INFECTION AND IMMUNITY, Sept. 1987, p. 2066-20730019-9567/87/092066-08$02.00/0Copyright © 1987, American Society for Microbiology

Activation of the Classical and Alternative Pathways of Complementby Treponema pallidum subsp. pallidum and Treponema vincentii

THOMAS J. FITZGERALDDepartment of Medical Microbiology and Immunology, School of Medicine, University of Minnesota at Duluth,

Duluth, Minnesota 55812

Received 16 March 1987/Accepted 29 May 1987

Both in vivo and in vitro studies have indicated that complement plays an important role in the syphiliticimmune responses. Few quantitative data are available concerning activation of the classical pathway byTreponema pallidum subsp. pallidum, and no information is available on treponemal activation of thealternative pathway. Activation of both pathways was compared by using T. pallidum subsp. pallidum and thenonpathogen T. vincentii. With rabbit and human sources of complement, both organisms rapidly activated theclassical pathway, as shown by hemolysis of sensitized sheep erythrocytes and by the generation of soluble C4a.With human sources of complement, both organisms also activated the alternative pathway, as shown byhemolysis of rabbit erythrocytes and by the generation of soluble C3a in the presence of magnesium ethyleneglycol-bis(,l-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). During incubation, organisms remainedactively mobile and did not lyse, indicating that activation was a function of complement reactivity with theintact outer treponemal surface. In addition, freshly harvested T. pallidum subsp. pallidum immediatelyactivated both pathways of complement; preincubation of organisms did not enhance complement reactivity.T. vincentii was a more potent activator of this pathway. T. pallidum subsp. pallidum contained almost fourtimes as much surface sialic acid as T. vincentii did. When sialic acid was enzymaticaliy removed from T.pallidum subsp. pallidum, enhanced activation of the alternative pathway was detected. It is proposed that T.pallidum subsp. pallidum retards complement-mediated damage by the alternative pathway through surface-associated sialic acid. This may be an important virulence determinant that enables these organisms to readilydisseminate through the bloodstream to infect other tissues.

A number of in vivo observations have suggested a role forcomplement in treponemal immune responses. Syphiliticlesions in C4-deficient guinea pigs, which lack a classicalpathway, were more pronounced and slower to heal than incontrols (46). When hamsters were infected with treponemapallidum subsp. endemicum or with T. pallidum subsp.pallidum and treated with cobra venom factor to depletecomplement, lesions appeared earlier and persisted longerthan in untreated controls (2, 42). Immune sera conferredprotection on hamsters infected with the yaws spirochete;this protection, however, was abrogated in animals pre-treated with cobra venom factor (42). Shortly after experi-mental treponemal infection, C3 levels were significantlylowered (3, 42). Circulating immune complexes containingcomplement fragments, antibody, and treponemal antigenshave been demonstrated in both human and experimentalrabbit syphilis (4, 28). Finally, C3 was detected on thesurface of freshly isolated organisms (1; unpublished obser-vations), and C3a and C5a were present within inflamedtissues, indicating activation of complement at the site ofinfection (40).

In 1949 Nelson and Mayer (34) initially proposed thatcomplement plays a role in the immune response to T.pallidum subsp. pallidum. Serum samples from syphiliticpatients, in conjunction with an undefined heat-leabile com-

ponent, immobilized the organisms in vitro (TPI reaction).Others have shown that this reaction was mediated primarilyby immunoglobulin G (6, 39) and that the heat-labile com-

ponent was in fact complement (39). A recent paper ex-

panded on the role of complement in immobilizing trepo-nemes (39). Loss of motility was accompanied by markeddecreases in complement levels. Immobilization required a

minimum of 2 h of in vitro exposure to antibody and

complement to sensitize the organisms, at which timetreponemes remained actively motile; the antibody andcomplement could then be removed, and immobilizationoccurred 4 to 6 h later. An intact classical pathway was

required for immobilization. Serum samples from C4-deficient guinea pigs did not show immobilization; whenexogenous C4 was added back to these deficient serum

samples, immobilizing activity was restored. Immobilizationdid not occur in the presence of magnesium ethylene glycol-bis (,-aminoethyl ether)-N,N,N',N'-tetraacetic acid(EGTA), which blocks the classical but not the alternativepathway. In addition, terminal complement componentswere required to immobilize T. pallidum. Serum samplesfrom C6-deficient rabbits failed to show immoblization;when the exogenous C4 was added to these deficient serumsamples, immobilizing activity was restored.A coating of hyaluronic acid on the surface of T. pallidum

subsp. pallidum (15-17, 49) interfered with the immobiliza-tion mediated by antibody and complement (20). Thehyaluronic acid did not appear to interfere with antibodybinding to the surface treponemal antigens; inhibitory effectswere not detected by radioimmunoassay or neutralizationreactions, both of which are complement independent (6, 14,20). On the basis of findings by others using complement-dependent lysis of sensitized erythrocytes (RBCs) (7, 8;N. S. Chang and R. J. Boackle, Fed. Proc. 42:1236, 1983), itwas proposed that hyaluronic acid inhibited the treponemalimmobilization reaction through anticomplementary effects(20).The above findings clearly indicate a role for complement

in treponemal immune responses. The purpose of this reportwas to expand on our previous findings to answer thefollowing two questions. (i) Does T. pallidum subsp. palli-

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dum immediately activate complement or is there an earlyperiod when it is refractory? (ii) Does the nonpathogen T.vincentii differ in its ability to activate complement?

MATERIALS AND METHODS

Treponemes. The Nichols strain of T. pallidum subsp.pallidum was maintained by intratesticular passage in rab-bits. A total of 2 x 107 to 4 x 107 organisms were injected pertestis. Animals were treated daily with cortisone acetate(Merck Sharp & Dohme, West Point, Pa.) at 6 mg/kg of bodyweight beginning on day 3 of infection. Organisms wereharvested after 9 to 11 days. Testicular tissue was extractedaerobically on a rotary shaker for 20 min at room tempera-ture. The treponemal extraction medium contained Eagleminimal essential medium (90%), heated normal rabbit se-rum (10%), dithiothreitol (1 mM), and HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) buffer (30 mM; pH7.2). The extract was centrifuged for 10 min at room tem-perature at 1,000 x g to sediment gross particulates. Thesupernatant containing the organisms was then centrifugedfor 30 min at 4°C at 12,000 x g. The pelleted treponemeswere suspended in the complement buffer (glucose-gelatin-Veronal buffer [GGVB]) at 2 x 108 to 4 x 108 treponemes perml. This high-speed centrifugation removes adherent glyco-saminoglycans from the surface of T. pallidum subsp. palli-dum (20). This is important because hyaluronic acid inhibitsactivation of the classical pathway (7, 8; Chang and Boackle,Fed. Proc., 1983). T. vincentii was grown to the mid-logarithmic stage as previously described (18). This nonpath-ogen was centrifuged for 30 min at 4°C at 12,000 x g, andpelleted organisms were suspended in GGVB at 2 x 108 to 4x 108 treponemes per ml.RBCs. Commercial preparations of sheep RBCs were

obtained from Wilfer Laboratories, Stillwater, Minn. RabbitRBCs were freshly isolated in our laboratory. Cells werewashed four times with saline and once with GGVB contain-ing 0.01 M EDTA. They were suspended in GGVB at 5 x 108cells per ml. To sensitize the sheep RBCs, an equal volumeof hemolysin (Texas Biological Laboratories, Fort Worth,Tex.) diluted 1/400 in 0.01 M EDTA was added to the cells,and the mixture was incubated at 37°C for 30 min and then at4°C for 30 min. RBCs were washed once with 0.01 M EDTAand three times with GGVB and suspended in GGVB at 5 x108 cells per ml. Rabbit RBCs were not sensitized. SheepRBCs were used to determine the activation of the classicalpathway, and rabbit RBCs were used to determine theactivation of the alternative pathway.Complement sources. Freshly prepared samples of either

human or rabbit whole serum provided sources of comple-ment. Blood was clotted by incubation at room temperaturefor 30 min and then at 4°C for 20 min. After centrifugation,serum samples were immediately aliquoted, quick-frozen inethanol-dry ice, and stored at -70°C. Just before eachexperiment, the serum samples used for the complementsources were thawed in a 37°C water bath and used onlyonce. Human serum samples from four or five individualswithout previous exposure to syphilis were pooled; theserum samples from all individuals were nonreactive in therapid plasma reaginic test. Rabbit serum samples from threeanimals not previously exposed to T. pallidum were pooled;these serum samples were nonreactive in the rapid plasmareaginic test.

Classical pathway. Complement lyses sensitized sheepRBCs via the classical pathway. A 1-ml portion of T.pallidum subsp. pallidum or T. vincentii in GGVB or a 1-ml

portion of GGVB (buffer control) was incubated with com-plement at 37°C for 1 h to activate the complement cascade.Then 1 ml of sensitized sheep RBCs at 5 x 108 cells per mlwas added, and the mixture was incubated for an additional1 h at 37°C to elicit hemolysis. All samples were immersed inan ice-water bath to block further complement activation.The preparations were centrifuged at 1,000 x g for 10 min at4°C to pellet the RBCs. The optical density of the superna-tant was read at 541 nm. In all experiments the positivecontrol (100% lysis) consisted of 5 x 108 RBCs suspended in2 ml of distilled water; the negative control (0 to 4% lysis)consisted of 5 x 108 RBCs suspended in 2 ml of GGVB. Thepercent hemolysis was determined by comparing opticaldensities of the test preparations with the optical density ofthe positive lysed control. Treponemes remaining in thesesupernatants did not interfere with optical densities. Asnegative controls, no hemolysis occurred when either non-sensitized RBCs or heat-inactivated complement (56°C, 30min) was used.

Alternative pathway. Human serum lyses unsensitizedrabbit RBCs via the alternative pathway (36). A 1-ml portionof T. pallidum subsp. pallidum or T. vincentii in GGVB or a1-ml portion of GGVB (buffer control) was incubated withcomplement at 37°C for 1 h in the presence of magnesiumEGTA, which blocks activation of the classical pathway andallows activation of the alternative pathway. A 1-ml portionof rabbit RBCs at 5 x 108 cells per ml was then added, andthe mixture was incubated at 37°C for an additional 10 min.All samples were immersed in an ice-water bath to blockfurther complement activation. After centrifugation the op-tical density of the supernatant was read at 541 nm. Positiveand negative controls and the determination of the percenthemolysis were as described above for the classical path-way. As another negative control, heat-inactivated serumdid not produce hemolysis.C3a and C4a assays. The generation of C3a and C4a was

quantitated with Upjohn radioimmunoassay diagnostic kits(Upjohn Diagnostics, Kalamazoo, Mich.). Only human se-rum samples were used as sources of complement. Rabbitserum in the assays did not always provide reproducibleresults. For C4a evaluation as a determinant of activation ofthe classical pathway, organisms were incubated with 4%human serum for 1, 8, and 15 min. For C3a evaluation as adeterminant of activation of the alternative pathway, organ-isms were incubated with 4% human serum for 1, 8, and 15min in the presence of magnesium EGTA to block theclassical pathway.

Blockage of activation. EDTA blocks the classical andalternative pathways; magnesium EGTA blocks only theclassical pathway. EDTA was dissolved in distilled water at100 mM and diluted to a final concentration of 10 mM.EGTA was dissolved in 1 N NaOH at 100 mM and thenbrought to pH 7.2 with HCI; the final concentration was 8mM (13). MgSO4 was dissolved in distilled water at 20 mMand used at a final concentration of 4 mM with the EDTA.When EDTA was added to sensitized sheep RBCs pluscomplement, no lysis occurred; magnesium EGTA alsoblocked activation of the classical pathway. When EGTAwas added to the unsensitized rabbit RBCs plus humancomplement, no lysis occurred; magnesium EGTA did notprevent lysis in this system, indicating normal functioning ofthe alternative pathway. These two controls were includedin each set of all reported experiments to ensure properfunctioning of each pathway.

Sialic acid determination. Sialic acid on the surface of T.pallidum subsp. pallidum or T. vincentii was quantitated by

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using the sialic acid radioimmunoassay test kit from Boeh-ringer Mannheim Biochemicals, Indianapolis, Ind. Proce-dures were carried out as specified by the manufacturer,with the following modification. Treponemes were pelletedvia high-speed centrifugation, and the supernatant was dis-carded. The initial test reagent was then added directly to thepelleted organisms.Neuraminidase treatment. T. pallidum subsp. pallidum at 2

x 108 to 4 x 108 organisms per ml was suspended inphosphate-buffered saline at pH 5.4. Neuraminidase fromClostridium perfringens was obtained from Sigma ChemicalCo., St. Louis, Mo. A final enzyme concentration of 0.5 to1.0 U/ml was added to the treponemes. After 1 h at 37°C, theorganisms were not lysed. They were washed in 20 volumesof GGVB by centrifugation. The enzyme-treated organismswere suspended in GGVB at 2 x 108 to 4 x 108 treponemesper ml. For controls, treponemes were suspended in phos-phate-buffered saline at pH 5.4 without neuraminidase andtreated as above.

Statistics. Data were analyzed for differences betweenmeans by the Student t test.

RESULTS

The lack of published quantitative data on treponeme-complement interaction made it necessary to optimize anumber of in vitro parameters. In terms of complementsources, rabbit serum contained variable levels of comple-ment activity. Serum was isolated from 10 rabbits and testedfor hemolytic activity by using sensitized sheep RBCs. Theconcentration of serum required to lyse 50% of the sheepcells (L50) varied from 1.3 to 8.4%. To minimize variabilityproblems, serum samples from three rabbits were pooled.Similar variations in complement levels also occurred inindividual human serum samples, although the ranges werenot as widespread. Human serum samples from four or fiveindividuals were pooled.

Difficulties with pool-to-pool variation in complementlevels could be minimized by harvesting one large pool andstoring it for long periods. Experiments were performed todetermine the stability of rabbit complement during storageat -70°C. Activity was evaluated after 0, 1, 2, 3, 4, and 8weeks. Slight decreases in activity were detected beyond 3weeks. Stored human complement was more stable thanrabbit complement. To avoid long-term storage problems,fresh pools of rabbit and human serum were isolated every 3weeks. As indicated above, the complement activity variedslightly from pool to pool. For experiments requiring onlyone concentration of complement, the equivalent of at least2 L50s was used to ensure excess levels of complement.To ensure complement interaction with an intact outer

surface during investigation of treponemal-mediated comple-ment activation, viable organisms were required. To main-tain viability, T. pallidum subsp. pallidum is usuallysuspended in extraction medium. Early preliminary experi-ments suggested that this medium might be anticomplemen-tary. Complement (4% rabbit serum) was added to theextraction medium or to GGVB and incubated without RBCsor organisms at 37°C in air. After 0, 1, 2, 3, 4, 10, and 20 h ofincubation, samples were removed and added to sensitizedsheep RBCs to quantitate residual complement activity.Figure 1 summarizes the results of six independent experi-ments. Two observations are noteworthy. First, the extrac-tion medium was anticomplementary for the first 3 h ofincubation. Thereafter, complement activity was similar to

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FIG. 1. Anticomplementary behavior of extraction medium.Freshly prepared treponemal extraction medium or GGVB wasincubated with 4% rabbit serum at 37°C in air for 0, 1, 2, 3, 4, 10, and20 h. Sensitized sheep RBCs were added, and after 1 h hemolysiswas determined as a function of residual complement activity. Barsrepresent one half of the standard error of the mean (SEM) of sixindependent experiments. Symbols: U, treponemal extraction me-dium; *, GGVB control.

that in the GGVB buffer control. Subsequent research iden-tified the inhibitory substance as dithiothreitol. Complementcomponents C3 and C4 have intrachain disulfide bonds (31,41) which are sensitive to reducing agents. We have previ-ously shown that during in vitro incubation, dithiothreitoloxidizes fairly rapidly (19). This oxidation in turn couldexplain the restoration of complement activity beyond 3 h.All further experiments were performed with treponemessuspended in GGVB. This buffer was not toxic for T.pallidum subsp. pallidum; organisms retained good motilityfor at least 24 h and did not lyse. This is important in thatsubsequent complement activation indicates outer surfacereactivity with viable organisms.A second point about the data in Fig. 1 is that the

complement in GGVB was activated during incubation in theabsence of exogenous stimulants. Within 6 h, only half of theoriginal activity remained. This presumably results from theprocedures used for harvesting serum (45). All further ex-periments were performed with short-term (<2-h) incuba-tions.

In the next series of experiments, complement activationas a function of treponemal concentration was investigated.T. pallidum subsp. pallidum at 5 x 106, 1 X 107, 5 X 107, 1x 108, 2 x 108, 4 x 108 organisms per ml was tested by using4% rabbit serum and hemolysis of sheep RBCs. At lowtreponemal concentrations, minimal complement was acti-vated during the 2-h incubation. As treponemal numbersincreased, more complement was correspondingly activated.At the highest concentrations of 2 x 108 and 4 x 108organisms per ml, rapid activation was detected. For allfurther experiments 2 x 108 to 4 x 108 treponemes per mlwere used.Complement activation was then determined as a function

of time. In the above experiments, organisms were incu-bated with complement for 1 h. T. pallidum subsp. pallidumor GGVB were incubated with 4% rabbit serum and sensi-tized sheep RBCs for 0, 10, 20, 30, 40, 50, and 60 min. Theresults of five independent experiments are shown in Fig. 2.Treponemes rapidly activated complement, and differenceswere apparent within 20 min. In related experiments, freshlyharvested organisms were compared with organisms prein-cubated for 24 h. Both fresh and aged treponemes activatedcomplement in a similar fashion. For the buffer control, RBC

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FIG. 2. Determination of complement activation as a function oftime. T. pallidum subsp. pallidum or GGVB was incubated with 4%rabbit serum. After 0, 10, 20, 30, 40, 50, and 60 min, samples wereremoved for determination of complement activity. Bars representone half of the SEM of five independent experiments.

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FIG. 4. Classical pathway activation. T. pallidum subsp. palli-dum, T. vincentii, or buffer was incubated with 4% human serum for1, 8, and 15 min. Soluble C4a was determnined as a function ofactivation of the classical pathway. Bars represent one half of theSEM of five experiments.

hemolysis peaked after 50 to 60 min. In all further experi-ments, treponemes or GGVB were incubated initially for 60min with complement; RBCs were then added for an addi-tional 60 min to maximize hemolysis.

After optimizing these in vitro parameters, we comparedcomplement activation by T. pallidum subsp. pallidum withactivation by the nonpathogen T. vincentii. Both treponemeswere preincubated for 1 h with different concentrations ofeither human or rabbit serum. Sensitized sheep RBCs werethen added to determine the level of residual complement.The data in Fig. 3 summarize results of five to eight exper-iments for each serum type. Both organisms activated com-plement. The L50 for human serum in the buffer control was4.5%, contrasted to 28.6% for T. pallidum subsp. pallidum.T. vincentii was more effective, and even with 40% serum(data not shown), less than 10% lysis occurred. Similar

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observations were recorded for the rabbit serum. The L50 inthe buffer control was approximately 1.8%, contrasted to12.6% for T. pallidum subsp. pallidum. Again, T. vincentiiwas more effective in activating complement.C4a was then quantitated as a direct reflection of activa-

tion of the classical pathway. It was possible that thealternative pathway was being activated by the organisms,thereby depleting later complement components and indi-rectly influencing the subsequent hemolysis of sheep RBCsvia the classical pathway. Figure 4 presents the averages offive independent experiments with 4% human serum as asource of complement (rabbit serum was not tested owing tocross-reactivity problems). After incubation for 8 min, thebuffer control contained 1,610 ng/ml, compared with 2,330ng/ml for T. pallidum subsp. pallidum and 2,600 ng/ml for T.vincentii. The last two numbers are statistically significant (P

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% HUMAN SERUM % RABBIT SERUMFIG. 3. Complement activation by treponemes. T. pallidum subsp. pallidum, T. vincentii, or buffer was incubated with different

concentrations of human or rabbit serum for 1 h. Sensitized sheep RBCs were added for an additional 1 h. Hemolysis was determined as afunction of complement activation. Bars represent one half of the SEM of eight experiments with human serum samples and five experimentswith rabbit serum sample.

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FIG. 5. Alternative pathway activation. T. pallidum subsp. pal-lidum, T. vincentii, or buffer was incubated with different concen-

trations of human serum in the presence of magnesium EGTA for 1h. Rabbit RBCs were added for an additional 10 min. Hemolysis was

determined as a function of activation of the alternative pathway.Bars represent 0.5 SEM of five experiments.

< 0.05). As observed for the hemolytic assay, some com-

plement was activated in the buffer control.T. pallidum subsp. pallidum and T. vincentii were each

preincubated for 1 h with different concentrations of humanserum. Rabbit RBCs were then added for 10 min as the testsystem for alternative-pathway activation. These experi-ments were performed in the presence of magnesium EGTA,which prevents activation of the classical pathway. Figure 5presents the averages of five independent experiments. T.pallidum subsp. pallidum slightly activated the alternativepathway; the differences at 9 and 11% serum were statisti-cally significant (P < 0.05). The L50s for the buffer controland T. pallidum subsp. pallidum were 8.6 and 10.6%, respec-tively. In contrast, the percent hemolysis with T. vincentiiremained less than 10%, even at the highest serum concen-

tration. Thus the nonpathogen was a better activator of thispathway.

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FIG. 6. Generation of C3a as indicator of alternative pathway. T.pallidum, subsp. pallidum, T. vincentii, or buffer was incubated with4% human serum in the presence of magnesium EGTA for 1, 8, and15 min. Soluble C3a was determined as a function of activation ofthe alternative pathway. Bars represent 0.5 SEM of five ex-

periments.

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% HUMAN SERUM

FIG. 7. Effect of sialic acid removal on activation of alternativepathway. T. pallidum subsp. pallidum, neuraminidase-treatedT. pallidum subsp. pallidum, or buffer was incubated with differentconcentrations of human serum in the presence of magnesiumEGTA for 1 h. Rabbit RBCs were added for an additional 10min. Hemolysis was determined as a function of activation ofthe alternative pathway. Bars represent 0.5 SEM of five experi-ments.

In related experiments the generation of C3a in the pres-ence of magnesium EGTA was also quantitated as anotherindicator of the alternative pathway. In confirmation of theabove results, T. vincentii rapidly activated this pathway.Figure 6 presents the averages of five independent experi-ments with 4% human serum as a source of complement.After 15 min of incubation, the buffer control contained1,620 ng/ml, the suspension of T. pallidum subsp. pallidumcontained 4,000 ng/ml, and the suspension of T. vincentiicontained 7,000 ng/ml.

Sialic acid interacts with C3b and the H regulatory factorto inhibit activation of the alternative pathway (11, 35).Experiments were performed to determine whether sialicacid was present on the treponemal surface. Different con-centrations of T. pallidum subsp. pallidum were comparedwith T. vincentii. The pathogen contained almost four timesas much sialic acid. As a control in these experiments, T.pallidum subsp. pallidum was incubated with neuramini-dase. Sialic acid concentrations were at baseline levels of thecontrol medium (100 to 120 ng/ml), indicating that underthese conditions neuraminidase effectively removed the sur-face-associated sialic acid.The observation that T. pallidum subsp. pallidum was not

as potent an activator of the alternative pathway may berelated to its higher amounts of surface sialic acid. Experi-ments were performed to determine whether enzymaticremoval of the surface sialic acid would increase activationof the alternative pathway. After neuraminidase exposure,treponemal numbers were not decreased. Thus the enzymedid not lyse the organisms, and subsequent complementactivation reflected direct interaction with the outer surfaceof the treponemes. Figure 7 shows the averages of fiveindependent experiments. Neuraminidase treatment in-creased complement activation of the alternatiye pathway.The L50s for the buffer control and T. pallidum subsp.pallidum were 12.2 and 14.8%, respectively. Importantly,the enzyme-treated treponemes were better activators thanthe nontreated organisms, and the L50 was greater than 20%,which was statistically significant (P < 0.05). This suggestedthat the surface-associated treponemal sialic acid retardsactivation of the alternative pathway.

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DISCUSSION

Studies of complement-treponeme interactions are com-

plicated by two inherent problems. Preparations of T. palli-dum subsp. pallidum totally devoid of antibody and comple-ment are not available. Organisms are passaged in rabbittestes and isolated after 9 to 11 days; at this time inflamma-tion is well developed. Freshly harvested treponemes con-

tain some surface-associated antibody (23, 27, 29, 30, 38, 40,48). Complement has also been activated at the time of invivo harvest as shown by treponemal surface C3 (1) and bysoluble C3a and C5a within the testicular extract (40). Asecond inherent problem is the complement source. Humanserum from nonsyphilitic patients contains antibodies thatcross-react with and immobilize T. pallidum subsp. pallidum(P. A. Hanff and J. N. Miller, Abstr. Annu. Meet. Am. Soc.Microbiol. 1979, B66, p. 26). Rabbit serum from animals notpreviously exposed to T. pallidum subsp. pallidum alsocontains treponemal antibodies (47; A. Jakubowski, K.Wicher, V. Wicher, and L. Grady, Abstr. Annu. Meet. Am.Soc. Microbiol. 1986, B136, p. 126). Thus the complementsource may supply exogenous antibodies.

In the experiments reported in this paper, treponemeswere incubated for a maximum of 2 h in GGVB. Importantly,this buffer, with its added serum as a complement source,was not toxic, and organisms remained actively motile for atleast 24 h and did not lyse. Therefore activation was a

function of direct complement interaction with intact trepo-nemal surface components of viable organisms.

Freshly harvested T. pallidum subsp. pallidum organismsimmediately activated complement. For syphilitic serologictests such as immobilization, neutralization, and agglutina-tion, incubation for 6 to 24 h is required for positivereactions. Furthermore, the fluorescent treponemal antibodyreaction involves fixation with acetone or methanol; unfixedorganisms are either nonreactive or poorly reactive. It hasbeen suggested that T. pallidum subsp. pallidum has an outerprotective layer that has to dissipate before the organismbecomes seroreactive (5, 9, 22, 32, 33, 43, 44). The resultspresented in the present paper indicate that if this outer layerdoes exist, it does not inhibit complement activation. C4a, as

a function of the classical pathway, and C3a, as a function ofthe alternative pathway, were detected after 8 min of incu-bation. In addition, complement activation by freshly har-vested T. pallidum subsp. pallidum organisms was similar toactivation by organisms preincubated for 24 h.The findings in the report represent the first demonstration

of treponemal activation of the alternative pathway. Both T.pallidum subsp. pallidum and T. vincentii activated thispathway, with the latter organism being a better activator.This observation may have application to the infectiousprocess in vivo. T. pallidum subsp. pallidum organismsreadily disseminate to other tissues via the bloodstream,whereas nonpathogenic treponemes do not. After enteringthe bloodstream, T. pallidum subsp. pallidum must bypassrapid activation of the alternative pathway long enough todisseminate to other tissues. The inability of the nonpatho-gens to disseminate may result from rapid killing in thebloodstream, mediated by the alternative pathway. Sialicacid on the surface of T. pallidum subsp. pallidum could beprotective by interfering with the H regulatory factor thatcurtails C3b activity (11, 35), thereby minimizing activationof the alternative pathway. In this paper it was shown that T.pallidum subsp. pallidum has four times as much sialic acidas T. vincentii and that enzymatic removal of treponemalsialic acid accelerated activation of the alternative pathway.

Highly invasive organisms such as group B streptococci,groups B and C Neisseria meningitidis, and Escherichia coliKl contain large amounts of surface sialic acid. Theseorganisms do not activate the alternative pathway, and it hasbeen suggested that the sialic acid facilitates their invasive-ness (12, 37). In like manner, the sialic acid on the surface ofT. pallidum subsp. pallidum may be an important immuneevasion mechanism that facilitates treponemal invasivenessand subsequent dissemination to other tissues.The specific mechanism of complement-mediated killing

of T. pallidum subsp. pallidum is unknown. Many organismshave evolved different mechanisms to retard complementactivity (21). Streptococcus pneumoniae is not lysed bycomplement; the membrane attack complex forms but is notable to penetrate the thick peptidoglycan layer (24). Salmo-nella minnesota S218 is resistant to complement; the termi-nal components fail to insert into the hydrophobic outermembrane domains and are shed from the surface of theorganism (25). Serum-resistant Neisseria gonorrhoeae bindsthe C5-C9 complex in an altered configuration that interfereswith the usual bacterial killing of serum-sensitive N. gonor-rhoeae (26). Trypanosoma brucei activates the alternativepathway but is not lysed; this parasite interrupts the cascadeat the C3 convertase level (10).

T. pallidum subsp. pallidum susceptibility to complementis unusual in that organisms are killed but not lysed. Inaddition, incubation with antibody and complement for atleast 2 h is required; thereafter the antibody and complementcan be removed, and killing occurs 4 to 6 h later. Experi-ments are in progress to determine whether T. pallidumsubsp. pallidum transiently sheds antibody and complementat least for the initial 1 to 2 h of in vitro incubation.

ACKNOWLEDGMENTS

I gratefully acknowledge the expert technical assistance providedby Barb Elmquist and Brad Ingersoll. Their careful and patient helpwas most important in performing these experiments. I also thankJean Regal, Department of Pharmacology, University of Minnesotaat Duluth School of Medicine, for valuable insights into complementactivation and for developing laboratory techniques for measuringcomplement activity. Her help greatly facilitated this research.Finally, I thank James N. Miller, Department of Medical Microbi-ology and Immunology, University of California Los AngelesSchool of Medicine, for critical evaluation of this manuscript.

This work was supported by Public Health Service grants Al16585 and Al 18619 from the National Institute of Allergy andInfectious Diseases.

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