genitourin med the importance components

Genitourin Med 1993;69:280-285

The importance of different components ofnormal human serum and lysozyme in the rapidimmobilisation of purified Treponema pallidum,Nichols strain

H J H Engelkens, M Kant, P C Onvlee, E Stolz, J J van der Sluis

AbstractObjectives-To study the role of differentcomponents in normal human serumand the role of lysozyme in rapid immo-bilisation of Percoll purified T pallidum(Nichols).Materials and methods-The immobili-sation of Percoll purified Tpallidum wasstudied after pre-incubations with differ-ent serum fractions (Fr) of normalhuman serum (Fr 1, containing IgM; Fr2, containing IgG and a low level ofhaemolytic complement, and Fr 1 (abs),depleted of IgG). A guinea-pig serumpool was used as a complement source inthe immobilisation experiments. Theinfluence was studied of removal oflysozyme from guinea-pig serum on theimmobilisation reactions. Further exper-iments were performed, using a fluores-cence technique, to detect C3bdepositions on fixed treponemes and tre-ponemes in suspension.Results-Rapid immobilisation ofPercoll-purified treponemes by the NHSserum fractions occurred only after pre-incubation with Fr 1 and Fr 2 simultane-ously. This was largely dependent on thepresence of a small amount ofhaemolytic C in Fr 2. Removal oflysozyme reduced this rapid rate ofimmobilisation. In fluorescence experi-ments it was demonstrated that C3bdeposition on fixed (that is damaged)treponemes occurred upon their incuba-tion with Fr 2 or the combination of Fr 1and 2. However, on treponemes in sus-pension C3b deposition occurred onlyafter incubation with the combination ofFr 1 and 2.Conclusion-The rapid immobilisationof Percoll purified treponemes by serumfractions from normal human serumrequires antibodies of the IgM and IgGclass, together with complement andlysozyme. Omission of one of these reac-tants slows immobilisation. Our experi-ments suggest that the reactants act insequence: the loss of integrity of theouter membrane by an attack by IgM andC offers the opportunity for lysozyme tohydrolyse the peptidoglycan layer sur-rounding the cytoplasmic membrane ofthe treponemes, which then is accessiblefor attack by antibodies and C.

(Genitourin Med 1993;69:280-285)

IntroductionThe eradication of Treponema pallidum sub-species pallidum (T pallidum), the causativeorganism of syphilis, in patients is oftenincomplete. This may lead to chronic infec-tion and in some cases to tertiary syphilis. Inexperimental syphilis in rabbits the persis-tence of treponemes has also been docu-mented, despite the development of chancreimmunity.The mechanisms of survival of the tre-

ponemes for many years in the host are notfully understood. Several hypotheses havebeen proposed. It may relate to a poorimmune response, but experimental resultsare contradictory. Recently, it was hypo-thesised that an early down-regulation of theimmune response could allow the survival ofa small number of treponemes.1 Alternativelyit was postulated that coverage of the tre-ponemal outer membrane with mucopolysac-charides2 3 or host serum proteins4 confersprotection against attack by the host'sdefences upon the treponemes. Although thismight explain the presence of pathogenictreponemes together with high titre trepone-mal antibodies in a host, evidence for thepresence of a cover offering protection to thetreponemes is lacking. It may be that anabsence of antigenicity, due to the presenceof only few transmembrane particles in thetreponemal outer membrane, may provide amechanism by which the treponemes evadethe host immune response.56

In vitro immobilisation of rabbit-derivedtreponemes by antibodies and complementtakes a long time. It was hypothesised thattreponemes had first to lose their protectivecover before antibodies could gain access tothe treponemal surface and complementcould be activated to eventually lyse the tre-ponemes. The presence of lysozyme acceler-ates immobilisation. The location of thesubstrate of this enzyme has been a subject ofdiscussion.7-9 Blanco et al in 199010 demon-strated that it was complement-activation andnot antibody-binding which was rate limitingin the immobilisation process. The formercorrelated with the antibody-mediated aggre-gation of the rare outer membrane protein.Recently, we demonstrated that treponemes,when harvested from rabbit testicles and puri-fied by Percoll centrifugation, were quicklyimmobilised by normal human serum(NHS)." The immobilisation could be inhib-ited by fluids from infected and non-infectedrabbit testicles. We investigated which com-

Department ofDermatology andVenereology,University HospitalRotterdam - Dijkzigtand ErasmusUniversity Rotterdam,The NetherlandsH J H EngelkensM KantP C OnvleeE StolzJ J van der SluisAddress for correspondence:H J H Engelkens, MD,Departnent of Dermatologyand Venereology, UniversityHospital Rotterdam -Dijkzigt, Dr Molewaterplein40, 3015 GD Rotterdam,The Netherlands.Accepted for publication12 February 1993

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The importance ofdifferent components ofnormal human serum and lysozyme

ponents of NHS participate in the rapidimmobilisation of purified treponemes.

Materials and methodsPropagation and extraction of T pallidum.Propagation and extraction of T pallidum(Nichols) were performed as previouslydescribed." Briefly, the testes were mincedand 1 ml of serum free basal reduced medium(BRM) without dithiothreitol was added pergram of wet testicular tissue. The mixturewas shaken for 45 minutes at room tempera-ture in an atmosphere of 5% carbon dioxideand 95% nitrogen, and centrifuged for 10minutes (800 g) to sediment gross particulatematter. The fluid layer containing the tre-ponemes was collected and centrifuged at12 000 g at 4°C for 10 minutes to pellet thetreponemes. The supernatant was removedand the treponemes were resuspended infresh BRM and subjected to Percoll(Pharmacia, Uppsala, Sweden) density gradi-ent centrifugation (43% Percoll in BRM) for30 minutes at 37 000 g.l2 The layer contain-ing the treponemes was collected and usedfor further experiments.

Enumeration of treponemes. The treponemeswere counted using microslides (path length0.05 mm, Camlab Limited, Cambridge,England, ref: 5005) and the density of tre-ponemes was calculated as previouslydescribed. 3

Human serum pool. A serum pool was pre-pared from blood samples from 150 blooddonors with negative TPHA results. The poolwas stored in aliquots at - 70°C. Portions of10 ml were used to prepare antibody-contain-ing serum fractions.

Guinea-pig serum pool. Guinea-pigs werebled and their serum was tested individuallyfor the capacity to sustain the viability ofPercoll purified treponemes in an assay simi-lar to the immobilisation assay describedbelow. Only those specimens in which tre-ponemal viability was better than 70% after22 h were used to prepare a serum pool. Thispool was stored in aliquots at - 70C andwas used as the complement source in immo-bilisation experiments. Samples used in theseexperiments were thawed only once.

Preparation of serum fractions from normalhuman serum (NHS). Approximately 10 mlportions from the human serum pool weresubjected to Sephadex G-200 gel filtration.The IgM-containing 19S and the IgG-con-taining 7S fractions (designated Fr 1 and Fr 2respectively) were collected and concentratedto the volume of serum initially applied to thecolumn in Amicon concentration cellsequipped with PM10 membranes. After dial-ysis for 24 h against Earles balanced salt solu-tion (one change), the fractions were storedin small portions at - 70C until use.

Estimation of complement activity.Complement activity of sera and serum frac-tions was determined as previouslydescribed. 14

SDS-PAGE and Western blotting. Sodiumdodecyl sulphate-polyacrylamide gel elec-

trophoresis (SDS-PAGE) and Western blot-ting, including the blocking and staining pro-cedures of the strips were performed asdescribed previously.1' Ten,pl of Fr 1, Fr 1(abs) (see below), and human IgG (Nordic,Tilburg, the Netherlands, lot nr. 1-169) as areference, were appropriately diluted in sam-ple buffer and applied to different slots of thegel after heating in a boiling water bath forfour minutes. After completion of the elec-trophoresis run and the blotting procedurethe strips were incubated with conjugate,consisting of the affinity purified gold-labelledIgG fraction from a goat antiserum againstheavy and light chains of human IgG (JanssenLife Sciences Products, Beerse, Belgium) for2 hours, followed by silver enhancement.Separate strips were stained according theAuroprobe staining procedure (Janssen LifeSciences Products) to visualise the polypep-tides. The low-molecular weight standardsfrom Pharmacia (Uppsala, Sweden) wereused in estimating the size of the visualisedpolypeptides.

Depletion of IgG. Depletion of IgG from Fr1 was accomplished by absorption withStaphylococcus aureus, strain Cowan 1. (Serva,Heidelberg, Germany, lot nr. 16082c). 2-5 mlof Fr 1 was incubated with 0.05 g dry weightof pre-washed bacteria for 1 h at 4°C. Thebacteria were pelleted by centrifugation at27 000 g at 4°C. The supernatant, designatedFr 1 (abs) was collected and stored inaliquots at -70°C. The efficacy of theabsorption procedure was controlled by SDS-PAGE electrophoresis and immunoblotting.

Estimation of lysozyme. The lysozyme con-tent of guinea-pig serum and bentonite-absorbed guinea-pig serum, expressed asunits/ml and of chicken egg white lysozyme(Sigma, St Louis, USA, lot nr. 89F8275),expressed as units/mg, was determined by thelysis of Micrococcus lysodeikticus (Sigma, lotnr. 109F68081) according to the instructionsof the supplier. The latter results were used tocalculate the amounts of chicken egg whitelysozyme to be added to bentonite-absorbedserum to attain the specified number of unitsfinally present in the mixtures for the study ofimmobilisation.

Depletion of lysozyme. Guinea-pig serumwas depleted of lysozyme by absorption withbentonite-SF (Serva, Heidelberg, Germany,cat.nr. 14515) according to Wardlaw.'5 Aftercompletion of the absorption procedure theabsorbed serum was checked for its lysozymeand complement contents. Lysozyme couldno longer be detected. The complement levelhad decreased by 25% as compared with pre-absorption levels.

Immobilisation of treponemes. The Percollpurified treponemes were used in a final den-sity of 2 x 107 treponemes/ml. A sufficientnumber of treponemes were mixed with Fr 1or Fr 2 (final content 10% v/v) or with a 1:1mixture of Fr 1 and Fr 2 (final content 20%v/v). These mixtures were supplemented tothree-quarters of the final volume with BRMand pre-incubated for 15 minutes at roomtemperature. Finally, pooled guinea-pig

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serum or bentonite-absorbed pooled guinea-pig serum was added to a final content of25% (v/v). Aliquots of 0.5 ml of these mix-tures were placed in small tubes, which wereloosely plugged with cottonwool and incu-bated in a reduced oxygen atmosphere (4%)at 34WC.'6 The percentage of mobile tre-ponemes was determined in wet mounts after0, 1, 2, 3.5 and 5.5 h by observing at least100 treponemes in randomly selected micro-scopic darkfields. Control tubes were set upby adjusting a volume containing 4 x 107 tre-ponemes from the various suspensions to 1-5ml with BRM. After the pre-incubationperiod 0.5 ml pooled guinea-pig serum wasadded. These tubes were treated further asdescribed above. A similar set-up was used tostudy the capacity of serum from individualguinea-pigs to support the mobility of the tre-ponemes. Here, 4 x 107 treponemes, sus-pended in 1.5 ml BRM, were pre-incubatedand mixed with 0.5 ml of individual guinea-pig serum. Aliquots of these mixtures werestored and read as described above with addi-tional readings after 22 h.

Fluorescence. Three types of fluorescenceexperiments were performed to detect C3bdepositions on treponemes. In the first type50 ,u from a Percoll purified treponeme sus-pension (density 2 x 107) was applied toglass slides, which were air-dried and heat-fixed. It was demonstrated previously thatthis procedure damages the treponemal outermembrane."' These slides were overlaid withtwo drops of Fr 1, Fr 2 or a 1:1 mixture of Fr1 and Fr 2 for 30 minutes at room tempera-ture. The second type of experiments wasperformed on treponemes which had beenincubated in suspension with Fr 1, Fr 2 ortheir 1:1 mixture. In the third type of experi-ments the treponemes in the tubes with thereaction mixtures for immobilisation wereused. In the type 2 and type 3 experimentsthe contents of the tubes were supplemented

percenta

9e

1 2 3.5 5.5

hours

E Fr Fr1 +2 []Fr 2 EControl

Figure 1 Immobilisation ofPercoll purified treponemes. Treponemal mobility afterpreincubation with fraction 1, fraction 2, the mixture offractions 1 and 2, orBRM( = control). Pooled guinea-pig serum was used as a complement source throughout theseexperiments. Results are expressed in percentages of mobile treponemes. Means andstandard deviations of the results ofexperiments with six treponemal suspensionsoriginatingfrom different rabbits are shown.

with 10 ml BRM and centrifuged at 12 000 gat 4°C for 10 minutes. The supematant wasremoved and the pelleted treponemes wereresuspended in 1 ml BRM. The suspensionwas pipetted into a Petri dish equipped with acoverglass. After centrifugation at 800 g forfive minutes, the coverglasses were rinsedwith BRM. In type 1 and type 2 experimentsthe treponemes were incubated with twodrops of a FITC labelled IgG fraction from agoat anti-human C3b antiserum (CentraalLaboratorium van de Bloedtransfusiedienst,Amsterdam), in type 3 experiments the tre-ponemes were incubated with a similar frac-tion from a goat anti-guinea-pig C3bantiserum (Kirkegaard & Perry Laboratories,Inc, Gaithersburg, USA) for 30 minutes atroom temperature. After rinsing, the slideswere covered with a coverglass. The cover-glasses with adhering treponemes from thePetri dishes were laid upside down on micro-scopic slides. The preparations were sealedwith nail polish and read within 3 h as previ-ously described.'7

ResultsA pre-incubation of Percoll purified tre-ponemes with the IgG-containing Fr 2resulted, after the addition of guinea-pigserum, in a survival of 88% of the treponemesafter two hours and of 50% after 5.5 h. Insimilar experiments with the IgM-containingFr 1 a mean of 88% survived after 2 h and22% after 5.5 h. A rapid immobilisation ofthe treponemes was noted only when the tre-ponemes had been pre-incubated with thecombined Fr 1 and Fr 2. Two hours after theaddition of guinea-pig serum almost all tre-ponemes had been immobilised (fig 1). In thecontrol tubes, a mean of 97% of the Percollpurified treponemes survived after 5.5 h.SDS-PAGE electrophoresis and immuno-

blotting showed that Fr 1 contained someIgG, which was no longer detectable afterabsorption of this fraction with Staphylococcusaureus, strain Cowan 1. The immobilisationof the purified treponemes after preincuba-tion with Fr 1 (abs) occurred more slowlythan the immobilisation after pre-incubationwith Fr 1. After 5.5 h, 51% of the treponemesthat had been pre-incubated with Fr 1(abs)survived; after pre-incubation with Fr 1 thiswas 8%. This indicates a role for IgG in theimmobilisation process.An analysis of the complement content of

Fr 1 and Fr 2 showed that only Fr 2 pro-duced a haemolysis of sensitised sheep ery-throcytes just above control values, whichdemonstrated that Fr 2 contained a low levelof haemolytic complement. A 1:1 mixture ofFr 1 and Fr 2 did not increase the haemolysisof sensitised erythrocytes, indicating that noseparation of complement components hadoccurred during the preparation of serumfractions.

Percoll purified treponemes which hadbeen pre-fixed onto glass slides showed adeposition of human C3b after they had beenoverlaid with Fr 2 or the combination of Fr 1

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Percentage of differently treated treponemes showing deposition ofC3b after incubationwith Sephadex G-200 separatedfractions from NHS and their mixture(Fr 1: 19Sfraction, Fr 2: 7Sfraction; int. = intensity offluorescence)

Fr2 Frl + 2Fr )

Antigen % pos % pos int. % pos int.

Fixed treponemes 0 100 1-2 100 2-3Treponemes in suspension 0 0 - 100 1-2

and Fr 2, followed by conjugate. No C3bdeposition was observed on pre-fixed tre-ponemes that had been overlaid with Fr 1and conjugate, emphasising the absence of Cfrom this fraction and demonstrating the non-reactivity of the conjugate with the tre-ponemes. After incubation of purifiedtreponemes in suspension with each fractionor their combination a deposition of C3b wasobserved only after incubation with the com-bined Fr 1 and Fr 2 (table). These resultsshow firstly the different reactivities of theserum fractions towards damaged and intacttreponemes, and secondly that in addition toFr 2, the presence of Fr 1 is essential for thedeposition of C3b on treponemes which hadbeen incubated in suspension.

Parallel with their immobilisation, the C3bdeposition on. the treponemes was studied.All treponemes which had been preincubatedwith the mixture of Fr 1 and Fr 2 showed adeposition of guinea-pig C3b of a 3 + to 4 +(strong to very strong) intensity, as early as

11

percenta

9e

1 2 3.5 5.5hours

*Fr1+2 Frabs *Fr+10U Fr+50U

E Control Fr + 5 U Fr + 25 U OIIJ Fr + 100 U

Figure 2 The role oflysozyme in the immobilisation of Percoll purified Tpallidum.Treponemal mobility after preincubation with the mixture offractions 1 and 2 (Fr 1 + 2),(orBRM = control), incubated with guinea-pig serum or bentonite-absorbed (Fr abs)guinea-pig serum. Reconstitution of the absorbed guinea-pig serum with lysozyme was

performed by addition ofgraded amounts ofchicken egg white lysozyme (from 5 U to100 U). Results are expressed in percentages of mobile treponemes. Means and standarddeviations of the results of experintents with three treponemal suspensions originatingfromdifferent rabbits are shown.

1 h after incubation. At later times the num-ber of treponemes decreased sharply in thefluorescence preparations. After pre-incuba-tion with Fr 1 or Fr 1 (abs) all treponemesshowed a very weak fluorescence for C3b of aspeckled character, which did not changetowards the end of the experiments. Thesame was true of a majority of approximately60% of the treponemes that had been pre-incubated with Fr 2.

It became clear that the rate of immobilisa-tion of the Percoll purified treponemes afterpre-incubation with the mixture of Fr 1and Fr 2 was greatly reduced when Fr 2was replaced by heat-inactivated Fr 2.Replacement of Fr 1 by heat-inactivated Fr 1in this mixture hardly influenced the rapidityof the immobilisation as compared with theimmobilisation after pre-incubation with themixture of Fr 1 and Fr 2.The role of lysozyme is demonstrated in fig

2. As before, the immobilisation of purifiedtreponemes which had been preincubatedwith the mixture of Fr 1 and Fr 2 proceededrapidly after the incubation with guinea-pigserum. However, when bentonite-absorbedguinea-pig serum was used the immobilisa-tion proceeded much more slowly: after 5.5 h51% of the treponemes still showed goodmobility. The participation of lysozyme in therapid immobilisation was further demon-strated by the reconstitution of the absorbedguinea-pig serum with graded amounts ofchicken egg white lysozyme. In the presenceof the lowest amount of lysozyme added(5 U) almost no mobile treponemes were leftafter 5.5 h (fig 2). Reconstitution of theabsorbed guinea-pig serum with 25 U oflysozyme resulted in an immobilisation asrapid as with unabsorbed guinea-pig serum.Higher amounts of lysozyme did not influ-ence the rate of immobilisation any further.Moreover, these results indicate that the slowimmobilisation with bentonite-absorbedguinea-pig serum was indeed due to thedepletion of lysozyme and not to a loss ofcomplement from the guinea-pig serum.To establish a possible reaction sequence

of the fractions, pre-incubations were per-formed with Fr 1 (abs) or Fr 2 and after 1 h ofincubation with guinea-pig serum, the miss-ing fraction was added to half of the reactionmixtures. These results are demonstrated infig 3. In all four types of reaction mixtures themobility of the treponemes hardly changedduring the first hour when one or both frac-tions were present. At the end of the experi-ments the immobilisation was greatest in thetubes which had been pre-incubated with Fr1 (abs) and to which Fr 2 was added later(survival 36%). In the tubes which had beenpre-incubated with Fr 2, with Fr 1 (abs)added later, the survival of the treponemeswas 56%. The survival of the treponemes inthe tubes containing only one fraction wasapproximately 70%. In parallel experimentsbentonite-absorbed guinea-pig serum wasused. Here, in all four types of reaction mix-tures approximately 90% of the treponemessurvived after 5.5 h.

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1

per

centa

g

e

2 3.5hours

*Fr l +Fr2 3Fr 1 [] Fr2+Frl

Figure 3 Experiments to study a possible reaction sequence of the fractions.Preincubations were performed with Fr 1 (abs) or Fr 2. After one hour of inculguinea-pig serum the missingfraction was added to one-half of the reaction mi(Fr 1(abs) + Fr 2, or Fr 2 + Fr 1(abs)), or incubation was continued with o;only (Fr 1 (abs) or Fr 2). Results are expressed in percentages of mobile treponMeans and standard deviations of the results of experiments with five treponensuspensions originating from different rabbits are shown.

DiscussionThe rapid immobilisation of puiponemes by the serum fractionsNHS pool occurred only whenponemes had been pre-incubatedand Fr 2 simultaneously and was

nied by a strong deposition of C3bponemal surface. The rapid immowas largely dependent on presenceamount of haemolytic complemenRemarkably, only a minor part oponemes were immobilised withinpre-incubation with Fr 2, despiteence of anti-treponemal IgG andfraction. This can possibly be ex;

the results of the experiments inC3 depositions on fixed treponemtreponemes in suspension were coI

was shown that C3b deposition on

ponemes occurred upon their incubFr 2 or the combined Fr 1 and 2.C3b deposition on treponemes in soccurred only when they were inculthe combined Fr 1 and 2. Thesephenomena may be a reflection ofent requirements for various classbodies, which lead to the activaticlassical complement pathway: a s

molecule present in an antigercomplex can bind C lq to start the (For IgG, however, at least a doubmolecules, sufficiently near to eac

needed for Clq binding.'8 Apparfixed treponemes (that is, damponemes) offer a sufficiently close 1the epitopes to allow C-activatiorThese results show that C-activaticFr 2" is possible. This does not c

treponemes in suspension. Here,containing fraction is needed to a

C3b deposition. As previously denby others,5 619 the outer membranelidum shows a scarcity of epitopes.be the reason why IgM, but not

form antigen-antibody complexes on the tre-ponemal surface, which are capable of C-acti-vation. This may be due to the larger size ofthis class of antibody molecules, which willallow them to bridge larger distances betweenepitopes. A preference for the binding of IgMantibodies to adherent treponemes has beendemonstrated previously.20 This makes itplausible that the first step in the rapid immo-bilisation of the treponemes is an attack byIgM and C. However, the results of theexperiments performed with Fr 1(abs)demonstrate that presence of IgM alone doesnot result in rapid immobilisation. For this to

5.5 occur IgG is also needed, as emphasised bythe rapid immobilisation produced by themixture of Fr 1 and Fr 2 and the reduction in

Fr 2 the immobilisation rate after removal of IgGfrom Fr 1. The results of the study of the

bation with reaction sequence of IgM and IgG antibodiesixtures largely agree with this. In this set of experi-nefraction ments more rapid immobilisation was notednales. when the preincubation was performed with

Fr 1 (abs) as compared with a preincubationwith Fr 2. These results might point to aninhibition of IgM by IgG, due to a competi-tion for epitopes on the treponemal surface.

rifled tre- A role of lysozyme in the immobilisation offrom the treponemes was recognised three decadesthe tre- ago.7-9 From our experiments it is clear that

with Fr 1 removal of lysozyme reduces the rate ofaccompa- immobilisation. Reconstitution of the guinea-on the tre- pig serum with graded amounts of lysozymeobilisation restores the capacity of rapid immobilisationof a small (Fig 2). The results so far obtained demon-t in Fr 2. strate that rapid immobilisation can bef the tre- accomplished without presence of lysozyme5.5 h after during the pre-incubation steps. This showsthe pres- that the action of lysozyme does not occurC in this during the initial steps of immobilisation, butplained by probably only after the outer membrane ofwhich the the treponemes has been damaged by a com-es and on plement dependent immunological reaction.mpared. It This is in agreement with the conclusions offixed tre- Muller et al.9

)ation with Our findings demonstrate that the rapidHowever, immobilisation of purified treponemes by;uspension serum fractions from NHS requires antibod-bated with ies of the IgM and IgG classes, together withc different complement and lysozyme. Omission of onethe differ- of these reactants inhibits the rapid immobili-es of anti- sation. All available evidence indicates thation of the these reactants act in sequence in the rapid;ingle IgM immobilisation: the integrity of the outera-antibody membrane is first attacked by IgM and C.C-cascade. The loss of integrity of this membrane pro-let of IgG vides the opportunity for lysozyme to hydro-h other is lyse the peptidoglycan layer surrounding therently, the cytoplasmic membrane of the treponemes.aged tre- The latter is then accessible for attack bypacking of antibodies and C. Presumably IgG plays ai by IgG. major role in this process, since rapid immo-)n "within bilisation without IgG did not occur.3ccur withthe IgM

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2 Fitzgerald TJ, Johnson RC. Surface mucopolysaccharidesIgG, can of Treponema pallidum. Infect Immun 1979;24:244-5 1.

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4 Alderete JF, Baseman JB. Surface-associated host proteinson virulent Treponema pallidum. Infect Immun 1979;26:1048-56.

5 Radolf JD, Norgard MV, Schulz WW. Outer membraneultrastructure explains the limited antigenicity of viru-lent Treponema pallidum. Proc Nad Acad Sci USA1989;86:2051-5.

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20 van der Sluis JJ, Kant M, Onvlee PC, Stolz E. The inac-cessibility of the outer membrane of adherent Treponemapallidum (Nichols strain) to anti-treponemal antibodies,a possible role of serum proteins. Genitourin Med 1990;66:165-70.

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