JOURNAL OF BACTERIOLOGY, June 1977, p. 1333-1344 Copyright ©) 1977 American Society for Microbiology
Vol. 130, No. 3 Printed in U.S.A.
Scanning Electron Microscopy of Treponema pallidum (Nichols Strain) Attached to Cultured Mammalian Cells T. J. FITZGERALD,* P. CLEVELAND, R. C. JOHNSON, J. N. MILLER, AND J. A. SYKES Department of Microbiology, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455*; Treponemal Research Laboratory, Department of Microbiology and Immunology, School of Medicine, University ofCalifornia at Los Angeles, Los Angeles, California 90024; and Research Department, Southern California Cancer Center, California Hospital, Los Angeles, California 90015 Received for publication 3 February 1977
This paper describes the attachment of Treponema pallidum (Nichols strain) to cultured mammalian cells as visualized by scanning electron microscopy. Treponemes were incubated for 3 h with cultured cells derived from normal rabbit testes or human skin epithelium, then fixed, processed with critical-point drying, and examined with a Cambridge Mark 2A scanning electron microscope. Large numbers of treponemes became attached to the cultured cells without
altering the morphological integrity of the cultured cells. Attachment appeared a very close physical proximity of treponemes to the cultured cells; at the site of attachment, no changes such as swelling or indentation of the cultured cell surface were observed. The addition of ruthenium red to the fixatives produced a treponemal-associated surface precipitate. This material, which is probably mucopolysaccharide and/or phospholipid, may be important in protecting the organisms against host defense mechanisms; in addition, it may be involved in the serological unresponsiveness of freshly prepared suspensions of T. pallidum. to involve
weighing 4 to 6 lb (ca. 1.8 to 2.7 kg) were used in this study. They were housed at 19 to 22°C and given antibiotic-free food and water ad libitum. T. pallidum. The Nichols strains of T. pallidum was maintained by intratesticular passage and harvested as previously described (7). Each testis was inoculated with 1 x 107 to 3 x 107 treponemes. Animals received daily injections of cortisone acetate (Merck, Sharp and Dohme) at 6 mg/kg of body weight beginning the third day after testicular inoculation. After development of a good orchitis, usually requiring 10 to 14 days, animals were sacrificed, and the testes were removed, minced with a scissors, and extracted at room temperature in tissue culture medium for 10 to 20 min. The treponemal suspension was then centrifuged at 1,000 x g for 7 min at 240C to sediment particulate matter. Cultured cells. The cells were derived from normal rabbit testes as previously described (7). A cell line derived from human skin epithelial tissue was kindly supplied by Carl Seiter of the Reheis Chemical Co. The tissue culture medium contained Eagle minimal essential medium with Hanks balanced salts, 2 x vitamins, 2 x amino acids, and 4 mM NaHCO3 buffered with 30 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid). The medium was supplemented with 10% fetal bovine serum (vol/vol) that had been heat inactivated (56°C MATERIALS AND METHODS for 30 min). Fetal bovine serum was obtained from Animals. Healthy male New Zealand white rab- the Reheis Chemical Co. The interaction of actively motile T. pallidum bits with nonreactive Rapid Plasma Reagin circle card tests (Hynson, Wescott, and Dunning, Inc.) and with cultured cells was visualized by phase-contrast 1333
Cultured mammalian cells extend the in vitro survival of the Nichols strain of Treponema pallidum (6-8). Large numbers of organisms rapidly attach to the cultured cells. This attachment prolongs the time of retention of treponemal motility and virulence. The cultured cells do not appear to be damaged by the attachment of T. pallidum. We have observed as many as 70 to 100 attached treponemes per individual cell, and yet these cultured cells retain their morphological integrity and viability. The purpose of this work was to visualize the topographical features of treponemes attached to cultured cells, with emphasis on the site of treponemal attachment. One other report consisting of one scanning electron micrograph of T. pallidum has been published (32). We have used critical-point drying techniques to improve the quality of our preparations. Attached treponemes were also fixed in the presence of ruthenium red. This resulted in the demonstration of a treponemal-associated surface material that had been previously documented using transmission electron microscopy (33).
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microscopy using Sykes-Moore chambers as previ- drying artifacts by critical-point drying with Freon (4). ously described (7). After critical-point drying, the cover slips were Scanning electron microscopy. Suspensions of T. to 13-mm aluminum stubs with silver-conattached x 9 to 107 x 7 pallidum adjusted to approximately with carbon and gold in a 107 organisms/ml were inoculated into Sykes-Moore ducting paint and coatedevaporator. The specimens chambers containing cultured cells at approximately Denton DV-502 vacuum Mark 20 to 40% confluency. After a 3-h incubation at 360C, were examined at a 450 angle in aatCambridge kV. 20 microscope electron scanning the 2A to attached had treponemes of a large number Ruthenium red. Selected specimens were exposed cells. The chambers were flushed with 3 volumes of Five milligrams of tissue culture medium without serum to remove to ruthenium red during fixation. to each milliliter of unattached treponemes. Excess medium was dis- this inorganic dye was added and osmium tetroxide fixcarded. Cells with attached organisms were fixed by both the glutaraldehyde were dehydrated and specimens These solutions. ing with directly adding 2% glutaraldehyde (buffered 0.1 M sodium cacodylate to pH 7.4) to the chambers critical point-dried as described above. for 1 h at 20°C and then for 16 h at 4°C. The cover RESULTS slips containing the cultured cells and treponemes were removed from the chambers, washed twice Attachment. The electron micrographs with cacodylate buffer, and postfixed in 1% osmium tetroxide (buffered as above) for 1 h. The cover slips within this paper were prepared after a 3-h were then washed twice with distilled water and incubation of T. pallidum with cultured cells. dehydrated in a graded series of ethanol to 100%. The fixation and critical-point drying techThis was followed by a graded series of Freon TF in niques successfully preserved the morphologiethanol to 100% Freon TF. The specimens on the cal integrity of both the cultured cells and the cover slips were rapidly transferred to a Bomar critiwithout cal-point drying device and preserved against air- treponemes. The organisms attached
FIG. 1. Electron micrograph of a normal rabbit testes cell with attached T. pallidum. Note the intact morphological integrity of the cell. Bar represents 11 ,um.
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FIG. 2. Electron micrograph of a portion of a normal rabbit testes cell with attached T. pallidum. Note what appears to be an unwinding axial filament at the left, lower center (arrow). Bar represents 10 ,um.
producing any observable alteration in the topography of the cultured cells. With suspensions containing 7 x 107 to 9 x 107 treponemes/ ml and cells at 20 to 40% confluency, as many as 50 to 70 treponemes were attached per individually cultured cell (Fig. 1 and 2). Treponemes appeared to be randomly distributed on the cell surface. The organisms did not exhibit a predilection for one specific area of the cells, such as the nuclear region (slightly elevated interior part of the cell) or the thin lamellae of the peripheral edge. Although certain
areas of some cells contained larger groups of organisms, no patterns suggestive of a definite preference for one specific area emerged. T. pallidum attached at its distal end as shown in Fig. 3. Some of these micrographs may be misleading in that a number of the treponemes appear to be attached along their entire length. However, this is artifactual as revealed by phase-contrast microscopy of attached, actively motile organisms. The treponemes attach in a number of different planes and during fixation settle on the cell surface,
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FIG. 3. Electron micrograph of T. pallidum attached at distal ends to the edge of a normal rabbit testes cell. One treponeme is attached at both ends. Bar represents 5 ,um.
giving the appearance of full-length attachment. Occasionally, T. pallidum attached at both ends as shown in Fig. 3 and 4. This occurred with 5 to 10% of the attached organisms. These treponemes may represent two organisms attached at one end just prior to division or an individual organism attached at both ends. This observation was not an artifact inasmuch as it was routinely detected with phase-contrast microscopy of actively motile organisms. There were no detectable physical disruptions of the cultured cell surface at the site of treponemal attachment. The point of attachment appeared to involve a very close physical proximity (Fig. 3-5). With higher magnifications (x 100,000), no swelling or indentation of the cell surface was observed. Figures 1 to 5 demonstrate treponemal attachment to normal rabbit testes cells, which are essentially fibroblastic-like. T. pallidum also attached to epithelial cells, as shown in
Fig. 6 and 7 which depict attachment to human skin epithelial cells, a cell line derived from human skin epithelium. Numerous microvilli at the cell surface are evident; these are characteristic of epithelial cells and are not to be confused with the much longer treponemes. Treponemes. Treponemal morphology was well preserved. Flagella (axial filaments) were not apparent on the surface of intact organisms. This may have been due to either the surface coating of the carbon and gold and/or the resolving power of the microscope. Occasionally, flagella were detected as they unwound from partially degraded organisms. This occurred with a very small proportion of the total treponemes visualized (less than 0.1%). Part of an apparently unwinding flagella is evident in Fig. 2 (left, lower center [arrow]). The lack of treponemes on the surface of the glass, away from the cultured cells, was due to the washing procedures prior to fixation. This was apparent by phase-contrast microscopy of
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the preparations before and after washing. rather than actual differences. The organism Some of the treponemes that were attached to pictured in Fig. 8 is attached to a cultured cell. the cells and to the glass were damaged during Because of the high magnification, the cultured the fixation procedures. Cultured cell shrink- cell surface is not readily apparent. age caused by dehydration produced an artifacRuthenium red. When the cultured cells tual stretching of the organisms, with subse- with attached treponemes were fixed in the quent straightening of the spirals (Fig. 3 and presence of ruthenium red, a striking difference in the surface of T. pallidum was observed. In 4). Although T. pallidum usually attached at the absence of ruthenium red, the treponemes one end, we were unsuccessful in demonstrat- exhibited a smooth topography, with an occaing any difference between ends. Using the sional small bulge at the surface. In contrast, resolving power of this scanning electron micro- with ruthenium red, a distinct precipitate was scope, each end appeared to be morphologically detected, resulting in a very rough surface toindistinguishable from the opposite end. pography (Fig. 9 and 10). Most treponemes apWith different preparations of T. pallidum, a peared to be ruthenium red positive. The variation in shape was observed. In the first amount of precipitated material varied; some and third set of preparations, as represented by organisms contained only a few small areas, Fig. 1 to 7, almost all of the organisms gener- whereas others were uniformly coated with a ally exhibited blunt ends. In the second set of relatively thin layer of the precipitate. Ruthepreparations, as represented by Fig. 8, almost nium red-precipitated material seemed to be all of the organisms generally exhibited very randomly distributed along the length of the tapered ends. This may reflect artifactual treponemes.
FIG. 4. Electron micrograph of T. pallidum attached to a normal rabbit testes cell. Note the two organisms attached at both ends. Bar represents 5 ,um.
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FIG. 5. Electron micrograph of normal rabbit testes cells with attached treponemes. Note the close proximity of the organisms without physical disruption of the cultured cell surface. Bar represents I Am.
DISCUSSION The attachment of T. pallidum to cultured cells is similar to attachment of other microorganisms in vivo. Kirby (13) and Ball (1) reported that spirochetes within the gut of insects were attached at their very tips. They also observed organisms attached at both ends in a manner similar to T. pallidum shown in Fig. 3 and 4. Breznak (3) suggested that this may be related to multiplication of the organisms. Takeuchi and Zeller (29) observed end-on attachment of spirochetes in the cecal and colonic epithelium of monkeys; these organisms closely abutted the plasmalemma of the brush border cytoplasm without altering the apical cytoplasm. Savage and Blumershine (23) observed organisms inhabiting murine gastrointestinal epithelium. In certain areas, virtually all organisms were attached end-on. Many attached without appearing to disrupt the epithelial sur-
face. Some organisms attached to the epithelium via weblike filaments; other organisms were in close physical proximity to the epithelial surface and did not exhibit attachment ap-
pendages.
We were unable to visualize the mechanism of treponemal attachment to the cells. At the point of attachment, no physical derangement of either the cultured cell surface or the treponeme was observed. Furthermore, there was no detectable difference between the end of the organism that preferentially attached as opposed to the end that was unattached. Attachment appeared to involve a very close physical proximity of treponemes to the cells. The lack of a disruptive influence of attached T. pallidum on the cultured cells confirms our previous observations (6-8). Virulent treponemes were detected after incubation for 7 days. During this incubation, the cultured cells retained their viability, as demonstrated by trypan-blue exclusion.
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The results obtained with ruthenium red were interesting. The precise chemical specificity of this inorganic dye remains to be clarified. This dye precipitates certain polyanions and is especially useful for demonstrating extracellular materials that are probably acidic mucopolysaccharides (14, 28). In experimental syphilis, large amounts of hyaluronic acid and chondroitin sulfate, which are acidic mucopolysaccharides, have been found in conjunction with developing dermal and testicular infection (5, 25, 30, 31). It has been speculated that T. pallidum breaks down the ground substance of tissue (5, 25), which is composed of mucopolysaccharides. The organisms may then incorporate hyaluronic acid and/or chondroitin sulfate as part of a "slime layer" that may play a role in protection against host defense mechanisms (19, 20, 26, 30, 31). Ruthenium red is also intensely reactive toward acidic phospholipids such as phosphati-
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dyl serine, phosphatidyl ethanolamine, and cardiolipin (14). Both humans and experimentally infected rabbits produce antibodies to cardiolipin (reagin). If this antigen is a surface component of the treponemes, or is a host-derived by-product of treponemal infection that accumulates at the surface of the organisms, it should be observable with ruthenium red. Further research will be required to determine whether the ruthenium red material associated with the surface of T. pallidum is a polysaccharide, a lipid, or a combination of the two. These scanning electron micrographs appear to be similar to the recently published transmission electron micrographs of T. pallidum fixed in the presence of ruthenium red (33). The presence of a ruthenium red precipitate on and around T. pallidum that had been directly fixed within infected tissue (33) excludes the possibility that the ruthenium red precipitate in our preparations was derived from the tissue
FIG. 6. Electron micrograph of T. pallidum attached to human skin epithelial cells. Note the microvilli at the cell surface. These are characteristic of epithelial cells and are not to be confused with the much longer treponemes. Bar represents 5 ,um.
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FIG. 7. Electron micrograph of T. pallidum attached to human skin epithelial cells. At a higher magnification (x13,500), there is a better differentiation of treponemes and microvilli. Bar represents 1
Aim.
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FIG. 8. Electron micrograph of a treponeme attached to a normal rabbit testes cell. Note that both ends of the organism are distinctly tapered. In all other micrographs, the ends, in general, were blunt. Bar represents 1 ,um.
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FIG. 9. Electron micrograph o0! T. pallidum attached to a normal ratbit testes cell and exposea tO ruthenium red. Note the accumulation of a precipitate at the surface ofthe treponemes. Bar represents 1 ,um.m
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FIG. 10. Electron micrograph of T. pallidum in the presence of ruthenium red. Note the rough surface appearance of the treponemes compared to the smooth appearance of treponemes in Fig. 3, 5, and 8. Bar represents 1 ,um.
culture medium during in vitro incubation with the cultured cells. The previous, report of T. pallidum exposed to ruthenium red suggested that this treponemal-associated surface material dissipates during in vitro incubation (33). If this precipitate is a mucopolysaccharide, it should be susceptible to hyaluronidase, which acts on polymers of hyaluronic acid and chondroitin sulfate. Testicular tissue is a rich source of hyaluronidase, and it is likely that a relatively large amount of the enzyme is liberated during the extraction of treponemes. Others (10, 25) have speculated that hyaluronidase from testicular tissue may affect T. pallidum. The action of hyaluronidase in degrading this treponemal-associated surface material during in vitro incubation may
partially explain the requirement of preincubation for various serological tests such as TPI (21), FTA ABS (11), TPHA (22), agglutination (10), and neutralization (2). As others have suggested, this preincubation may correspond to the time required for the dissolution of the surface material (2, 10, 15-20, 24, 27, 34). A feasible explanation for the attachment of T. pallidum to cultured cells involves the ruthenium red-positive material. Other reports utilizing different microorganisms have related attachment to the presence of ruthenium redpositive material on the surface of the microbes. These other reports suggested that this material was a mucopolysaccharide (9, 12, 28). The ruthenium red surface-associated precipitate on T. pallidum may endow the organisms with
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adhesive properties. This adhesiveness may partially explain the relatively firm attachment of the treponemes without physically disturbing the cultured cell surface. Further research will be required to characterize the potential role of mucopolysaccharides in the attachment of T. pallidum to cultured cells. ACKNOWLEDGMENTS We would like to acknowledge the very capable technical assistance rendered by Elizabeth Thompson Wolff and Beth Davidian. We wish to thank the Scanning Electron Microscope Laboratory at the Space Science Center, University of Minnesota, for use of the facilities. We are also indebted to Kari Hovind-Hougen at the Statens Seruminstitut in Denmark for incisive critical suggestions about the content of this manuscript. 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. Ball, G. H. 1969. Organisms living
on and in protozoa, 565. In T. T. Chen (ed.), Research in protozoology, vol. 3. Pergamon Press Inc., Elmsford, N.Y. Bishop, N. H., and J. N. Miller. 1976. Humoral immunity in experimental syphilis. II. The relationship of neutralizing factors in immune serum to acquired resistance. J. Immunol. 117:197-207. Breznak, J. A. 1973. Biology of non-pathogenic, host associated spirochetes. Crit. Rev. Microbiol. 2:457489. Cohen, A. L., D. P. Marlow, and G. E. Garner. 1968. A rapid critical point method using fluorocarbons ("Freons") as intermediate and transitional fluids. J. Micros. 7:331-342. De Lamater, 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. Fitzgerald, T. J., R. C. Johnson, J. A. Sykes, and J. N. Miller. 1976. Interaction of Treponema pallidum (Nichols strain) with cultured mammalian cells: effects of oxygen, reducing agents, serum supplements, and different cell types. Infect. Immun. 15:444-452. Fitzgerald, T. J., J. N. Miller, and J. A. Sykes. 1975. Treponema pallidum (Nichols strain) in tissue cultures: cellular attachment, entry, and survival. Infect. Immun. 11:1133-1140. Fitzgerald, T. J., J. N. Miller, J. A. Sykes, and R. C. Johnson. 1976. Tissue culture and Treponema pallidum, p. 57-64. In R. C. Johnson (ed.), The biology of parasitic spirochetes. Academic Press Inc., New York. Fletcher, M., and G. D. Floodgate. 1973. An electron microscopic demonstration of an acidic polysaccharide involved in the adhesion of a marine bacterium to solid surfaces. J. Gen. Microbiol. 74:325-334. Hardy, P. H., and E. E. Nell. 1957. Study of the antigenic structure df T. pallidum by specific agglutination. Am. J. Hyg. 66:160-172. Hunter, E. F., N. E. Deacon, and P. Meyer. 1964. An improved FTA test for syphilis, the absorption procedure (FTA-ABS). Public Health Rep. 79:410-412. Jones, H. C., I. L. Roth, and W. M. Sanders. 1969. Electron microscopic study of a slime layer. J. Bacteriol. 99:316-325. Kirby, H. 1941. Organisms living on and in protozoa, p. 1009-1113. In G. N. Calkins and F. M. Summers (ed.), Protozoa in biological research. Columbia University Press, New York. p.
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J. BACTERIOL. 14. Luft, J. H. 1971. Ruthenium red and violet. II. Fine structural localization in animal tissues. Anat. Rec. 171:369-416. 15. Metzger, M. 1962. A study of the role of serum and tissue lysozyme upon the treponemal inmobilization reaction. Am. J. Hyg. 76:267-275. 16. Metzger, M., P. H. Hardy, and E. E. Nell. 1961. Influence of lysozyme upon the treponeme immobilization reaction. Am. J. Hyg. 73:236-244. 17. Metzger, M., and J. Podwinska. 1965. Studies of the mechanisms of development of agglutinability of pathogenic Treponema pallidum. Arch. Immun. Ther. Exp. 13:516-524. 18. Metzger, M., and J. Ruczkowska. 1964. Influence of lysozyme upon the reactivity of Treponema pallidum in the fluorescent antibody reaction. Arch. Immun. Ther. Exp. 12:702-708. 19. Miller, J. N. 1972. Development of an experimental syphilis vaccine. Med. Clin. North Am. 56:1217-1220. 20. Miller, J. N. 1973. Immunity in experimental syphilis. VI. Successful vaccination of rabbits with Treponema pallidum, Nichols strain, attenuated by y-irradiation. J. Immunol. 110:1206-1215. 21. Nelson, R. A., and M. M. Mayer. 1949. Immobilization of Treponema pallidum in vitro by antibody produced in syphilitic infection. J. Exp. Med. 89:369-393. 22. Rathlev, T. 1967. Haemagglutination test utilizing pathogenic Treponema pallidum for the sero-diagnosis of syphilis. Br. J. Vener. Dis. 43:181-185. 23. Savage, D. C., and R. V. H. Blumershine. 1974. Surface-surface associations in microbial communities populating epithelial habitats in murine gastro-intestinal ecosystem: scanning electron microscopy. Infect. Immun. 10:240-250. 24. Schmerald, N., and B. Deubner. 1954. Electronenmikroskopische untersuchungen an Reiter-spirochaetales und Nichols-treponemen. Hautarzt 5:511-513. 25. Scott, V., and G. J. Dammin. 1950. Hyaluronidase and experimental syphilis. III. Metachromasia in syphilitic orchitis and its relationship to hyaluronic acid. Am. J. Syph. 34:501-514. 26. Smibert, R. M. 1973. Spirochaetales, a review. Crit. Rev. Microbiol. 2:491-552. 27. Swain, R. A. A. 1955. Electron microscopic studies of the morphology of pathogenic spirochaetes. J. Pathol. Bacteriol. 69:117-132. 28. Szubinska, B., and J. H. Luft. 1971. Ruthenium red and violet. III. Fine structure of the plasma membrane and extraneous coats in amoebae (A. proteus and chaoschaos). Anat. Rec. 171:417-442. 29. Takeuchi, A., and J. A. Zeller. 1972. Ultrastructural identification of spirochetes and flagellated microbes at the brush border of the large intestinal epithelium of the Rhesus monkey. Infect. Immun. 6:1008-1018. 30. Turner, T. B., and D. H. Hollander. 1950. Cortisone in experimental syphilis. Johns Hopkins Hosp. Bull. 87:505-509. 31. 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. 32. Wiegand, S. E., P. L. Strobel, and L. H. Glassman. 1972. Electron microscopic anatomy of pathogenic Treponema pallidum. J. Invest. Dermatol. 58:186204. 33. 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. 34. Zinsser, H., J. G. Hopkins, and M. McBurney. 1916. Studies on Treponema pallidum and syphilis. IV. The difference in behavior in immune serum between cultivated non-virulent Treponema pallidum and virulent treponemata from lesions. J. Exp. Med. 23:341352.
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Scanning Electron Microscopy of Treponema pallidum (Nichols Strain) Attached to Cultured Mammalian Cells T. J. FITZGERALD,* P. CLEVELAND, R. C. JOHNSON, J. N. MILLER, AND J. A. SYKES Department of Microbiology, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455*; Treponemal Research Laboratory, Department of Microbiology and Immunology, School of Medicine, University ofCalifornia at Los Angeles, Los Angeles, California 90024; and Research Department, Southern California Cancer Center, California Hospital, Los Angeles, California 90015 Received for publication 3 February 1977
This paper describes the attachment of Treponema pallidum (Nichols strain) to cultured mammalian cells as visualized by scanning electron microscopy. Treponemes were incubated for 3 h with cultured cells derived from normal rabbit testes or human skin epithelium, then fixed, processed with critical-point drying, and examined with a Cambridge Mark 2A scanning electron microscope. Large numbers of treponemes became attached to the cultured cells without
altering the morphological integrity of the cultured cells. Attachment appeared a very close physical proximity of treponemes to the cultured cells; at the site of attachment, no changes such as swelling or indentation of the cultured cell surface were observed. The addition of ruthenium red to the fixatives produced a treponemal-associated surface precipitate. This material, which is probably mucopolysaccharide and/or phospholipid, may be important in protecting the organisms against host defense mechanisms; in addition, it may be involved in the serological unresponsiveness of freshly prepared suspensions of T. pallidum. to involve
weighing 4 to 6 lb (ca. 1.8 to 2.7 kg) were used in this study. They were housed at 19 to 22°C and given antibiotic-free food and water ad libitum. T. pallidum. The Nichols strains of T. pallidum was maintained by intratesticular passage and harvested as previously described (7). Each testis was inoculated with 1 x 107 to 3 x 107 treponemes. Animals received daily injections of cortisone acetate (Merck, Sharp and Dohme) at 6 mg/kg of body weight beginning the third day after testicular inoculation. After development of a good orchitis, usually requiring 10 to 14 days, animals were sacrificed, and the testes were removed, minced with a scissors, and extracted at room temperature in tissue culture medium for 10 to 20 min. The treponemal suspension was then centrifuged at 1,000 x g for 7 min at 240C to sediment particulate matter. Cultured cells. The cells were derived from normal rabbit testes as previously described (7). A cell line derived from human skin epithelial tissue was kindly supplied by Carl Seiter of the Reheis Chemical Co. The tissue culture medium contained Eagle minimal essential medium with Hanks balanced salts, 2 x vitamins, 2 x amino acids, and 4 mM NaHCO3 buffered with 30 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid). The medium was supplemented with 10% fetal bovine serum (vol/vol) that had been heat inactivated (56°C MATERIALS AND METHODS for 30 min). Fetal bovine serum was obtained from Animals. Healthy male New Zealand white rab- the Reheis Chemical Co. The interaction of actively motile T. pallidum bits with nonreactive Rapid Plasma Reagin circle card tests (Hynson, Wescott, and Dunning, Inc.) and with cultured cells was visualized by phase-contrast 1333
Cultured mammalian cells extend the in vitro survival of the Nichols strain of Treponema pallidum (6-8). Large numbers of organisms rapidly attach to the cultured cells. This attachment prolongs the time of retention of treponemal motility and virulence. The cultured cells do not appear to be damaged by the attachment of T. pallidum. We have observed as many as 70 to 100 attached treponemes per individual cell, and yet these cultured cells retain their morphological integrity and viability. The purpose of this work was to visualize the topographical features of treponemes attached to cultured cells, with emphasis on the site of treponemal attachment. One other report consisting of one scanning electron micrograph of T. pallidum has been published (32). We have used critical-point drying techniques to improve the quality of our preparations. Attached treponemes were also fixed in the presence of ruthenium red. This resulted in the demonstration of a treponemal-associated surface material that had been previously documented using transmission electron microscopy (33).
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microscopy using Sykes-Moore chambers as previ- drying artifacts by critical-point drying with Freon (4). ously described (7). After critical-point drying, the cover slips were Scanning electron microscopy. Suspensions of T. to 13-mm aluminum stubs with silver-conattached x 9 to 107 x 7 pallidum adjusted to approximately with carbon and gold in a 107 organisms/ml were inoculated into Sykes-Moore ducting paint and coatedevaporator. The specimens chambers containing cultured cells at approximately Denton DV-502 vacuum Mark 20 to 40% confluency. After a 3-h incubation at 360C, were examined at a 450 angle in aatCambridge kV. 20 microscope electron scanning the 2A to attached had treponemes of a large number Ruthenium red. Selected specimens were exposed cells. The chambers were flushed with 3 volumes of Five milligrams of tissue culture medium without serum to remove to ruthenium red during fixation. to each milliliter of unattached treponemes. Excess medium was dis- this inorganic dye was added and osmium tetroxide fixcarded. Cells with attached organisms were fixed by both the glutaraldehyde were dehydrated and specimens These solutions. ing with directly adding 2% glutaraldehyde (buffered 0.1 M sodium cacodylate to pH 7.4) to the chambers critical point-dried as described above. for 1 h at 20°C and then for 16 h at 4°C. The cover RESULTS slips containing the cultured cells and treponemes were removed from the chambers, washed twice Attachment. The electron micrographs with cacodylate buffer, and postfixed in 1% osmium tetroxide (buffered as above) for 1 h. The cover slips within this paper were prepared after a 3-h were then washed twice with distilled water and incubation of T. pallidum with cultured cells. dehydrated in a graded series of ethanol to 100%. The fixation and critical-point drying techThis was followed by a graded series of Freon TF in niques successfully preserved the morphologiethanol to 100% Freon TF. The specimens on the cal integrity of both the cultured cells and the cover slips were rapidly transferred to a Bomar critiwithout cal-point drying device and preserved against air- treponemes. The organisms attached
FIG. 1. Electron micrograph of a normal rabbit testes cell with attached T. pallidum. Note the intact morphological integrity of the cell. Bar represents 11 ,um.
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FIG. 2. Electron micrograph of a portion of a normal rabbit testes cell with attached T. pallidum. Note what appears to be an unwinding axial filament at the left, lower center (arrow). Bar represents 10 ,um.
producing any observable alteration in the topography of the cultured cells. With suspensions containing 7 x 107 to 9 x 107 treponemes/ ml and cells at 20 to 40% confluency, as many as 50 to 70 treponemes were attached per individually cultured cell (Fig. 1 and 2). Treponemes appeared to be randomly distributed on the cell surface. The organisms did not exhibit a predilection for one specific area of the cells, such as the nuclear region (slightly elevated interior part of the cell) or the thin lamellae of the peripheral edge. Although certain
areas of some cells contained larger groups of organisms, no patterns suggestive of a definite preference for one specific area emerged. T. pallidum attached at its distal end as shown in Fig. 3. Some of these micrographs may be misleading in that a number of the treponemes appear to be attached along their entire length. However, this is artifactual as revealed by phase-contrast microscopy of attached, actively motile organisms. The treponemes attach in a number of different planes and during fixation settle on the cell surface,
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FIG. 3. Electron micrograph of T. pallidum attached at distal ends to the edge of a normal rabbit testes cell. One treponeme is attached at both ends. Bar represents 5 ,um.
giving the appearance of full-length attachment. Occasionally, T. pallidum attached at both ends as shown in Fig. 3 and 4. This occurred with 5 to 10% of the attached organisms. These treponemes may represent two organisms attached at one end just prior to division or an individual organism attached at both ends. This observation was not an artifact inasmuch as it was routinely detected with phase-contrast microscopy of actively motile organisms. There were no detectable physical disruptions of the cultured cell surface at the site of treponemal attachment. The point of attachment appeared to involve a very close physical proximity (Fig. 3-5). With higher magnifications (x 100,000), no swelling or indentation of the cell surface was observed. Figures 1 to 5 demonstrate treponemal attachment to normal rabbit testes cells, which are essentially fibroblastic-like. T. pallidum also attached to epithelial cells, as shown in
Fig. 6 and 7 which depict attachment to human skin epithelial cells, a cell line derived from human skin epithelium. Numerous microvilli at the cell surface are evident; these are characteristic of epithelial cells and are not to be confused with the much longer treponemes. Treponemes. Treponemal morphology was well preserved. Flagella (axial filaments) were not apparent on the surface of intact organisms. This may have been due to either the surface coating of the carbon and gold and/or the resolving power of the microscope. Occasionally, flagella were detected as they unwound from partially degraded organisms. This occurred with a very small proportion of the total treponemes visualized (less than 0.1%). Part of an apparently unwinding flagella is evident in Fig. 2 (left, lower center [arrow]). The lack of treponemes on the surface of the glass, away from the cultured cells, was due to the washing procedures prior to fixation. This was apparent by phase-contrast microscopy of
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the preparations before and after washing. rather than actual differences. The organism Some of the treponemes that were attached to pictured in Fig. 8 is attached to a cultured cell. the cells and to the glass were damaged during Because of the high magnification, the cultured the fixation procedures. Cultured cell shrink- cell surface is not readily apparent. age caused by dehydration produced an artifacRuthenium red. When the cultured cells tual stretching of the organisms, with subse- with attached treponemes were fixed in the quent straightening of the spirals (Fig. 3 and presence of ruthenium red, a striking difference in the surface of T. pallidum was observed. In 4). Although T. pallidum usually attached at the absence of ruthenium red, the treponemes one end, we were unsuccessful in demonstrat- exhibited a smooth topography, with an occaing any difference between ends. Using the sional small bulge at the surface. In contrast, resolving power of this scanning electron micro- with ruthenium red, a distinct precipitate was scope, each end appeared to be morphologically detected, resulting in a very rough surface toindistinguishable from the opposite end. pography (Fig. 9 and 10). Most treponemes apWith different preparations of T. pallidum, a peared to be ruthenium red positive. The variation in shape was observed. In the first amount of precipitated material varied; some and third set of preparations, as represented by organisms contained only a few small areas, Fig. 1 to 7, almost all of the organisms gener- whereas others were uniformly coated with a ally exhibited blunt ends. In the second set of relatively thin layer of the precipitate. Ruthepreparations, as represented by Fig. 8, almost nium red-precipitated material seemed to be all of the organisms generally exhibited very randomly distributed along the length of the tapered ends. This may reflect artifactual treponemes.
FIG. 4. Electron micrograph of T. pallidum attached to a normal rabbit testes cell. Note the two organisms attached at both ends. Bar represents 5 ,um.
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FIG. 5. Electron micrograph of normal rabbit testes cells with attached treponemes. Note the close proximity of the organisms without physical disruption of the cultured cell surface. Bar represents I Am.
DISCUSSION The attachment of T. pallidum to cultured cells is similar to attachment of other microorganisms in vivo. Kirby (13) and Ball (1) reported that spirochetes within the gut of insects were attached at their very tips. They also observed organisms attached at both ends in a manner similar to T. pallidum shown in Fig. 3 and 4. Breznak (3) suggested that this may be related to multiplication of the organisms. Takeuchi and Zeller (29) observed end-on attachment of spirochetes in the cecal and colonic epithelium of monkeys; these organisms closely abutted the plasmalemma of the brush border cytoplasm without altering the apical cytoplasm. Savage and Blumershine (23) observed organisms inhabiting murine gastrointestinal epithelium. In certain areas, virtually all organisms were attached end-on. Many attached without appearing to disrupt the epithelial sur-
face. Some organisms attached to the epithelium via weblike filaments; other organisms were in close physical proximity to the epithelial surface and did not exhibit attachment ap-
pendages.
We were unable to visualize the mechanism of treponemal attachment to the cells. At the point of attachment, no physical derangement of either the cultured cell surface or the treponeme was observed. Furthermore, there was no detectable difference between the end of the organism that preferentially attached as opposed to the end that was unattached. Attachment appeared to involve a very close physical proximity of treponemes to the cells. The lack of a disruptive influence of attached T. pallidum on the cultured cells confirms our previous observations (6-8). Virulent treponemes were detected after incubation for 7 days. During this incubation, the cultured cells retained their viability, as demonstrated by trypan-blue exclusion.
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The results obtained with ruthenium red were interesting. The precise chemical specificity of this inorganic dye remains to be clarified. This dye precipitates certain polyanions and is especially useful for demonstrating extracellular materials that are probably acidic mucopolysaccharides (14, 28). In experimental syphilis, large amounts of hyaluronic acid and chondroitin sulfate, which are acidic mucopolysaccharides, have been found in conjunction with developing dermal and testicular infection (5, 25, 30, 31). It has been speculated that T. pallidum breaks down the ground substance of tissue (5, 25), which is composed of mucopolysaccharides. The organisms may then incorporate hyaluronic acid and/or chondroitin sulfate as part of a "slime layer" that may play a role in protection against host defense mechanisms (19, 20, 26, 30, 31). Ruthenium red is also intensely reactive toward acidic phospholipids such as phosphati-
1339
dyl serine, phosphatidyl ethanolamine, and cardiolipin (14). Both humans and experimentally infected rabbits produce antibodies to cardiolipin (reagin). If this antigen is a surface component of the treponemes, or is a host-derived by-product of treponemal infection that accumulates at the surface of the organisms, it should be observable with ruthenium red. Further research will be required to determine whether the ruthenium red material associated with the surface of T. pallidum is a polysaccharide, a lipid, or a combination of the two. These scanning electron micrographs appear to be similar to the recently published transmission electron micrographs of T. pallidum fixed in the presence of ruthenium red (33). The presence of a ruthenium red precipitate on and around T. pallidum that had been directly fixed within infected tissue (33) excludes the possibility that the ruthenium red precipitate in our preparations was derived from the tissue
FIG. 6. Electron micrograph of T. pallidum attached to human skin epithelial cells. Note the microvilli at the cell surface. These are characteristic of epithelial cells and are not to be confused with the much longer treponemes. Bar represents 5 ,um.
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FIG. 7. Electron micrograph of T. pallidum attached to human skin epithelial cells. At a higher magnification (x13,500), there is a better differentiation of treponemes and microvilli. Bar represents 1
Aim.
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FIG. 8. Electron micrograph of a treponeme attached to a normal rabbit testes cell. Note that both ends of the organism are distinctly tapered. In all other micrographs, the ends, in general, were blunt. Bar represents 1 ,um.
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FIG. 9. Electron micrograph o0! T. pallidum attached to a normal ratbit testes cell and exposea tO ruthenium red. Note the accumulation of a precipitate at the surface ofthe treponemes. Bar represents 1 ,um.m
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.
FIG. 10. Electron micrograph of T. pallidum in the presence of ruthenium red. Note the rough surface appearance of the treponemes compared to the smooth appearance of treponemes in Fig. 3, 5, and 8. Bar represents 1 ,um.
culture medium during in vitro incubation with the cultured cells. The previous, report of T. pallidum exposed to ruthenium red suggested that this treponemal-associated surface material dissipates during in vitro incubation (33). If this precipitate is a mucopolysaccharide, it should be susceptible to hyaluronidase, which acts on polymers of hyaluronic acid and chondroitin sulfate. Testicular tissue is a rich source of hyaluronidase, and it is likely that a relatively large amount of the enzyme is liberated during the extraction of treponemes. Others (10, 25) have speculated that hyaluronidase from testicular tissue may affect T. pallidum. The action of hyaluronidase in degrading this treponemal-associated surface material during in vitro incubation may
partially explain the requirement of preincubation for various serological tests such as TPI (21), FTA ABS (11), TPHA (22), agglutination (10), and neutralization (2). As others have suggested, this preincubation may correspond to the time required for the dissolution of the surface material (2, 10, 15-20, 24, 27, 34). A feasible explanation for the attachment of T. pallidum to cultured cells involves the ruthenium red-positive material. Other reports utilizing different microorganisms have related attachment to the presence of ruthenium redpositive material on the surface of the microbes. These other reports suggested that this material was a mucopolysaccharide (9, 12, 28). The ruthenium red surface-associated precipitate on T. pallidum may endow the organisms with
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FITZGERALD ET AL.
adhesive properties. This adhesiveness may partially explain the relatively firm attachment of the treponemes without physically disturbing the cultured cell surface. Further research will be required to characterize the potential role of mucopolysaccharides in the attachment of T. pallidum to cultured cells. ACKNOWLEDGMENTS We would like to acknowledge the very capable technical assistance rendered by Elizabeth Thompson Wolff and Beth Davidian. We wish to thank the Scanning Electron Microscope Laboratory at the Space Science Center, University of Minnesota, for use of the facilities. We are also indebted to Kari Hovind-Hougen at the Statens Seruminstitut in Denmark for incisive critical suggestions about the content of this manuscript. 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. Ball, G. H. 1969. Organisms living
on and in protozoa, 565. In T. T. Chen (ed.), Research in protozoology, vol. 3. Pergamon Press Inc., Elmsford, N.Y. Bishop, N. H., and J. N. Miller. 1976. Humoral immunity in experimental syphilis. II. The relationship of neutralizing factors in immune serum to acquired resistance. J. Immunol. 117:197-207. Breznak, J. A. 1973. Biology of non-pathogenic, host associated spirochetes. Crit. Rev. Microbiol. 2:457489. Cohen, A. L., D. P. Marlow, and G. E. Garner. 1968. A rapid critical point method using fluorocarbons ("Freons") as intermediate and transitional fluids. J. Micros. 7:331-342. De Lamater, 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. Fitzgerald, T. J., R. C. Johnson, J. A. Sykes, and J. N. Miller. 1976. Interaction of Treponema pallidum (Nichols strain) with cultured mammalian cells: effects of oxygen, reducing agents, serum supplements, and different cell types. Infect. Immun. 15:444-452. Fitzgerald, T. J., J. N. Miller, and J. A. Sykes. 1975. Treponema pallidum (Nichols strain) in tissue cultures: cellular attachment, entry, and survival. Infect. Immun. 11:1133-1140. Fitzgerald, T. J., J. N. Miller, J. A. Sykes, and R. C. Johnson. 1976. Tissue culture and Treponema pallidum, p. 57-64. In R. C. Johnson (ed.), The biology of parasitic spirochetes. Academic Press Inc., New York. Fletcher, M., and G. D. Floodgate. 1973. An electron microscopic demonstration of an acidic polysaccharide involved in the adhesion of a marine bacterium to solid surfaces. J. Gen. Microbiol. 74:325-334. Hardy, P. H., and E. E. Nell. 1957. Study of the antigenic structure df T. pallidum by specific agglutination. Am. J. Hyg. 66:160-172. Hunter, E. F., N. E. Deacon, and P. Meyer. 1964. An improved FTA test for syphilis, the absorption procedure (FTA-ABS). Public Health Rep. 79:410-412. Jones, H. C., I. L. Roth, and W. M. Sanders. 1969. Electron microscopic study of a slime layer. J. Bacteriol. 99:316-325. Kirby, H. 1941. Organisms living on and in protozoa, p. 1009-1113. In G. N. Calkins and F. M. Summers (ed.), Protozoa in biological research. Columbia University Press, New York. p.
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