Vol. 22, No. 3
INFECTION AND IMMUNITY, Dec. 1978, p. 689-697 0019-9567/78/0022-0689$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Influence of Oxygen Tension, Sulfhydryl Compounds, and Serum on the Motility and Virulence of Treponema pallidum (Nichols Strain) in a Cell-Free System STEVEN J. NORRIS,' * JAMES N. MILLER,' JOHN A. SYKES,2
AND
THOMAS J. FITZGERALD3
Treponemal Research Laboratory, Department ofMicrobiology and Immunology, UCLA School of Medicine, Los Angeles, California 90024;' Research Department, Southern California Cancer Center, California Hospital, Los Angeles, California 90015;' Department of Microbiology, University of Minnesota School of Medicine, Minneapolis, Minnesota 554553 Received for publication 11 September 1978
The motility and virulence of Treponema pallidum (Nichols strain) were monitored during incubation in a modified tissue culture medium to study the effects of oxygen tension and medium composition on survival of the organism. A basal medium of Eagle minimal essential medium with 50% fresh, heat-inactivated normal rabbit serum was used inasmuch as better survival occurred with 50% normal rabbit serum than with lower concentrations. Addition of 0.5 to 2.0 mM dithiothreitol or 2.0 mM dithioerythritol to the basal medium led to significantly longer retention of T. pGclidum viability in the presence of 3% oxygen than under aerobic or anaerobic conditions. The results of this investigation lend support to the classification of T. pallidum as a microaerophilic organism and provide direction for the design of potentially successful culture systems, with or without tissue culture cells.
The in vitro cultivation of Treponema pallidum, the causative agent of syphilis, continues to be an elusive goal. Numerous reports of successful cultivation emanated from early research, but attempts of confirmation yielded negative results (31); the so-called avirulent cultivatable strains of T. pallidum have recently been shown by DNA hybridization studies to be unrelated to virulent T. pallidum (20). Many investigators have studied the effects of medium components on the in vitro survival of T. pallidum. As a result, serum tissue extracts, sulfhydryl compounds, carbon dioxide, sodium pyruvate, glucose, and magnesium salts have been reported to contribute to the retention of motility and virulence by T. pallidum; in no instance, however, was an increase in the number of T. pallidum observed (11, 16, 17, 22, 30). Mammalian cells have been shown to extend the in vitro survival of T. pallidum and have been used widely in recent cultivation attempts (6-9, 15, 24, 26-28). Systems utilizing tissue culture cells and intermediate levels of oxygen have been shown to be particularly effective in maintaining the motility and virulence of T. pallidum (6, 9, 26). Jones et al. (15) reported the multiplication of T. pallidum in baby hamster kidney cell cultures; however, their results were equiv-
transient increases in the number of T. pallidum in a tissue culture system utilizing 3 to 4% oxygen. Based on its survival under anaerobic conditions and rapid death upon exposure to air, T. pallidum traditionally has been considered to be a strict anaerobe. However, recent studies have suggested that oxygen may play a major role in the metabolism of the bacterium (2, 5, 23). The purpose of the present study was to examine in detail the effects of oxygen, sulfhydryl compounds, and serum on the survival of virulent T. pallidum (Nichols strain) in a cell-free system so that our findings could be used in subsequent cultivation attempts. MATERIALS AND METHODS Rabbits. Adult male New Zealand white rabbits were housed in air-conditioned quarters at 18 to 20'C and fed antibiotic-free rabbit chow and water ad libitum. Rabbits with nonreactive VDRL and TPI tests whose clipped backs were initially free of irregular or heavy hair patterns were selected for virulence deter-
minations. Media components and preparation. All chemicals were of reagent grade. The F16 formulation of Eagle minimal essential medium (MEM, Grand Island Biological Co.) was dissolved in double-distilled water, supplemented with 20 mM HEPES (N-2-hydroxyocal and have not been confirmed (10). Sandok ethyl piperazine-N'-2-ethanesulfonic acid) (Calbiet al. (26) recently reported the observation of ochem), adjusted to pH 7.2, and sterilized by filtration. 689
690
NORRIS ET AL.
Fresh normal rabbit serum (NRS) was obtained from VDRL nonreactive rabbits 2 to 3 h before use and heat-inactivated at 560C for 30 min. When chemical reagents were added to the basal medium, concentrated solutions of the compounds were made in MEM or 0.85% NaCl, filter sterilized, and added aseptically to the MEM portion of the medium. In most experiments, solutions were prepared immediately before use; in some cases, the solutions were stored for a maximum of 2 weeks at -60'C. Just prior to the extraction of T. pallidum, the MEM components of the media containing the various compounds were combined with fresh, heat-inactivated NRS and equilibrated with an atmosphere of 95% N2:5% CO2 for 30 min at 34WC. Extraction of T. pallidurm The Nichols strain of T. pallidum was maintained by intratesticular passage in rabbits. Treponemes were obtained from rabbit testes with a firm orchitis which developed 7 to 10 days postinfection. The testes were removed aseptically, rinsed with sterile 0.85% saline, and each was sliced into eight sections. Unless otherwise noted, the tissue was evenly distributed in screw-capped test tubes (25 by 150 mm) containing 5 ml of the medium to be tested. After 3 to 15 min of extraction on a shaker, these suspensions containing 1 x 106 to 20 x 106 T. pallidum per ml were centrifuged at 1,000 x g for 7 min to remove tissue debris, and each was pipetted in 1-ml aliquots into sterile test tubes (13 x 100 mm) with gas-permeable closures. The time from removal of the testes to the beginning of the incubation period ranged from 30 to 60 min. Conditions of incubation. Treponemal suspensions were incubated at 340C under three atmospheric conditions: aerobic (21% oxygen), anaerobic, and 3% oxygen. Aerobic samples were incubated in the presence of air. Anaerobic conditions were established by placing the samples in an anaerobic jar (BBL), evacuating to -J
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FIG. 4. Results of a combined motility-virulence experiment in which suspensions of 1.0 x T. pallidum per ml in MEM-50%o NRS with or without 1.0 mMDTT were incubated concurrently under aerobic, 3% oxygen, or anaerobic conditions. Motility values represent the means of three determinations. Virulence was determined by the id. inoculation of five or ten rabbits at single sites with 0.1 ml of the suspensions and daily examination of the sites for lesion development; data represent the mean time of lesion development. Lesions occurred at all inoculation sites unless otherwise indicated (sites positive per total sites). Motility (A) and virulence (B) results were qualitatively similar; optimal retention of both parameters occurred when DTT was added to the medium and the suspensions were incubated in an atmosphere containing 3o oxygen.
167
of days. This change in appearance indicated that sublethal damage to the structural elements of T. pallidum had taken place prior to the loss of motility. Effect of medium and atmospheric conditions on mammalian cells. Since one of the goals of this study was to develop an incubation system which could be used in conjunction with mammalian cell cultures, the conditions found beneficial to the retention of T. pallidum viability were tested for their effect on NRT cells. Secondary NRT cultures were incubated in MEM-50% NRS + 1.0 mM DTT at 340C in either Sykes-Moore chambers (37), T-15 culture flasks, or roller bottles. Incubation under either aerobic or 3% 02 conditions yielded healthy cultures which grew to confluency at the expected rate. In contrast, identical cultures incubated in 95% N2:5% CO2 exhibited abnormal cell morphology with cellular retraction, extensive vacuolization, and cell death. Thus, 3% 02 conditions were compatible with NRT cell growth
whereas anaerobic conditions suitable.
were
clearly
un-
DISCUSSION Concurrent incubation of T. pallidum suspensions with different medium additives under varied oxygen tensions allowed examination of the toxic and beneficial effects of oxygen and the mechanism of action of various medium components. The three atmospheric conditions used in this study provided a range of oxygen tensions and thus of oxygen toxicities. Increasing NRS concentrations resulted in enhanced retention of motility under both anaerobic and 3% 02 conditions. Addition of sulfhydryl compounds prolonged survival of T. pallidum in oxygen-containing environments but had no beneficial effect under anaerobic conditions. Thus the effect of serum is independent of the presence of oxygen, whereas the effect of sulfhydryl compounds is oxygen dependent. Suspensions of T. pallidum incubated in MEM-50% NRS with DTT or DTE
VOL. 22, 1978
FACTORS INFLUENCING T. PALLIDUM IN VITRO
under 3% 02 conditions retained their viability for a longer period than under anaerobic conditions, suggesting that T. pallidum is probably a microaerophilic rather than an anaerobic organism. Increasing NRS levels from 10 to 50% resulted in an extension of motility under anaerobic and 3% 02 conditions (Fig. 1, Table 2). Since it is possible that essential serum components are depleted during long-term incubation and may lead to loss of viability (25, 30), an increase in serum concentration or the addition of serum during the incubation period may extend the availability of these components and thereby prolong survival. The toxic effect of oxygen upon T. pallidum was most clearly exhibited when treponemes suspended in MEM-50% NRS were incubated under different 02 tensions (Fig. 2). Under those conditions, in which no compounds were added to the medium to protect the bacteria from oxygen toxicity, the loss of treponemal motility and virulence was related to the concentration of oxygen in the gaseous phase. Certain sulfhydryl compounds are effective in counteracting the toxic effects of oxygen on T. pallidum (6, 7, 11, 22, 30). Weber (30) found that, in an aerobic incubation system utilizing a paraffin overlay to partially exclude oxygen, addition of thioglycolic acid, mercaptosuccinic acid, DL-homocysteine, L-cysteine, or reduced glutathione resulted in extension of the 50% motility end point from 11.5 h to 12 to 18 days. He suggested that the active sulfhydryl compounds did not act solely through a reduction of redox potential, but also had some metabolic or protective activity due to their sulfhydryl groups and polar nature. Examination of the effects of sulfhydryl compounds under different 02 tensions in the present study revealed that treponemal motility and virulence were extended by these compounds only under atmospheric conditions containing significant levels of oxygen (Table 1, Fig. 3 and 4). None of the sulfhydryl compounds tested had a consistent positive effect under anaerobic conditions. This result indicated that the activity of sulfhydryl compounds was somehow involved with oxygen, the most obvious interpretation being that the compounds protected T. pallidum from the toxic effects of oxygen. In contrast, previous reports have indicated that sulfhydryl compounds were effective in extending the survival of T. pallidum under anaerobic conditions (13, 26, 31, 33); this could be explained as an inadvertent exposure of the T. pallidum suspensions to significant levels of oxygen in these studies (2). The mode of action by which DTT and other sulfhydryl compounds lessen oxygen toxicity
695
and extend motility requires further study. Only reduced sulfhydryl compounds are effective in extending motility (30), implicating the hydrogen-donating sulfhydryl group in their activity. The oxidation of sulf'hydryl groups is thought to be mediated by oxygen radicals (14), and the observed protective effect may lie in their ability to scavenge these highly reactive and toxic oxygen derivatives (1). The fact that DTT and DTE possess unusually low redox potentials and yet oxidize more slowly than cysteine or glutathione (4) may in part account for their special ability to extend the survival of T. pallidum under 3% 02 conditions. The effectiveness of DTT under 3% 02 conditions was reduced by the addition of cysteine or glutathione (Table 1), suggesting that these compounds accelerated the oxidation of DTT through their own oxidation and subsequent reduction by DTT (4). Low levels of oxygen appeared to be beneficial to the maintenance of T. pallidum motility and virulence when the toxic effects of oxygen were counteracted by the presence of DTT. Prolonged retention of motility and high degrees of virulence were consistently observed for treponemal suspensions containing 0.5 to 2.0 mM DTT or 2.0 mM DTE incubated in 3% 02 as compared to suspensions incubated anaerobically either with or without these compounds. Another explanation is that oxygen modifies DTT to a form beneficial to T. pallidum, but this possibility was negated by the finding that the disulfide product of DTT oxidation (4) was ineffective in extending treponemal motility. That oxygen is, under special conditions, beneficial to the in vitro survival of T. pallidum is consistent with previous indications that it may play a role in the metabolism of the bacterium. Cox and Barber (5) reported that suspensions of T. pallidum consumed oxygen at a rate approximately half that of a known aerobic spirochete, Leptospira B16. Biochemical studies by Baseman et al. (2) and Nichols and Baseman (23) showed that C02 was evolved as a result of the oxidative degradation of glucose and pyruvate and that incorporation of tritiated amino acids or uridine into acid-precipitable material was maximal at gaseous 02 tensions of 10 to 20% as compared to higher and lower concentrations. Ultrasonic lysates of T. pallidum exhibit UV absorbtion spectra consistent with those of cytochromes and flavoproteins, suggesting that a terminal electron transport system may exist in the bacterium (18). Conflicting results have been obtained for the effects of azide and other inhibitors of oxidative phosphorylation upon the metabolism of T. pallidum (2, 5). Prolonged survival of T. pallidum under conditions of limited oxygen exposure has been
696
INFECT. IMMUN.
NORRIS ET AL.
Health Organization agreement V3/181/26, and the noted in tissue culture interaction studies. Fitz- World Soiland Cancer Foundation. S.J.N. is the recipient of gerald et al. (7) found that an atmosphere con- Albert predoctoral Public Health Service National Research Service taining 3% 02 was superior to aerobic and anaer- Award GM07185 in Cellular and Molecular Biology from the obic conditions in maintaining treponemal mo- National Institute of General Medical Sciences. tility and virulence in the presence of SflEp cells LITERATURE CITED and a medium containing cysteine, glutathione, S. Kanematsu. 1976. Reactivity of thiols 1. and Asada, K., and DTT. By utilizing an oxygen gradient syswith superoxide radicals. Agr. Biol. Chem. 40:1891tem with SflEp cells and a prereduced medium, 1892. Fieldsteel et al. (6) showed that attachment to 2. Baseman, J. B., J. C. Nichols, and N. S. Hayes. 1976. Virulent Treponema pallidum: aerobe or anaerobe. Inthe cell cultures and retention of viability of T. fect. Immun. 13:704-711. pallidum were optimal at the boundary of oxy3. Clark, J. W., Jr. 1962. Loss of virulence in vitro of motile gen penetration; virulent treponemes were presTreponema pallidum. Br. J. Vener. Dis. 38:78-81. ent at this interface up to 21 days following 4. Cleland, W. W. 1964. Dithiothreitol, a new protective reagent for SH groups. Biochemistry 3:480-482. inoculation. These studies suggest that low levels of oxygen are beneficial to T. pallidum. How- 5. Cox, C. D., and M. K. Barber. 1974. Oxygen uptake by Treponema pallidum. Infect. Immun. 10:123-127. ever, there exists the equally likely possibility 6. Fieldsteel, A. H., F. A. Becker, and J. G. Stout. 1977. that treponemal survival is optimal at the lowest Prolonged survival of virulent Treponema pallidum oxygen concentration which can maintain mam(Nichols strain) in cell-free and tissue culture systems. Infect. Immun. 18:173-182. malian cell viability. Thus no definitive concluT. J., R. C. Johnson, J. A. Sykes, and J. sions regarding the relationship between T. pal- 7. Fitzgerald, N. Miller. 1977. Further observations on the interaction lidum and oxygen can be drawn from tissue of Treponema pallidum (Nichols strain) with cultured culture interaction studies. mammalian cells: effects of oxygen, reducing agents, serum supplements, and other cell types. Infect. Immun. The role of oxygen in the biology of T. palli15:444452. dum has been discussed previously (2, 5, 9, 18, 8. Fitzgerald, T. J., J. N. Miller, and J. A. Sykes. 1975. 23). The finding that oxygen is both extremely Treponemapallidum (Nichols strain) in tissue cultures: cellular attachment, entry, and survival. Infect. Immun. toxic to and, under certain conditions, beneficial 11:1133-1140. to T. pallidum appears on its surface to be T. J., J. N. Miller, J. A. Sykes, and R. C. contradictory. However, it is well known that 9. Fitzgerald, Johnson. 1976. Tissue culture and Treponema pallioxygen, if present in sufficiently high concentradum, p. 57-64. In R. C. Johnson (ed.), The biology of tions, is toxic to all organisms from E. coli to parasitic spirochetes. Academic Press Inc., New York. humans (13, 32). Most organisms which live in 10. Foster, J. W., D. S. Kellogg, J. W. Clark, and A. Balows. 1977. The in vitro cultivation of Treponema the presence of oxygen have developed elaborate pallidum. Corroborative studies. Br. J. Vener. Dis. means of neutralizing oxygen radicals and their 53:338-339. products; superoxide dismutase, glutathione per- 11. Graves, S. R., P. L. Sandok, H. M. Jenkin, and R. C. Johnson. 1975. Retention of motility and virulence of oxidase, catalases, peroxidases, and alpha-toTreponema pallidum (Nichols strain) in vitro. Infect. copherol have all been implicated in this role Immun. 12:1116-1120. (12). Although T. pallidum thrives in well-oxy- 12. Halliwell, B. 1974. Superoxide dismutase, catalase, and genated tissues and seems to utilize oxygen in its glutathione peroxidase: solutions to the problems of living with oxygen. New Phytol. 73:1075-1086. metabolism, it has a very low tolerance for the toxic effects of oxygen. It is possible that T. 13. Haugaard, N. 1968. Cellular mechanisms of oxygen toxicity. Physiol. Rev. 48:311-373. pallidum is an obligate parasite and is difficult 14. Jocelyn, P. C. 1972. Biochemistry of the SH group: the to culture in vitro because of its reliance on the occurrence, chemical properties, metabolism, and biological function of thiols and disulfides. Academic Press host's ability to scavenge oxygen radicals. The Inc., New York. beneficial effects which tissue culture cells and R. H., M. A. Finn, J. J. Thomas, and C. Folger. sulfhydryl compounds have on the in vitro sur- 15. Jones, 1976. Growth and subculture of pathogenic T. paffidum vival of T. pallidum may in part be due to their (Nichols strain) in BHK-21 cultured tissue cells. Br. J. ability to provide this activity (9). Vener. Dis. 52:18-23. In our opinion, the results of this study are 16. Kimm, G. E., R. Allen, H. J. Morton, and J. F. Morgan. 1960. Enhancement of survival in vitro of Trepoconsistent with the classification of T. pallidum nema pallidum by addition of glucose and magnesium. as a microaerophilic organism and provide direcJ. Bacteriol. 80:726-727. tion for the design of potentially successful cul- 17. Kimm, G. E., R. H. Allen, H. J. Morton, and J. F. Morgan. 1962. Studies on the in vitro survival of viruture systems, with or without tissue culture cells. ACKNOWLEDGMENTS We thank Mark Falkenbach, Frank Fazzan, Ruth Elwell, and Renee Norris for their invaluable assistance. This work was supported by the Office of Naval Research contract N00014-76-C-0148, Public Health Service contract AI-42538 from the National Institute of Allergy and Infectious Diseases,
lent Treponema pallidum. I. Methodology and basal synthetic medium. Am. J. Hyg. 75:339-346. 18. Lysko, P. G., and C. D. Cox. 1977. Terminal electron transport in Treponema pallidum. Infect. Immun.
16:885-890.
19. Magnuson, H. J., H. Eagle, and R. Fleischman. 1948. The minimal infectious dose of Spirochaeta pallida (Nichols strain) and a consideration of its rate of mul-
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FACTORS INFLUENCING T. PALLIDUM IN VITRO
tiplication in vivo. Am. J. Syph. 32:1-18. 20. Miao, R., and A. H. Fieldsteel. 1978. Genetics of Treponema: relationship between Treponema pallidum and five cultivatable treponemes. J. Bacteriol. 133:101107. 21. Miller, J. N. (ed.). 1971. Spirochetes in body fluids and tissues: manual of investigative methods. Charles C. Thomas, Springfield, Illinois. 22. Nelson, R. A. 1948. Factors affecting the survival of Treponema pallidum in vitro. Am. J. Hyg. 48:120-131. 23. Nichols, J. C., and J. B. Baseman. 1978. Ribosomal ribonucleic acid synthesis by virulent Treponema pallidum. Infect. Immun. 19:854-860. 24. Rathlev, T. 1975. Investigations on in vitro survival and virulence of T. pallidum under aerobiosis. Br. J. Vener. Dis. 51:296-300. 25. Rice, F. A. H., and R. A. Nelson. 1951. The isolation from beef serum of a survival factor for Treponema pallidum. J. Biol. Chem. 191:35-41. 26. Sandok, P. L., H. M. Jenkin, S. R. Graves, and S. T. Knight. 1976. Retention of motility of Treponema pallidum (Nichols strain) in an anaerobic cell culture sys-
697
tem and in a cell-free system. J. Clin. Microbiol.
3:72-74. 27. Sandok, P. L., H. M. Jenkin, H. M. Matthews, and M. S. Roberts. 1978. Unsustained multiplication of Treponema pallidum (Nichols virulent strain) in vitro in the presence of oxygen. Infect. Immun. 19:421-429. 28. Sandok, P. L., S. T. Knight, and H. M. Jenkin. 1976. Examination of various cell culture techniques for coincubation of virulent Treponema pallidum (Nichols I strain) under anaerobic conditions. J. Clin. Microbiol. 4:360-371. 29. Sykes, J. A., and E. B. Moore. 1960. A simple tissue culture chamber. Tex. Rep. Biol. Med. 18:288-297. 30. Weber, M. D. 1960. Factors influencing the in vitro survival of Treponema pallidum. Am. J. Hyg. 71:401-417. 31. Willcox, R. R., and T. Guthe. 1966. Treponema pallidum: a bibliographical review of the morphology, culture, and survival of T. pallidum and related organisms. Bull. WHO 35(Suppl.):1-169. 32. Yost, F. J., and I. Fridovich. 1976. Superoxide and hydrogen peroxide in oxygen damage. Arch. Biochem. Biophys. 175:514-519.
INFECTION AND IMMUNITY, Dec. 1978, p. 689-697 0019-9567/78/0022-0689$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Influence of Oxygen Tension, Sulfhydryl Compounds, and Serum on the Motility and Virulence of Treponema pallidum (Nichols Strain) in a Cell-Free System STEVEN J. NORRIS,' * JAMES N. MILLER,' JOHN A. SYKES,2
AND
THOMAS J. FITZGERALD3
Treponemal Research Laboratory, Department ofMicrobiology and Immunology, UCLA School of Medicine, Los Angeles, California 90024;' Research Department, Southern California Cancer Center, California Hospital, Los Angeles, California 90015;' Department of Microbiology, University of Minnesota School of Medicine, Minneapolis, Minnesota 554553 Received for publication 11 September 1978
The motility and virulence of Treponema pallidum (Nichols strain) were monitored during incubation in a modified tissue culture medium to study the effects of oxygen tension and medium composition on survival of the organism. A basal medium of Eagle minimal essential medium with 50% fresh, heat-inactivated normal rabbit serum was used inasmuch as better survival occurred with 50% normal rabbit serum than with lower concentrations. Addition of 0.5 to 2.0 mM dithiothreitol or 2.0 mM dithioerythritol to the basal medium led to significantly longer retention of T. pGclidum viability in the presence of 3% oxygen than under aerobic or anaerobic conditions. The results of this investigation lend support to the classification of T. pallidum as a microaerophilic organism and provide direction for the design of potentially successful culture systems, with or without tissue culture cells.
The in vitro cultivation of Treponema pallidum, the causative agent of syphilis, continues to be an elusive goal. Numerous reports of successful cultivation emanated from early research, but attempts of confirmation yielded negative results (31); the so-called avirulent cultivatable strains of T. pallidum have recently been shown by DNA hybridization studies to be unrelated to virulent T. pallidum (20). Many investigators have studied the effects of medium components on the in vitro survival of T. pallidum. As a result, serum tissue extracts, sulfhydryl compounds, carbon dioxide, sodium pyruvate, glucose, and magnesium salts have been reported to contribute to the retention of motility and virulence by T. pallidum; in no instance, however, was an increase in the number of T. pallidum observed (11, 16, 17, 22, 30). Mammalian cells have been shown to extend the in vitro survival of T. pallidum and have been used widely in recent cultivation attempts (6-9, 15, 24, 26-28). Systems utilizing tissue culture cells and intermediate levels of oxygen have been shown to be particularly effective in maintaining the motility and virulence of T. pallidum (6, 9, 26). Jones et al. (15) reported the multiplication of T. pallidum in baby hamster kidney cell cultures; however, their results were equiv-
transient increases in the number of T. pallidum in a tissue culture system utilizing 3 to 4% oxygen. Based on its survival under anaerobic conditions and rapid death upon exposure to air, T. pallidum traditionally has been considered to be a strict anaerobe. However, recent studies have suggested that oxygen may play a major role in the metabolism of the bacterium (2, 5, 23). The purpose of the present study was to examine in detail the effects of oxygen, sulfhydryl compounds, and serum on the survival of virulent T. pallidum (Nichols strain) in a cell-free system so that our findings could be used in subsequent cultivation attempts. MATERIALS AND METHODS Rabbits. Adult male New Zealand white rabbits were housed in air-conditioned quarters at 18 to 20'C and fed antibiotic-free rabbit chow and water ad libitum. Rabbits with nonreactive VDRL and TPI tests whose clipped backs were initially free of irregular or heavy hair patterns were selected for virulence deter-
minations. Media components and preparation. All chemicals were of reagent grade. The F16 formulation of Eagle minimal essential medium (MEM, Grand Island Biological Co.) was dissolved in double-distilled water, supplemented with 20 mM HEPES (N-2-hydroxyocal and have not been confirmed (10). Sandok ethyl piperazine-N'-2-ethanesulfonic acid) (Calbiet al. (26) recently reported the observation of ochem), adjusted to pH 7.2, and sterilized by filtration. 689
690
NORRIS ET AL.
Fresh normal rabbit serum (NRS) was obtained from VDRL nonreactive rabbits 2 to 3 h before use and heat-inactivated at 560C for 30 min. When chemical reagents were added to the basal medium, concentrated solutions of the compounds were made in MEM or 0.85% NaCl, filter sterilized, and added aseptically to the MEM portion of the medium. In most experiments, solutions were prepared immediately before use; in some cases, the solutions were stored for a maximum of 2 weeks at -60'C. Just prior to the extraction of T. pallidum, the MEM components of the media containing the various compounds were combined with fresh, heat-inactivated NRS and equilibrated with an atmosphere of 95% N2:5% CO2 for 30 min at 34WC. Extraction of T. pallidurm The Nichols strain of T. pallidum was maintained by intratesticular passage in rabbits. Treponemes were obtained from rabbit testes with a firm orchitis which developed 7 to 10 days postinfection. The testes were removed aseptically, rinsed with sterile 0.85% saline, and each was sliced into eight sections. Unless otherwise noted, the tissue was evenly distributed in screw-capped test tubes (25 by 150 mm) containing 5 ml of the medium to be tested. After 3 to 15 min of extraction on a shaker, these suspensions containing 1 x 106 to 20 x 106 T. pallidum per ml were centrifuged at 1,000 x g for 7 min to remove tissue debris, and each was pipetted in 1-ml aliquots into sterile test tubes (13 x 100 mm) with gas-permeable closures. The time from removal of the testes to the beginning of the incubation period ranged from 30 to 60 min. Conditions of incubation. Treponemal suspensions were incubated at 340C under three atmospheric conditions: aerobic (21% oxygen), anaerobic, and 3% oxygen. Aerobic samples were incubated in the presence of air. Anaerobic conditions were established by placing the samples in an anaerobic jar (BBL), evacuating to -J
50
20 -
0)
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_\
>i4/5
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230-I
l
4
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100
~~~Anaerobic
< >kK
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_
Anoerobic
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-
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2/5
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10
FIG. 4. Results of a combined motility-virulence experiment in which suspensions of 1.0 x T. pallidum per ml in MEM-50%o NRS with or without 1.0 mMDTT were incubated concurrently under aerobic, 3% oxygen, or anaerobic conditions. Motility values represent the means of three determinations. Virulence was determined by the id. inoculation of five or ten rabbits at single sites with 0.1 ml of the suspensions and daily examination of the sites for lesion development; data represent the mean time of lesion development. Lesions occurred at all inoculation sites unless otherwise indicated (sites positive per total sites). Motility (A) and virulence (B) results were qualitatively similar; optimal retention of both parameters occurred when DTT was added to the medium and the suspensions were incubated in an atmosphere containing 3o oxygen.
167
of days. This change in appearance indicated that sublethal damage to the structural elements of T. pallidum had taken place prior to the loss of motility. Effect of medium and atmospheric conditions on mammalian cells. Since one of the goals of this study was to develop an incubation system which could be used in conjunction with mammalian cell cultures, the conditions found beneficial to the retention of T. pallidum viability were tested for their effect on NRT cells. Secondary NRT cultures were incubated in MEM-50% NRS + 1.0 mM DTT at 340C in either Sykes-Moore chambers (37), T-15 culture flasks, or roller bottles. Incubation under either aerobic or 3% 02 conditions yielded healthy cultures which grew to confluency at the expected rate. In contrast, identical cultures incubated in 95% N2:5% CO2 exhibited abnormal cell morphology with cellular retraction, extensive vacuolization, and cell death. Thus, 3% 02 conditions were compatible with NRT cell growth
whereas anaerobic conditions suitable.
were
clearly
un-
DISCUSSION Concurrent incubation of T. pallidum suspensions with different medium additives under varied oxygen tensions allowed examination of the toxic and beneficial effects of oxygen and the mechanism of action of various medium components. The three atmospheric conditions used in this study provided a range of oxygen tensions and thus of oxygen toxicities. Increasing NRS concentrations resulted in enhanced retention of motility under both anaerobic and 3% 02 conditions. Addition of sulfhydryl compounds prolonged survival of T. pallidum in oxygen-containing environments but had no beneficial effect under anaerobic conditions. Thus the effect of serum is independent of the presence of oxygen, whereas the effect of sulfhydryl compounds is oxygen dependent. Suspensions of T. pallidum incubated in MEM-50% NRS with DTT or DTE
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FACTORS INFLUENCING T. PALLIDUM IN VITRO
under 3% 02 conditions retained their viability for a longer period than under anaerobic conditions, suggesting that T. pallidum is probably a microaerophilic rather than an anaerobic organism. Increasing NRS levels from 10 to 50% resulted in an extension of motility under anaerobic and 3% 02 conditions (Fig. 1, Table 2). Since it is possible that essential serum components are depleted during long-term incubation and may lead to loss of viability (25, 30), an increase in serum concentration or the addition of serum during the incubation period may extend the availability of these components and thereby prolong survival. The toxic effect of oxygen upon T. pallidum was most clearly exhibited when treponemes suspended in MEM-50% NRS were incubated under different 02 tensions (Fig. 2). Under those conditions, in which no compounds were added to the medium to protect the bacteria from oxygen toxicity, the loss of treponemal motility and virulence was related to the concentration of oxygen in the gaseous phase. Certain sulfhydryl compounds are effective in counteracting the toxic effects of oxygen on T. pallidum (6, 7, 11, 22, 30). Weber (30) found that, in an aerobic incubation system utilizing a paraffin overlay to partially exclude oxygen, addition of thioglycolic acid, mercaptosuccinic acid, DL-homocysteine, L-cysteine, or reduced glutathione resulted in extension of the 50% motility end point from 11.5 h to 12 to 18 days. He suggested that the active sulfhydryl compounds did not act solely through a reduction of redox potential, but also had some metabolic or protective activity due to their sulfhydryl groups and polar nature. Examination of the effects of sulfhydryl compounds under different 02 tensions in the present study revealed that treponemal motility and virulence were extended by these compounds only under atmospheric conditions containing significant levels of oxygen (Table 1, Fig. 3 and 4). None of the sulfhydryl compounds tested had a consistent positive effect under anaerobic conditions. This result indicated that the activity of sulfhydryl compounds was somehow involved with oxygen, the most obvious interpretation being that the compounds protected T. pallidum from the toxic effects of oxygen. In contrast, previous reports have indicated that sulfhydryl compounds were effective in extending the survival of T. pallidum under anaerobic conditions (13, 26, 31, 33); this could be explained as an inadvertent exposure of the T. pallidum suspensions to significant levels of oxygen in these studies (2). The mode of action by which DTT and other sulfhydryl compounds lessen oxygen toxicity
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and extend motility requires further study. Only reduced sulfhydryl compounds are effective in extending motility (30), implicating the hydrogen-donating sulfhydryl group in their activity. The oxidation of sulf'hydryl groups is thought to be mediated by oxygen radicals (14), and the observed protective effect may lie in their ability to scavenge these highly reactive and toxic oxygen derivatives (1). The fact that DTT and DTE possess unusually low redox potentials and yet oxidize more slowly than cysteine or glutathione (4) may in part account for their special ability to extend the survival of T. pallidum under 3% 02 conditions. The effectiveness of DTT under 3% 02 conditions was reduced by the addition of cysteine or glutathione (Table 1), suggesting that these compounds accelerated the oxidation of DTT through their own oxidation and subsequent reduction by DTT (4). Low levels of oxygen appeared to be beneficial to the maintenance of T. pallidum motility and virulence when the toxic effects of oxygen were counteracted by the presence of DTT. Prolonged retention of motility and high degrees of virulence were consistently observed for treponemal suspensions containing 0.5 to 2.0 mM DTT or 2.0 mM DTE incubated in 3% 02 as compared to suspensions incubated anaerobically either with or without these compounds. Another explanation is that oxygen modifies DTT to a form beneficial to T. pallidum, but this possibility was negated by the finding that the disulfide product of DTT oxidation (4) was ineffective in extending treponemal motility. That oxygen is, under special conditions, beneficial to the in vitro survival of T. pallidum is consistent with previous indications that it may play a role in the metabolism of the bacterium. Cox and Barber (5) reported that suspensions of T. pallidum consumed oxygen at a rate approximately half that of a known aerobic spirochete, Leptospira B16. Biochemical studies by Baseman et al. (2) and Nichols and Baseman (23) showed that C02 was evolved as a result of the oxidative degradation of glucose and pyruvate and that incorporation of tritiated amino acids or uridine into acid-precipitable material was maximal at gaseous 02 tensions of 10 to 20% as compared to higher and lower concentrations. Ultrasonic lysates of T. pallidum exhibit UV absorbtion spectra consistent with those of cytochromes and flavoproteins, suggesting that a terminal electron transport system may exist in the bacterium (18). Conflicting results have been obtained for the effects of azide and other inhibitors of oxidative phosphorylation upon the metabolism of T. pallidum (2, 5). Prolonged survival of T. pallidum under conditions of limited oxygen exposure has been
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Health Organization agreement V3/181/26, and the noted in tissue culture interaction studies. Fitz- World Soiland Cancer Foundation. S.J.N. is the recipient of gerald et al. (7) found that an atmosphere con- Albert predoctoral Public Health Service National Research Service taining 3% 02 was superior to aerobic and anaer- Award GM07185 in Cellular and Molecular Biology from the obic conditions in maintaining treponemal mo- National Institute of General Medical Sciences. tility and virulence in the presence of SflEp cells LITERATURE CITED and a medium containing cysteine, glutathione, S. Kanematsu. 1976. Reactivity of thiols 1. and Asada, K., and DTT. By utilizing an oxygen gradient syswith superoxide radicals. Agr. Biol. Chem. 40:1891tem with SflEp cells and a prereduced medium, 1892. Fieldsteel et al. (6) showed that attachment to 2. Baseman, J. B., J. C. Nichols, and N. S. Hayes. 1976. Virulent Treponema pallidum: aerobe or anaerobe. Inthe cell cultures and retention of viability of T. fect. Immun. 13:704-711. pallidum were optimal at the boundary of oxy3. Clark, J. W., Jr. 1962. Loss of virulence in vitro of motile gen penetration; virulent treponemes were presTreponema pallidum. Br. J. Vener. Dis. 38:78-81. ent at this interface up to 21 days following 4. Cleland, W. W. 1964. Dithiothreitol, a new protective reagent for SH groups. Biochemistry 3:480-482. inoculation. These studies suggest that low levels of oxygen are beneficial to T. pallidum. How- 5. Cox, C. D., and M. K. Barber. 1974. Oxygen uptake by Treponema pallidum. Infect. Immun. 10:123-127. ever, there exists the equally likely possibility 6. Fieldsteel, A. H., F. A. Becker, and J. G. Stout. 1977. that treponemal survival is optimal at the lowest Prolonged survival of virulent Treponema pallidum oxygen concentration which can maintain mam(Nichols strain) in cell-free and tissue culture systems. Infect. Immun. 18:173-182. malian cell viability. Thus no definitive concluT. J., R. C. Johnson, J. A. Sykes, and J. sions regarding the relationship between T. pal- 7. Fitzgerald, N. Miller. 1977. Further observations on the interaction lidum and oxygen can be drawn from tissue of Treponema pallidum (Nichols strain) with cultured culture interaction studies. mammalian cells: effects of oxygen, reducing agents, serum supplements, and other cell types. Infect. Immun. The role of oxygen in the biology of T. palli15:444452. dum has been discussed previously (2, 5, 9, 18, 8. Fitzgerald, T. J., J. N. Miller, and J. A. Sykes. 1975. 23). The finding that oxygen is both extremely Treponemapallidum (Nichols strain) in tissue cultures: cellular attachment, entry, and survival. Infect. Immun. toxic to and, under certain conditions, beneficial 11:1133-1140. to T. pallidum appears on its surface to be T. J., J. N. Miller, J. A. Sykes, and R. C. contradictory. However, it is well known that 9. Fitzgerald, Johnson. 1976. Tissue culture and Treponema pallioxygen, if present in sufficiently high concentradum, p. 57-64. In R. C. Johnson (ed.), The biology of tions, is toxic to all organisms from E. coli to parasitic spirochetes. Academic Press Inc., New York. humans (13, 32). Most organisms which live in 10. Foster, J. W., D. S. Kellogg, J. W. Clark, and A. Balows. 1977. The in vitro cultivation of Treponema the presence of oxygen have developed elaborate pallidum. Corroborative studies. Br. J. Vener. Dis. means of neutralizing oxygen radicals and their 53:338-339. products; superoxide dismutase, glutathione per- 11. Graves, S. R., P. L. Sandok, H. M. Jenkin, and R. C. Johnson. 1975. Retention of motility and virulence of oxidase, catalases, peroxidases, and alpha-toTreponema pallidum (Nichols strain) in vitro. Infect. copherol have all been implicated in this role Immun. 12:1116-1120. (12). Although T. pallidum thrives in well-oxy- 12. Halliwell, B. 1974. Superoxide dismutase, catalase, and genated tissues and seems to utilize oxygen in its glutathione peroxidase: solutions to the problems of living with oxygen. New Phytol. 73:1075-1086. metabolism, it has a very low tolerance for the toxic effects of oxygen. It is possible that T. 13. Haugaard, N. 1968. Cellular mechanisms of oxygen toxicity. Physiol. Rev. 48:311-373. pallidum is an obligate parasite and is difficult 14. Jocelyn, P. C. 1972. Biochemistry of the SH group: the to culture in vitro because of its reliance on the occurrence, chemical properties, metabolism, and biological function of thiols and disulfides. Academic Press host's ability to scavenge oxygen radicals. The Inc., New York. beneficial effects which tissue culture cells and R. H., M. A. Finn, J. J. Thomas, and C. Folger. sulfhydryl compounds have on the in vitro sur- 15. Jones, 1976. Growth and subculture of pathogenic T. paffidum vival of T. pallidum may in part be due to their (Nichols strain) in BHK-21 cultured tissue cells. Br. J. ability to provide this activity (9). Vener. Dis. 52:18-23. In our opinion, the results of this study are 16. Kimm, G. E., R. Allen, H. J. Morton, and J. F. Morgan. 1960. Enhancement of survival in vitro of Trepoconsistent with the classification of T. pallidum nema pallidum by addition of glucose and magnesium. as a microaerophilic organism and provide direcJ. Bacteriol. 80:726-727. tion for the design of potentially successful cul- 17. Kimm, G. E., R. H. Allen, H. J. Morton, and J. F. Morgan. 1962. Studies on the in vitro survival of viruture systems, with or without tissue culture cells. ACKNOWLEDGMENTS We thank Mark Falkenbach, Frank Fazzan, Ruth Elwell, and Renee Norris for their invaluable assistance. This work was supported by the Office of Naval Research contract N00014-76-C-0148, Public Health Service contract AI-42538 from the National Institute of Allergy and Infectious Diseases,
lent Treponema pallidum. I. Methodology and basal synthetic medium. Am. J. Hyg. 75:339-346. 18. Lysko, P. G., and C. D. Cox. 1977. Terminal electron transport in Treponema pallidum. Infect. Immun.
16:885-890.
19. Magnuson, H. J., H. Eagle, and R. Fleischman. 1948. The minimal infectious dose of Spirochaeta pallida (Nichols strain) and a consideration of its rate of mul-
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tiplication in vivo. Am. J. Syph. 32:1-18. 20. Miao, R., and A. H. Fieldsteel. 1978. Genetics of Treponema: relationship between Treponema pallidum and five cultivatable treponemes. J. Bacteriol. 133:101107. 21. Miller, J. N. (ed.). 1971. Spirochetes in body fluids and tissues: manual of investigative methods. Charles C. Thomas, Springfield, Illinois. 22. Nelson, R. A. 1948. Factors affecting the survival of Treponema pallidum in vitro. Am. J. Hyg. 48:120-131. 23. Nichols, J. C., and J. B. Baseman. 1978. Ribosomal ribonucleic acid synthesis by virulent Treponema pallidum. Infect. Immun. 19:854-860. 24. Rathlev, T. 1975. Investigations on in vitro survival and virulence of T. pallidum under aerobiosis. Br. J. Vener. Dis. 51:296-300. 25. Rice, F. A. H., and R. A. Nelson. 1951. The isolation from beef serum of a survival factor for Treponema pallidum. J. Biol. Chem. 191:35-41. 26. Sandok, P. L., H. M. Jenkin, S. R. Graves, and S. T. Knight. 1976. Retention of motility of Treponema pallidum (Nichols strain) in an anaerobic cell culture sys-
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tem and in a cell-free system. J. Clin. Microbiol.
3:72-74. 27. Sandok, P. L., H. M. Jenkin, H. M. Matthews, and M. S. Roberts. 1978. Unsustained multiplication of Treponema pallidum (Nichols virulent strain) in vitro in the presence of oxygen. Infect. Immun. 19:421-429. 28. Sandok, P. L., S. T. Knight, and H. M. Jenkin. 1976. Examination of various cell culture techniques for coincubation of virulent Treponema pallidum (Nichols I strain) under anaerobic conditions. J. Clin. Microbiol. 4:360-371. 29. Sykes, J. A., and E. B. Moore. 1960. A simple tissue culture chamber. Tex. Rep. Biol. Med. 18:288-297. 30. Weber, M. D. 1960. Factors influencing the in vitro survival of Treponema pallidum. Am. J. Hyg. 71:401-417. 31. Willcox, R. R., and T. Guthe. 1966. Treponema pallidum: a bibliographical review of the morphology, culture, and survival of T. pallidum and related organisms. Bull. WHO 35(Suppl.):1-169. 32. Yost, F. J., and I. Fridovich. 1976. Superoxide and hydrogen peroxide in oxygen damage. Arch. Biochem. Biophys. 175:514-519.
