Studies on the chemical composition and immunologic properties of a polysaccharide from the reiter treponeme

Iraraunochemistry. Pergamon Press 1966. Vol. 3, pp. 233-245. Printed in Great Britain

STUDIES ON THE CHEMICAL COMPOSITION AND IMMUNOLOGIC PROPERTIES OF A POLYSACCHARIDE FROM THE REITER TREPONEME E. ELLEN NELL and PAUL H. HARDY Department of Microbiology, T h e Johns Hopkins University, School of Medicine, Baltimore, Maryland

(Received 13 September 1965) Abstract--A polysaccharide was isolated from the Reiter strain of Treponeraa pallidum by fractionation of an alkaline digest of treponeme ghosts. Chemical analysis showed that the polysaccharide was composed of simple sugars, amino sugars and a small amount of amino acid residues. Immunologic homogeneity was established by precipitin and complement fixation tests, gel diffusion and immunoelectrophoresis." Three Kazan strains of T. pallidura, two oral treponemes and one strain of Borrelia were found to possess an identical or serologically indistinguishable antigen. Antisera to the Noguchi and apathogenic Nichols strains, as well as immune sera to the pathogenic Nichols strain, were non-reactive with the Reiter polysaccharide.

INTRODUCTION STUDIm on the immunology of treponemes have long been motivated by the need for better methods of diagnosing syphilis and other spirochetal infections. However, direct investigation of the immunologically active components of pathogenic treponemes has been hampered by the inability to cultivate these organisms in vitro. Some investigators~1~ have attempted to circumvent this difficulty by substituting cultivable, non-pathogenic spirochetes, especially those strains allegedly isolated from syphilitic lesions ~4~and, therefore, presumed to be avirulent strains of Tmponema pallidum. Early studies with these organisms were disappointing, however, and interest in this approach waned until D'Alessandro, Oddo, Comes and Dardanoni ~5~ reported that a protein component of the Reiter treponeme was reactive in serologic tests with serum from syphilitic individuals. This observation, which has been substantiated repeatedly, stimulated a renewed interest in immunochemical studies of cultivable spirochetes, the Reiter treponeme in particular. The potential diagnostic value of the protein antigen has made it the component of primary interest in the Reiter treponeme. Less attention has been given to a polysaccharide, another constituent of this and other spirochetes, which also may have considerable serologic significance. The presence of such an antigen in the Reiter treponeme was first reported by D'Alessandro et aL in 1949~5~ and since that time studies concerned with this antigenic component have been described by other investigators/6-10~ Several have claimed that the polysaccharide, as well as the protein antigen, is reactive with syphilitic sera,~6,81 but others have failed to confirm this observation.~9,1~,1~ D'Alessandro, Zaffiro, Fichera and di Chiara ~ls~ are of the opinion that the polysaccharide and its corresponding antibody comprise the reactants responsible for immune treponemal immobilization, and it has been postulated a4~ that this antigen may possess species specificity. 233

234

E. ELLEN NELL and PAUL H. HARDY

The aforementioned studies suggest that the spirochetal polysaccharide may provide a basis for serological classification of this poorly studied group of microorganisms. In addition, such studies may eventually offer a means for determining which, if any, of the cultivable spirochetes are really avirulent forms of T. pallidum. In some of the previously reported studies on the Reiter treponeme polysaccharide the product under investigation does not appear to have been critically evaluated with respect to immunologic purity. This may explain some of the contradictory findings that have been observed. Therefore, the present investigation was initiated in an attempt to obtain a serologically homogeneous polysaccharide which might contribute further to the evaluation of this spirochetal component. A new method for extraction and purification of the polysaccharide is described, together with observations on its chemical composition and its immunologic properties. MATERIALS AND M E T H O D S

Growth of the Reiter treponeme Cultures were grown in banks of Florence flasks (6 1. capacity each) filled to the neck with USP alternate thioglycollate broth* containing 10% inactivated normal calf serum. Each flask was inoculated with approximately 5 ml of an actively growing culture, tightly stoppered and placed over a magnetic mixer to ensure continuous agitation during 4 days' incubation at 35 °. The organisms were collected by centrifugation at 22,000 g in a Spinco Model K continuous flow centrifuge and subsequently washed three times with physiologic saline. The washed, packed cells were stored at --20 ° until used for fractionation. Treponeme yields averaged 1.6 g wet weight, or 0.2 g dried weight, per 1.

Physical and chemical analyses Ultraviolet absorption spectra were determined in a Beckman DU spectrophotometer and analytical centrifugation was performed in a Spinco Model E ultracentrifuge. Total nitrogen was determined by the micro-Kjeldahl method and protein by the Lowry modification of the Folin-Ciocalteu procedure. ~15~ Phosphorus was estimated by the method of Fiske and Subbarow. ~161 The general reaction with cysteine and H2SO4 (17~ was used as a preliminary test for detecting the presence of various classes of sugars. Carbohydrates, exclusive of amino sugars, were estimated by the orcinol-HeSOa (16~ and indol reactions, (19~ hexoses by the anthrone 120~ and carbazole (z~ reactions, ketoses by the diphenylamine reaction (~9~ and hexosamines by the method of Dische and Borenfreund. (z~) Carbazole (za~ and naphthoresorcino1124) methods were used to determine uronic acids and the procedure described by Weissbach and Hurwitz (zSI for sialic acid. Glucostat and galactostat, Worthington Biochemical Corp., were utilized for the quantitative determination of glucose and galactose, respectively.

Hydrolysis of sample A 10 mg portion of the polysaccharide in 1 ml 0.5 N H~SOa was hydrolysed in sealed ampoules at 100 ° for 6 hr. After heating, the sample was neutralized with * Sterility Test Medium (BBL).

Reiter Treponeme Polysaccharide

235

solid BaCO3 and the precipitate removed by centrifugation. Increasing the time of heating, or the use of 2 N H2SO4, did not result in further separation of amino acids or amino sugars. Hydrolysis of the polysaccharide with 6 N HC1 was attempted but caramelization of the sugars rendered the sample unsatisfactory for chromatography.

Paper and thin-layer chromatography Paper chromatograms were prepared on Whatman No. 1 filter paper and developed in an ascending chamber. Phenol-H20 (3:1) and n-butanol-H~Oacetic acid (4:3: 1) gave good separation of sugars. The chromatographic solvent used for amino acids was n-butanol-H20-pyridine-acetic acid (5:4: 3:1). Sugars were stained with p-anisidine and amino acids with ninhydrin. Thin-layer chromatography was carried out according to the method of Stahl as described by Randerath.126~ Sugars were separated on cellulose (Zellulosepulver, Camag) or Silica Gel G (Brinkman) in (1)n-butanol-pyridine-H20 (6:4:3), and (2) phenol-water (3: 1). The spots were detected by alkaline AgNO~ ~7~ on both media, and also by anisaldehyde-H~SO4 {2s~ on Silica Gel G. Amino acids were chromatographed on Silica Gel G in the above solvent systems, and also in chloroform-methanol-17% NH3 (2:2:1). Spots were revealed by Moffat and Lytle's ~9~ polychromatic modification of ninydrin reagent. Amino sugars were recognized by reactivity with the nin~drin and AgNO~ reagents and failure to react with anisaldehyde-H~SO4.

Sephadex chromatography A 10 mg portion of the polysaccharide, in 2 ml of saline, was chromatographed on a Sephadex G-100 column, 2.5 ×45 cm. The procedure was carried out at 4 ° and 5 ml samples were collected. Polysaccharide was measured by the orcinolH~SO4 method.

Serologic analyses Quantitative precipitin tests were performed accordin.g to the method developed by Heidelberger and associates as described in Kabat and Mayer. I~0) Complement fixation reactions were carried out by the method of Osler, Strauss and Mayer. ~31~ Double immunodiffusion precipitin studies in agar gel were performed according to the technique of Feinberg 1~2}and for comparison of various sera the immunoplate of Hyland Laboratories was utilized. Immunoelectrophoresis of 1 mg antigen/ml was carried out in 0.5 # barbiturate-acetate-HC1 buffer, pH 8.6, for 2 hr at a potential of 40 V; precipitin lines were developed at room temperature for 24 hr.

Preparation of immune sera Antisera to the following strains of cultured treponemes were used in the present study: Reiter, Nichols, Noguchi, Kazan A, Kazan 2, Kazan 5, FM, N9 and PKOT. The origin, description and medium for optimum growth of these strains have been described previously.~ Organisms to be used for immunization were grown in the appropriate medium enriched with normal rabbit serum. Treponemes from full grown cultures were collected, washed free of culture medium and suspended to a concentration of

236

E. E,J~'~ NELL and PAUL H. HARDY

10t0 organisms/ml in 0.15 M saline containing 0"005 M MgCI2. After pre-immunization testing to ensure absence of treponemal antibodies, rabbits were injected intravenously on 4 consecutive days per week for 3 weeks, rested for 2 weeks, then subjected to a second 3 weeks' course of immunization. The dosage was 0.5 ml per injection the first week and was increased by this amount each succeeding week. Providing sample bleedings showed adequate immunization, the rabbits were bled by cardiac puncture and the sera from animals immunized with the same antigen were pooled. All serum pools had a macroscopic agglutination titer of 1/1280 or above when tested with the homologous organism. RESULTS The results presented in this communication are part of an investigation undertaken to develop methods for the isolation and purification of the major antigenic components of anaerobic spirochetes. In preliminary experiments, emphasis was placed upon recovery of the protein antigen described by D'Alessandro, Oddo, Comes and Dardanoni)5~ This antigen was found to be a cytoplasmic constituent of treponemes, and therefore could be extracted only by cellular disruption. However, because of the unusual physical dimensions and marked plasticity of spirochetes, most accepted methods for rupturing bacteria proved inefficient with these organisms. Best results were achieved by means of lysis by alkaline shock. A thick suspension of washed treponemes in a small volume of cold distilled water (equivalent to 60 mg dry weight per ml) was placed in an icebath and while stirring vigorously, N/100 NaOH was added until pH 11.6 was reached. Stirring was continued for an additional 5-10 min after which all the organisms were uniformly dispersed and lysed, as determined microscopically. The suspension was then neutralized with N/100 HC1 and the treponeme ghosts removed by centrifugation. Almost all of the protein antigen, together with the bulk of the nucleic acids to which it was attached, was recovered in the lysate. The major portion of the other antigenic constituents, including the polysaccharide, remained associated with the ghosts..

Isolation and purification of the polysaccharide Mild extraction procedures failed to solubilize the treponemal polysaccharide and therefore more severe treatment could not be avoided. Thus, the ghosts, were subjected to alkaline hydrolysis in a fashion similar to the procedure described by Meyer and Hahnel ~4~ for the isolation of a mucopo.lysaccharide from gram positive organisms. A diagramatic flow sheet is presented in Fig. 1 and shows the steps in isolation and purification of one polysaccharide preparation, 60-2D2. Following the same scheme, a second batch was made, 65-D~, which was comparable in every respect to the first. Washed lysed cells were suspended in 0.5 N NaOH, stirred until the suspension was homogenous and then stored under an atmosphere of nitrogen at room temperature for 4 days. After digestion, microscopic examination revealed no intact spirochetal forms. Particulate matter consisted of occasional fine thread-like fibers and a few hexagonal and needle shaped crystals. The latter increased many fold following neutralization of the extraction mixture with glacial acetic acid and overnight storage in the cold; they were removed by centrifugation. Proteins were

Reiter Treponeme Polysaccharide

237

then precipitated in the cold by addition of glacial acetic acid to pH 4.5. The clear, pale yellow supernatant fluid contained the polysaccharide. Addition of 4 volumes of cold ethyl alcohol resulted in the appearance of a fine white precipitate which, after standing overnight in the cold, was easily packed by centrifugation. The precipitate was washed twice with alcohol and then dissolved in a small volume of distilled water. Residual proteins were removed from the polysaccharide solution by extraction with aqueous phenol.tas~ Enough 89.3 % recrystallized phenol was added to give a final concentration of 50 per cent, and the mixture was shaken vigorously for 2 hr. After centrifugation the upper aqueous phase was removed and the inter-phase cake re-extracted once with a small volume of water. The aqueous layers were pooled and dialysed against distilled water. For every 100 ml, 10 g of sodium acetate and ~6 g Treponeme$-~et we~g~t

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FI~. 1. Flow diagram giving the steps in preparation of Reiter polysaccharide.

1 ml of acetic acid were added and the polysaccharide reprecipitated with 1.5 volumes of ethyl alcohol. When this precipitate was taken up in distilled water, a small amount of insoluble material remained which was removed by filtration through a medium sintered glass filter. The water soluble fraction was again precipitated with alcohol as before, packed and washed successively with alcohol, acetone and diethyl ether. The final product was dried to constant weight in vacuo over P205. Recovery of polysaccharide represented 2.95 per cent of the total calculated treponemal dry weight. Physical and chemical properties The dried material was a white powder that dissolved readily in water and physiologic saline, yielding a water clear solution. Ultraviolet absorption spectra of a 1.5% aqueous solution, over wave lengths of 220-330 m/~, showed no absorption maxima corresponding to nucleic acids or aromatic amino acids. Sedimentation

238

E. ELLEN NELL and PAUL H. HARDY

studies in t h e ultracentrifuge revealed a single peak which, however, was not entirely symmetrical. A trailing edge indicated the presence of a smaller component along with the major constituent, the molecular weight of which was estimated to be about 30,000. Since a shoulder did not develop with time in the ultracentrifugal analysis it seemed probable that smaller fragments of the antigen were responsible for the trailing. This interpretation was confirmed by chromatography on Sephadex (G-100) where two distinct carbohydrate fractions of like chemical composition and immunologic specificity were separated. These data are presented below. Chemical analyses were carried out on the unfractionated polysaccharide. The P content was found to be 2.5 per cent, all organically bound; however, identification of the P containing components was never achieved. No protein could be detected by the Folin-Ciocalteu procedure, but, as indicated in Table 1, total N determinations revealed a content of 4 per cent. Amino sugars, expressed as glucosamine, amounted to 27 per cent of the antigen but accounted for only half of the N, suggesting the presence of non-carbohydrate material, presumably amino acids. TABLE 1. CHEMICALCOMPOSITIONOF REITERPOLYSACCHARIDE Component Nitrogen Phosphorus Total carbohydrates (excluding hexosamines) Hexosamine Amino acids Total

Per cent of total weight 4.0 2.5 42-64" 27

12.5 c 84-106

Compounds identified A m i n o sugars a n d a m i n o acids .

.

.

Glucose (16.5%), galactose (26%), rhamnose, hydroxymethyl furfural Glucosamine, galactosamineb Alanine, lysine, glycinea

a Results vary with method employed. b One additional compound reactive with ninhydrin and AgNOa. c Calculated from total N less hexosamine N. a Three additional unidentified compounds reactive with ninhydrin. Exclusive of amino sugars, total carbohydrate calculated as glucose, accounted for 42 per cent of the total weight by the indol reaction and 64 per cent by the orcinol reaction. Conventional methods for detecting various classes of sugars indicated the polysaccharide contained hexoses and methyl pentose. Initial ketose determinations on the intact polysaccharide were negative. However, following the chromatographic identification of hydroxymethyl furfural in a hydrolysed sample, a similar sample was analysed for ketose and a value of 18.5 per cent, calculated as fructose, was obtained. It may be noted that Fichera, Del Carpio and Di Chiara t0~ also reported the presence of ketose in a hydrolysed sample of their preparation of Reiter treponeme polysaccharide. Heptoses, 2-deoxypentoses, uronic acids or sialic acids were not present. Of the several sugars identified chromatographically, glucose and galactose could be quantitated accurately. The former was found to comprise 16.5 per cent and the latter 25 per cent of a hydrolysed sample of the polysaccharide.

Reiter Treponeme

239

Polysaccharide

In preliminary studies, the individual components in hydrolysates of the polysaccharide were separated by paper chromatography. Thereafter, thin-layer chromatography was used since considerably smaller samples were required. Hydroxymethyl furfural was the only component of the polysaccharide identified on paper which did not appear on the thin layer chromatograms. The sugars identified by thin-layer chromatography were easily detectable in 10-20/zg samples, whereas ten times this quantity was required to detect the amino acids. This confirmed the impression that the latter were present in low concentration, calculated to be about 12.5 per cent of the total weight. In all solvents used, three components which migrated with glucose, galactose and rhamnose were readily identified (Fig. 2). In addition, two amino sugars migrating in the same position as glucosamine and galaetosamine were found (Fig. 3). The presence of alanine, lysine and glycine was established (Fig. 3), and four additional ninhydrin reacting compounds were detectable, none of which moved with any of the common amino acids. One of these reacted with AgNOs, as well as ninhydrin and therefore, was presumed to be an amino sugar. The possibility that it might be muramic acid was considered, but could not be confirmed. Inadequate hydrolysis, resulting in incomplete breakdown of peptide chains, could aacount for the other unidentified amino compounds, but attempts to establish this by additional hydrolysis of the polysaccharide were without success.

Immunologicproperties Figure 4 shows the curve obtained in a quantitative precipitin analysis of the polysaccharide with antiserum to the Reiter treponeme. Increasing quantities of antigen were added to ml aliquots of the antiserum; after 3 days in the cold, the .~50 -.

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FIG. 4. Quantitative analysis of antibody nitrogen precipitated from 1 ml rabbit anti-Reiter serum with increasing amounts of polysaccharide. Reaction mixtures kept 3 days at 4°. precipitates were packed, washed and the N content determined. Antibody N was calculated as total N precipitated less that contributed by the polysaccharide. The shape of the curve was similar to those of other polysaccharide antigen-antibody systems. Examination of the supernatants for presence of excess antibody or antigen, revealed the absence of both in the tube that received 200/~g of polyo saccharide. Supernatants from tubes where less polysaccharide was added all

2~0

E. ELLI~ NELL and PAUL H. HARDY

contained excess antibody, whereas excess antigen was demonstrable where antigen exceeding 200 t~g was added. Thus, the precipitin analysis indicated that the polysaccharide behaved as a single antigen. T h e complement fixing activity of the polysaccharide was assayed in a checkerboard block titration with antiserum to the whole organisms. T h e results are presented in Table 2. Optimal antigen activity was obtained at a concentration of 0.02 t~g per test mixture and, as typical of single antigen-antibody systems, little fixation of C' occurred below the maximum activity concentration. TABLE 2. TWO-DIMENSIONAL COMPLEMENT FIXATION TEST WITH REITER POLYSACCHARIDE ANTIGEN AND ANTI-REITER SERUM

Serum dilution a Antigen (#g) ~ 2.5 1.25 0.625 0.312 0.156 0.078 0.039 0.02 0.01 0.005 Buffer control

160 320 640 0 0 0 0 0 0 0 0 0 0 3 4

0 0 0 0 0 0 0 0 0 0 4 4

a Expressed as reciprocal Serum---0.2 ml C'--5 C'H50/0.25 ml Antigen--0.2 ml

1 0 0 0 0 0 0 0 0 0 4 4

1280 2560 4 2 0 0 0 0 0 0 0 1 4 4

4 4 4 1 0 0 0 0 0 2 4 4

5120

10,240

20,480

Buffer control

4 4 4 4 4 2 0 0 0 3 4 4

4 4 4 4 4 4 4 3 2 4 4 4

4 4 4 4 4 4 4 4 4 4 4 4

4 4 4 4 4 4 4 4 4 4 4 4

Fixation--18 hr at 4 ° Sensitized cells---0.1 ml Lysis--60 min at 37 ° 0--No lysis 4--Complete lysis

Further indication that the polysaccharide was a single reacting antigen was obtained by double diffusion in gel where the antigen precipitated as a single line with Reiter immune serum (Fig. 5). In immunoelectrophoretic studies as well, o n l y one band appeared with the developing antiserum (Fig. 6). T h e antigen moved toward the cathode.

Cross reactions between Reiter polysaccharide fraction and other treponemal immune sera Using the optimum concentration of antigen as determined in the homologous system, antisera against nine other strains of treponemes were examined for complement fixing antibody to the Reiter polysaeeharide. Table 3 shows that antisera to FM, N9, P K O T and the three Kazan strains contained such antibody. However, antisera to the non-pathogenic Nichols and Noguchi strains, as well as immune serum from rabbits infected with the pathogenic Nichols strain, were devoid of complement fixing antibodies to this antigen. Since it is generally accepted that the coalescence of bands of precipitate between two sera and one antigen in double diffusion in gel represent identical antibodies, this technique was used to determine whether the antibodies in the various sera were identical, or of a cross-reacting nature. When Reiter polysaceharide antigen

10,240

Reiter

5120

5120

Kazan 2 4000

PKOT 2560

N9 1280

FM

0 Serum dilution expressed as reciprocal (antigen concentration = 0.02 gg per test mixture).

10,240

Kazan 5

<40

Nichols"

<40

Nogue'hi"

F I X A T I O N TITERS OF REITER POLYSACCHARIDE A N T I G E N W I T H VARIOUS SPIROCHETAL ANTISERA

Kazan A

COMPLEMENT

a Serum anticomplementary in lower dilution.

Titer~

Antiserum

TABLE 3.

<40

Path~ Nichols

o

~o

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~o

242

E. ELLEN NELL and PAUL H. HAND?

was placed in the center reservoir (500/~g/ml) and different treponemal antisera in the surrounding reservoirs, a line of precipitate formed between the antigen and each of the antisera that had reacted in the complement fixation tests (Fig. 7). Moreover, the different precipitin lines fused both with that formed by the Reiter immune serum and with one another. It was concluded, therefore, that the following strains of treponemes contain a common polysaccharide antigen: Reiter, Kazan A, Kazan 2, Kazan 4, Ng, PKOT and FM. The Noguchi and cultivable Nichols strain, as well as pathogenic T. pallidum must contain polysaccharides of a different composition.

Sephadex chromatography When the polysaccharide was passed through a Sephadex G-100 column two discrete peaks of carbohydrate content were obtained. The sample at the top of each peak, designated S16 (first peak) and S27 (second peak), was analysed chemically and immunologically. Total carbohydrate of the two fractions was 117 y/ml and 72 y/ml, respectively. Assuming total carbohydrate to be 64 per cent of the polysaccharide, as in the unfractionated material, calculated values of polysaccharide in the 516 fraction was 183 7/ml and of the $27 fraction, 112 7/ml. Accurate values, as determined from dry weight, could not be obtained with the limited quantities. Aliquots of the two fractions were dialysed against distilled H20 and hydrolysed. The samples were analysed for glucose and galactose content and the values obtained were in accord with those found in the unfractionated material. Thin layer chromatography revealed that both S16 and $27 contained glucose, galactose and rhamnose, present in the unfractionated polysaccharide (Fig. 2). The amino sugars, glucosamine and galactosamine, were also identified. However, insufficient quantity prevented adequate concentration to determine if the amino acids were present in one or both fractions. Capillary precipitin tests showed that both fractions possessed the same immunologic specificity as the unfractionated material. Sera which reacted with the complete antigen gave a positive precipitin reaction with S16 and $27. Conversely, sera which failed to precipitate with the unfractionated antigen were also non-reactive with both fractions. Analysis by double diffusion in gel showed fusion of lines with the complete antigen. In addition, both fractions were capable of fixing complement in the presence of anti-Reiter serum, $27 giving a 2-fold lesser serum titer than the larger molecular weight material, S16. DISCUSSION The data presented extend the findings of previous investigators 15-ta~ who have extracted an antigen of a polysaccharide nature from the Reiter strain of Treponema pallidum. The isolation and purification of a polysaccharide fraction of high serologic activity has been accomplished by alkaline digestion of the treponeme ghosts. This material differs from the preparations previously described in that it has been shown to consist of a single reacting antigen, free from contaminating proteins or nucleo-proteins. Before meaningful antigenic studies and chemical analyses can be carried out, chemical and immunologic homogeneity must be established. A number of

Reiter Treponeme Polysaccharide

243

approaches were considered in exploring the homogeneity of the polysaccharide antigen isolated in the present study. By gel diffusion, immunoelectrophoresis, complement fixation and precipitin tests with antiserum prepared against the homologous whole organism, the antigen proved to be serologically homogeneous. Although the antigen was separated into two fractions of different molecular weight, both fractions possessed the same immunologic specificity and the same chemical composition, as far as could be determined. Therefore, partial breakdown of the macromolecule was responsible for the different particle sizes, rather than the presence of an unlike substance. As spirochetal antigens of known purity are isolated, a classification of these organisms will eventually evolve. The Reiter polysaccharide is one such antigen which may be used to study strain differences and similarities. The purified polysaccharide did not react with antisera to the Noguchi and apathogenic Nichols strains. Serum from rabbits infected with the pathogenic Nichols strain were also non-reactive. It is likely that protein or nucleoprotein impurities were responsible for the positive reactions of some syphilitic sera with the lipopolysaccharides described by D'Alessandro and Del Carpio ~6~ and by De Bruijn, ~8~ as previously suggested by Christiansen. 17) In contrast, the reactivity of the presently described antigen with antisera to three Kazan strains, two oral treponemes and one strain of Borrelia indicate that these spirochetes contain the same polysaccharide antigen as Rieter. It is interesting to note that previous investigators have found essentially the same strain relationships by other methods. Utilizing whole organisms in agglutination and complement fixation reactions, Eagle and Germuth la6~ placed Reiter, Kazan and two saprophytic mouth treponemes into one serologic group, and the Noguchi and non-pathogenic Nichols into another group. Similar serologic strain relationships were reported by Robinson and Wichelhausen ~aT~ who employed a formamide extracted spirochetal antigen in precipitin tests. Whether different polysaccharides will prove to be unique to groups of spirochetes and be useful as a means of classification can only be determined by isolation and immunologic studies of such antigens from other strains. The specificity of the Reiter polysaccharide and the demonstration by De Bruijn ~la~ that Treponerna zuelzerae contains yet another specific carbohydrate antigen warrants pursuit of these investigations. Furthermore, the participation of a polysaccharide antigen in the immobilization of the Reiter treponeme by immune sera as shown by D'Alessandro et aL ~ suggests that a specific diagnostic test for treponemal diseases may be attained when it is possible to isolate such an antigen from the pathogenic organisms. In addition to the practical significance of antigenic studies of spirochetes, the characterization of the major constituents may also lead to a better concept of the structure of these organisms. The non-rigidity of the spirochetes sets them apart from bacteria with rigid cell walls and it has been assumed that they lack a structure of this nature. However, the recent demonstration by Ginger 13sl that muramic acid is present in two members of the Spirochaetales, Leptospira and Borrelia, indicates that this group of organisms may not be devoid of a cell wall-like substance. The structure containing muramic acid was not located, but if the morphology of the spirochete is maintained by a double 'membrane' with cell wall and cell membrane

244

E. ELLEN N~LL and PAUL H. I-I.~vY

features surrounding the protoplasmic cylinder as suggested for Treponema microdentium~} it is not unreasonable to predict that constituents similar to those of the bacterial cell wall, if present, will be found in this portion of the organism. T h e polysaccharide was isolated from treponeme ghosts and this, together with the results of a chemical analysis supports the hypothesis that material comparable to bacterial cell wall may reside in the inner skeletal portion of these organisms. T h e major constituents of the mater~al were found to be glucose, galactose, rhamnose, glucosamine, galactosamine and an unidentified amino sugar. In addition, a limited number of amino acids were found, with the identification of alanine, lysine and glycine. These results are reminiscent of cell wall composition of some gram positive bacteria. {40~ However, muramic acid as well as glutamic acid, invariably present in gram positive bacteria, could not be detected. T h e presence of 2.5 per cent organically bound P in the Reiter treponeme polysaccharide is significant and is greater than that generally found in cell walls of gram positive bacteria. Attempts to identify any P containing compound were without success. From the results of the present study, therefore, it is impossible to say whether the polysaecharide does have structural significance in the treponeme. More gentle extraction methods that will yield undegraded cellular components for study must be found before this question will be resolved. Acknowledgement--This work was supported by Public Health Service Research Grant

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