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Journal of General Microbiology (1 989, 131, 2349-2357.

Printed in Great Britain 2349

The Outer Membrane of Treponemapallidum: Biological Significance and


Biochemical Properties
By C . W . P E N N , * A . C O C K A Y N E A N D M . J . B A I L E Y
Department of’ Microbiology, University of’ Birmingham, Birmingham B15 2TT, UK

(Receiued 6 February 1985; revised 7 May 1985)

Rabbits infected intravenously with Treponema pallidum were not markedly febrile, and the
pyrogenicity of treponeme preparations administered intravenously to rabbits was negligible.
The antibiotic polymyxin B did not induce any ultrastructural changes on the treponemal surface
and was not lethal (immobilizing) for T. pallidum, which was, however, highly susceptible to
detergents such as SDS. Extraction of treponemes with Triton X-100 removed the outer
membrane (despite the presence of Mg2+)as shown by electron microscopy, and solubilized a
limited number of proteins detectable by SDS-PAGE, including a dominant antigen (47 kDal)
demonstrated by immunoblotting. None of the proteins were heat-modifiable. Periodic acid-
silver staining of polyacrylamide gels for carbohydrate together with protease K digestion did
not demonstrate major carbohydrate components in whole treponemes, or in the Triton-soluble
fraction. Surface iodination of intact treponemes revealed very little surface exposure of
treponemal proteins, although a protein which co-migrated with host albumin was labelled and
appeared to be associated with the treponemal surface. Many treponemal proteins were,
however, labelled when iodination was done in the presence of Triton. These observations
indicate that the outer membrane of T. pallidurn differs significantly from those of many Gram-
negative pathogens.

INTRODUCTION
Syphilis is essentially a chronic infection and the causative organism, Treponema pallidum
does not elicit host reactions in many stages of the disease. Clearly the treponemes are capable at
these times either of sequestration in sites where host reactions cannot be activated, or of some
form of ‘disguise’ so that they are not recognised by the host as foreign substances, or of active
suppression of host defensive responses.
Treponemes may be found intracellularly in human and rabbit infections; this possibly
protects them from host defence mechanisms and favours their persistence in the face of an
immune response (Sykes & Miller, 1971; Sykes et al., 1974; Turk, 1979). However, in early
lesions treponemes are predominantly extracellular, often near blood vessels, and clearly
accessible to host defences (Drusin et al., 1969; Hasegawa, 1969; Penn, 1981; Penn & Clay,
1982). Thus, while intracellular survival may promote long term persistence of small numbers of
organisms, it cannot explain the initial extracellular multiplication of treponemes without
excitation of host defences.
The ‘disguise’ of pathogens to avoid immunological recognition may comprise surface
acquisition of host substances, or similarity between surface constituents of the pathogen and
those of the host. The former has been suggested for T.pallidurn by the finding of host proteins,
including albumin and immunoglobulins, associated with the surface of organisms taken from
infected rabbits (Alderete & Baseman, 1979). The significance of this in pathogenicity is yet to
be determined, however: other pathogens growing in viuo may bind host proteins (Finn et al.,
1982), but these would not necessarily disguise the foreign antigens of the organism and alter its
mode of pathogenicity. Similarity between the surfaces of T.pallidum and the host might result

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from a mucopolysaccharide capsule, possibly responsible for the mucoid exudate found in
syphilis lesions (Fitzgerald, 1981). The treponemal origin of this material is disputed, however,
and recent work by Strugnell et al. (1984) indicates that the material is synthesized by the host.
T. pallidurn exhibited enhanced reactivity with antibody after treatment with Triton X-100,
concomitant with the removal of the outer membrane (Penn & Rhodes, 1982; Penn & Lichfield,
1982).Hence the Triton-soluble outer membrane and associated layer(s) appeared crucial to the
antigenic crypticity of intact treponemes. The nature and composition of the intact treponemal
surface and the Triton-soluble fraction are now described further.

METHODS
T. pallidum strain. The Nichols strain of T .pallidum was maintained and propagated in rabbits, and harvested
for study in ritro as described previously (Penn, 1981; 1983). Organisms suspended in extraction medium were
killed by addition of 0.1 % (w/v) sodium azide and storage at 4 "C for at least 24 h. They were used within 48-72 h.
Pyrogenicity testing. Treponemes were concentrated and washed by centrifugation at IOOOOg for 20 min and re-
suspension in phosphate buffered saline pH 7.2 (PBS: 7.2 mM-NazHPO,, 2.8 mM-NaH2P0,, 0.15 M-NaCl) to a
density of 1 x lo9 ml-' determined by total visual counts on the original suspensions in a counting chamber. They
were then fragmented by ultrasonication at 20 kHz for 5min (Penn & Rhodes, 1982). For comparison, a
suspension of gonococci (strain BS4; Penn et al., 1976)grown on complex agar medium containing peptone, yeast
extract and serum was treated similarly. Bacterial suspensions (0.1 or 1.0 ml), or diluent alone, were injected
intravenously into healthy adult male Californian rabbits which had shown stable (k0.2 "C), normal rectal
temperatures for at least 1 beforehand. Rectal temperatures were monitored at 1 h intervals for 5-6 h.
Reactions of' organisms with polymyxin B. Bacteria were incubated with polymyxin B (Sigma) as described by
Wiegel & Quandt (19821, with the addition to the buffer of 0.15 M-NaCl when the organisms were to be fixed and
sectioned, to stabilize them osmotically. Organisms were centrifuged at lOOOOg for 20 min and suspended in
polymyxin B at a density of 1 x los ml-' for treatment. Salmonella typhimurium strain 395 MS (obtained from Dr
E. Kihlstrom, Gothenburg) was similarly treated as a positive control organism known to contain
lipopolysaccharide (LPS). Processing for electron microscopy, including post-fixation with uranyl acetate, was as
described by Penn & Lichfield (1982).
Ejects ojpolymy.uin B and S D S on treponemal motility. Viable treponeme suspensions, in the medium in which
they had been extracted from rabbit testes, were treated immediately by the addition of 10% (v/v) of solutions
of polymyxin B or SDS in PBS to give final concentrations of 1000 U polymyxin B ml-I or 1, 0.1 or 0.01 % (w/v)
SDS in the treponemal suspension. Treponemal motility in test and control (addition of PBS alone) samples
was assessed after incubation at 37 "C for 1 h.
Treatment with Triton X-IOO.Azide-killed treponemes (batches of 1-5 x lolo) were centrifuged at lOOOOg for
20 min, washed by suspending in 10 ml PBS and re-centrifuged. The sedimented treponemes were then re-
suspended in PBS containing 10 mM-MgCl? and 0.2% (v/v) Triton X-100 to a density of 2 x lo1* ml-I. After
30 min incubation at 37 "C, treponemes were centrifuged as above and both the supernate and the pellet
(resuspended in the same volume of PBS) were retained for polyacrylamide gel electrophoresis. Some organisms
from the pellet were also suspended in 2% (w/v) potassium phosphotungstate, pH 7.2, in water, for the preparation
of negatively stained samples for electron microscopy.
Electrophoretic analysis. SDS-PAGE was done as described by Laemmli (1970). Molecular weight standards
were obtained from BioRad. Samples (25 pl) of Triton-treated treponemes, soluble fraction (supernate) or re-
suspended pellet were applied to 10 mm wide slots in 1 mm thick slab gels. In some experiments the same volume
of a suspension of whole treponemes (2 x 1 O l o ml-I) was also run. Periodic acid-silver staining for carbohydrate
and digestion of protein with protease K (Boehringer) were done by the method of Hitchcock & Brown (1983). LPS
(from Escherichia coli, type 0 1 1 I ) was obtained from Sigma and samples of 10 pg were run for positive control
staining.
Immunoblotting was done by the method of Towbin et al. (1979) with an electroblot apparatus (BioRad), and
antigenic components were detected by reaction of blots with pooled hyperimmune antiserum from rabbits which
had recovered from syphilitic orchitis and had been boosted by immunization with sonicated treponemes (Penn &
Rhodes, 1982). The visualization of immuno-reactive components was done with alkaline phosphatase conjugated
to goat anti-rabbit serum (Miles) by the method of O'Connor & Ashman (1982) with ASMX phosphate and Fast
Red (Sigma).
Surface iodination. Two 1.5 ml portions of freshly harvested, live treponemes, extracted in a minimal volume of
medium to obtain counts of at least 5 x lo8 ml-l, were centrifuged at 200g for 5 min to remove host cells, and
were then centrifuged for 5 min at 1 1600 g on a microcentrifuge to sediment the treponemes. These were then re-
suspended in 0-1 ml testis extraction medium (Hanks' solution with 20 mM-HEPES and 2 mM-dithiothreitol, pH
7.4) with or without 0.2% (v/v) Triton X-100. To this was added 10 pl [approx. 100 pCi (3.7 Mgq)] Nalt51

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Outer membrane of Treponema pallidurn 235 1
(Amersham) in 0.01 M-NaOH, and 1 Iodobead (Pierce & Warriner). Iodination was allowed to proceed for 15 min
at room temperature with occasional gentle agitation, and was terminated by removal of the Iodobead. The
suspension was centrifuged at 1 1 600 g.Material iodinated in the presence of Triton was retained as supernate and
pellet (resuspended in the same volume of medium). The reaction supernate of the material iodinated without
Triton was retained, and the pellet resuspended in medium with Triton. After 15 min at room temperature, this
was again centrifuged to provide supernate and pellet, and the latter was resuspended as above. All samples were
analysed by SDS-PAG E followed by autoradiography of the dried gels.

RESULTS
Fever in T. pallidurn infection and pyrogenicity of the organism
Rectal temperatures of a group of acutely orchitic rabbits infected with T. pallidurn, and
uninfected animals housed in the same conditions, are shown in Fig. 1. The mean temperature of
the normal rabbits was 39.15 "C, SD 0.16 "C, and in infected rabbits 39.5 "C, SD 0.51 "C.Thus
there was no statistically significant difference in temperatures between the two groups.
However, some infected rabbits undoubtedly had elevated temperatures, although they were
seldom severely pyretic ; other acutely orchitic rabbits had normal temperatures.
Intravenous injection of ultrasonically disintegrated T . pallidurn produced only a slight fever
in comparison with an equivalent dose of Neisseria gonorrhoeae (Fig. 2). A tenfold increase in the
number of T .pallidurn injected led to some increase in the degree of fever, but the kinetics of this
response did not accord with the typical dual peaks at 1 and 3 h characteristic of bacterial LPS
(Milner & Finkelstein, 1966), and seen here after injection of N . gonorrhoeae.
Efect of polyrnyxin B on treponemal ultrastructure
T. pallidurn treated with polymyxin B, at either 200 or 1000 U ml-l, were macroscopically
agglutinated but light microscopy did not reveal any other change in their appearance. Control
S . typhimurium cells were similarly affected. Ultrastructurally, negative staining showed that
characteristic surface structures, often annular in appearance, were prominent on the surface of
S. typhimurium but were not seen on T. pallidurn (Fig. 3). In thin section, the surface of T.
pallidurn appeared unaffected, but S . typhirnurium showed the surface structures visible in
negatively stained preparations (Fig. 4); in section these appeared to be vesicular projections of
the bacterial surface, adjacent to the outer membrane. The membrane itself did not appear to be
substantially disrupted.
Eject oj'polyrnyxin B and SDS on treponemal motility
T.pallidurn was not immobilized by exposure to polymyxin B (1 000 U ml-l) for 1 h. Tests with
SDS showed immobilization within a few minutes at concentrations as low as 0.01% (w/v).
Solubilizaton of the outer membrane of T . pallidurn by Triton X-100
Treponemes treated with Triton X-100 in the presence of Mg2+underwent the morphological
changes which were previously shown to accompany removal of the outer membrane (Penn &
Lichfield, 1982). These are shown in Fig. 5.
Electrophoretic analysis of the Triton-soluble .fraction
SDS-PAGE of the Triton-soluble fraction showed that a minority of the total treponemal
polypeptides were significantly extracted into this fraction, notably those of approximately 60,
47 and 40 kDal (Fig. 6a). Of these, only the 47 kDal polypeptide, and two or more minor
components, including one of about 33 kDal and another of about 16 kDal near the dye front,
which was poorly resolved under these conditions, appeared to be extracted almost completely,
suggesting their location in the outer membrane. The position of the largest major polypeptide
on this gel at 70 kDal (starred) differed from its position (80 kDal) when organisms not treated
with Triton were examined (unpublished observation). The reason for this is not known.
Periodic acid-silver staining for carbohydrate components stained many bands in the various
untreated treponemal fractions (Fig. 6b). Those which were stained strongly with Coomassie
blue did not generally stain well with the silver stain, and indeed clear unstained areas were often
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2352 C. W . P E N N , A . COCKAYNE A N D M . J . BAILEY

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Fig. 1 Fig. 2
Fig. 1. Rectal temperatures of identically housed normal rabbits and rabbits acutely infected
intratesticularly with T. pallidurn.
Fig. 2. Fever curves of groups of 4-5 rabbits injected intravenously with 1 x los (0) or 1 x lo9 ( 0 )
sonicated T. pallidum or 1 x lo8 ( 0 )sonicated N . gomrrhoeae. Error bars indicate SD. Mean
temperatures of two control rabbits which received diluent alone (I )
are also shown.

Fig. 3. Negatively stained untreated (a) and polymyxin B-treated (b)S . typhimurium, and polymyxin B-
treated T .pallidum (c). The arrow indicates the annular appearance of surface vesicle on S . typhimurium
after reaction with polymyxin B. Bars represent 0-2pm.

present in these positions. A sample of 10 pg LPS from E. coli was stained well by this method.
After treatment of treponemal components with protease K, no silver-stainable bands
remained, except at the dye front.
Western blotting (Fig. 6 c ) of whole treponemes and the Triton-soluble fraction showed that
an immunodominant antigen was extracted by Triton treatment. Examination of a section of the
blot stained for protein with amido black, and of the original polypeptide profiles in
polyacrylamide gel, showed this immunodominant polypeptide to be the major, 47 kDal
polypeptide of the Triton-soh ble fraction.

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Outer membrane o j Treponema pallidurn 2353

Fig. 4. Ultrathin section of mixed T . paffidumand S . typhimurium after treatment with polymyxin B.
Note vesicles on the surface of S . typhimurium ( l ) , and unaffected outer membrane of T. paffidum( 2 ) .
Bar represents 0.2 pm.

Fig. 5. Negatively stained T.pallidurn, intact (a),or treated with Triton in the presence of Mg2+(b).The
arrow indicates cytoplasmic filaments, made more visible by Triton treatment. Bars represent 0.2 pm.

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Fig. 6. (a) SDS-PAGE analysis of the Triton-soluble fraction (I), cell residue after Triton extraction (2), Triton-treated but unseparated
treponeme suspension (3) and molecular mass markers (4). The arrows indicate polypeptides substantially solubilized by Triton with their
molecular weights. (b) Periodic acid-silver stained SDS-PAGE gel, with 10 pg E. coli LPS (l), whole treponemes (2, 5 ) , Triton-extracted
residue (3,6) and Triton-soluble fraction (4, 7). Material in lanes 2 4 was untreated; material in lanes 5-7 was digested with protease K. ( c )
Western blot of whole treponemal cells ( I ) and Triton-soluble fraction (2), reacted with hyperimmune rabbit serum. The arrow indicates the
major 47 kDal antigenic polypeptide, extractable into the Triton-soluble fraction.
Outer membrane of' Treponerna pallidurn 2355

Fig. 7. SDS-PAGE analysis of radio-iodinatedtreponemes. The gel was stained with Coomassie blue
(a),or autoradiographed (b).Treponemeswere iodinated either in testis extraction medium alone (lanes
1-3) or in the same medium in the presence of Triton (lanes 4 and 5). Lanes 1 and 4, supernatant of
iodination reaction mixture after removal of treponemes by centrifugation; lane 2, Triton-soluble
fraction of pelleted treponemes iodinated in the absence of Triton; lanes 3 and 5, pelleted treponemes
after Triton extraction (3) or removal of reaction supernatant (5). The arrows in (a)indicate the Triton-
extractable polypeptides; the arrow in (b) indicates the albumin-like, 66 kDal polypeptide.

Surface labelling
Surface iodination of intact treponemes, viable until addition of the iodinatiog reagents, led to
incorporation of significant amounts of label into only one polypeptide as analysed by SDS-
PAGE (Fig. 7, lanes 1 and 2). This polypeptide was shown in other experiments to co-migrate
with bovine serum albumin, and its absence from the washed treponeme preparations analysed
as shown in Fig. 6 (a) suggests its loose association with the surface of the unwashed organisms
used for surface labelling. No significant presence of label in the cell pellet remaining after
Triton extraction was seen (lane 3). After iodination in the presence of Triton, considerable
incorporation of label into a variety of treponemal proteins was observed (Fig. 7, lanes 4 and 5).
Both the Triton-soluble fraction (lane 4) and the insoluble residue (lane 5) had incorporated
much label. The apparently greater incorporation into the albumin-like protein in the presence
of Triton (lane 1 compared with lane 4) did not appear to be due merely to the presence of Triton
since control iodination of solutions of bovine serum albumin in the presence and absence of
Triton showed no difference in incorporation.

DISCUSSION
T.pallidurn establishes foci of infection but not acute inflammatory responses resulting in pus
formation. Thus reactive components such as LPS may be absent from T.pallidurn. The limited
availability of T.pallidurn precludes the use of many standard analyses of cell wall composition.
Therefore biological and ultrastructural investigations are important in the elucidation of the
surface structure.
Fever is not prominent in syphilis. Since the pyrogenicity of Gram-negative bacteria results
partly from their LPS content, fever in T . pallidurn infection in rabbits and the pyrogenicity of
treponemes might indicate presence of LPS. The fever observed in some acutely orchitic rabbits
was less than 1 "C, and depression of body temperature occurred in some animals. The low
pyrogenicity of treponemes in comparison with gonococci suggests the absence of typical Gram-

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2356 C . W . P E N N . A . COCKAYNE A N D M . J . B A I L E Y

negative LPS from T.pallidurn. This conclusion was supported by the absence of surface vesicles
from T. pallidum following treatment with polymyxin B. Vesicle formation, as seen on S .
typhimurium treated with polymyxin B, is characteristic of Gram-negative bacteria (Wiegel &
Quandt, 1982), and LPS appears to be the target for action of polymyxin B (Perkins, 1983). The
resistance of T. pallidum to killing (immobilization) by polymyxin B reinforces the
morphological evidence for the lack of effect of this antibiotic. The SDS susceptibility of T.
pallidum also suggests the absence of at least a smooth type of LPS, since the relative resistance
of many wild-type Gram-negative enteric bacteria to detergents is attributed to LPS of the
smooth type which presents a hydrophilic exterior of the bacterium (Wright & Tipper, 1979).
Additional evidence for the absence of LPS activity in T.pallidum has recently been reported by
Hardy & Levin (1983), who failed to detect any Limulus amoebocyte lysate gelation activity
associated with this organism. In other studies (Bailey et al., 1985) we have demonstrated by
immunoblotting a ladder profile of protease-resistant antigenic material characteristic of LPS in
the non-pathogenic Reiter treponeme, but no protease-resistant antigens were detected in T.
pallidum.
The removal of the outer mqnbrane of T. pallidum by Triton in Tris/HCl buffer has been
described previously (Penn & Qehfield, 1982). In Gram-negative enteric bacteria, the outer
membrane is often resistant to%$lubilization by such detergents unless divalent cations are
removed (Schnaitman, 1971). The susceptibility of the outer membrane of T. pallidum to
solubilization by Triton X-100 even in the presence of Mg2+again suggests the absence of LPS.
Analysis by SDS-PAGE of Triton-soluble material from the surface of T.pallidum showed that
only three polypeptides appeared to be completely solubilized. These are likely to be outer
membrane components, since this layer appeared to be completely removed ultrastructurally.
None of the polypeptides detected showed heat modification as determined by a comparison of
the molecular mass on SDS-PAGE of material solubilized by boiling with the molecular mass of
material held at 37 "C for 30 min. In this respect T. pallidurn differs from many pathogenic
Gram-negative bacteria. Lack of reactivity with the periodic acid-silver stain after protease
digestion indicated that carbohydrates are not prominent components of this organism, but
staining reactions alone cannot show that LPS or other carbohydrates were completely absent.
The many bands silver-stained before protease digestion could have been glycoproteins or
proteins (Hitchcock & Brown, 1983).
The almost total absence of iodination of proteins in intact treponemes, compared with the
promotion by Triton X-100 of the iodination of a large number of protein species, indicated that
the intact outer membrane shields the majority of treponemal proteins from surface exposure.
This conflicts with other reports of surface iodination of a number of treponemal proteins
(Alderete & Baseman, 1980 ;Jones et al., 1984), a discrepancy possibly resulting from variations
in treatment of treponemes before iodination. In our hands, long exposures during
autoradiography demonstrated low levels of iodination of other polypeptides, but this was
insignificant in comparison with the level of incorporation of label in the presence of Triton.
Neither the Triton-extractable 40 kDal polypeptide, which resembled a 39 kDal polypeptide
shown by Norris & Sell (1984) to be labelled after surface-iodination of purified, washed
treponemes, nor the 47 kDal Triton-extractable polypeptide, appeared to be accessible for
surface iodination in the intact, unwashed organism. The labelling of presumed albumin
associated with the treponemal surface agrees with other observations of host protein binding by
T.pallidum (Alderete & Baseman, 1979), but whether this protects against host defences remains
unclear. Enhanced iodination of the albumin-like protein in the presence of Triton suggests that
binding of albumin to the treponemal surface may be sufficiently intimate to afford some
protection against its iodination in the native configuration.
These observations indicate many differences in outer membrane properties of T. pallidum
from those of other Gram-negative pathogens, which may contribute to the low reactivity
towards the host of this pathogen and its adaptation to long term survival in chronic infection
despite host immune responses to internal antigens. The latter may become accessible to the host
immune system after damage to or death of some, but presumably not all treponemes in the
course of infection.

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Outer membrane of Treponema pallidum 2357

We are grateful to J. Rhodes, C. Easthope, J. Lichfield and D. Ruffles for technical assistance. The work was
supported in part by the Wellcome Trust.

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