INFEcriON AND IMMUNITY, May 1975, p. 1133-1140Copyright 0 1975 American Society for Microbiology

Vol. 11, No. 5Printed in U.S.A.

Treponema pallidum (Nichols Strain) in Tissue Cultures:Cellular Attachment, Entry, and Survival

T. J. F1TZGERALD,l* J. N. MILLER, AND J. A. SYKESTreponemal Research Laboratory, Department of Microbiology and Immunology, UCLA School of Medicine,Los Angeles, California 90024, and Research Department, Southern California Cancer Center, California

Hospital Medical Center, Los Angeles, California 90015

Received for publication 21 October 1974

The interaction of Treponema pallidum (Nichols strain) with cultured cellswas investigated under aerobic conditions. Cell monolayers derived from rabbittesticular tissue extended the survival of treponemes as indicated by activemotility. Large numbers of organisms rapidly attached to cultured cells. Within 3h, one to twelve actively motile treponemes were attached to 25 to 50% of thecells. In addition, T. pallidum attained intracellularity as early as 30 min afterinoculation of the cell monolayers. In sharp contrast, T. phagedenis biotypeReiter and T. denticola did not attach and did not enter cultured cells. Mostimportantly, intracellular and/or attached T. pallidum retained virulence for atleast 24 h. Similar observations of attachment and retention of virulence weredetected with ME-180, a cell line derived from a human cervical carcinoma.Preliminary studies with superoxide dismutase indicated that this enzymeprolonged treponemal motility and retention of virulence in the presence ofcultured cells. These data provide guidelines for further investigations of in vitrocultivation of T. pallidum.

Previous attempts to culture Treponemapallidum in vitro have been uniformly unsuc-cessful. The few reported successes have beeneither nonreproducible or have involved relatednonpathogenic treponemes. Further, elabora-tion of factors or parameters that prolong invitro survival has not led to subsequent culti-vation.

Reports based on maintenance of motilityhave indicated that viable tissue significantlyprolonged the survival of T. pallidum in vitro.Steinhardt observed motile treponemes for 25days in a hanging drop preparation of rabbittesticular tissue (13). Similar extensions ofsurvival were reported by Shaffer, using normalrabbit tissue, and by Gammel and Ecker, usingsyphilitic rabbit tissue (3, 11).

In addition to the use of tissue fragments, invitro growth of T. pallidum has been attemptedin the presence of cultured tissue cells. Perrymaintained the motility of T. pallidum for 10days by the addition of testicular epitheliumfrom new-born rabbits in a microaerophilicenvironment (10). Bessemans and de Geest (2)were unable to grow T. pallidum in the presenceof cultures of fibroblastic testicular cells; Kastand Kolmer (5) were unsuccessful using pri-mary cultures of syphilitic and normal testes.

Generally, attempts to culture the organismI Present address: Department of Microbiology, Medical

School, University of Minnesota, Minneapolis, Minn. 55455.

in vitro have been based on the assumption thatT. pallidum requires anaerobiosis. There islittle doubt that the cultivable treponemes areanaerobes, or that an anaerobic or microaero-philic environment prolongs the survival of T.pallidum (9, 12). The requirements for growthand multiplication of T. pallidum, however,appear to be more complex than those favorableto survival. Krause, in fact, has urged a reevalu-ation of the prevailing dogma of strict anaerobi-osis for T. pallidum (6). The circumstances of invivo infection strongly support such a reevalua-tion. In primary syphilis, T. pallidum usuallyinfects highly vascularized tissues that containhigh concentrations of oxygen; however, whenthe organisms are removed from the infectedhost, similar concentrations of oxygen becometoxic. It appears, therefore, that host tissuesmust effectively neutralize the toxicity of oxy-gen for T. pallidum.Any attempt at cultivation in vitro should

consider the intracellular location of T.pallidum within infected tissues (1, 14, 16).Azar initially described treponemes withinplasma cells obtained from a human chancre(1). Sykes and Miller reported intracellular T.pallidum within fibroblasts, interstitial cells,spermatocytes, and Leydig cells from infectedrabbit testes (14); Sykes et al. detected trepo-nemes within white cells, fibroblasts, epithelial,and endothelial cells from human primary le-

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FITZGERALD, MILLER, AND SYKES

sions (16). The presence of T. pallidum withinsuch diverse tissue cells suggests that intracel-lularity might be of importance in growth andmultiplication.The purpose of this study was to determine

whether the in vitro cultivation of T. pallidumcould be accomplished by using cultured cellsderived from rabbit testes under aerobic condi-tions. Attempts were made to relate survival ofthe organisms to intracellular location; survivalof organisms was based on motility, as deter-mined by dark-field microscopy, and virulence,as determined by the development of pathog-nomic lesions in the rabbit.

MATERIALS AND METHODSRabbits. Adult male New Zealand white rabbits

with nonreactive venereal disease research laboratorytests were used in all experiments. The animals weremaintained at 18 to 20 C.

Source of treponemes. Rabbits were inoculatedintratesticularly with the Nichols strain of T.pallidum. Organisms were harvested after the devel-opment of a satisfactory orchitis, usually within 9 to11 days. Testes were removed aseptically, placed in asuitable medium, and sliced. After extraction for 5 to30 min, the suspension was centrifuged at 1,000 x gfor 7 min to sediment gross tissue debris. The super-natant containing the organisms was removed and thenumber of treponemes was determined according tothe method described by Miller et al. (8).

Treponema phagedenis biotype Reiter andTreponema denticola were grown and maintained aspreviously described (15).

Reagents. All chemicals used in this study werereagent grade. Superoxide dismutase derived frombovine erythrocytes was obtained through thecourtesy of Miles Laboratories (Research Division)and from Truett Laboratories.

Source and maintenance of cultured cells. Nor-mal rabbit testes were removed aseptically, placed in0.14 M saline, equilibrated with 95% nitrogen-5%carbon dioxide, and transported within 30 min to theResearch Department of the Southern CaliforniaCancer Center. The testes were rinsed in 0.14 Msaline, cut and minced into small fragments, and thenplaced in 0.25% trypsin-0.01% ethylenediaminetetraa-cetic acid in phosphate-buffered saline at pH 7.8. Thetissue fragments were continuously agitated on amagnetic stirrer for 25 min at 22 C. The resulting cellsuspension was centrifuged at 1,000 x g for 10 min tosediment the cells; the supernatant was discarded.The cells were resuspended in a basal tissue culturemedium (BM) with heat-inactivated fetal calf serum(FCS) at a final concentration of 20%. The BMconsisted of Eagle minimal essential medium withHanks balanced salt solution supplemented with 2xamino acids and vitamins with 20 mM N-2-hydroxy-ethyl-piperazine-N'-2-ethanesulfonic acid as a buffer-ing agent. The cells were grown at 35 C and the me-dium was changed as necessary. Primary cultureswhich had attained confluency were used to estab-lish secondary cultures. An epithelial cell line (ME-

180) derived from a human cervical carcinoma (18)was also tested under the same conditions as thecells derived from rabbit testes.

Cell harvesting. After inoculation of treponemesand the appropriate incubation of the monolayers at35 C, the culture medium was discarded. The mono-layers were washed three times with 8 ml of BM. Afterthe third washing, 3 ml of BM containing FCS ornormal rabbit serum (NRS) was added to the mono-layers and the cells were removed from the glass bygentle scraping with a rubber policeman. To break upcell clumps, the suspensions were rapidly pipettedfive times with a 5-ml pipette. Cells harvested in thisfashion retained their viability as measured by vitalstaining with trypan blue.

Treponemal virulence and concentration. Todetermine the number of virulent treponemes withinor attached to the cultured cells, control suspensionsof T. pallidum containing 107, 105, and 103 organismsper ml were prepared in BM with NRS and inoculatedintradermally into the shaved backs of the samerabbits. Animals were observed daily for the initialappearance of characteristic lesions. The graph shownin Fig. 1 relates the number of treponemes injected to

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FIG. 1. Time of appearance of erythema and indu-ration as a function of the number of inoculated T.pallidum (Nichols). Inocula were made intrader-mally in a volume of 0.1 ml into the shaved backs ofwhite New Zealand rabbits housed at 20 C. Thesedata represent mean values for five separate experi-ments.

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T. PALLIDUM IN TISSUE CULTURES

the day of appearance of pathognomonic erythemaand induration.

Preparation of cells for electron microscopy. Thecells were fixed in 3.6% glutaraldehyde (cacodylatebuffered) either as suspended cells or in situ as a cellmonolayer. After exposure to glutaraldehyde for 15min, the suspended cells were pelleted by centrifuga-tion, the supernatant was discarded, and fresh 3.6%glutaraldehyde was added for 1 h. Cells fixed in situwere exposed to 3.6% glutaraldehyde for 15 min. Thecells were removed from the culture flasks by scrapingwith a rubber policeman; fixation was allowed tocontinue for 1 h. In both cases, the cells were thenpelleted, fixed with 1% osmic acid, and processed asdescribed for syphilitic rabbit testicular tissue (14).

RESULTSThe initial studies were designed to deter-

mine the influence of testicular tissue compo-nents on the motility of T. pallidum in a normalatmosphere. Infected rabbit testes were ex-tracted in heat-inactivated NRS either un-diluted or diluted 50% (vol/vol) with 0.14 Msaline. After extraction, the tissue suspensionwas centrifuged at 1,000 x g for 7 min tosediment blood cells, small fragments of tissue,and spermatozoa. After centrifugation, half ofthe supematant was removed and placed in asterile tube, and the remaining portion wasmixed with the pelleted tissue components. Inthis way, the identical preparations of T.pallidum were suspended in medium with andwithout tissue components.The presence of tissue constituents extended

the duration of motility of T. pallidum (Fig. 2).The time required for a 50% reduction in themotility of organisms (t50) in the presence oftissue constitutents was 22 h as opposed to 4 hfor organisms suspended in the cell-free me-dium. Loss of all detectable motility (to) wasobserved after 30 h in the presence of tissuecomponents, and after 5 h in the absence oftissue components. Other viable cell systemsalso prolonged treponemal survival. Motilitywas extended by the addition of leucocytes,erythrocytes, large (2 cm3) fragments of testes,and by contaminating bacteria.

In similar experiments in which an atmos-phere of 95% nitrogen-5% carbon dioxide wasused to provide an anerobic milieu, no enhance-ment of treponemal motility in the presence oftissue constituents was observed.Monolayers of cells derived from normal rab-

bit testes, in T-30 flasks, were inoculated with 3ml of T. pallidum suspended in BM containing10 to 50% heat-inactivated NRS. At the sametime, flasks not containing cultured cells wereinoculated with treponemes. The data from sixexperiments indicate that the presence of cellmonolayers extended the persistence of trepone-

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FIG. 2. Decrease in the motility of T. pallidum(Nichols) with time. Organisms were suspended inheat-inactivated normal rabbit serum either un-diluted or diluted 50%o (vol/vol) with 0.14 M salinewith and without testicular constituents. Symbols:

with testicular constituents; (- ) withouttesticular constituents. These data represent meanvalues for 10 separate experiments.

mal motility (Fig. 3). The t50 for organismsincubated with cultured cells was 5.0 h incontrast to a t50 of 3.0 h for treponemes incu-bated in the absence of cells; the to was 10.4 hwith cells and 5.5 h without cells.To determine whether products released by

the cultured cells were responsible for extendingmotility, medium was obtained from uninocu-lated cell cultures following contact with thecells for 0, 1, 5, 10, and 24 h. After centrifuga-tion at 2,000 x g for 10 min to remove anydetached tissue cells, all samples were adjustedto pH 7.2, (with 0.1 N NaOH) and inoculatedwith T. pallidum. No differences in motilitywere detected, indicating that the cells wereresponsible for the observed enhancement ofmotility, rather than released cellular products.

In observing treponemal motility in the pres-ence of cultured cells, a distinct drop in thenumber of organisms was detected. In a series ofsix experiments, utilizing an inoculum contain-ing an average of 6.4 x 107 treponemes per ml,an immediate drop in the number of organismswas observed (Fig. 4). Within 10 min the countdecreased 31% to 4.4 x 107 treponemes per ml.After this rapid initial decline, a more gradual

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FIG. 3. Decrease in motility of T. pallidum (Ni-chols) with time. Organisms suspended in BM con-taining 10 to 50% heat inactivated normal rabbitserum were inoculated into T-30 flasks of cell culturesderived from rabbit testes. Symbols: ( ) withcultured cells; (-----) without cultured cells. Thesedata represent mean values for six separate experi-ments.

decreased occurred. Six hours after inoculation,the count had dropped to 2.7 x 107 treponemesper ml, representing a decrease of 58%. Inidentical flasks not containing cell monolayers,no decrease in numbers was recorded during thesame time period, suggesting that the drop incount was not due to lysis of the organisms, butrather to an attachment and/or entry of thetreponemes into the cultured cells.To explore this possibility, ME-180 cell cul-

tures in Sykes-Moore chambers (17) were inocu-lated with T. pallidum and observed by phasecontrast microscopy. Three hours after inocula-tion, actively motile treponemes (two to six percell) were attached to 70% of the cells.

Similar observations were made with cul-tured cells derived from normal rabbit testes.Three hours after inoculation, actively motiletreponemes (one to twelve per cell) were at-tached to the cells. A wide range of cell involve-ment was detected. The numbers of cells withattached organisms varied from 25 to 50%. Thisvariation was not as pronounced with ME-180cells. This may be a reflection of the heterogene-ity of primary cultures relative to the homoge-neity of cells of a cell line. With both types ofcells, it appeared as if some of the treponemeswere intracellular. This apparent observation islimited by the technical difficulties of docu-menting intracellularity by phase contrast mi-croscopy.

To evaluate the specificity of this trepone-mal-cellular interaction, chambers containingcultured cells derived from normal rabbit testeswere inoculated with approximafe 107 per mlsuspensions of T. pallidum, heat-killed T. pal-lidum, T. denticola, and T. phagedenis biotypeReiter. In contrast to the attachment of virulenttreponemes, heat-killed T. pallidum and thetwo nonpathogens did not attach to the cells.These findings suggest that attachment is nei-ther a phagocytic nor a pinocytotic function ofthe cultured cells, but rather an active trepone-mal process associated with the pathogenicquality of organisms.

Viability of cultured cells, as assessed by vitalstaining with trypan blue, was unaffected by thepresence of virulent T. pallidum. After 24 h, 85to 90% of cells exposed to treponemes wereviable. Identical percentages of viable cells weredetected in cultures not exposed to treponemes.

Sykes and Miller (14) and Sykes et al. (16)reported the intracellular location of T.pallidum in tissue from infected rabbit testesand in tissue from human primary syphiliticlesions. To examine the possibility of intracellu-larity in tissue cultures, monolayers of cellsderived from rabbit testes were inoculated withT. pallidum in BM with FCS or NRS containing6 x 107 organisms. After various periods ofincubation, cells, after washing with BM to

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FIG. 4. Numbers of T. pallidum (Nichols) ob-served by dark-field microscopy (x600) in the pres-ence and absence of cultured cells. T-30 flasks wereinoculated with an average of 6.4 x 107 organisms.Symbols: ( ) with cultured cells; (---- ) withoutcultured cells. These data represent mean values forsix separate experiments.

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remove unattached organisms, were fixed forelectron microscopy. Intracellular treponemeswere observed 30 min after inoculation (Fig. 5).This finding of rapid entry correlates with thedata represented in Fig. 4, in which a rapiddecrease in treponemal numbers (31%) wasdemonstrable immediately after inoculation ofcultured cells. Intracellular, morphologicallyintact organisms were observed at 1, 2, 4, 6, 11,and 18 h. Additionally, intact organisms weredemonstrated both intra- and extracellularly 4(Fig. 6) and 7 days after inoculation.In related experiments using T. denticola and

T. phagedenis biotype Reiter, no intracellularorganisms were detected.An important consideration in these studies

was the assessment of virulence of intracellularor attached organisms. At various times afterinoculation of monolayers, the cells werewashed and 0.1 ml of washed cell suspensionwas inoculated intradermally into the shavedbacks of rabbits.Table 1 summarizes the data obtained from

10 experiments of this type. The estimatednumber of virulent treponemes based on thetime of appearance of erythema and induration(Fig. 1) is presented. These studies demonstrateunequivocally that the intracellular and/or at-tached treponemes retained their infectivity.Cell monolayers that had been inoculated with2 x 107 to 3 x 107 organisms per ml contained107 organisms per ml within 30 min. Thesefindings correlate with data of Fig. 4 whichindicated a rapid decline in numbers of extra-cellular treponemes after inoculation of cul-tured cells. After incubation for 24 h, 104treponemes per ml were detected.

In parallel experiments, no virulent orga-nisms were detected in the absence of culturedcells after 9 h of incubation.

Nonspecific reactions to uninfected culturedcells derived from rabbit testes were not de-tected.

Related studies using cultures of the humancell line ME-180 revealed a similar interactionof treponemes with cultured cells. Cell attach-ment and/or penetration of 3 x 102 virulent T.pallidum per 0.1 ml of inoculum was demon-strable 16 h after cell-treponeme interaction. Asexpected, nonspecific side reactions did occurwith ME-180 cells; control cell suspensionswithout T. pallidum produced areas of ery-thema 5 to 9 mm in diameter with slightinduration between days 6 and 15. After day 15,these reactions regressed.

Preliminary experiments were undertaken todetermine the effect of superoxide dismutase onthe cell-treponeme interaction. Six thousandunits of the enzyme in BM with NRS were

added to cell monolayers. Identical culturesreceived BM with NRS without the enzyme.These cell monolayers were then inoculatedwith 5 x 106 treponemes and incubated for 24 hat 35 C. This inoculum is lower than that usedin previous experiments listed in Table 1. Whenwashed cell suspensions from the enzyme-con-taining cultures were inoculated intradermally,treponemal skin lesions became apparent in 21days. In contrast, no skin lesions were producedafter inoculation of cells not exposed to theenzyme. Besides extending retention of viru-lence of T. pallidum, superoxide dismutase alsoprolonged active motility of the organisms.

DISCUSSIONThe use of aerobiosis, although representing a

departure from previous efforts to culture T.pallidum, was considered appropriate since itmore closely approximates environmental con-ditions during growth and multiplication of theorganisms in vivo. Justification for this ap-proach appeared to be confirmed by the findingthat large numbers of treponemes attached to,and entered, cultured cells; these organismsretained their virulence for at least 24 h.The reports of in vivo intracellularity of T.

pallidum by Sykes and Miller (14) and by Sykeset al. (16) support further research utilizing cellculture systems. The presence of organismswithin such a diversity of cell types suggeststhat an intracellular location was more thanchance occurrence, and might play an integralrole in the pathogenesis of both human andexperimental syphilis. T. pallidum may entercells to escape from an unfavorable environ-ment; or perhaps some stage of treponemalgrowth may require an intracellular residence.Whatever the reason(s), cell cultures shouldprovide a system that will permit correlationbetween in vitro studies and observed in vivophenomenon.

In contrast to T. pallidum, T. denticola andT. phagedenis biotype Reiter, when inoculatedintratesticularly into rabbits, were not seenwithin the cells of the intact organ; furthermore,no evidence of an intracellular location wasdetected within cell cultures derived from rab-bit testes. This major difference between thesetwo nonpathogenic treponemes and T. pallidumfocused attention on the possible importance ofintracellularity in the pathogenesis of T.pallidum infection.The failure of killed T. pallidum to attach to

cultured cells indicated that attachment was anactive treponemal function unassociated withphagocytosis or pinocytosis. T. pallidum ap-peared to be specifically attracted to culturedcells. Within 3 h, numerous attached trepo-

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FIG. 5. Electron micrograph of part of a cell fixed 30 min after infection with T. pallidum (Nichols). The cellswere from an aerobic secondary culture derived from rabbit testes. Part of a treponeme (T) lying close to the cellnucleus (N) has been cut across a bend. Three axial filaments link the two oblique cross sections. The cytoplasmof the cell contains mitochondria (M) with some degeneration of cristae. There is some swelling of theendoplasmic reticulum and vacuolation of the golgi complex. The bar in both electron micrographs represents 1

pm. Magnification x30,000.FIG. 6. Electron micrograph of parts of two cells from a culture fixed 4 days after infection with T. pallidum

(Nichols). The cells were from a secondary culture of rabbit testes that had been maintained under an

atmosphere of N2 95%-CO2 5%. The nucleus (N) indicates a maturing cell and the cytoplasm of the lower cellshows some minor swelling of the rough endoplasmic reticulum. Parts of a morphologically intact treponeme(T) are lying between the cells. Magnification x30,000.

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T. PALLIDUM IN TISSUE CULTURES

TABLE 1. Virulence of T. pallidum (Nichols) in thepresence of cultured cells of normal rabbit testes

Development of intra- EstimatedNo. of dermal lesions no. of

Time rab- virulent(h) in bits Sites pos- Day of EIa treponemesculture inocu- itive/ appearance in 0.1 ml

lated sites in- of inoc-oculated Average Range ulum°

0.5 2 4/4 5.0 5 1061.0 4 8/8 6.2 5-7 2 x 1053.0 2 2/2 7.0 7 8 x 1046.0 8 12/13 8.0 5-11 3 x 1049.0 4 8/8 7.6 7-10 4 x 10412.0 2 4/4 8.0 7-9 3 x 10414.0 2 4/4 10.0 10 6 x 10324.0 2 6/6 12.0 7-21 103

a Erythema and induration.'See Fig. 1. Cultures were inoculated with 2 x 107

to 3 x 107 treponemes per ml.

nemes were demonstrable by phase contrastmicroscopy at the surface of 70% of ME-180cells and 25 to 50% of rabbit testicular cells.This multiple cell involvement was also evidentin the virulence studies. Cell monolayers de-rived from rabbit testes, in T-30 flasks, con-tained approximately 3 x 106 cells. Thirty min-utes after inoculation, 3 x 107 treponemeswere attached to the washed cells. Further-more, this treponemal interaction occurredwithout significant killing of cultured cells.Of considerable interest was the susceptibil-

ity of ME-180, a cell line derived from a humantumor, to infection with T. pallidum. A humancell line could be used to prepare clean prepara-tions of treponemes that would facilitate immu-nological studies directed toward antigenicstructure, immune response mechanisms, andvaccine efficacy. After attachment of trepo-nemes and subsequent washing, the organismscould be forced to detach in a controlled fash-ion, yielding a relatively clean preparation freeof contaminating rabbit constituents.

In some of the tissue culture studies, itappeared that a "shedding" of attached orintracellular T. pallidum, with subsequent reat-tachment or reentry, was occurring. After re-placement of the culture medium with freshmedium, and further incubation, there was asignificant decrease in virulent treponemes. Thefluctuating extracellular treponemal count inthe presence of cell monolayers also suggested a"shedding" phenomenon. The initial 6-h incu-bation revealed a gradual decline in the numberof extracellular organisms (Fig. 4). Thereafter,there was a slight but reproducible fluctuationin numbers up to 24 h.The BM used was relatively toxic in that T.

pallidum did not survive beyond 5 h in theabsence of cultured cells (Fig. 3). In view of this"shedding" of treponemes, this toxicity as-sumes major importance. Oxygen, which isdetrimental to the organisms in vitro, may bethe primary toxic factor in this cell culturesystem. The extension of treponemal survival inthe presence of viable cells indicated that thesecells provided a degree of detoxification. Inas-much as oxygen at high concentrations is toxicto all cells, the cells must possess an effectivemechanism(s) for neutralization of atmosphericoxygen toxicity.

Superoxide dismutase may be one of themajor mechanisms for neutralization of thistoxicity. This enzyme prevents the accumula-tion of highly destructive superoxide anions.The presence of superoxide dismutase withinerythrocytes, brain, lung, liver, heart, andtestes tissue, and within yeasts and bacteria,demonstrates its ubiquitous occurrence in na-ture. Recently, McCord et al. (7) proposed anenzyme-based theory of anaerobiosis related tothe presence of superoxide dismutase withinmicroorganisms. They found that aerobes con-tained large amounts of the enzyme, strictanaerobes contained none, and microaerophilicorganisms contained low levels of the enzyme.They proposed that superoxide dismutase wasthe primary factor that enabled organisms tosurvive in the presence of oxygen. In an exten-sion of this work, Gregory et al. (4) successfullyprotected Escherichia coli from the lethality ofsuperoxide anions by addition of superoxidedismutase.This theory, which postulates the necessity of

superoxide dismutase for survival of T.pallidum in an aerobic environment, raisessome interesting possibilities. Within infectedtestes, superoxide dismutase could conceivablyprevent oxygen toxicity and allow treponemesto grow and multiply in a favorable environ-ment. Removal of T. pallidum from infectedtissue might result in an accumulation of lethalsuperoxide anions. Addition of a viable cellsystem, such as testicular tissue, could providea transient source of superoxide dismutase thatwould prolong survival of the treponemes untildeath of the tissues and loss of enzyme activity.Preliminary studies using superoxide dismu-

tase have been encouraging; they have shownthat addition of the enzyme prolonged themotility and virulence of T. pallidum withinthis culture medium in the presence of culturedcells. These cells probably contain an endoge-nous source of superoxide dismutase, but thepreliminary data have indicated that this levelwas inadequate to maintain extracellular trepo-nemal survival. An exogenous source of super-

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FITZGERALD, MILLER, AND SYKES

oxide dismutase appears, therefore, to be essen-

tial.In view of the possible "shedding" of orga-

nisms, any reduction in culture medium toxic-ity should enhance the possibility of successfulin vitro cultivation of T. pallidum. If thetoxicity shown in these studies was primarilydue to oxygen, the advantages of adding super-

oxide dismutase are readily apparent.

ACKNOWLEDGMENTS

We gratefully acknowledge the excellent technical assist-ance provided by F. P. Fazzan, L. Briggs, and N. Parry. Wewould like to thank Russell C. Johnson for constructivecritical evaluation.

This work was supported by the Office of Naval Researchgrant N00014-69-A-0200-4039, by Public Health Service grantNO1 AI 42538 from the National Institute of Allergy andInfectious Diseases, and by the World Health Organizationgrant V3-181-26.

LITERATURE CITED

1. Azar, H. A., T. D. Pham, and A. T. Kurban. 1970. Anelectron microscopic study of a syphilitic chancre.Arch. Pathol. 90:143-150.

2. Bessemans, A., and B. de Geest. 1934. Essais de culturein vitro du treponeme pale en symbiose avec du tissutesticulaire de lapin. Rev. Belge Sci. Med. 6:28-36.

3. Gammel, J. A., and E. E. Ecker. 1931. The virulence ofSpirocheta pallida in culture. Arch. Dermatol. Syph.23:439-444.

4. Gregory, E. M., F. J. Yost, and I. Fridovich. 1973.Superoxide dismutases of Escherichia coli; intracellu-lar localization and functions. J. Bacteriol. 115:987-991.

5. Kast, C. C., and J. A. Kolmer. 1933. On the cultivation ofSpirochaeta pallida in living tissue media. Am. J.Syph. 17:529-532.

6. Krause, R. M. 1972. Workshop on the biology of thetreponemes. J. Infect. Dis. 125:332-336.

7. McCord, J. M., B. G. Keele, and I. Fridovich. 1971. Anenzyme-based theory of obligate anaerobiosis: thephysiological function of superoxide dismutase. Proc.Natl. Acad. Sci. U.S.A. 68:1024-1027.

8. Miller, J. N., S. J. Whang, and F. P. Fazzan. 1963.Studies on immunity in experimental syphilis. I. Im-munological response in rabbits immunized with Reiterprotein antigen and challenged with virulentTreponema pallidum. Br. J. Vener. Dis. 39:195-198.

9. Nelson, R. A. 1948. Factors affecting the survival ofTreponema pallidum in vitro. Am. J. Hyg. 48:120-132.

10. Perry, W. L. M. 1948. The cultivation of Treponemapallidum in tissue culture. J. Pathol. Bacteriol.60:339-342.

11. Shaffer, L. W. 1926. Cultured methods for increasing thenumbers of Spirochaeta pallida in fresh syphilitictissue. Arch. Pathol. Lab. Med. 2:50-58.

12. Smibert, R. M. 1971. Isolation, cultivation, and charac-terization of anaerobic treponemes. WHONVDT/Res/71.242.

13. Steinhardt, E. 1913. A preliminary note on Spirochaetapallida and living tissue cells in vitro. J. Am. Med.Assoc. 61:1810.

14. Sykes, J. A., and J. N. Miller. 1971. Intracellular locationof Treponema pallidum (Nichols strain) in rabbittestes. Infect. Immun. 4:307-314.

15. Sykes, J. A., and J. N. Miller. 1973. Ultrastructuralstudies of treponemes: location of axial filaments andsome dimensions of Treponema pallidum (Nicholsstrain), Treponema denticola, and Treponema reiteri.Infect. Immun. 7:100-110.

16. Sykes, J. A., J. N. Miller, and A. J. Kalan. 1974.Treponema pallidum within cells of a primary chancrefrom a human female. Br. J. Vener. Dis. 50:40-44.

17. Sykes, J. A., and E. B. Moore. 1960. A simple tissueculture chamber. Texas Rep. Biol. Med. 18:288-297.

18. Sykes, J. A., J. Whitescarver, P. Jernstrom, J. F. Nolan,and P. Byatt. 1970. Some properties of a new epithelialcell line of human origin. J. Natl. Cancer Inst.45:107-122.

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