-100 solubilizes the cytoplasmic membrane of E. coli cell envelopes, one-third of the outer membrane phospholipid is solubilized without release of the outer membrane protein (27). Therefore, if the analogy between the stabilities of the E. coli outer membrane and -the T. pallidum cytoplasmic membrane are valid, TX-114 would be more likely to solubilize phospholipid than protein components of the cytoplasmic membrane. Although our results do not rigorously preclude the possibility that some of the periplasmic flagella are released from and cosediment with the cytoplasmic cylinders after TX-114 treatment, our electron micrographs suggest that flagella remain specifically associated, by one end, with the cytoplasmic cylinders. At present, however, we have no other means available for commenting on the fate of additional periplasmic constituents during the removal of the T. pallidum outer membrane with TX-114, and we cannot rule out the possibility that some of the proteins released by the detergent are derived from the periplasm, not from the outer membrane. TX-114 treatment of T. pallidum consistently results in the release of the same small set of polypeptides. Two of these polypeptides, the 47- and the 190-kDa (4D) polypeptides, have been studied extensively. Antibodies to the 4D ordered ring (7) have treponemicidal activity in the T. pallidum immobilization test (9), and 4D has been associated with the surface of T. pallidum by immunoelectron microscopy (25). Rabbits immunized with recombinant 4D have manifested an altered course of lesion development, consistent with partial protection in experimental syphilis (5a). The native structure of 4D in T. pallidum is that of a disulfide bond-linked assembly of ordered rings too large to enter an SDSpolyacrylamide gel (23). Penn et al. reported that a 47-kDa protein was released from T. pallidum as a result of treatment with Triton X-100 (19), and Jones et al. have identified a murine monoclonal antibody that has activity in both the T. pallidum immobilization and in vitro-in vivo neutralization tests and that recognizes a 47-kDa T. pallidum surfaceassociated protein (15). In this study we found that TX-114 treatment did not release 4D from the cytoplasmic cylinder fraction unless a reducing agent was present. Using the monoclonal antibody of Jones et al. (15) as a probe, we found that full disassociation of the 47-kDa protein from the cytoplasmic cylinders occurred only with the addition of a reducing agent. Factors such as covalent linkage of the 190- and 47-kDa proteins to a subsurface structure such as peptidoglycan, very high molecular weight, and/or limited solubility of their native structures in TX-114 could explain this observation. A 38-kDa antigen also was released by TX-114 treatment and partitioned into the warm pellet of the hydrophobic phase with the 47-kDa antigen. The correspondence of the 38-kDa molecule to a recombinant surface-associated 38- T. PALLIDUM OUTER MEMBRANE 5795 kda molecule which we have described previously (8) is under investigation. Creation of the warm pellet provides a simple means for purification of the 38- and 47-kDa proteins from T. pallidum. Polypeptides of 33 to 35 kda partitioned into the hydrophobic detergent phase and were reactive with syphilitic serum. These antigens may have been previously unnoticed on one-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting because of the presence of the relatively abundant periplasmic flagellum proteins of similar molecular masses. The phase partitioning possible with Triton X-114 has proven useful in the isolation of certain membrane proteins from eucaryotic cells (5) and, more recently, from mycoplasmas (26). We have found, using cell envelopes of E. coli treated with EDTA and Triton X-114, that proteins whose molecular masses corresponded to those of OmpA and OmpF/C were partially solubilized and were found in the hydrophobic phase after partitioning (T. M. Cunningham and M. A. Lovett, unpublished observations). However, the appearance of a specific set of T. pallidum proteins in the hydrophobic TX-114 phase, including the surface-associated 190- and 47-kDa proteins, although strongly suggestive, does not prove an outer membrane origin. Recently, Radolf et al. concluded on the basis of examination of whole-mount electron micrographs of detergent-treated organisms that TX-114 removed the T. pallidum outer membrane, and they reported that the outer membrane could be released under conditions (0.1% TX-114) which released very little protein; organisms treated with 0.5% TX-114 were judged to have removal of large segments of the cytoplasmic membranes and extrusion of cytoplasmic contents (24). These results were thought to be consistent with the idea that the T. pallidum outer membrane is a protein-deficient phopholipid bilayer, a possibility also considered by Stamm et al. (28). Our conclusions differ in several ways from those of Radolf et al. (24). We believe that our use of electron microscopy of thin sections has provided a reliable means of judging both outer membrane and cytoplasmic membrane integrity; our results clearly show that 1% TX-114 does not disrupt the appearance of the cytoplasmic membrane. Demonstration that T. pallidum penicillin-binding proteins are not released by TX-114 corroborates this conclusion. Our finding that complete release of the 190- and 47-kDa proteins by TX-114 requires treatment with a reducing agent suggests that these proteins are linked covalently to a structure which is not solubilized by TX-114. Although it is possible that the proteins which partition into the hydrophobic TX-114 phase, including the very abundant 47-kDa protein, are largely derived from the periplasmic space, it is more likely that they are constituents of the T. pallidum outer membrane but have limited surface exposure. We believe that epitopes of T. pallidum outer membrane proteins could be transiently exposed to the surface during the bending and flexing that accompany treponemal motility. The susceptibility of T. pallidum endoflagella to bactericidal antibodies (4) is in accord with this hypothesis. ACKNOWLEDGMENTS This study was supported by Public Health Service research grants AI and AI from the National Institute for Allergy and Infectious Diseases to M.A.L. and J.N.M., respectively. We thank David Blanco, Denee Thomas, Lee Borenstein, and Susan Thompson for valuable discussions and Fred Urquhart for expert technical assistance. We thank Michael Norgard, University of Texas Health Science Center, Dallas, for providing monoclonal antibody to the 47-kDa protein.

8 5796 CUNNINGHAM ET AL. ADDENDUM IN PROOF We have completed freeze-fracture studies of the outer membrane of T. pallidum which are relevant to understanding the origin of the polypeptides released by TX-114. The particle density per square micrometer of the E face of this membrane is 70, and that of its P face is 100. Assuming that each particle represents a protein complex, in contrast the corresponding protein densities for the E and P faces of the E. coli outer membrane are 6,000 to 10,000 and 500 to 700, respectively. This strikingly low protein composition of the outer membrane of T. pallidum indicates that while the molecules released by TX-114 must include the outer membrane protein, most if not all the polypeptides we identified are probably derived from the periplasmic space or the cytoplasmic membrane. The low protein composition of its outer membrane may explain the relative antigenic inertness of the surface of virulent T. pallidum. LITERATURE CITED 1. Alderete, J. 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