Electron Microscopic Anatomy of Pathogenic Treponema Pallidum · Th e microsco pic anatomy of t h e Treponema pallidum (T. pallidum) is ummari ze d in schematic form. L iterature

THE JounNAL or l Nvf:STIGAl'IYE DEitMATOLOGY

Copy rij!hl © 1972 by The Williams & Wilkins Co . Vol. 58, No. 4

Print ed in U.S.A.

ELECTRON MICROSCOPIC ANATOMY OF PATHOGENIC TREPONEMA PALLIDUM*

S. E . WIEGAND, M.S. , M.D., t P. L. STROBEL, M.S.:j: AND L. H. GLASSMAN, M.S .§

ABSTRACT

The microsco pic anatomy of t he Treponema pallidum (T. pallidum) is summari zed in

schematic form. L iterature is reviewed and an electron micrograph is presented s uggesting: that m esosom es protruding through the cell envelope may account for t he lupus erythema­tosus sera beading phenomenon seen in the "false p ositive" F luorescent Treponema! Anti­

body Absorption Test; that mucoid coat m ay inhibit protective immunologic recognition

of T . pallidum; a nd, that axia l filam ents contribute significantly to the three-dimensional configuration of t he o rga nis m.

The subcellula r a natom y of the treponemes has been gradually elucidated during the last two decades. In this article the anatomy of the Trepo­ne.ma pallidum (T. pallidum) and the techn iques used to study it are described . Some thoughts about the function of the subcellular strudures are discussed ,

T . pallidum is a spirochete 6- 22.iL lon g (ll,IL av e rage) a nd about 0.21L wide (outer envelope diam eter in the fixed state) (Fig. 1). It has about 10 coils with a wave length of 1)!, coil to coil, a nd a n ampli tude of 0.21! to 0.71L. The organis m cons ists of a protoplas mic cylinder containing deep fila ­m ents, ribosomes, vacuoles, " mesoso mes" and poss ibly nucleoid materi a l. The semirigid cyl­inder is surrounded by an intermediate layer con­taining axial fi la m ents and possibly stored nu­trients. There is a nose piece at each end of the protoplas mic cylinder. The entire organism is covered by a nonrigid envelope whi ch may be covered by a mucoid layer in vivo.

MATERIALS AND METHODS

A clarkfield positive, three-week-old chancre on a human penis (Rapid Plasma Reagin card test positive) was aspirated with a modified syringe. The transudate obtained was diluted wi th 0.01 M phosphate buffered saline (pH 7.3) and fil te red in succes ion t hrough two 0.5J.L Nucleopore (General E lectri c Co. Irradiation Proc­essing Operation) membrane filters. The filtrate was fixed in 4% paraformaldehyde for 1 hour at 5° C and centrifuged at 35,000 Relative Centrifuga l Force (RCF) (g) for 20 minutes. The supernatant was discarded and the T. pallidum were resupsended in a cup of parafor­maldehyd e.

Nichols pathogenic T . pallidum were inoculated in to

Received September 23, 1971; accepted for publica­tion November 16, 1971 .

* From the Venerea l Disease Research Laboratory, Venereal Disease Branch and the Clinica l Laboratory Branch, Pathology Section ,:\: State and Com munity Services Divis ion, Center for Disease Control, Health Services and Mental Health Administra tion , U.S. De­partment of Hea lth, Education, and Welfare, Atlanta, Georgia 30333.

t Present address: 6500 Vernon Woods D ri ve, Suite D-8, (Sandy Springs), Atlanta, Georgia 30328.

§Ana lytica l Instrumentation Laborato ri es, Engi­neering Experiment Station, Georgia Institute of Tech­nology.

rabbit testes . One month later the rabbi t's testes were removed through a sterile abdominal incision. T . Pal. lidum were extracted from the rabbit testes and filtered through an 811 Millipore fil te r followed by a 0.51L Nu. cleopore filter. The fil t rate was fixed in paraforma\. dehyde a nd centrifuged at 35,000 RCF (g) for 20 min . utes.

Whole mounts. Part of the paraforma ldehyde suspe11 •

sions of both the human and the Nichols T. pallidum were each mixed with an equa l amount of 1% phospho. tungstic acid (PTA). Drops of the mixtures were picked up on Formvar-coated copper grids. The negative ly stained T. pallidum were examined in a Philips 200 transmission electron microscope (TEMJ operated at 60 and 80 Kv with two condenser lenses.

Thin sections. The remainder of the paraformal­dehyde-fixed human T. pallidum suspension was post­fixed in buffered 1% osmium tet rox ide solution and sus­pended in warm agar by the method of Ke llenberger (1). The solid agar pellet was dehydrated in graded a lcohols satura ted with propylene oxide, and gradually infil: t rated with Maraglas. The spec imen was embedded in Maraglas and sectioned on an LKB ultra microtome. The si lver-gray sections (600- 900 Al were picked up on copper grids, sta ined with satura ted uranyl acetate (in methanol) and aqueous lead cit rate (2) , and exa mined in a Philips 200 TEM .

186

Carbon replicas. T. pallidum, Nichols pathogenic strain, were dialyzed against phosphate buffered saline (pH 7.2). The d.ia lyzed suspension was centrifuged at 48,000 RCF (g) for 30 minutes, resuspended in buffered 5% glutera ldehyde and again centrifuged at 48,000 RCF (g) for 30 minutes. The centrifugate was twice resus. pended in dist ill ed water a nd recentrifuged.

A portion of the fina l centrifugate was dried onto a rectangu lar glass sli de. Unwashed, 0.5J.L Nucleopore fil­tered, human chan cre T. pallidum were also applied to glass slides. A platinum preshadowed ca rbon replica was prepared by evaporating platinum from a tungsten fi lament. This shadowing layer was then covered with carbon evapora ted from a carbon arc.

The carbon replica was 11oated from the glass slide in 48% hydrol1uoric acid (HF) and t ransferred to distilled water. Several methods were t ried in an attempt to remove the T . pallidum from the repli ca. After the carbon was floated free in HF, the fi lms were treated ul t rason ica lly for 10- 30 seconds. Enzymatic digestion with trypsin , lysozyme and N-butanol in phosphate buf­fer, pH 7.5, was a lso tried (3). As an alternative to flota­tion in HF, the replica was soa ked in 2.5 N NaOH for two days, then dried and !1oated on distilled water. None of these methods was successful. The resul ting pseudo- replicas were exa mined and photographed in a Philips 200 TEM at 60 I<v . Stereo micrographs were taken usin g 12° tilt sepa ration between sets.

Carbon-metal coated specimens. A portion of the centrifugate of T. pallidum prepared for the stereo

ELECTRON MICROSCOPY OF T .. pallidum 187

FIG. 1. T. pallidum , removed from a human chancre and negatively sta ined with 1% phosphotungstic acid; by TEM ( x 11,416) .

carbon replication technique was applied to glass cover s lips. The specimens were placed into a vacuum chamber and coated with a thin layer of carbon and an outer thin coating of gold -pallad ium. These specimens were then examined and photographed under a Cam­bridge scanning electron microscope (SEM) .

RESULTS

TEM of negatively stained whole mounts. The organisms revea l a centra lly placed protoplasm which is bound by two or three membranes. Be­neath the inner membrane, longitud inally-ori ­ented, parallel, deep filaments can be seen throughout the length of the cylinder. The deep filaments appear to be about 75 A wide (Fig. 2). The cylinder varies in diameter and density along its course. Protruding from the cylinder are at least three distinct types of bodies. One type is the multilaminated body which shows layers and whorls of parallel 30 A dense membranes sepa­rated by 30 A electronlucent spaces (Fig. 3). These lami nated bodies vary in size from 0.021-l to 0.17 in depth and to more than 1J.l in length, causing a protrusion in the envelope in the fixed state. They appea r to project through an opening in t he envelope in some views (Fig. 4, A). Occa­s ionally the la minated bodies appear to have 200

A openings in to the cavity of the cylinder (Fig. 5, A) . A second type of protruding body is seen as an elevated, granu lar-surfaced nodule extending from the surface of the cylinder (Fig. 6, A) . A third type of protruding body is composed of a serpen t ine " in testi ne- like" 300- 400 A wide t ract coiled into an a rea of about 1J.l x 0.2J.l x 0.2J.l

(Fig. 7, A). In addition to these three types, there are occasionally seen round 0.1J.l diameter bodies (subterminal bodies) at the ends of the cy linder and subjacen t to the nose piece (Fig. 4, B). Elec­t ron- lucen t areas 100 A x 125 A surrounded by electron-dense areas 250 A x 400 A are a lso ran­domly seen as distinct structures along the proto­plasmic cy linder cell wall (Fig. 6, B). Similar structures are seen as subte rmina l insertion points of axia l fi laments. These filaments (100-150 A in diameter) occur in parallel groups of 3 to 7, fo llowing a helica l course outside the proto­plasmic cylinder ce ll wall but inside the outer envelope and causing a bulge in this intermediate layer in the fixed state (Fig. 8) .

On the envelope there are large ba lloon-shaped extensions with narrow connecting n ecks whi ch may originate in t he protoplasmic wa ll -membrane (Fig. 9, A). Large, very dense objects, 0.2J.l x 0.4J.l,

ap paren tly att~ched to the envelope are of uncer­ta in origin (Figs. 1 a nd 7, B). The nose piece at each end of the sp irochete occas ionall y appears to be separated from the protop lasmic cylinder by a 100 A electron-lucen t space a nd is surrounded by t he continuous outer envelope (Fig. 5, B). T he nose pieces of the organisms are usually tapered and rounded (Fig. 4) , but in some cases may be fl attened or f1ange -like (Figs. 5 and 7).

TEM of thin sections. The protoplasmic cy l­inder in cross section appea rs as a round mass of osmiophi lie particles that vary in density and dis ­tr ibu t ion (Fig. 10, A). Oblong deep fi laments, (Fig. 10) 75 A x 120 A, are located in parallel

188 THE JOURNAL OF INVE STIGATIVE DERMATOLOGY

FIG. 2. A portion of the midbody of negatively stained whole mount of a T. pallidum demonstrating the axia l fil­aments (A) and the deep filam ents (B); by TEM (x 163,000).

ELECTRON MICROS COPY OF T : pallidum 189

F IG. 3. A port ion of the midbody of a negat ive ly sta ined whole moun t of a T. pallidum demonstrating a la mi ­nated mesoso me; by TE M ( x 336,000).

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194 TH E JO URNAL OF INVESTIGATIVE DERMATOLOGY

F IG. 8. A portion of the midbody of a negatively stained whole mount of a T . pallidu.m demonstrating the axial fil aments; by TEM (x 133,000).

rows just beneath the tripl e- layered protoplasmic cell membrane (Fig. 10, D). Round axial fil a ments (150 A diameter) a re seen outside the membrane (Fig. 10, C). The envelope was not found to be intact in our s pecimens unless it was in t he form of the a bove ment ioned ba lloon structures (Fig. 11 , B). Axia l fil amen ts were seen to follow a long the body of the protoplas mi c cy linder in a heli ca l fashion in longitudina l and ta ngent ia l sections (Fig. 11, A).

S tereo T EM of carbon replicas . Carbon replica­t ion is a means of examining the outside surface

of an object. the organi sm is ideally removed from the cast prior to examination of the cast wa ll. Replicas of the extensively processed organism revealed , by TEM, typica lly serpen tine proto­plasmic cy linders wi th numerous protuberance and ma ny axial fil a ments, ruptured and unrup ­tured. The fila ments were usually in groups of 3 , to 7 a long any part of the orga nism (Fig. 12). In segments of the T . pallidum , where the fila men t were intac t, the organism in stereo could be seen standing in a t hree dimensiona l flattened cork­screw configurat ion. H owever, where the fila-

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196 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

FIG. 10. A thin cross section of a T. pallidum, stained with uranyl acetate and lead citrate, demonstrating the protoplasmic cy li nder (A), the protoplasmic cell membrane (B), the axial filaments (C), and the deep filaments (D); by TEM (x 123,000).

ments were ruptured free of t he protoplasmic cy l­inder t he cylinder lay in a completely nat but serpent ine (two dimensional) fashion.

Pseudo-casts made of unwashed fresh orga­nisms, filtered t hrough 0.5Jl Nuclepore filters, re­vea led a thick outs id e dia meter, 0.4J.L to 6J.L , with a morphous materia l surrounding the protoplasmic cylinders (Figures 13 and 14).

SEM ofT. pallidum. T he extens ive ly processed orga ni s ms appeared as protoplasmic cy lind ers with numerous protuberances giving the cylinders

a n earthworm -like appearance (Fig. 15). The axial filaments were ruptured a nd the organisms lay in a two-dimensiona l serpentine fashion.

DISCUSSION

Electron microscopy has contributed s ignifi­ca ntly to our understanding of t he structure ofT. pallidum. However, the lack of applied histo­chemistry in t his fie ld has left the fun ction of t hese anatom ical parts conjectura l. Because nu-

ELECTRON MICROSCOPY OF T. pallidum 197

198 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

F IG. 12. A ca rbon replica of a port ion of a T .· pallidum demonstrating multiple round axial filam ents; by TEM ( X 132,000).

merous authors have described t he same struc­t ures under diffe ren t d esignations, a list of syn ­onyms is presented in the Table.

Protoplasmic Cy linder

The protoplasmic cylinder (F ig. 17) has been co nsidered to be bounded by several se mirigid limi t ing membranes (4), somewhat analogous to the usual cell wall of bacteri a (5, 6). The proto­plasmic cylinder cell wall may conta in mura mic acid (6, 7). In fi xed specimens, the protoplasmic cylinder reta ins the characte ristic wave length and a mpli tud e of li ve t reponemes. The proto­plasmic cylinder wid th is about 0.1511 to 0.2011 in the fix ed, dehydrated state, occasionally wi t h a slight ta pering of the distal end . Recent observa­t ions suggest t ha t t he protoplasmic cylinder may be bounded by two triple-layered membranes (8) (Fig. 16). This latte r concept is compatible wi th but not documented by our electron micrographs.

Attached to the insid e of the cylinder cell me mbra ne, a nd ori en te d in se milongit udina l bundles, are the deep fil a ments (9) (Figs. 2, 10, 17, and 18). They cont ribute to the structura l in ­tegrity (10) and elasticity of the protoplasmic cyl­inder, and perhaps to one of the types of motion seen in vivo (10, 11) .

~------------- -- --- -----------------·-

Distributed within the interior of the proto­plasmic cylinder a re numerous osmiophili c bo d ­ies, 25 A in di a meter, thought to be ribosomes (4 8, 10, 11, 12). '

Ovc innikov (13) and Ka wata (6) have observed wi t hin the interior of t he cylinder, centra lly placed s tructures with multiple fin ger-like projec­t ions. These structures, about 25 A in dia meter and not bound by a limiting membrane, a r e ca lled nuclea r fibril s (14) a nd could be the equiva­lent of a nucleus conta ining the geneti c informa­t ion for t he cell. They do not becom e more promi ­nen t a t the t ime of cell di vision by binary fission (1 0) .

Also wi thin the protoplas mic cylinder a re non ­membrane limited areas which are electron -lu­cent, lac king both nu cleoid and ribosomal mate­rial (4, 10, 11). These areas, which are ca lled vac­uoles, may be related to ingestion or may be arti ­facts. The deep fil a ments perhaps enter one of the nucl ea r vac uoles (9). Our thin secti on electron microgra phs do not confirm or contest the exist­ence of nuclea r strands or vacuoles.

At tached to the protoplasmic cell wall and pro­truding both inward into the cylinder a nd out ­wa rd into the in termedia te space are mul t ila m i­nat ed bodi es (4, 6) consisting of whorl s a nd layers

ELECTRON MICROSCOPY OF T : pallidum 199

F IG. 13. A pseudo- repli ca of a T . pallidum demonstrating amorphous material surroundi ng the protoplas mic cyl­ind er ; by T E M (x 14,000) .

of typica l double- layered membra nes. If these bodies a re ana logous to the mesosomes of some other bacte ria, their fun ction may be t he genera­tion of new cell me mbra ne for fu ture cell di vision. Man y of these structures conta in a 100- 200 A cir­cula r opening in to the protoplas mi c cylinder. T he la minated bodi es a lso occas iona ll y prot rud e thro ugh a ppa rent openin gs in the envelope. It may be that the beadin g phenomenon, seen wi th fl u o r esce in-tagged ant i-ga mma globulin attached to t h e globulins in some lupus erythematosus (LE ) patients ' sera, can be accoun ted for by the a dh erence of a ntinuclear a nt ibodies to these rela ­tively ex posed sites a long t he body of the T . pal­lidum (15). It will require addi t iona l resea rch to cla ri fy t his poin t. D NA is defini te ly attached to membra nes in some bacteria and is occasiona lly

de monstrated only in la minated mesosomes (16). The proto plas mic cylinder of T. pallidu.m is nor­ma ll y surrounded by the in ter mediate laye r, the · en velope, and perhaps a mucoid layer.

Granular bodies a re a lso seen as protrusions a long the s ide of the protoplas mic cylinder (10, 11), and their fun ction is unknown. Protoplasmic cylinder protuberances which conta in a n " in tes­t ine-like" body are a lso seen (7) and a re best il ­lustrated in the work of Kawata (6). The " in tes­t ine- li ke" bodies a re sa id to open both to t he out­side and in to the protoplas mic cylinder and their function may poss ibly be ingestion of nut rients or eliminatio n of waste materia l. The " intestine­like" bodi es may be lam inated mesosomes d ilated by different osmotic cond it ions ( 16) .

Occasiona ll y there will be seen a long the wall of

200 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

FIG. 14. A higher magnification of part of Fi g-ure 13; by TEM ( x 62.900).

t he protoplas mi c cy linder an electron-dense oval rin g concentric to an electron-lucent ova l ri ng. T hey have been ca ll ed " dark circumscribed bod­ies" in Leptospira and their function is unknown. They may be a reas from which axial filaments have detached since it has been suggested tha t the axia l fi la ments may occas iona lly attach other than subterminally ( 13) .

The Nose Piece

The structure seen at both ends of a t reponeme has been ca ll ed the nose piece and is insid e the outer envelope and outside the protoplasmic cy l-

inder. It a ppears to have a rather amorphous structure. S in ce the T . pallidum has been re­ported to have been located both intracellular! (9) and extracellularly (4), it is possible that t he nose piece may be in some way related to t he ab ility of the organism to enter a cell. However, some authors believe t hat the macrophage ac­t ively phagocytizes the passive T. pallidum by surrounding it with a plas ma membrane (9). Ov­cinnikov believes that the nose piece may be a nuclear exc retory organ. It is a lso possible that t he nose piece represents redundant envelope from wh ich the ends of t he protoplasmic cylinder have retracted (F igure 16).

ELECTRON MICROSCOPY OF T. pallidum 201

FIG .. 15. Seve ra l extensively washed T. pallidum with ruptured axia l filaments; by SEM ( x 13,000).

The Axial Filam ents

The axia l filaments are a para ll el series of round fibers that traverse t he len gth of the proto­plasm ic cy lind er in a helica l arrange ment (9). The fi laments may be 160-220 A in midbody, 120 A average diameter a nd about 80 A at their inser­tion . At their insertion, the filaments ma ke a 90° bend a nd insert in to a disk-shaped segment of t he protoplas mic cylind er (4) . The ax ial filament may be composed of 5-7 subfibrils of about 30 A diam­eter (4, 6, 10) . However, it see ms reasonable to the authors that the ax ial filaments of pathogenic T. pallidum a re like those demonstrated in the non-pathogenic Nichols T. pallidum as described in t he work of Jackson (8) a nd illustrated in our schematic drawings (Figures 18 and 19). Jepsen believes that these a re three ax ia l filaments at­tached to each end of the organism a nd t hat they overlap in the middl e, accoun ting for t he six par­allel fil a ments seen in the photographed midbody of some T. pallidum (4). However, we have seen, as have others, orga nisms with six parallel fila­ments in a subtermina l location. It see ms possible that some of these fila men ts traverse t he entire organism (17), but this has not been proven. Two

insertion paints have not been shown for a s ingle filament. It has been conjectured t hat ax ia l fila ­ments are essentia ll y endoflagellar, having a sub ­structure s imilar to that of bacterial flage lla (4), a nd that they provide the peculiar motility of the orga ni sm (5, 6, 14, 17) .

Envelope

The T. pallidum outer envelope is a t ripl e- lay­ered membrane (4) which encases the entire orga­nism with t he exception of occasiona I open ings over protruding bod ies from the protoplasmic cy l- . inder (9). Mucopeptides may be stored in the in ­termed iate laye r between the enve lope a nd the protoplasmic cy lind er cell wall (4). The envelope is probably more pliable and is more eas ily rup ­tured tha n the cylinder ce ll wall (5, 6, 10) . Ryter and Pillot we re unab le to demonstrate succ inic dehydrogenase activity in the envelope of spiro­chetes (18) . Occasionally, large balloon bodies (19, 20) (projections rese mbling pseudopodia (12, 21)) with a narrow neck, are seen protruding from the outer en velope or t he protoplasmic cy linder. Their origin may be related to a n adverse os motic environment (17), or may be a means of multipli -

202 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

TABLE I

List of synonyms in the literature on Treponema{ structure

[

Nuclear strands Nuclear fibril s Nuclear apparatus Nucleoid bodies

[Vacuoles Hyalanoplasts

lMultilaminated bodies Laminated bodies Mesomeres Blebs Mesoso mes*

[

Dark circumscribed bodies

Electron-dense oval concentric ring*

[

Balloon structures • Balloon-shaped exten­

sions Ba lloon bodies Projections rese mblin g

pseudopodia

[

Nose piece* Head structure Dense body Spongy mass

Basa l body Di sk-shaped segment of

the protoplasmic cyl­inder

Basa l granule Terminal knob Blepharoplast Annular swelling Disk-like attachment Insertion point*

[

Protoplasm ic cylinder* Cytoplasmic cylinder

Deep filaments* Microtubules Inner fibrils

* Name preferred by authors.

mucoid 1

'------------------ 3-1 aye red ce 11 wa 11

FIG. 16. A schematic drawing of a combined transverse and saggital section of a T. pallidum.

cation (ll). Also, under adverse conditions such as the add ition of small amounts of penicillin, the lack of nutrients, the addition of sera (particu­larly immune sera (ll)), the envelope may intra-

cellularly or extracellularly (9) form a cyst (3, 22, 23) . This cyst envelops t he T. pallidum coi led into a ball-shaped structure. With the cyst form present, the antigenic stimulus for the serologic

ELECTRON MICROSCOPY OF T. pallidum 203

cell membrane (3 layer ) ] protoplasmic flt'lHH---cell wall (3 l ayer) cylinder

mucoid l ayer

0 .1 5-.25 1'

FIG. 17. A schematic drawing of a cross section ofT. paltidum with various structures designated.

T. PA LLIDUM,NICHOLS STRAIN

membrane

F IG. 18. A schematic drawing of one end of a T. pallidum Nichols Strain illustra t ing the nose piece and various anatomi ca l relationships as labeled.

tests for syphilis may pers ist (9) and the envelope may serve a protective function (10, 17, 22).

Mucoid Layer

Numerous authors m the past have theorized

that an a morphous layer or casing surrounds the T. pallidum (4) isolated from human disease but not those isolated from spontaneous spirochetosis in rabbi ts (24, 25). Our observations with SEM of the cast replicas of unwashed organisms tend to

204 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

oo~e poert 1 ,.~,.,. t ln<l po• Ill 4' '"' r 1 l<l•• •n t • ontf'\ tin~ 1 ""'\0\0..,. l•d I loon bod¥

F tc. 19. A schematic dfawing of a T. pallidum without a mucoid layer, illustrating various ana to mical stru c­tures.

support the presence of an a morphou s laye r a round virulent T . pallidum from huma n les ions. Such a coverin g might be contribu te d by either the orga ni s m (10, 11) or the host (9). This layer is a ppa rently lac kin g in T . p ertenue (10). This mu­coid layer might explain the host's poor protec­tive a ntibody respo nse to the organism s ince it wou ld tend to protect the potential a n t igeni c characte ristics of t he enve lope , axia l fil a ments and protoplas mic cylinder. It has been observed that fresh T. pallidum wi t h the mucous layer still intact, will not tluoresce in the Fluorescent Tre­ponema ! Antibody- Abso rpti on (FTA-ABS) test a nd are unaffected by sy philitic sera in the T . pallidum Immobili zation (TPI) test. Therefore, the test organis m s used in t he FTA-ABS test a re prepared (26) a nd dried onto t he surface of the s lide before se rving as ad equate a n t igens, a nd t he organi s ms used in the TPI test must be at least 24 hours o ld before immobili zation ca n b e in ­duced by reactive se ra.

CONCLUS IONS

Althou gh e lectron mi c roscopi c techniques a re definite ly limi ted in their abili ty to de lineate the d yna mics of living o rga nis m s, some poss ibilities ca n b e proposed:

1. Protop las mi c cylinders do not of t he mselves account for t he semi-rigid corkscrew confi gurat ion of T. pallidum. In tact axial filaments probably contribute s ignificantly to this characteristic.

2. A mucoid laye r is perha ps present a round unwashed T. pallidum a nd may shield t he o rga­nis m from protective immunologica l recognition.

3. M esoso ma l protuberances through the enve­lope of washed T . pallidum possibly account for the LE sera's beading fluorescence pattem be­cause of t he exposed antigenic s ites .

Our thanks to Jeanne F. Ryan of the Creative Arts Department, CDC, for the schematic drawings. We also thank Dr. Sally J ackson and Dr. S. H. Black, Baylor College of Medicine, Houston, Texas, for helpful discus­sions.

REFERENCES 1. Pease, D. C.: Histological T echniques for Electron

Microscopy. Academic Press, New York, 1964. 2. Reynolds, E. S.: The use of lead citrate at high pH

as an electronopaque stain in electron micro­scopy. J. Cell Bioi. , 17: 208, 1963.

3. J ones, R. H. , Nev in, T. A., Guest, W. J. : Lytic ef­fect of tryps in , lysozyme and complement on Treponema pallidum. Brit. J. Vener. Dis ., 44: 193, 1968.

4. J epsen, 0 . B., Hougen, K. H. and Birch-Ander­sen , A.: E lect ron microscopy of Treponema pal­lidum Nichols. Acta Path. Microbia l. Scand ., 74: 241, 1968.

5. Pi llot, J . and Ryter, A.: Structure des spirochetes. I. Etude des gentres Treponema, Borrelia et

Leptospira au microscope electronique. Ann Inst. Pasteur. (Paris) , 108: 791, 1965. ·

6. Kawata, 1'. and Inoue, T .: Fine structure of the Reiter treponeme as revealed by electron micros­copy u sing thin secti?nin~ and negati ve staining techmques. J ap. J. M!crobtol. , 8: 49, 1964.

7. Ginger, C. D.: Isolation and characterization of muramic ac id from two spirochaetes: Borrelia duttonii and Heptospira biflexa. Nature, 199· ~~ 1~. .

8. J ackson, S.: The fin e structure of Treponema pa[_ lidum, Nichols strain. Ph.D. Dissertation, Bay_ lor College of Medicine, 1969.

9. Oveinnikov , N. M. and Delektorskij, V. V.: Ultra­fin e structure of the cell elements in a hard chancre and their interrelationship with Trep 0 _

nema pallidum. Bull . WHO, 42: 437, 1970. 10. Ovcinnikov, M. M. and Delektorskij, V. V.: Tre­

ponema pertenue under the electron microscope. WHO/VDT/RES, 69: 184 , 1969.

11. Ovcinnikov, N. M. and Delektorskij, V. V.: Fur­ther study of ultrathin sections of Treponema pallidum under the electron microscope. Brit . J . Vener. Dis .. 44: 87, 1969.

12. Hasegawa, T.: Ult rastructure of Nichols spirochetes Nippon Acta Radio!. , 77: 1, 1967. ·

13. Ovc inn ikov, N. M. and Delektorskij , V. V.: Mor­phology of Treponema pallidum. Bull. WHO, 35 · 223, 1966. .

14. Listgarten, M. A. and Socransky, S. S.: E lectron mi crosco py of ax ia l fibril s, outer envelope and cell division of certa in oral spirochetes. J . Bact., 88 · 1087, 1964 . .

15. Kraus, S. J ., Hase rick, J., Logan, L. C. and Bul la rd J. C.: Atypica l fluorescence in the fluorescent tre: ponema l-antibody-absorption (FTA-ABS) test related to deoxyribonucl eic ac id (DNA) anti­bodies. J . lmmunol. , 106: 1665, 1971.

16. Rogers, H. J .: Bacterial growth and the cell enve_ lope. Bact. Rev., 34: 194, 1970.

17. Pillot, J. : Contribu t ion a !'etude du genre Trep0 _ nema: structures anatomique et anti ge nique . Thesis. Paris, 1965.

18. Ryter, A. and Pillot, J .: Etude au microscope elect­ronique de Ia structure externe et interne du t r e­poneme Reite r. Ann. Inst. Pasteur. (Paris), 104: 496, 1963.

19. Miller, N. G. and Wilson, R. B.: In vivo and in vitro observat ions of Leptospira pomona by electron microscopy. J . Bact., 84: 569, 1962.

20. Gelperin , A.: Morphol ogy, cultural characteristics and a method for mass cul tivation of the Reiter spirochete . Amer. J. Syph., 33: 101, 1949.

21. Bladen, H. A. and Hampp, E. G.: Ultrastructure of Treponema microdentium and Borellia vin.centii. J . Bact., 87: 1180, 1964.

22. Ovcinnikov, N. M. and Delekto rskij , V. V.: Electron mi croscopy of Treponemata. Vestn. Derm . Vener. , 10: 51, 1967.

23. Ovcinnikov, N. M. and Delektorskij, V. V.: E lectron mi croscopy of Treponemata. Vestn. Derm . Vener., 1: 50, 1965.

24. Turner, T . and Hollander, D.: Cortisone in experi­mental syphilis. Bull. Johns Hopkins Hosp., 87: 505, 1950.

25. Turner, T . and Hollander, D.: Studies on the mech­anism of action of cort isone in experimental syph­ilis. Amer. J. Syph., 38: 371, 1954.

26. SeroLo{fic T ests for SyphiLis . USPHS Publication No. 411: 72, 1964.