Vol. 24, No. 1
INFECTION AND IMMUNITY, Apr. 1979, p. 244-251 0019-9567/79/04-0244/08$02.00/0
Surface Mucopolysaccharides of Treponema pallidum T. J. FITZGERALD* AND R. C. JOHNSON Department of Microbiology, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455
Received for publication 26 December 1978
The viscous mucoid fluid that accumulates within syphilitic lesions may be due to breakdown of host tissue during infection, or may be synthesized by Treponema pallidum. Experiments were performed to investigate the acidic mucopolysaccharides that occur at the surface of T. pallidum (Nichols strain). These mucopolysaccharides were demonstrated by reaction with acidified bovine serum albumin and by agglutination with wheat germ agglutinin and soybean agglutinin. The polycations ruthenium red and toluidine blue influenced treponemal survival. Concentrations of both compounds at 200 tg/ml inhibited survival, whereas concentrations at 0.1 /Lg/ml enhanced survival. The mucopolysaccharide concentration within the mucoid fluid that accumulates during intratesticular infection was determined by reaction with acidified bovine serum albumin; it ranged from 10,000 jig/ml to less than 8 jtg/ml. The addition of this mucoid fluid to treponemal suspensions resulted in differing effects on T. pallidum survival. Some preparations were inhibitory, and others were stimulatory. Commercial preparations of hyaluronic acid and chondroitin sulfate at 400, 200, 100, and 50 ytg/ml were detrimental to treponemal survival. The organisms exhibited pronounced clumping in the presence of the higher concentrations of hyaluronic acid. These clumps of treponemes were comprised of mucopolysaccharides as shown by acidified bovine serum albumin and toluidine blue reactions and by hyaluronidase degradation. Results are discussed in terms of the derivation and potential role of acidic mucopolysaccharides at the surface of T. pallidum.
Mucoid fluid accumulates within dermal and testicular lesions produced by Treponema pallidum (1, 9, 13-15, 25-27, 29, 31-35). This material is partially comprised of the acidic mucopolysaccharides hyaluronic acid and chondroitin sulfate (9, 26, 27, 31-34). In certain diagnostic tests for syphilis, T. pallidum exhibits a relatively slow serological reactivity. It has been suggested that the organisms have an outer surface layer that has to dissipate prior to seroreactivity (8, 17, 22, 23, 31, 34). Zeigler et al. (36) and Fitzgerald et al. (10) demonstrated a surface layer on T. pallidum (Nichols strain) that reacted with ruthenium red. This suggested that the nature of this material was acidic mucopolysaccharide (20). It is important to characterize the derivation of viscous mucoid fluid within lesions since it appears to be directly related to virulence. Turner (31) and Turner and Hollander (34) observed that infections with more virulent strains of T. pallidum were associated with increased accumulations of mucoid material. In addition, Fitzgerald and Johnson (Br. J. Vener. Dis., in press) have found that this mucoid material exhibited immunosuppressive activity. The purpose of this report was to further elaborate on the surface mucopolysaccharide of T. pallidum.
MATERIALS AND METHODS T. pallidumL The Nichols strain of T. pallidum
was maintained by intratesticular passage in healthy Dutch Belt rabbits weighing 4 to 5 pounds (ca. 1.8 to 2.3 kg). The animals were housed at 19 to 22°C and given antibiotic-free food and water ad libitum. Approximately 1 x 107 to 3 x 107 treponemes were inoculated per testis. Daily intramuscular injections of cortisone acetate (Merck, Sharpe, and Dohme, West Point, Pa.) at 6 mg/kg of body weght were initiated 3 days postinoculation. After 9 to 13 days, the animals were sacrificed by intracardial injection of sodium pentabarbitol, and the testes were removed and placed in physiological saline. The testes were sliced and extracted aerobically for 20 to 30 min in tissue culture medium containing Eagle minimal essential medium supplemented with 4 mM NaHCO3, 10% (vol/vol) fetal bovine serum, 1 mM dithiothreitol, 4 mM glutathione, and 1 mM cysteine. This medium was buffered with 30 mM HEPES (N-2-hydroxyethyl piperazine-N'-2ethanesulfonic acid) at pH 7.2. After extraction, the treponemal suspension was centrifuged at 1,000 x g for 7 min to sediment particulate matter. For testing the influences of various compounds, suspensions were adjusted to 2 x 107 to 5 x 107 treponemes per ml and placed into test tubes with loose-fitting caps or Sykes-Moore chambers (30). Incubation was performed at 30°C in desiccator jars that were evacuated and flushed five times with 2.5% oxygen-92.5% nitrogen-5% carbon dioxide. 244
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SURFACE MUCOPOLYSACCHARIDES OF T. PALLIDUM
Photography. Treponemes were placed in SykesMoore chambers. After reaction with acidified bovine serum albumin (acid BSA) or plant lectins, 3% glutaraldehyde-1% TAPO (tris-1-aziridenyl phosphine oxide) in physiological saline was added for 30 min. The fixative was removed, and the treponemes were photographed using a Leitz phase-contrast microscope with an attached Leitz automatic exposure timing device. The film used was Kodak Tri-X pan for black and white prints. Chemicals. Crystalline type IV bovine testicular hyaluronidase, hyaluronic acid from human umbilical cord, chondroitin sulfate from whale and shark cartilage, soybean agglutinin, wheat germ agglutinin, and ruthenium red were obtained from Sigma (St. Louis, Mo.). Crystalline toluidine blue was obtained from Pharmaceutical Laboratories (New York, N.Y.).
RESULTS Acid BSA reacts with acidic mucopolysaccharides by producing a precipitate (16, 18). This procedure was adapted to T. pallidum to demonstrate the acidic mucopolysaccharides on the treponemal surface. One drop of treponemal suspension was added to 1 drop of acid BSA. This resulted in an immediate, beaded-type precipitate at the surface of the organisms. With some organisms, the entire length was heavily coated with the precipitate, whereas with others only a slight surface precipitate was observed. Control preparations of T. pallidum that were mixed with acidified saline did not exhibit surface precipitation. The percentage of treponemes reacting with acid BSA within freshly harvested suspensions prepared from different rabbits was variable. In some preparations, 90% of the organisms were acid BSA positive; in other preparations, 50% of the organisms were acid BSA positive; in still other preparations, only 5% were acid BSA positive. The specificity of the acid BSA reaction was tested by adding bovine hyaluronidase (16, 18). The addition of this enzyme neutralizes the acid BSA reaction, preventing the development of the precipitate. No precipitate was observed at the surface of the treponemes after addition of acid BSA and hyaluronidase. Plant lectins were used to determine whether the acidic mucopolysaccharides hyaluronic acid and chondroitin sulfate were present on the outer surface of T. pallidum. Wheat germ agglutinin agglutinates substances containing N-acetyl-D-glucosamine, a major constituent of hyaluronic acid; soybean agglutinin agglutinates substances containing N-acetyl-D-galactosamine, a major constituent of chondroitin sulfate. Each lectin was dissolved in saline and added to freshly harvested preparations of T. pallidum. After 24 h, a slight clumping of organisms was detected in control preparations treated with saline (Fig. 1A). In the two lectin-treated prep-
245
arations, pronounced agglutination was observed. Figure 1B shows agglutination resulting from 100 ug of wheat germ agglutinin per ml; Fig. 1C shows agglutination resulting fom 250 ,Lg of soybean agglutinin per ml. The specificity of these lectin reactions was tested by adding related sugars at 5 mg/ml and incubating for 24 h. The treponemal agglutination produced by wheat germ agglutinin was prevented by N-acetyl-D-glucosamine, but not by N-acetyl-D-galactosamine, glucose, sucrose, or lactose. The treponemal agglutination produced by soybean agglutinin was prevented by N-acetyl-D-galactosamine, but not by N-acetylD-glucosamine, glucose, sucrose, or lactose. Inasmuch as acidic mucopolysaccharides have polyanionic characteristics (2), attempts were made to influence the outer surface of the organisms by using polycations. Ruthenium red and toluidine blue dissolved in tissue culture medium were added to freshly harvested preparations of T. pallidum. Controls received an equivalent volume of tissue culture medium. Directly opposing effects were detected depending on the concentration of polycation. The data in Table 1 show the influences of concentrations of 200 ,ug/ml versus 0.1 ,ug/ml on retention of treponemal motility. The higher concentration of both polycations inhibited treponemal survival. At 200 ,ug/ml, motile treponemes were not detected after 17 h with toluidine blue and after 41 h with ruthenium red, contrasted to 165 h for the control preparation. The lower concentration extended treponemal survival. At 0.1 ,tg/ml, 50% motility of the treponemes was detected after 75 h with toluidine blue and after 114 h with ruthenium red, contrasted to approximately 58 h for the control preparation. During testicular infection with T. pallidum, a mucoid testicular fluid (TF) accumulates. This fluid was removed by needle aspiration. Acid BSA titrations revealed variable quantities of acidic mucopolysaccharides within TF. These quantities ranged from 10,000 to less than 8 ,ug/ ml. The treponemes present in TF were actively motile, and their movement was apparently affected by the viscosity of TF. Treponemes on a microscopic slide do not move far from their original position in spite of active flexing and rotating motion. In the viscous TF, however, the organisms exhibited smooth translational motion, gliding backwards and forwards, and moving rapidly across areas of the viewing field in a type of "directed" motility. Attempts were made to produce a similar directed treponemal motility by the addition of viscous agents. Each agent was dissolved in tissue culture medium. Directed motility was not
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VOL. 24, 1979
SURFACE MUCOPOLYSACCHARIDES OF T. PALLIDUM
TABLE 1. Effects of ruthenium red and toluidine blue on retention of motility of T. pallidum (Nichols strain) % Motility Time (h)
0 17 41 75 114 137 165
Control without polycations
96 88 68 30 16 2 0
Ruthenium red
Toluidine blue
200
0.1
200
0.1
sg/m, 96 76 0 0 0 0 0
/g/nml
Ag/ml
92 76 72 60 52 20 0
96 0 0 0 0 0 0
ig/m1 96 80 72 52 40 12 4
detected in Ficoll (10 to 30%), agar (0.2 to 0.3%), gelatin (6 to 10%), chondroitin sulfate (4.2 to 42 mg/ml), hyaluronic acid (1 to 10 mg/ml), or glycerol (25 to 50%). Directed motility was observed in methyl cellulose at 0.1 to 0.4%. Experiments were performed to assess the influence of TF on treponemal survival during in vitro incubation. The organisms within TF retained motility for 1 to 2 days at room temperature in air. Thus, TF samples were used 2 days after isolation. TF at 25, 5, 0.1, and 0.05% (vol/vol) was added to freshly harvested preparations of T. pallidum. The control received 25% tissue culture medium. The test mixtures containing 5, 0.1, and 0.05% TF received 20, 24.9, and 24.95% tissue culture medium, respectively. Samples of TF isolated from 20 different infected rabbits were tested. Both stimulatory (10 experiments) and inhibitory effects (8 experiments) were observed. The data in Table 2 reflect a representative experiment in which TF enhanced treponemal survival, as indicated by retention of motility. Concentrations of 0.05 and 0.1% were slightly effective, and 5 and 25% were more effective in extending survival of T. pallidum. The data in Table 3 reflect a representative experiment in which TF was detrimental to treponemal survival as indicated by retention of motility. In the control preparation, motility was retained for much longer periods than in the four TF-containing preparations. Commercial sources of acidic mucopolysaccharides were then tested. Freshly harvested preparations of T. pallidum were added to hyaluronic acid and chondroitin sulfate dissolved in tissue culture medium at 400, 200, 100, and 50 ,ug/ml. The control preparation received an equivalent volume of tissue culture medium. Four separate experiments were performed, and
247
the data from a representative experiment are presented in Table 4. Both mucopolysaccharides were detrimental at all four concentrations. Hyaluronic acid appeared to be more inhibitory than chondroitin sulfate. The detrimental effect of hyaluronic acid was abolished by prior incubation with bovine and streptomyces hyaluronidase. When hyaluronic acid at 1,000,ug/ml was pretreated with this enzyme for 20 h, and then added to T. pallidum, it did not inhibit treponemal survival relative to control preparations. In the presence of hyaluronic acid, the treponemes had a tendency to form small clumps consisting of 10 to 25 treponemes per clump. Pronounced clumping was detected at 400 jig of hyaluronic acid per ml; very slight clumping was detected at 50 and 100 jug of hyaluronic acid per ml. Figure 2A shows treponemes incubated for 3 days with hyaluronic acid at 400 fig/ml. Figure 2B shows the control preparation incubated for 3 days without hyaluronic acid. Similar observations of clumping were detected when TF was TABLE 2. Beneficial effects of TF on retention of motility of T. pallidum (Nichols strain) % Motility Time (h)
18 41 53 65 90 123 138 162 188 212
Control tissue culture medium
25%
5%
0.1%
0.05%
92 72 48 56 16 4 4 0 0 0
92 100 84 92 84 72 56 32 12 0
92 76 56 56 26 20 44 28 20 4
84 76 36 68 36 24 0 0 8 8
72 68 52 60 52 4 4 12 12 0
Mucoid TF
TABLE 3. Detrimental effects of TF on retention of motility of T. pallidum (Nichols strain) % Motility Time (h)
0 16 40 66 94 113 141 159
Control tissue culture medium
25%
5%
0.1%
0.05%
96 88 80 60 52 24 28 0
96 88 64 8 0 0 0 0
96 84 72 36 0 0 0 0
96 92 84 52 4 8 0 0
96 88 68 36 16 0 0 0
Mucoid TF
FIG. 1. T. pallidum exposed to (A) saline, (B) wheat germ agglutinin in saline, or (C) soybean agglutinin in saline. Bar, 8 pm.
248
FITZGERALD AND JOHNSON
INFECT. IMMUN.
TABLE 4. Effects of acidic mucopolysaccharides on retention of motility of T. pallidum (Nichols strain) % Motility
Time (h)
mucopolysac20 45 72 93 117 140 164 188
98 92 84 84 60 42 28 0
400 jg/ ml
200 fig/ ml
100 ,g/ ml
50 tig/ ml
98 99 94 42 36 8 4 0
96 97 88 53 33 10 2 0
94 97 82 69 37 8 0 0
98 97 82 75 37 8 0 0
used as a source of acidic mucopolysaccharides. The organisms within clumps were actively motile and exhibited directed motility. Individual organisms, besides flexing and rotating, rapidly moved forwards and backwards within the observable area of the clump. Treponemes not within clumps exhibited the usual motility but not directed motility. This phenomenon was observed only during day 1 of incubation. Thereafter, the clumped organisms remained actively motile but did not exhibit directed motion. Additionally, treponemes that were heat or air inactivated did not exhibit clumping during subsequent incubation with hyaluronic acid at 400
[tg/ml.
Hyaluronic acid
Chondroitin sulfate
Control without
Three observations suggested that these clumps of organisms contained acidic mucopolysaccharides. The addition of toluidine blue (0.5%) resulted in metachromasia (3-5) within the clumps; the addition of acid BSA resulted in a heavy precipitate within the clumps; and the addition of bovine hyaluronidase (1,200 National Federation units per ml) and streptomyces hyaluronidase (3 turbidity-reducing units per ml) partially degraded these clumps. DISCUSSION The addition of acid BSA to suspensions of T. pallidum resulted in a precipitate, presumably acidic mucopolysaccharide, at the surface of the organisms. The only previous method for observing treponemal mucopolysaccharides involved ruthenium red staining and electron microscopy (10, 36). The acid BSA reaction provides an alternative method for characterizing treponemal mucopolysaccharides which is much faster and more practical than electron microscopy. The resulting treponemal surface precipitate was unevenly distributed along the length of the organisms. Some treponemes were heavily coated with mucopolysaccharide, whereas others were devoid of this material, or contained very little. The percentage of organisms exhibiting the acid BSA precipitate varied within prep-
400 jg/
200 Ag/
mnl
100 Ag/ ml
0
98 99 56 23 10 0 0 0
98 99 60 34 18 2 2 0
ml 96 89 14 1 0 0 0
50 tig/
ml 98 100 90 51 35 8 2 0
arations of T. pallidum isolated from different rabbits. In some preparations as many as 90% of the organisms were positive, and in others as few as 5% were positive. The agglutination of T. pallidum by lectins was probably due to surface-associated mucopolysaccharides. Wheat germ agglutinin agglutinates substances containing N-acetyl-D-glucosamine, and soybean agglutinin agglutinates substances containing N-acetyl-D-galactosamine. Hyaluronic acid is comprised of polymers of N-acetyl-D-glucosamine-D-glucuronic acid; chondroitin sulfate is comprised of polymers of N-acetyl-D-galactosamine-D-glucuronic acid. These two acidic mucopolysaccharides have been identified as primary components of the accumulated mucoid material within syphilitic lesions (9, 26, 27, 31-34). The surface-associated mucopolysaccharide of T. pallidum might be a complex of hyaluronic acid-chondroitin sulfate, or very closely related acidic mucopolysaccharides. The integrity of the mucopolysaccharide layer appeared to be in delicate balance. Certain TF preparations, hyaluronic acid, chondroitin sulfate, and high concentrations of polycations were detrimental to treponemal survival. Different preparations of TF and lower concentrations of polycations were beneficial to treponemal survival. The reasons for these differing effects may involve a stabilization of the mucopolysaccharide layer and/or an interference with the mucopolysaccharidase enzyme of T. pallidum (11). Two other reports have shown that certain acidic mucopolysaccharides interfere with hyaluronidase activity (7, 21). Within treponemal suspensions, some clumping of organisms is usually observed during in vitro incubation. In contrast to the slight clumping in control preparations, pronounced clumping occurred in preparations incubated with hyaluronic acid. These treponemal clumps contained acidic mucopolysaccharides as shown by reaction with acid BSA, metachromasia with
VOL. 24, 1979
SURFACE MUCOPOLYSACCHARIDES OF T. PALLIDUM
249
FIG. 2. T. pallidum incubated with (A) hyaluronic acid or (B) control medium. Bar, 20 jim.
toluidine blue, and partial degradation by hyaluronidase. Treponemes exhibited directed motility within viscous preparations of TF and within viscous preparations of methyl cellulose (34). This directed motility was also observed within the clumps of organisms during the first 24 h of incubation. The exogenous source of hyaluronic acid may have stimulated the mucopolysaccharidase activity of the treponemes (11), enhancing mucopolysaccharide synthesis with consequent
clumping of organisms within the mucopolysaccharide capsular material. The directed motility could then be attributed to the viscous nature of the acidic mucopolysaccharides within the clumps. It is unlikely that the hyaluronic acid merely coated the organisms, producing a stickiness that resulted in clumping, since treponemes that were heat or air inactivated did not heavily clump when incubated with hyaluronic acid. The acidic mucopolysaccharides could be syn-
250
FITZGERALD AND JOHNSON
thesized by T. pallidum, or they could be hostderived constituents that merely attach to the outer surface of the organisms. The observations within this paper, in conjunction with related published observations (12, 23, 33, 34), are consistent with synthesis by T. pallidum. This would clarify some previous findings. Acidic mucopolysaccharides, especially hyaluronic acid, are very water soluble (2, 6), and it is conceivable that they would readily dissipate from the surface of the organisms. This dissolution of treponemal synthesized mucopolysaccharide would account for the accumulation of the mucoid material within syphilitic lesions, the accumulation of mucopolysaccharides on the surface of cultured mammalian cells during incubation with T. pallidum (12), and the occurrence of acidic mucopolysaccharides in the serum of the infected animals (11). Acidic mucopolysaccharides at the surface of T. pallidum may be beneficial to the organisms by restricting the access of antitreponemal antibodies (8, 17, 22, 23, 31, 33, 34). Mucopolysaccharides have an excluded volume which limits or retards penetration of large molecules such as antibodies (19). Furthermore, the addition of lysozyme to preparations of T. pallidum enhances seroreactivity (22). It was assumrred that this enzyme degraded surface mucopeptides, thereby hastening the antibody reactions. An alternative possibility is related to the surfaceassociated treponemal mucopolysaccharides. Lysozyme specifically precipitates acidic mucopolysaccharides (6). This might result in removal or depolymerization of the treponemal mucopolysaccharides, which in turn would hasten antibody reactions. Lastly, there may be a relationship between treponemal mucopolysaccharides and oxygen. Acidic mucopolysaccharides undergo irreversible depolymerization in the presence of oxygen and oxidation-reduction substances (24, 28). This is termed oxidative-reductive depolymerization. The depolymerization of the acidic mucopolysaccharides on the surface of T. pallidum could explain the oxygen sensitivity of the organisms and the involvement of certain reducing agents in neutralizing oxygen toxicity. In certain diagnostic tests for syphilis, T. pallidum exhibits relatively slow serological reactivity. A number of reports have proposed that T. pallidum possesses an outer protective layer that has to dissipate prior to seroreactivity. Christiansen (8) suggested that it was comprised of polysaccharides and lipoproteins derived from host tissue. Turner and Hollander (31, 34), Hardy and Nell (17), and Metzger et al. (22) referred to the possibility of a mucopolysaccharide capsule or slime layer of T. pallidum. Miller
INFECT. IMMUN.
(23) postulated that the outer layer of treponemal mucopolysaccharide was an important immunogen. Zeigler et al. (36) and Fitzgerald et al. (10) were the first to specifically demonstrate the existence of an outer surface layer of T. pallidum (Nichols strain). Fitzgerald et al. (12) showed that T. pallidum (Nichols strain) produced acidic mucopolysaccharides during incubation with cultured cells. Scott and Dammin (26, 27) and Turner and Hollander (31-34) observed that the accumulation of mucoid material within treponemal lesions paralleled the infective process. These postulations and findings, in conjunction with the studies within this paper, strongly imply that T. pallidum synthesizes acidic mucopolysaccharides. Unequivocal proof of this, however, will require further research. ACKNOWLEDGMENTS We gratefully acknowledge the expert technical assistance rendered by Elizabeth Thompson Wolff. This investigation was supported by Public Health Service grants AI-08124 and AI-12978 from the National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Akutsu, A. 1921. Histopathy of the scrotal chancre. Z. Jap. Mikrobiol. Ges. 15:205-210. 2. Balazs, E. A. 1970. Chemistry and molecular biology of the intercellular matrix, vol. 2. Academic Press, New
York. 3. Belanger, L. F., and A. Hartnett. 1960. Persistent toluidine blue metachromasia. J. Histochem. 8:75. 4. Belanger, L. F., and B. B. Migicovsky. 1961. A comparison between different mucopolysaccharide stains as applied to chick epiphyseal cartilage. J. Histochem. 9: 73-86. 5. Blumenkrantz, N. 1957. Microtest for mucopolysaccharides by means of toluidine blue. Clin. Chem. 3:696702. 6. Brimacombe, J. S., and J. M. Webber. 1964. Mucopolysaccharides, chemical structure, distribution, and isolation. Elsevier Publishing, Amsterdam. 7. Cameron, E. 1966. Hyaluronidase and cancer. Pergamon Press, Oxford. 8. Christiansen, S. 1963. Protective layer covering pathogenic treponemata. Lancet i:423-425. 9. DeLamater, E. D., V. R. Saurino, and F. Urbach. 1952. Studies on the immunology of spirochetoses. I. Effect of cortisone on experimental spirochetosis. Am. J. Syph. 36:127-139. 10. Fitzgerald, T. J., P. Cleveland, R. C. Johnson, J. N. Miller, and J. A. Sykes. 1976. Scanning electron microscopy of Treponema pallidum (Nichols strain) attached to cultured mammalian cells. J. Bacteriol. 130: 1333-1344. 11. Fitzgerald, T. J., and R. C. Johnson. 1978. Mucopolysaccharidase of Treponema pallidum. Infect. Immun. 24:261-268. 12. Fitzgerald, T. J., R. C. Johnson, and E. T. Wolff. 1978. Mucopolysaccharide material resulting from the interaction of Treponema pallidum (Nichols strain) with cultured mammalian cells. Infect. Immun. 22:575-584. 13. Graetz, F., and E. Delbanco. 1914. Beitrage zum Studien der Histopathologie der experimentellen kaninchen Syphilis. Med. Klin. 10:375-420. 14. Graetz, F., and E. Delbanco. 1914. Weitere Beitrage
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Studien der Histopathologie der experimentellen kaninchen Syphilis. Derm. Wochenschr. 58:6-28. Gregoriew, P. S., and K. G. Jarisheva. 1928. The histological structure of syphilitic lesions of rabbits. Am. J. Syph. 12:67-81. Grossfield, H. 1958. Studies on production of hyaluronic acid in tissue culture. The presence of hyaluronidase in embryo extract. Exp. Cell Res. 14:213-216. Hardy, P. H., and E. E. Nell. 1957. Study of the antigenic structure of Treponema pallidum by specific agglutination. Am. J. Hyg. 66:160-172. Ibrahim, A. N., and M. M. Streitfeld. 1973. The microassay of hyaluronic acid concentration and hyaluronidase activity by capillary turbidity (CT) and capillary turbidity reduction (CTR) tests. Anal. Biochem. 56: 428-434. Laurent, T. C., and A. G. Ogston. 1963. The interaction between polysaccharides and other macromolecules. IV. The osmotic pressure of mixtures of serum albumin and hyaluronic acid. Biochem. J. 89:249-253. Luft, J. H. 1971. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat. Rec. 171:347-368. Matthews, M. B., and A. Dorfman. 1955. Inhibition of hyaluronidase. Physiol. Rev. 35:381-402. Metzger, M., P. H. Hardy, and E. E. Nell. 1961. Influence of lysozyme upon the treponeme immnobilization reaction. Am. J. Hyg. 73:236-244. Miller, J. N. 1972. Development of an experimental syphilis vaccine. Med. Clin. N. Am. 56:1217-1220. Pigman, W., and S. Rizvi. 1959. Hyaluronic acid and the ORD reaction. Biochem. Biophys. Res. Commun. 1:3943. Scott, V., and G. J. Dammin. 1949. Experimental syphilis in the rabbit. The relationship of metachromasia to fibrinoid degeneration of collagen and the localization zum
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of spirochaetes in the testis. J. Lab. Clin. Med. 34:17481755. Scott, V., and G. J. Dammin. 1950. Hyaluronidase and experimental syphilis. III. Metachromasia in syphilitic orchitis and its relation to hyaluronic acid. Am. J. Syph. 34:501-514. Scott, V., and G. J. Dammin. 1954. Morphologic and histochemical sequences in syphilitic and tuberculous orchitis in the rabbit. Am. J. Syph. 38:189-202. Skanse, B., and L. Sundblad. 1943. Oxidative breakdown of hyaluronic and chondroitin sulfuric acid. Acta Physiol. Scand. 6:37-51. Strempel, R., and G. Armuzzi. 1926. Histobiologie der ersten Inkubationsperiode der Kaninchensyphilis. Exp. Untersuch. Derm. Z. 46:267-288. Sykes, J. A., and E. B. Moore. 1960. A simple tissue culture chamber. Tex. Rep. Biol. Med. 18:288-297. Turner, T. B. 1970. Syphilis and the treponematoses, p. 346-390. In S. Mudd (ed.), Infectious agents and host reactions. W. B. Saunders, Philadelphia. Turner, T. B., and D. H. Hollander. 1950. Cortisone in experimental syphilis. Johns Hopkins Hosp. Bull. 87: 505-509. Turner, T. B., and D. H. Hollander. 1954. Studies on the mechanism of action of cortisone in experimental syphilis. Am. J. Syph. 38:371-387. Turner, T. B., and D. H. Hollander. 1957. Biology of the treponematoses. WHO Monograph Series no. 35. Uhlenhuth, P., P. Mulzer, and M. Koch. 1912. Uber die histopathologischen Veranderungen bei der experimentellen kaninchen Syphilis. Dtsch. Med. Wochenschr. 38:1079-1081. Zeigler, J. A., A. M. Jones, R. H. Jones, and K. M. Kubica. 1976. Demonstration of extracellular material at the surface of pathogenic T. pallidum cells. Br. J. Vener. Dis. 52:1-8.
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Surface Mucopolysaccharides of Treponema pallidum T. J. FITZGERALD* AND R. C. JOHNSON Department of Microbiology, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455
Received for publication 26 December 1978
The viscous mucoid fluid that accumulates within syphilitic lesions may be due to breakdown of host tissue during infection, or may be synthesized by Treponema pallidum. Experiments were performed to investigate the acidic mucopolysaccharides that occur at the surface of T. pallidum (Nichols strain). These mucopolysaccharides were demonstrated by reaction with acidified bovine serum albumin and by agglutination with wheat germ agglutinin and soybean agglutinin. The polycations ruthenium red and toluidine blue influenced treponemal survival. Concentrations of both compounds at 200 tg/ml inhibited survival, whereas concentrations at 0.1 /Lg/ml enhanced survival. The mucopolysaccharide concentration within the mucoid fluid that accumulates during intratesticular infection was determined by reaction with acidified bovine serum albumin; it ranged from 10,000 jig/ml to less than 8 jtg/ml. The addition of this mucoid fluid to treponemal suspensions resulted in differing effects on T. pallidum survival. Some preparations were inhibitory, and others were stimulatory. Commercial preparations of hyaluronic acid and chondroitin sulfate at 400, 200, 100, and 50 ytg/ml were detrimental to treponemal survival. The organisms exhibited pronounced clumping in the presence of the higher concentrations of hyaluronic acid. These clumps of treponemes were comprised of mucopolysaccharides as shown by acidified bovine serum albumin and toluidine blue reactions and by hyaluronidase degradation. Results are discussed in terms of the derivation and potential role of acidic mucopolysaccharides at the surface of T. pallidum.
Mucoid fluid accumulates within dermal and testicular lesions produced by Treponema pallidum (1, 9, 13-15, 25-27, 29, 31-35). This material is partially comprised of the acidic mucopolysaccharides hyaluronic acid and chondroitin sulfate (9, 26, 27, 31-34). In certain diagnostic tests for syphilis, T. pallidum exhibits a relatively slow serological reactivity. It has been suggested that the organisms have an outer surface layer that has to dissipate prior to seroreactivity (8, 17, 22, 23, 31, 34). Zeigler et al. (36) and Fitzgerald et al. (10) demonstrated a surface layer on T. pallidum (Nichols strain) that reacted with ruthenium red. This suggested that the nature of this material was acidic mucopolysaccharide (20). It is important to characterize the derivation of viscous mucoid fluid within lesions since it appears to be directly related to virulence. Turner (31) and Turner and Hollander (34) observed that infections with more virulent strains of T. pallidum were associated with increased accumulations of mucoid material. In addition, Fitzgerald and Johnson (Br. J. Vener. Dis., in press) have found that this mucoid material exhibited immunosuppressive activity. The purpose of this report was to further elaborate on the surface mucopolysaccharide of T. pallidum.
MATERIALS AND METHODS T. pallidumL The Nichols strain of T. pallidum
was maintained by intratesticular passage in healthy Dutch Belt rabbits weighing 4 to 5 pounds (ca. 1.8 to 2.3 kg). The animals were housed at 19 to 22°C and given antibiotic-free food and water ad libitum. Approximately 1 x 107 to 3 x 107 treponemes were inoculated per testis. Daily intramuscular injections of cortisone acetate (Merck, Sharpe, and Dohme, West Point, Pa.) at 6 mg/kg of body weght were initiated 3 days postinoculation. After 9 to 13 days, the animals were sacrificed by intracardial injection of sodium pentabarbitol, and the testes were removed and placed in physiological saline. The testes were sliced and extracted aerobically for 20 to 30 min in tissue culture medium containing Eagle minimal essential medium supplemented with 4 mM NaHCO3, 10% (vol/vol) fetal bovine serum, 1 mM dithiothreitol, 4 mM glutathione, and 1 mM cysteine. This medium was buffered with 30 mM HEPES (N-2-hydroxyethyl piperazine-N'-2ethanesulfonic acid) at pH 7.2. After extraction, the treponemal suspension was centrifuged at 1,000 x g for 7 min to sediment particulate matter. For testing the influences of various compounds, suspensions were adjusted to 2 x 107 to 5 x 107 treponemes per ml and placed into test tubes with loose-fitting caps or Sykes-Moore chambers (30). Incubation was performed at 30°C in desiccator jars that were evacuated and flushed five times with 2.5% oxygen-92.5% nitrogen-5% carbon dioxide. 244
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SURFACE MUCOPOLYSACCHARIDES OF T. PALLIDUM
Photography. Treponemes were placed in SykesMoore chambers. After reaction with acidified bovine serum albumin (acid BSA) or plant lectins, 3% glutaraldehyde-1% TAPO (tris-1-aziridenyl phosphine oxide) in physiological saline was added for 30 min. The fixative was removed, and the treponemes were photographed using a Leitz phase-contrast microscope with an attached Leitz automatic exposure timing device. The film used was Kodak Tri-X pan for black and white prints. Chemicals. Crystalline type IV bovine testicular hyaluronidase, hyaluronic acid from human umbilical cord, chondroitin sulfate from whale and shark cartilage, soybean agglutinin, wheat germ agglutinin, and ruthenium red were obtained from Sigma (St. Louis, Mo.). Crystalline toluidine blue was obtained from Pharmaceutical Laboratories (New York, N.Y.).
RESULTS Acid BSA reacts with acidic mucopolysaccharides by producing a precipitate (16, 18). This procedure was adapted to T. pallidum to demonstrate the acidic mucopolysaccharides on the treponemal surface. One drop of treponemal suspension was added to 1 drop of acid BSA. This resulted in an immediate, beaded-type precipitate at the surface of the organisms. With some organisms, the entire length was heavily coated with the precipitate, whereas with others only a slight surface precipitate was observed. Control preparations of T. pallidum that were mixed with acidified saline did not exhibit surface precipitation. The percentage of treponemes reacting with acid BSA within freshly harvested suspensions prepared from different rabbits was variable. In some preparations, 90% of the organisms were acid BSA positive; in other preparations, 50% of the organisms were acid BSA positive; in still other preparations, only 5% were acid BSA positive. The specificity of the acid BSA reaction was tested by adding bovine hyaluronidase (16, 18). The addition of this enzyme neutralizes the acid BSA reaction, preventing the development of the precipitate. No precipitate was observed at the surface of the treponemes after addition of acid BSA and hyaluronidase. Plant lectins were used to determine whether the acidic mucopolysaccharides hyaluronic acid and chondroitin sulfate were present on the outer surface of T. pallidum. Wheat germ agglutinin agglutinates substances containing N-acetyl-D-glucosamine, a major constituent of hyaluronic acid; soybean agglutinin agglutinates substances containing N-acetyl-D-galactosamine, a major constituent of chondroitin sulfate. Each lectin was dissolved in saline and added to freshly harvested preparations of T. pallidum. After 24 h, a slight clumping of organisms was detected in control preparations treated with saline (Fig. 1A). In the two lectin-treated prep-
245
arations, pronounced agglutination was observed. Figure 1B shows agglutination resulting from 100 ug of wheat germ agglutinin per ml; Fig. 1C shows agglutination resulting fom 250 ,Lg of soybean agglutinin per ml. The specificity of these lectin reactions was tested by adding related sugars at 5 mg/ml and incubating for 24 h. The treponemal agglutination produced by wheat germ agglutinin was prevented by N-acetyl-D-glucosamine, but not by N-acetyl-D-galactosamine, glucose, sucrose, or lactose. The treponemal agglutination produced by soybean agglutinin was prevented by N-acetyl-D-galactosamine, but not by N-acetylD-glucosamine, glucose, sucrose, or lactose. Inasmuch as acidic mucopolysaccharides have polyanionic characteristics (2), attempts were made to influence the outer surface of the organisms by using polycations. Ruthenium red and toluidine blue dissolved in tissue culture medium were added to freshly harvested preparations of T. pallidum. Controls received an equivalent volume of tissue culture medium. Directly opposing effects were detected depending on the concentration of polycation. The data in Table 1 show the influences of concentrations of 200 ,ug/ml versus 0.1 ,ug/ml on retention of treponemal motility. The higher concentration of both polycations inhibited treponemal survival. At 200 ,ug/ml, motile treponemes were not detected after 17 h with toluidine blue and after 41 h with ruthenium red, contrasted to 165 h for the control preparation. The lower concentration extended treponemal survival. At 0.1 ,tg/ml, 50% motility of the treponemes was detected after 75 h with toluidine blue and after 114 h with ruthenium red, contrasted to approximately 58 h for the control preparation. During testicular infection with T. pallidum, a mucoid testicular fluid (TF) accumulates. This fluid was removed by needle aspiration. Acid BSA titrations revealed variable quantities of acidic mucopolysaccharides within TF. These quantities ranged from 10,000 to less than 8 ,ug/ ml. The treponemes present in TF were actively motile, and their movement was apparently affected by the viscosity of TF. Treponemes on a microscopic slide do not move far from their original position in spite of active flexing and rotating motion. In the viscous TF, however, the organisms exhibited smooth translational motion, gliding backwards and forwards, and moving rapidly across areas of the viewing field in a type of "directed" motility. Attempts were made to produce a similar directed treponemal motility by the addition of viscous agents. Each agent was dissolved in tissue culture medium. Directed motility was not
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VOL. 24, 1979
SURFACE MUCOPOLYSACCHARIDES OF T. PALLIDUM
TABLE 1. Effects of ruthenium red and toluidine blue on retention of motility of T. pallidum (Nichols strain) % Motility Time (h)
0 17 41 75 114 137 165
Control without polycations
96 88 68 30 16 2 0
Ruthenium red
Toluidine blue
200
0.1
200
0.1
sg/m, 96 76 0 0 0 0 0
/g/nml
Ag/ml
92 76 72 60 52 20 0
96 0 0 0 0 0 0
ig/m1 96 80 72 52 40 12 4
detected in Ficoll (10 to 30%), agar (0.2 to 0.3%), gelatin (6 to 10%), chondroitin sulfate (4.2 to 42 mg/ml), hyaluronic acid (1 to 10 mg/ml), or glycerol (25 to 50%). Directed motility was observed in methyl cellulose at 0.1 to 0.4%. Experiments were performed to assess the influence of TF on treponemal survival during in vitro incubation. The organisms within TF retained motility for 1 to 2 days at room temperature in air. Thus, TF samples were used 2 days after isolation. TF at 25, 5, 0.1, and 0.05% (vol/vol) was added to freshly harvested preparations of T. pallidum. The control received 25% tissue culture medium. The test mixtures containing 5, 0.1, and 0.05% TF received 20, 24.9, and 24.95% tissue culture medium, respectively. Samples of TF isolated from 20 different infected rabbits were tested. Both stimulatory (10 experiments) and inhibitory effects (8 experiments) were observed. The data in Table 2 reflect a representative experiment in which TF enhanced treponemal survival, as indicated by retention of motility. Concentrations of 0.05 and 0.1% were slightly effective, and 5 and 25% were more effective in extending survival of T. pallidum. The data in Table 3 reflect a representative experiment in which TF was detrimental to treponemal survival as indicated by retention of motility. In the control preparation, motility was retained for much longer periods than in the four TF-containing preparations. Commercial sources of acidic mucopolysaccharides were then tested. Freshly harvested preparations of T. pallidum were added to hyaluronic acid and chondroitin sulfate dissolved in tissue culture medium at 400, 200, 100, and 50 ,ug/ml. The control preparation received an equivalent volume of tissue culture medium. Four separate experiments were performed, and
247
the data from a representative experiment are presented in Table 4. Both mucopolysaccharides were detrimental at all four concentrations. Hyaluronic acid appeared to be more inhibitory than chondroitin sulfate. The detrimental effect of hyaluronic acid was abolished by prior incubation with bovine and streptomyces hyaluronidase. When hyaluronic acid at 1,000,ug/ml was pretreated with this enzyme for 20 h, and then added to T. pallidum, it did not inhibit treponemal survival relative to control preparations. In the presence of hyaluronic acid, the treponemes had a tendency to form small clumps consisting of 10 to 25 treponemes per clump. Pronounced clumping was detected at 400 jig of hyaluronic acid per ml; very slight clumping was detected at 50 and 100 jug of hyaluronic acid per ml. Figure 2A shows treponemes incubated for 3 days with hyaluronic acid at 400 fig/ml. Figure 2B shows the control preparation incubated for 3 days without hyaluronic acid. Similar observations of clumping were detected when TF was TABLE 2. Beneficial effects of TF on retention of motility of T. pallidum (Nichols strain) % Motility Time (h)
18 41 53 65 90 123 138 162 188 212
Control tissue culture medium
25%
5%
0.1%
0.05%
92 72 48 56 16 4 4 0 0 0
92 100 84 92 84 72 56 32 12 0
92 76 56 56 26 20 44 28 20 4
84 76 36 68 36 24 0 0 8 8
72 68 52 60 52 4 4 12 12 0
Mucoid TF
TABLE 3. Detrimental effects of TF on retention of motility of T. pallidum (Nichols strain) % Motility Time (h)
0 16 40 66 94 113 141 159
Control tissue culture medium
25%
5%
0.1%
0.05%
96 88 80 60 52 24 28 0
96 88 64 8 0 0 0 0
96 84 72 36 0 0 0 0
96 92 84 52 4 8 0 0
96 88 68 36 16 0 0 0
Mucoid TF
FIG. 1. T. pallidum exposed to (A) saline, (B) wheat germ agglutinin in saline, or (C) soybean agglutinin in saline. Bar, 8 pm.
248
FITZGERALD AND JOHNSON
INFECT. IMMUN.
TABLE 4. Effects of acidic mucopolysaccharides on retention of motility of T. pallidum (Nichols strain) % Motility
Time (h)
mucopolysac20 45 72 93 117 140 164 188
98 92 84 84 60 42 28 0
400 jg/ ml
200 fig/ ml
100 ,g/ ml
50 tig/ ml
98 99 94 42 36 8 4 0
96 97 88 53 33 10 2 0
94 97 82 69 37 8 0 0
98 97 82 75 37 8 0 0
used as a source of acidic mucopolysaccharides. The organisms within clumps were actively motile and exhibited directed motility. Individual organisms, besides flexing and rotating, rapidly moved forwards and backwards within the observable area of the clump. Treponemes not within clumps exhibited the usual motility but not directed motility. This phenomenon was observed only during day 1 of incubation. Thereafter, the clumped organisms remained actively motile but did not exhibit directed motion. Additionally, treponemes that were heat or air inactivated did not exhibit clumping during subsequent incubation with hyaluronic acid at 400
[tg/ml.
Hyaluronic acid
Chondroitin sulfate
Control without
Three observations suggested that these clumps of organisms contained acidic mucopolysaccharides. The addition of toluidine blue (0.5%) resulted in metachromasia (3-5) within the clumps; the addition of acid BSA resulted in a heavy precipitate within the clumps; and the addition of bovine hyaluronidase (1,200 National Federation units per ml) and streptomyces hyaluronidase (3 turbidity-reducing units per ml) partially degraded these clumps. DISCUSSION The addition of acid BSA to suspensions of T. pallidum resulted in a precipitate, presumably acidic mucopolysaccharide, at the surface of the organisms. The only previous method for observing treponemal mucopolysaccharides involved ruthenium red staining and electron microscopy (10, 36). The acid BSA reaction provides an alternative method for characterizing treponemal mucopolysaccharides which is much faster and more practical than electron microscopy. The resulting treponemal surface precipitate was unevenly distributed along the length of the organisms. Some treponemes were heavily coated with mucopolysaccharide, whereas others were devoid of this material, or contained very little. The percentage of organisms exhibiting the acid BSA precipitate varied within prep-
400 jg/
200 Ag/
mnl
100 Ag/ ml
0
98 99 56 23 10 0 0 0
98 99 60 34 18 2 2 0
ml 96 89 14 1 0 0 0
50 tig/
ml 98 100 90 51 35 8 2 0
arations of T. pallidum isolated from different rabbits. In some preparations as many as 90% of the organisms were positive, and in others as few as 5% were positive. The agglutination of T. pallidum by lectins was probably due to surface-associated mucopolysaccharides. Wheat germ agglutinin agglutinates substances containing N-acetyl-D-glucosamine, and soybean agglutinin agglutinates substances containing N-acetyl-D-galactosamine. Hyaluronic acid is comprised of polymers of N-acetyl-D-glucosamine-D-glucuronic acid; chondroitin sulfate is comprised of polymers of N-acetyl-D-galactosamine-D-glucuronic acid. These two acidic mucopolysaccharides have been identified as primary components of the accumulated mucoid material within syphilitic lesions (9, 26, 27, 31-34). The surface-associated mucopolysaccharide of T. pallidum might be a complex of hyaluronic acid-chondroitin sulfate, or very closely related acidic mucopolysaccharides. The integrity of the mucopolysaccharide layer appeared to be in delicate balance. Certain TF preparations, hyaluronic acid, chondroitin sulfate, and high concentrations of polycations were detrimental to treponemal survival. Different preparations of TF and lower concentrations of polycations were beneficial to treponemal survival. The reasons for these differing effects may involve a stabilization of the mucopolysaccharide layer and/or an interference with the mucopolysaccharidase enzyme of T. pallidum (11). Two other reports have shown that certain acidic mucopolysaccharides interfere with hyaluronidase activity (7, 21). Within treponemal suspensions, some clumping of organisms is usually observed during in vitro incubation. In contrast to the slight clumping in control preparations, pronounced clumping occurred in preparations incubated with hyaluronic acid. These treponemal clumps contained acidic mucopolysaccharides as shown by reaction with acid BSA, metachromasia with
VOL. 24, 1979
SURFACE MUCOPOLYSACCHARIDES OF T. PALLIDUM
249
FIG. 2. T. pallidum incubated with (A) hyaluronic acid or (B) control medium. Bar, 20 jim.
toluidine blue, and partial degradation by hyaluronidase. Treponemes exhibited directed motility within viscous preparations of TF and within viscous preparations of methyl cellulose (34). This directed motility was also observed within the clumps of organisms during the first 24 h of incubation. The exogenous source of hyaluronic acid may have stimulated the mucopolysaccharidase activity of the treponemes (11), enhancing mucopolysaccharide synthesis with consequent
clumping of organisms within the mucopolysaccharide capsular material. The directed motility could then be attributed to the viscous nature of the acidic mucopolysaccharides within the clumps. It is unlikely that the hyaluronic acid merely coated the organisms, producing a stickiness that resulted in clumping, since treponemes that were heat or air inactivated did not heavily clump when incubated with hyaluronic acid. The acidic mucopolysaccharides could be syn-
250
FITZGERALD AND JOHNSON
thesized by T. pallidum, or they could be hostderived constituents that merely attach to the outer surface of the organisms. The observations within this paper, in conjunction with related published observations (12, 23, 33, 34), are consistent with synthesis by T. pallidum. This would clarify some previous findings. Acidic mucopolysaccharides, especially hyaluronic acid, are very water soluble (2, 6), and it is conceivable that they would readily dissipate from the surface of the organisms. This dissolution of treponemal synthesized mucopolysaccharide would account for the accumulation of the mucoid material within syphilitic lesions, the accumulation of mucopolysaccharides on the surface of cultured mammalian cells during incubation with T. pallidum (12), and the occurrence of acidic mucopolysaccharides in the serum of the infected animals (11). Acidic mucopolysaccharides at the surface of T. pallidum may be beneficial to the organisms by restricting the access of antitreponemal antibodies (8, 17, 22, 23, 31, 33, 34). Mucopolysaccharides have an excluded volume which limits or retards penetration of large molecules such as antibodies (19). Furthermore, the addition of lysozyme to preparations of T. pallidum enhances seroreactivity (22). It was assumrred that this enzyme degraded surface mucopeptides, thereby hastening the antibody reactions. An alternative possibility is related to the surfaceassociated treponemal mucopolysaccharides. Lysozyme specifically precipitates acidic mucopolysaccharides (6). This might result in removal or depolymerization of the treponemal mucopolysaccharides, which in turn would hasten antibody reactions. Lastly, there may be a relationship between treponemal mucopolysaccharides and oxygen. Acidic mucopolysaccharides undergo irreversible depolymerization in the presence of oxygen and oxidation-reduction substances (24, 28). This is termed oxidative-reductive depolymerization. The depolymerization of the acidic mucopolysaccharides on the surface of T. pallidum could explain the oxygen sensitivity of the organisms and the involvement of certain reducing agents in neutralizing oxygen toxicity. In certain diagnostic tests for syphilis, T. pallidum exhibits relatively slow serological reactivity. A number of reports have proposed that T. pallidum possesses an outer protective layer that has to dissipate prior to seroreactivity. Christiansen (8) suggested that it was comprised of polysaccharides and lipoproteins derived from host tissue. Turner and Hollander (31, 34), Hardy and Nell (17), and Metzger et al. (22) referred to the possibility of a mucopolysaccharide capsule or slime layer of T. pallidum. Miller
INFECT. IMMUN.
(23) postulated that the outer layer of treponemal mucopolysaccharide was an important immunogen. Zeigler et al. (36) and Fitzgerald et al. (10) were the first to specifically demonstrate the existence of an outer surface layer of T. pallidum (Nichols strain). Fitzgerald et al. (12) showed that T. pallidum (Nichols strain) produced acidic mucopolysaccharides during incubation with cultured cells. Scott and Dammin (26, 27) and Turner and Hollander (31-34) observed that the accumulation of mucoid material within treponemal lesions paralleled the infective process. These postulations and findings, in conjunction with the studies within this paper, strongly imply that T. pallidum synthesizes acidic mucopolysaccharides. Unequivocal proof of this, however, will require further research. ACKNOWLEDGMENTS We gratefully acknowledge the expert technical assistance rendered by Elizabeth Thompson Wolff. This investigation was supported by Public Health Service grants AI-08124 and AI-12978 from the National Institute of Allergy and Infectious Diseases.
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