Vol. 170, No. 12JOURNAL OF BACTERIOLOGY, Dec. 1988, p. 5789-57960021-9193/88/125789-08$02.00/0Copyright (© 1988, American Society for Microbiology

Selective Release of the Treponema pallidum Outer Membrane andAssociated 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,* Universityof California at Los Angeles, Los Angeles' California 90024

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% concentrationof Triton X-114 resulted in cell surface blebbing followed by lysis of blebs and a decrease in diameter from 0.25-0.35 ,um to 0.1-0.15 ,im. Examination of thin sections of untreated Percoll-purified T. pallidum showedintegrity of outer and cytoplasmic membranes. In contrast, thin sections of Triton X-114-treated treponemesshowed integrity of the cytoplasmic membrane but loss of the outer membrane. The cytoplasmic cylindersgenerated by detergent treatment retained their periplasmic flagella, as judged by electron microscopy andimmunoblotting. Recently identified T. pallidum penicillin-binding proteins also remained associated with thecytoplasmic cylinders. Proteins released by Triton X-114 at 4°C were divided into aqueous and hydrophobicphases 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 beenassociated 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 palli-dum remains a major goal of research focused on relating thestructure of this noncultivable pathogen to the pathogenesisof syphilis. Application of conventional methods for deter-mining the structure of the gram-negative cell envelope havenot yielded consistent results, despite the availability ofvirulent treponemes purified from rabbit host tissue debris(10). Several properties which distinguish T. pallidum fromother groups of bacteria have been major factors in compli-cating this basic structural analysis. Unlike other bacteria,freshly extracted virulent T. pallidum does not readily bindspecific antibodies to its surface (13, 14, 25, 28). Themolecular basis of this relative antigenic inertness of thetreponemal surface is unknown, but could reflect eithercoating with host tissue components (1) or an innate struc-tural property of the treponemal surface. Only with 4 h ormore of incubation in vitro does the organism becomesusceptible to complement-dependent serum bactericidalactivity (2), raising the possibility that surface antigensbecome exposed only when the treponemal surface becomesaltered on in vitro incubation (28). Although the antigenicinertness of the treponemal surface may explain in part howthe organism can evade the host immune response in vivo, ithas complicated identification of surface molecules by directantibody binding. The antigenic inertness of the surface isparalleled by the finding that it has not been possible toidentify surface proteins of T. pallidium by radioiodination(19) or radioimmunoprecipitation (28) techniques.Another factor in complicating the basic structural analy-

sis of T. pallidum has been the difficulty encountered inconsistently demonstrating an outer membrane by electron

* Corresponding author.t Present address: Division of Bacterial Products, Food and Drug

Administration, Bethesda, MD 20892.

microscopy of thin sections. The absence of such a reliableultrastructural means of judging cell envelope integrity hascomplicated attempts to develop treponemal cell fractiona-tion protocols. In this report, using modified procedures forpreparing thin sections of T. pallidum which consistentlydemonstrate outer membrane structure, we provide ultra-structural evidence that the nonionic detergent Triton X-114(TX-114) removes the outer membrane of T. pallidum with-out altering the bilayer appearance of the cytoplasmic mem-brane. We describe the partitioning of the detergent-ex-tracted polypeptides into the aqueous and hydrophobicphases formed by TX-114 at temperatures above its cloudpoint. We also show that the periplasmic flagella and re-cently described penicillin-binding proteins (6) of T. palli-dum remain with the cytoplasmic cylinder after detergenttreatment.

(This work was originally presented at the University ofCalifornia Los Angeles Symposium on Bacteria-Host CellInteraction, at Park City, Utah, on 15 February 1987 and atthe 87th Annual Meeting of the American Society for Micro-biology, 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 forthe Ph.D. degree from the University of California LosAngeles, 1987.)

MATERIALS AND METHODS

Bacteria. The Nichols strain of T. pallidum subsp. palli-dum was cultivated by passage in New Zealand Whiterabbits injected intramuscularly with 10 mg of cortisoneacetate (Merck Sharpe & Dohme, Rahway, N.J.) per kg ofbody weight. Rabbits were housed individually, maintainedat 18 to 20°C, and given antibiotic-free food and water.Treponemes were extracted from infected testes in phos-phate-buffered saline with 10% heat-inactivated normal rab-

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Percoll purifiedTreponema pallidum

4 degrecs C 1% Triton X-114

4 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 partitioningprocedure. Intact T. pallidum was extracted in 1% TX-114 at 4°C asdescribed in Materials and Methods. Particulate material was re-moved by centrifugation at 4°C (cold pellet). After incubation ofTX-114-extracted material at 37°C, centrifugation resulted in theformation of an aqueous phase, a TX-114 phase, and a pellet termedthe warm pellet.

bit serum (56°C for 30 min). After two low-speed centrifu-gations (400 x g for 7 min), the organisms were purified fromhost components by Percoll density gradient centrifugationas described previously (10).

Antisera. Specific antiflagellar serum was a gift of DavidBlanco and was prepared against the periplasmic flagellaisolated from T. pallidum or T. phagedenis biotype Reiter(3). Antibodies to the 19-kilodalton (kDa) monomer ofrecombinant 4D were affinity purified on a recombinant 4DReacti-gel 6X (Pierce Chemical Co., Rockford, Ill.) columnas previously described (23). A monoclonal antibody reac-tive with a 47-kDa T. pallidum protein (15) was a gift ofMichael V. Norgard, University of Texas Health ScienceCenter, Dallas, Tex. Sera from patients with secondarysyphilis 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 or-ganisms were suspended at a concentration of 2.5 x 109/mlat 4°C in TX-114 extraction solution (1% TX-114, 10 mM Tris[pH 7.5], 5 mM EDTA). The suspension was placed on arotating platform for 4 h at 4°C. In some cases T. pallidumwas extracted as above but with the addition of 50 mMdithiothreitol (DTT). After extraction, the suspensions werecentrifuged 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 forphase partitioning.

Phase partitioning with TX-114. The 20°C cloud point ofTX-114 facilitates phase partitioning of detergent-solubilizedproteins and recovery of proteins with hydrophobic charac-ter (5, 21). TX-114-extracted samples of T. pallidum wereincubated at 37°C for 30 min in a water bath and thencentrifuged at 25°C at 50,000 x g for 1 h to separate thephases. A lower TX-114 phase and an upper aqueous phasewere formed; a pellet also resulted from the centrifugationand was termed the warm pellet, reflecting its solubility inTX-114 at 4°C but not at 25 or 37°C. The warm pellet was

suspended directly in electrophoresis sample buffer for anal-ysis. After the upper aqueous phase was removed, the lowerTX-114 phase was decanted and washed twice with 20% ofits volume in 10 mM Tris (pH 7.5)-5 mM EDTA to removeany aqueous material remaining at the detergent-aqueous-phase interface. Each of the fractions was precipitated with10 volumes of acetone on ice for 45 min. These fractionswere centrifuged at 12,600 x g at 4°C for 30 min, and thepellets were air dried prior to sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis.

SDS-polyacrylamide gel electrophoresis and immunoblot-ting. Sodium dodecyl sulfate (SDS)-polyacrylamide gel elec-trophoresis and immunoblotting were performed as previ-ously described (9). Samples were suspended in final samplebuffer (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-waterbath for 10 min before being loaded. After electrophoresis onan SDS-12.5% polyacrylamide gel, the proteins were stainedwith Coomassie blue or transferred to nitrocellulose mem-branes for immunological analysis with immune sera and125I-protein A. Autoradiography was performed with KodakXAR 5 X-ray film (Eastman Kodak Co., Rochester, N.Y.)and intensifying screens at -70°C. The amount of eachsample used for SDS-polyacrylamide gel electrophoresisanalysis was chosen to reflect the same molar proportion ofthe unfractionated organisms.

Electron microscopy. Samples of T. pallidum (15 pJ) wereapplied to Formvar (Ted Pella, Inc., Redding, Calif.)-coated,carbon-stabilized cooper grids (200 mesh) for 4 min at 23°Cin a moist chamber. The grids were then dried by beingblotted on filter paper. Further treatment of the treponemeswas carried out by floating grids on drops of the followingsolutions in a moist chamber. The grids were exposed for 30s, 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 icebath. Control grids were processed without TX-114 treat-ment. Following treatment, the grids were fixed in 1%glutaraldehyde in sodium cacodylate buffer (pH 7.2), washedby serial passage through 10 drops of phosphate-bufferedsaline (pH 7.2), treated for 15 s with an aqueous solution of50 ,ug of bacitracin (Sigma Chemical Co., St. Louis, Mo.) perml as a wetting agent, and negatively stained in 1% aqueousuranyl acetate. Excess stain was absorbed by blotting withfilter paper, and the grids were allowed to air dry. The gridswere observed with a 100 CX II electron microscope (JEOLU.S.A. Inc., Peabody, Mass.). In experiments to assess theultrastructural effects of Percoll purification and TX-114extraction, T. pallidum was extracted from infected rabbittestes in 50% serum-saline and centrifuged at 400 x g for 15min to remove gross tissue debris. A portion of the freshsuspension containing 5.0 x 109 organisms was processedfor thin sectioning as described below. The remainder of thesuspension was purified by the Percoll procedure (10) andwas divided into a control sample and a second sample for a4-h treatment with 1% TX-114 before being fixed for thinsectioning.Samples were fixed in suspension with a final concentra-

tion of 1.25% paraformaldehyde, 2.0% glutaraldehyde,0.03% picric acid, and 3.0 mM calcium chloride in 0.025 Mcacodylate buffer (pH 7.4) and then pelleted by centrifuga-tion at 10,000 x g. They were postfixed in 1.0% osmiumtetroxide in 0.1 M cacodylate buffer (pH 7.4) with 3.0 mMcalcium chloride added, suspended in 2.0% lonagar (OxoidColab Laboratories, Inc., Chicago Heights, Ill.), cut into1-mm3 blocks, and stained en bloc with 1.0% uranyl acetate

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T. PALLIDUM OUTER MEMBRANE 5791

A B

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FIG. 2. Effects of TX-114 treatment on T. pallidum ultrastructure. Electron micrographs of T. pallidum before (A) and after treatment withTX-114 for 15 min (B) and 30 (D). TX-114-treated and untreated organisms were mixed on the same grid (C), demonstrating that differencesin diameters were not artifacts of processing on separate grids for electron microscopy. Parallel striations in panel A are adherent periplasmicflagella (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 anacetone series and embedded in Spurr epoxy resin. Silver togray sections were prepared with an ultramicrotome (IvanSorvall, Inc., Norwalk, Conn.), placed on Formvar-coatedcopper keyhole grids, and stained with 1.0% aqueous uranylacetate and Reynold lead citrate before being observed witha 100 CX II electron microscope.

RESULTS

Release of the T. pallidum outer membrane by TX-114. Theeffect of TX-114 on the structure of freshly isolated, Percoll-purified virulent T. pallidum was examined by whole-mountelectron microscopy. Untreated control organisms exhibitedno blebbing of the outer membrane, retained tightly adherentperiplasmic flagella on their cytoplasmic cylinders, and haddiameters ranging from 0.25 to 0.35 p.m (Fig. 2A). In

contrast, 15-min TX-114 treatment unwound but did notrelease the periplasmic flagella from the morphologicallyintact cytoplasmic cylinders and reduced the diameters ofthe organisms to 0.1 to 0.15 jm (Fig. 2B); identical resultswere observed after a 4-h treatment with TX-114 (results notshown). The difference in diameter between freshly isolatedand TX-114-treated T. pallidum was further demonstratedwhen detergent-treated and untreated organisms wereplaced on the same grid (Fig. 2C). Exposure of the organismto TX-114 for 30 s resulted in blebbing (Fig. 2D). Immediateblebbing, followed by lysis of blebs, was also observedduring a similar time course when treponemes were treatedin suspension with TX-114 and monitored by darkfieldmicroscopy (results not shown).We examined thin sections of T. pallidum to directly

determine the fate of the outer and cytoplasmic membranesafter treatment with TX-114. Figure 3 shows representative

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5792 CUNNINGHAM ET AL.

A200

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4 3

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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 frac-

tions 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-ex-

tracted 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.

@' ~~~~~~~~~~~7*::-'.T :

t~ ~ ~ ~~4Af.Lt

FIG. 3. Release of the T. pallidum outer membrane by TX-114.Transmission electron micrographs show thin sections of freshlyextracted (A) and Percoll-purified (B) T. pallidum cells beforeTX-114 detergent treatment and Percoll-purified T. pallidum cellsafter TX-114 treatment (C). Outer (arrowhead) and cytoplasmic(arrow) membranes were present on organisms not treated withTX-114. Only the cytoplasmic membrane remained on organismstreated with TX-114. Bar, 1 p.m.

thin sections indicating that a readily discernable outermembrane found on motile T. pallidum prior to Percollpurification (Fig. 3A) was unaltered by Percoll purification(Fig. 3B). TX-114 treatment of the Percoll-purified trepo-nemes resulted in loss of outer membrane structure, whilethe appearance of the cytoplasmic membrane was un-changed (Fig. 3C). We therefore conclude that the decreased

diameter of TX-114-treated T. pallidum shown in Fig. 2resulted from selective release of the outer membrane.

Composition of fractions derived from TX-114 extractionand phase partitioning of T. pallidum. As described in Mate-rials and Methods, Fig. 1 outlines the major steps in extrac-tion of Percoll-purified T. pallidum with Triton X-114. Figure4 is a Coomassie blue-stained gel, which shows the results ofTX-114 fractionation and phase partitioning of T. pallidumproteins under reducing conditions. Most of the cellularproteins of the T. pallidum cytoplasmic cylinders remainedin the insoluble cold pellet (Fig. 4, lane INSOL) after TX-114treatment. However, polypeptides of 57, 47, 38, 23, 19, 16(doublet), 14, and 12 kDa were consistently TX-114 ex-tracted (lane SOL). Smaller amounts of at least six polypep-tides 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. TheTX-114 phase contained polypeptides of 57, 47, 35, 33, 16,and 14 kDa (lane TX-114). The 35-, 33-, 16-, and 14-kDapolypeptides which partitioned into TX-114 were enrichedrelative to their molar representation in the TX-114-ex-tracted 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 withthe cytoplasmic cylinders after TX-114 extraction. T. pallidumpenicillin-binding proteins were labeled with 500 nM and 5

,uM [35S]benzylpenicillin as described previously (6). Thelabeled treponemes were extracted with TX-114 and frac-tionated as shown in Fig. 1. Figure 5 is an autoradiogram ofan SDS-polyacrylamide gel on which samples of whole,unextracted treponemes (lanes WH), the insoluble cold

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T. PALLIDUM OUTER MEMBRANE 5793

5 micromolarWH INSOL SOL

miR,M_~,~f

-ama .A

41-

24-

FIG. 5. Fate of T. pallidum penicillin-binding proteins in TX-114fractionation. T. pallidum was incubated in two different concentra-tions of [35S]benzylpenicillin (left lanes, 500 nM; right lanes, 5 ,uM)to allow covalent binding of penicillin. Labeled organisms were thenextracted with TX-114, yielding insoluble cold-pellet (lanes INSOL)and TX-114-extracted (lanes SOL) fractions (Fig. 1). Lanes WHrepresent whole T. pallidum. Molecular mass markers are shown inkilodaltons (kd).

WH INSOL SOL

47-38-35-

a-31-mo

6

pellet (lanes INSOL), and the TX-114-extracted (lanes SOL)fractions were separated. The penicillin-binding proteins, asdetected by using either concentration of labeled penicillin,remained with the cytoplasmic cylinder cold pellet. Thefinding is consistent with the electron-microscopic evidencepresented above that TX-114 does not destroy the integrityof the cytoplasmic membrane of T. pallidum.

Association of T. pallidum endoflagella with the cytoplasmiccylinder after TX-114 extraction. An immunoblot was pre-pared that contained samples of whole T. pallidum and thefractions derived from TX-114 extraction and phase parti-tioning. Figure 6 shows the results of probing the immuno-blot with antiflagellar serum at a 1:200 dilution. Endoflagellarbands were detected only in whole T. pallidum (Fig. 6, laneWH) and in the insoluble cold pellet (lane INSOL), whichcontainis the cytoplasmic cylinders. This result is consistentwith the electron micrograph presented in Fig. 2B, whichshowed that the periplasmic endoflagella were not releasedfrom the cytoplasmic cylinders by TX-114.

Fate of T. pallidum antigens in TX-114 extraction and phasepartitioning. The immunoblot of the TX-114-extracted T.pallidum fractions used to detect endoflagella (Fig. 6) wastreated with glycine hydrochloride (pH 2.5) to remove boundantibodies and reprobed with human syphilitic serum diluted1:100. Figure 7 shows that antibodies in syphilitic serumbound to all the major polypeptide constituents of theTX-114-extracted phase, the aqueous phase, the TX-114phase, and the warm pellet, identified in Fig. 4. In addition,several polypeptides, ranging in molecular mass from 33 to35 kDa, which partitioned into the TX-114 phase were veryantigenic relative to their molar representation in this frac-tion. Although this molecular mass range overlaps that of T.pallidum endoflagella, it is clear that these 33- to 35-kDapolypeptide antigens are distinct from the endoflagellarantigens which were shown not to be extracted by TX-114(Fig. 6).

AO TXSOL WP

WH INSOL SOL AG TXSOL WP

*sSw^ ^

FIG. 6. Fate of endoflagella in TX-114 extraction. An immuno-blot of the following TX-114 fractions derived from extraction andphase partitioning (Fig. 1) was probed with monospecific antiendo-flagellar antibodies. Lanes: WH, whole T. pallidum; INSOL, thecold pellet of cytoplasmic cylinders; SOL, TX-114-extracted poly-peptides; 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 andphase partitioning. The immunoblot described in the legend to Fig.6 was treated with glycine hydrochloride as described in the text toremove bound antibody and then reprobed with human syphiliticserum. Lanest WH, whole T. pallidum; INSOL, the cold pellet ofcytoplasmic cylinders; SOL, TX-114-extracted polypeptides; AQ,aqueous phase; TXSOL, TX-114 phase; WP, warm pellet. Molecu-lar mass markers are shown in kilodaltons.

500 nanomolarWH INSOL SOL

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(+50mmn DTT}WH SOL INSOL TXSOL WP AO

0

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FIG. 8. Fate of the 190- and 47-kDa antigens in TX-114 fraction-ation. 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; theright panel shows fractions derived from TX-114 extraction in thepresence of 50 mM DTT. Samples: lanes WH, whole T. pallidum;lanes SOL, the TX-114-extracted fraction; lanes INSOL, the coldpellet; lanes TXSOL, the TX-114 phase; lanes WP, the warm pellet;lanes AQ, the aqueous phase. Molecular mass markers are shown inkilodaltons.

Effect of disulfide bonding on the 190-kDa (4D) and 47-kDasurface antigens in TX-114 fractionation. Immunoblots were

prepared that contained samples of T. pallidum extractedwith TX-114 in the presence or absence of 50 mM DTT andfractionated by the phase partitioning procedure. Figure 8shows the results when the immunoblots were probed withaffinity-purified anti-4D antiserum (23) mixed with an anti-47-kDa monoclonal antibody (15). The 4D antigen, detectedas a 19-kDa monomer and 34-kDa dimer, remained with theinsoluble cytoplasmic cylinders (Fig. 8, left panel, laneINSOL) in the absence of DTT. However, when TX-114extraction was performed in the presence of DTT, 4D was

fully extracted (right panel, lane SOL) and subsequentlypartitioned into the aqueous phase (right panel, lane AQ).The amount of the 4D antigen, which is less than that ofother polypeptides extracted by TX-114 in these samples, isexpected, given our previous calculation that there is about2 ,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 panellanes SOL and INSOL), the addition of DTT to TX-114resulted in its complete extraction (right panel, lanes SOLand INSOL). The partitioning of the 47-kDa antigen into theTX-114 phase and warm pellet was not affected by thepresence of DTT. Extraction with TX-114 in the presence ofDTT did not otherwise alter the polypeptide composition or

antigenicity of the TX-114-extracted fraction or the integrityof the cytoplasmic membrane (data not shown).

DISCUSSION

T. pallidum is highly evolved for coexistence with itshosts. Perhaps reflecting this adaptation, certain surfaceproperties of the virulent organism are distinct from those ofother bacteria. It has been appreciated for three decades thatfreshly extracted intact treponemes are not reactive in

serological tests (11, 17). It has also been shown by immu-noelectron microscopy that the surface of the virulent or-ganism is resistant to the binding of antitreponemal antibod-ies in the absence of complement (13, 14, 25). The timerequired for in vitro complement-dependent serum bacteri-cidal reactions, 4 h or longer in the T. pallidum immobiliza-tion and in vitro-in vivo neutralization tests (2), is probablyalso indicative of this resistance to antibody binding. Furtherevidence demonstrating the unusual nature of the surface ofT. pallidum has been provided by Stamm and co-workers(28). Their attempts to immunoprecipitate surface proteinsidentified periplasmic flagella as being the most susceptibleto antibody binding; they also reported that the outer mem-brane could be removed by 0.04% SDS with the release onlyof periplasmic flagellar protein (28). Implicit in such obser-vations is the suggestion that the outer membrane is rela-tively devoid of protein. Other possibilities to explain theantigenic inertness of the surface and its resistance toradioiodination include a model in which the surface iscoated with host molecules lacking the substrates for rad-ioiodination and/or a model of outer membrane architecturewherein epitopes of its composite proteins are only tran-siently exposed owing to the motility of the organism andmembrane fluidity. In support of the idea that a fluid outermembrane allows at least transient surface exposure ofsubsurface structures, we have found that antibodies to T.pallidum endoflagella have treponemicidal activity (4).These unusual surface properties of living T. pallidum cellshave prompted the use of the term surface associated, ratherthan the term outer membrane, to describe the cellularlocation of proteins demonstrated on the surface of theorganism by immunoelectron microscopy under the condi-tions of the T. pallidum immobilization test (8, 25).

In this study we have used modified methods for thin-section electron microscopy to readily and consistentlydemonstrate the T. pallidum outer membrane. Our finding ofthe integrity of the outer membrane after Percoll purificationcorroborates our earlier report on the usefulness of thisprocedure for purifying virulent treponemes from host tissuecomponents (10). We have found that the nonionic detergentTX-114 removes the outer membrane without visually ap-parent damage to the lipid bilayer-type structure of thecytoplasmic membrane. Whole-mount electron microscopyof treponemes treated for 30 s with 1% TX-114 showed thatremoval of the outer membrane is preceded by an extensiveblebbing process followed by lysis of blebs in the detergentsolution. These results are in contrast to the well-describedsusceptibility of the cytoplasmic membrane of isolated Esch-erichia coli cell envelopes to nonionic detergent solubiliza-tion (27). In E. coli, covalent and strong noncovalent forceslink outer membrane proteins to peptidoglycan and thusstabilize 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 layerwhose appearance is thought to be consistent with that ofpeptidoglycan juxtaposed to the outer leaflet of the cytoplas-mic membrane (12). The stability of the cytoplasmic mem-brane of T. pallidum following TX-114 treatment may bebased on tight links to peptidoglycan or its equivalent, in amanner analagous to the association of the E. coli outermembrane with peptidoglycan, or on strong protein-proteininteractions. If treponemal peptidoglycan were tightly boundto the spirochetal outer membrane, movement mediated bythe periplasmic flagella might be impossible.

This study has demonstrated the utility of the endoflagellaand penicillin-binding proteins of T. pallidum in cell fraction-

(Non-reduced)WH SOL INSOL TX SOL WP AO

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ation studies. Penicillin-binding proteins known to be an-chored in the cytoplasmic membranes of many bacterialspecies (29) have been identified recently in T. pallidum (6).Our finding that T. pallidum penicillin-binding proteins re-main associated with the cytoplasmic cylinder followingTX-114 treatment provides additional evidence that TX-114treatment does not solubilize the T. pallidum cytoplasmicmembrane. We recognize the possibility that some cytoplas-mic membrane proteins are sensitive to TX-114 solubiliza-tion, but it seems improbable that there could be extensivesolubilization of cytoplasmic membrane protein withoutconcomitant disruption of phospholipid bilayer appearance.Schnaitman has shown that under conditions where TritonX-100 solubilizes the cytoplasmic membrane of E. coli cellenvelopes, one-third of the outer membrane phospholipid issolubilized without release of the outer membrane protein(27). Therefore, if the analogy between the stabilities of theE. coli outer membrane and -the T. pallidum cytoplasmicmembrane are valid, TX-114 would be more likely to solu-bilize phospholipid than protein components of the cytoplas-mic membrane.Although our results do not rigorously preclude the pos-

sibility that some of the periplasmic flagella are releasedfrom and cosediment with the cytoplasmic cylinders afterTX-114 treatment, our electron micrographs suggest thatflagella remain specifically associated, by one end, with thecytoplasmic cylinders. At present, however, we have noother means available for commenting on the fate of addi-tional periplasmic constituents during the removal of the T.pallidum outer membrane with TX-114, and we cannot ruleout the possibility that some of the proteins released by thedetergent are derived from the periplasm, not from the outermembrane.TX-114 treatment of T. pallidum consistently results in the

release of the same small set of polypeptides. Two of thesepolypeptides, the 47- and the 190-kDa (4D) polypeptides,have been studied extensively. Antibodies to the 4D orderedring (7) have treponemicidal activity in the T. pallidumimmobilization test (9), and 4D has been associated with thesurface of T. pallidum by immunoelectron microscopy (25).Rabbits immunized with recombinant 4D have manifested analtered course of lesion development, consistent with partialprotection in experimental syphilis (5a). The native structureof 4D in T. pallidum is that of a disulfide bond-linkedassembly of ordered rings too large to enter an SDS-polyacrylamide gel (23). Penn et al. reported that a 47-kDaprotein was released from T. pallidum as a result of treat-ment with Triton X-100 (19), and Jones et al. have identifieda murine monoclonal antibody that has activity in both the T.pallidum immobilization and in vitro-in vivo neutralizationtests and that recognizes a 47-kDa T. pallidum surface-associated protein (15). In this study we found that TX-114treatment did not release 4D from the cytoplasmic cylinderfraction unless a reducing agent was present. Using themonoclonal antibody of Jones et al. (15) as a probe, we foundthat full disassociation of the 47-kDa protein from thecytoplasmic cylinders occurred only with the addition of areducing agent. Factors such as covalent linkage of the 190-and 47-kDa proteins to a subsurface structure such aspeptidoglycan, very high molecular weight, and/or limitedsolubility of their native structures in TX-114 could explainthis observation.A 38-kDa antigen also was released by TX-114 treatment

and partitioned into the warm pellet of the hydrophobicphase with the 47-kDa antigen. The correspondence of the38-kDa molecule to a recombinant surface-associated 38-

kDa molecule which we have described previously (8) isunder investigation. Creation of the warm pellet provides asimple means for purification of the 38- and 47-kDa proteinsfrom T. pallidum. Polypeptides of 33 to 35 kDa partitionedinto the hydrophobic detergent phase and were reactive withsyphilitic serum. These antigens may have been previouslyunnoticed on one-dimensional SDS-polyacrylamide gel elec-trophoresis and immunoblotting because of the presence ofthe relatively abundant periplasmic flagellum proteins ofsimilar molecular masses.The phase partitioning possible with Triton X-114 has

proven useful in the isolation of certain membrane proteinsfrom eucaryotic cells (5) and, more recently, from mycoplas-mas (26). We have found, using cell envelopes of E. colitreated with EDTA and Triton X-114, that proteins whosemolecular masses corresponded to those of OmpA andOmpF/C were partially solubilized and were found in thehydrophobic phase after partitioning (T. M. Cunninghamand M. A. Lovett, unpublished observations). However, theappearance of a specific set of T. pallidum proteins in thehydrophobic TX-114 phase, including the surface-associated190- and 47-kDa proteins, although strongly suggestive, doesnot prove an outer membrane origin. Recently, Radolf et al.concluded on the basis of examination of whole-mountelectron micrographs of detergent-treated organisms thatTX-114 removed the T. pallidum outer membrane, and theyreported that the outer membrane could be released underconditions (0.1% TX-114) which released very little protein;organisms treated with 0.5% TX-114 were judged to haveremoval of large segments of the cytoplasmic membranesand extrusion of cytoplasmic contents (24). These resultswere thought to be consistent with the idea that the T.pallidum outer membrane is a protein-deficient phopholipidbilayer, 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 electronmicroscopy of thin sections has provided a reliable means ofjudging both outer membrane and cytoplasmic membraneintegrity; our results clearly show that 1% TX-114 does notdisrupt the appearance of the cytoplasmic membrane. Dem-onstration that T. pallidum penicillin-binding proteins arenot released by TX-114 corroborates this conclusion. Ourfinding that complete release of the 190- and 47-kDa proteinsby TX-114 requires treatment with a reducing agent suggeststhat these proteins are linked covalently to a structure whichis not solubilized by TX-114. Although it is possible that theproteins which partition into the hydrophobic TX-114 phase,including the very abundant 47-kDa protein, are largelyderived from the periplasmic space, it is more likely thatthey are constituents of the T. pallidum outer membrane buthave limited surface exposure. We believe that epitopes ofT. pallidum outer membrane proteins could be transientlyexposed to the surface during the bending and flexing thataccompany treponemal motility. The susceptibility of T.pallidum endoflagella to bactericidal antibodies (4) is inaccord with this hypothesis.

ACKNOWLEDGMENTS

This study was supported by Public Health Service researchgrants AI-21352 and AI-12601 from the National Institute for Allergyand 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 forexpert technical assistance. We thank Michael Norgard, Universityof Texas Health Science Center, Dallas, for providing monoclonalantibody to the 47-kDa protein.

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5796 CUNNINGHAM ET AL.

ADDENDUM IN PROOF

We have completed freeze-fracture studies of the outermembrane of T. pallidum which are relevant to understand-ing the origin of the polypeptides released by TX-114. Theparticle density per square micrometer of the E face of thismembrane is 70, and that of its P face is 100. Assuming thateach particle represents a protein complex, in contrast thecorresponding protein densities for the E and P faces of theE. coli outer membrane are 6,000 to 10,000 and 500 to 700,respectively. This strikingly low protein composition of theouter membrane of T. pallidum indicates that while themolecules released by TX-114 must include the outer mem-brane protein, most if not all the polypeptides we identifiedare probably derived from the periplasmic space or thecytoplasmic membrane. The low protein composition of itsouter membrane may explain the relative antigenic inertnessof the surface of virulent T. pallidum.

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18. Norgard, M. V., N. R. Chamberlain, M. A. Swancutt, and M. S.Goldberg. 1986. Cloning and expression of the major 47-kilo-dalton (kDa) surface immunogen of Treponema pallidum inEscherichia coli. Infect. Immun. 54:500-506.

19. Penn, C. W., A. Cockayne, and M. J. Bailey. 1985. The outermembrane of Treponema pallidum: biological significance andbiochemical properties. J. Gen Microbiol. 131:2349-2357.

20. Penn, C. W., and J. G. Rhodes. 1982. Surface-associatedantigens of Treponema pallidum concealed by an inert outerlayer. Immunology 46:9-16.

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22. Radolf, J. D., D. R. Blanco, J. N. Miller, and M. A. Lovett. 1986.Antigenic relationship between endoflagella of Treponema pha-gedenis biotype Reiter and Treponema pallidum (Nichols). II.Molecular characterization of endoflagellar proteins. Infect.Immun. 54:626-634.

23. Radolf, J. D., L. A. Borenstein, J. A. Kim, T. E. Fehniger, andM. A. Lovett. 1987. Role of disulfide bonds in the oligomericstructure and protease resistance of recombinant and nativeTreponema pallidum surface antigen 4D. J. Bacteriol. 169:1365-1371.

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25. Radolf, J. D., T. E. Fehniger, F. Silverblatt, J. N. Miller, andM. A. Lovett. 1986. The surface of virulent Treponema pallidumresistance to antibody binding in the absence of complementand surface association of recombinant 4D antigen. Infect.Immun. 52:579-585.

26. Riethman, H. C., M. J. Boyer, and K. S. Wise. 1987. TritonX-114 phase fractionation of an integral membrane surfaceprotein mediating monoclonal antibody killing of Mycoplasmahyorhinis. Infect. Immun. 55:1094-1100.

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