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 Emerging Infectious Diseases * Volume 3 * Number 1 * January-March 1997
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Synopses

Surface Antigens of the Syphilis Spirochete and Their Potential as
Virulence Determinants

David R. Blanco, James N. Miller, and Michael A. Lovett
UCLA School of Medicine, Los Angeles, California
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      A unique physical feature of Treponema pallidum, the
      venereally transmitted agent of human syphilis, is that its
      outer membrane contains 100-fold less membrane-spanning
      protein than the outer membranes of typical gram-negative
      bacteria, a property that has been related to the chronicity
      of syphilitic infection. These membrane-spanning T. pallidum
      rare outer membrane proteins, termed TROMPs, represent
      potential surface-exposed virulence determinants and targets
      of host immunity. Only recently has the outer membrane of T.
      pallidum been isolated and its constituent proteins
      identified. Five proteins of molecular mass 17-, 28-, 31-,
      45-, and 65-kDa were outer membrane associated. The 17- and
      45-kDa proteins, which are also present in greater amounts
      with the T. pallidum inner membrane protoplasmic cylinder
      complex, had been previously characterized lipoproteins and
      are, therefore, not membrane-spanning but rather
      membrane-anchored by their lipid moiety. In contrast, the 28-,
      31-, and 65-kDa proteins are exclusively associated with the
      outer membrane. Both the purified native and an Escherichia
      coli recombinant outer membrane form of the 31-kDa protein,
      designated Tromp1, exhibit porin activity, thereby confirming
      the membrane-spanning outer membrane topology of Tromp1. The
      28-kDa protein, designated Tromp2, has sequence
      characteristics in common with membrane-spanning outer
      membrane proteins and has also been recombinantly expressed in
      E. coli, where it targets exclusively to the E. coli outer
      membrane. The 65-kDa protein, designated Tromp3, is present in
      the least amount relative to Tromps1 and 2. Tromps 1, 2, and 3
      were antigenic when tested with serum from infection and
      immune syphilitic rabbits and humans. These newly identified
      TROMPs provide a molecular foundation for the future study of
      syphilis pathogenesis and immunity.

Despite the fact that Treponema pallidum subsp. pallidum has remained
sensitive to penicillin for more than four decades, syphilis remains an
important cause of sexually transmitted disease, accounting for more than
14,000 new cases in 1995 in the United States alone (1). Although syphilis
is not an emerging infectious disease, its reemergence, from 1986 to 1990,
culminated in more than 100,000 reported cases in 1990 and an 80% increase
in the reported rate per 100,000 population. The reemergence of syphilis
has also been associated in certain areas with the use of crack cocaine,
and syphilis continues to be an important risk factor for acquiring and
transmitting human immunodeficiency virus.

Syphilis is characterized by months of clinical disease followed by years
of latency with the potential for relapse to debilitating or lethal late
disease if left untreated. The chronicity of infection may relate to a
striking property of the T. pallidum outer membrane, namely that it
contains 100-fold less membrane-spanning protein than the outer membranes
of typical gram-negative bacteria (2,3). This article reviews recent
progress in identifying and characterizing the rare outer membrane proteins
(TROMPs) of T. pallidum.

The interaction of host and pathogen leading to the establishment of
infection generally proceeds through the interactions of their respective
surface molecules. An understanding of the molecular basis for syphilis
pathogenesis and the identification of corresponding T. pallidum surface
molecules have long been hampered by the inherent difficulties associated
with the study of this organism. In contrast to that of many other
pathogenic spirochetes from the genera Treponema, Borrelia, and Leptospira,
the in vitro multiplication of T. pallidum is unsuccessful in artificial
media and is limited and impractical in tissue culture (4). Further, only
limited numbers of T. pallidum are attainable from testicular cultivation
in rabbits. Organisms acquired from rabbit infection are also bound with
host material including albumin (5), fibronectin (6), and
glycosaminoglycans (7); therefore, the separation and purification of T.
pallidum molecules are complicated. Host material associated with T.
pallidum has also been the basis for past theories of molecular mimicry
during syphilitic infection (6), of T. pallidum adherence to host cells and
tissues (8), and of the long recognized antigenic inertness of the surface
of this organism.

Past studies showed that the surface of T. pallidum behaves as if it lacks
proteins and antigens. These findings included the inability to detect
antibody bound to the surface of structurally intact treponemas by
immunofluorescence (9) or by immunoelectron microscopy (10,11), the limited
radiolabeling of surface proteins (12,13), and the observation that only
with extended in vitro incubation could proteins of intrinsically
radiolabeled T. pallidum bind specific antibody (14). One hypothesis
generated by these observations was that the surface of T. pallidum was
antigenically inert because of a limited number of available
surface-exposed antigens.

The inability to apply conventional gram-negative cell fractionation
procedures to T. pallidum to isolate its outer membrane and identify its
constituent proteins further hampered the identification of surface-exposed
proteins. As a result, alternative methods, which included detergent
solubilization, were used to achieve this goal. The detergent Triton X114
(TX114) was used with particular interest because of its inherent property
of a low temperature cloud point that results in phase separation yielding
both aqueous and detergent phases. It was found that 0.1% to 2% TX114 would
selectively solubilize the T. pallidum outer membrane and result in the
recovery of several prominent detergent-phase proteins (15,16), which were
speculated, at the time, to be part of the outer membrane and potentially
surface exposed. These proteins were subsequently found to be lipoproteins
(17) and were strongly antigenic when tested with serum from syphilitic
rabbits and humans (17). While surface exposed lipoproteins have been
demonstrated for other pathogenic spirochetes, including Borrelia (18) and
Leptospira (19), a surface exposed location for a strongly antigenic T.
pallidum lipoprotein was inconsistent with the surface antigenic inertness
of this organism.

          [Figures not available in ASCII version]
          Figure 1. Freeze-fracture electron microscopy of
          Treponema pallidum subsp. pallidum (A), and
          Escherichia coli (B). Concave ([concave symbol]) and
          convex ([convex symbol]) outer membrane (OMF) and
          inner membrane (IMF) fracture faces.

A major advance in the understanding of the T. pallidum outer membrane and
cell surface followed the results of freeze-fracture electron microscopy in
1989. The observation that the fracture faces of the T. pallidum outer
membrane contained only 170 particles/mm2 (2,3), which is approximately
100-fold less than what is contained in typical gram-negative bacterial
outer membranes (Figure 1), together with the finding of freeze-etch
analysis (3), has provided the most plausible explanation for the antigenic
inertness of the T. pallidum surface. The rare particles revealing T.
pallidum rare outer membrane proteins were designated TROMPs (2,20), and
because of their membrane-spanning topology, they were distinct from
lipoproteins, which do not span membranes to form particles in
freeze-fracture analyses (21). Moreover, a membrane-spanning protein by
definition should possess several surface exposed regions, suggesting that
TROMPs had surface exposed antigenic sites. This theory was confirmed by
freeze-fracture analysis when it was observed that incubation of T.
pallidum in serum from syphilitic rabbits immune to reinfection resulted in
the aggregation of TROMPs (20) (Figure 2). It was found that 8 to 16 hours
of incubation was required for this aggregation to occur, suggesting that
antibody-mediated aggregation of TROMPs is the rate limiting step for
complement activation and killing of T. pallidum (20).

       [Figures not available in ASCII version]
       Figure 2. Freeze-fracture electron microscopy of T. pallidum
       subsp. pallidum demonstrating TROMP aggregation. Concave (
       [concave symbol]) and convex ([convex symbol]) outer membrane
       fracture faces (OM). T. pallidum incubated in
       heat-inactivated normal rabbit serum for 16 hours (A), in
       immune rabbit serum for 2 hours (B), and in immune rabbit
       serum for 16 hours (C) and (D). Arrows show individual (A&B)
       and aggregated (C&D) TROMPs. Bar in each micrograph
       represents 0.1mm. Photograph reprinted with the permission of
       the Journal of Immunology, copyright 1990, The American
       Association of Immunologists.

The findings of a potentially protective immunogen in low molar amounts on
the outer membrane surface of T. pallidum have provided a further
attractive explanation for the prolonged period required for the induction
of acquired protective immunity during syphilitic infection. This is also
in accord with the repeated immunizations required for protective immunity
with T. pallidum attenuated by gamma irradiation (22), the only method of
successful vaccination ever reported. Thus, the identification and study of
TROMPs has become essential in understanding syphilis pathogenesis and
immunity in molecular terms.

Isolation of the T. Pallidum Outer Membrane

In 1994, we developed a procedure to isolate the outer membrane of T.
pallidum without detergents (23). This method used three key features: 1) a
Ficoll step gradient to purify T. pallidum from host contaminating
material; 2) octadecyl rhodamine B chloride salt, a lipid-conjugated
chromophore that intercalates into membranes and provides a visual marker
to monitor outer membrane release and recovery; and 3) a hypotonic 0.05 M
sodium citrate buffer, pH 3.0, which was found to selectively release the
T. pallidum outer membrane. Since it has been suggested that endoflagellar
filaments physically interact with the outer membrane in the process of
motility, the probable mechanism for outer membrane release by this
procedure is the depolymerization of endoflagella at low pH.

Purification of T. pallidum outer membrane vesicles in sucrose density
gradients showed that the membrane banded at the very low density of 1.03
g/ml (7% sucrose). This, however, was expected for properties of a membrane
that lacks lipopolysaccharide (24) and contains a small amount of protein.
Freeze-fracture electron microscopy demonstrated that the purified membrane
vesicles contained extremely rare intramembranous particles consistent with
the low particle density observed for the native outer membrane of T.
pallidum. The selective isolation of the T. pallidum outer membrane was
shown by the inability to detect T. pallidum penicillin binding proteins, a
marker of the cytoplasmic membrane. Further support for the selectivity of
the outer membrane isolation was the complete absence of a previously
characterized 19-kDa periplasmic protein termed 4D (15) and the abundant
47-kDa lipoprotein (17) and the presence of only trace amounts of
endoflagellar protein. The absence of the 47-kDa lipoprotein was
particularly important since it is widely accepted that this very abundant
protein is exclusively anchored to the inner membrane (25). The finding
that the 47-kDa lipoprotein was not detected in the purified outer membrane
material indicates that inner membrane anchored lipoproteins were not
released by this procedure.

Two dimensional immunoblot analysis was used to identify the protein
constituents present in purified T. pallidum outer membrane. Two strongly
antigenic species were found by syphilitic immune rabbit serum, one very
basic protein (pI > 7.0) at 17-kDa and the other having a pI of
approximately 4.5 at 45-kDa. The observation that the 17-kDa protein was
very basic, showed higher oligomeric forms, and was selectively partitioned
into the hydrophobic phase after Triton X114 detergent extraction was
consistent with properties reported for the 17-kDa lipoprotein of T.
pallidum (26). With specific monoclonal antibodies, the 45-kDa protein was
identified as the previously characterized lipoprotein termed TmpA (27). It
was also found that most of these two lipoproteins, well over 90%, are
associated with the inner membrane protoplasmic cylinder complex. It would
appear, however, that the association of the 17- and 45-kDa lipoproteins
with the outer membrane is specific, given the absence of the normally
abundant 47-kDa lipoprotein. The function of these two outer membrane
lipoproteins remains to be determined.

In addition to the strongly antigenic 17- and 45-kDa lipoproteins,
gold-stained two-dimensional blots of outer membrane showed four additional
T. pallidum proteins, including one each at 28- and 65-kDa and two at
31-kDa, with close but distinct pI migration patterns. All of these
proteins, including the 17- and 45-kDa lipoproteins, were present in
approximately equal amounts in the outer membrane. However, in contrast to
the 17- and 45-kDa lipoproteins, these four additional proteins required
very low dilutions (1:25) of immune syphilitic rabbit serum for detection
by immunoblot analysis. Moreover, when compared with stained
two-dimensional blots of total T. pallidum proteins, these four additional
outer membrane proteins were either not detectable, as was the case for the
65-kDa protein and the more basic of the two 31-kDa proteins, or
represented extremely minor species, as was the case for the 28-kDa protein
and the more acidic 31-kDa protein. In view of the paucity of TROMPs
determined from freeze-fracture analysis, these four proteins, which were
clearly enriched in our outer membrane preparation, became the leading
candidates for membrane-spanning outer membrane proteins of T. pallidum.

In 1995, a second T. pallidum outer membrane isolation method that used
plasmolysis of organisms in 20% sucrose was reported by Radolf et al. (28).
Outer membrane isolated by this method was also shown by freeze-fracture
analysis to have an extremely low density of membrane-spanning protein.
These studies showed that the lipid composition of the T. pallidum outer
membrane was similar to that of the protoplasmic cylinder inner membrane
complex, with the exception of cardiolipin, which was prominently detected
only in protoplasmic cylinders. Of further interest was the finding that
outer membrane phospholipids and glycolipids did not react with antibody
from infection-derived immune serum, suggesting that only outer membrane
proteins are target antigens. However, in contrast to the limited number of
protein species that we observed, silver stained gels of their outer
membrane material showed more than 20 proteins, including the 47-kDa
lipoprotein. One interpretation of these results is that many of the
proteins identified in their outer membrane preparation are possibly
contaminants of the periplasm or inner membrane. Thus, we believe that the
small number of protein species identified by our isolation method
represent a more accurate account of the outer membrane protein composition
of T. pallidum.

While lipoproteins are integral membrane proteins, their membrane
association extends only to integration of the lipid moiety in the bilayer.
In contrast to membrane-spanning proteins, the T. pallidum lipoproteins do
not span membrane lipid bilayers and, therefore, do not form particles upon
freeze-fracture electron microscopy (21). By comparison, one type of
membrane-spanning protein common to virtually all gram-negative outer
membranes are porins, which form water-filled channels through membranes
allowing for transport of small molecular weight solutes (29). Porin
proteins, like almost all outer membrane proteins, span membranes in a
betapleated sheet topology rather than in alphahelical hydrophobic regions,
which are common to inner membrane-spanning proteins (30). Porin activity
can be detected by several methods (29), including radioisotope efflux or
substrate uptake in proteoliposomes, liposome swelling, and the black lipid
bilayer assay, which measures the conductivity of ions through porin
channels. In the black lipid bilayer assay, a set voltage is applied across
an artificial lipid bilayer which, if a porin protein has been inserted,
results in an increase in electrical conductance. The degree of conductance
increase for many insertional events is used to calculate the average pore
channel size. When the black lipid bilayer assay was used with Triton X100
detergent solubilized proteins from our purified T. pallidum outer
membrane, porin activity was demonstrated (23). Two distinct average
conductance increases were observed at 0.4 and 0.76 nanosiemens (nS),
corresponding to pore channel sizes of 0.35 and 0.68 nm, respectively,
which are similar in size to porin channels found for other gram-negative
bacteria (29). At the time, the two distinct conductance measurements
suggested two different porin species, or alternatively, that the larger
activity could be the result of dimeric aggregates of the smaller activity
or the smaller activity could be a substate of the larger channel, possibly
caused by the application of a voltage (31). Regardless of the exact number
of T. pallidum porin species, these findings confirmed that at least some
of the particles observed by freeze-fracture electron microscopy of the T.
pallidum outer membrane were membrane-spanning porin proteins. This finding
was of particular importance since several gram-negative bacterial porins
play a role in pathogenesis by acting as adhesins (32,33) and as surface
targets of bactericidal antibody (34-37). Thus, the search for an outer
membrane T. pallidum porin protein was initiated to identify a first TROMP
species.

T. Pallidum Rare Outer Membrane Protein 1 (Tromp1): an Outer Membrane Porin

Since most porin proteins have molecular masses of 28- to 48-kDa (29), our
focus was directed toward isolating the 28- and 31-kDa proteins identified
in purified T. pallidum outer membranes. Internal amino acid sequences from
the native 31-kDa protein were used to clone the encoding structural gene,
designated tromp1 (38). Antiserum generated to a recombinant Tromp1 fusion
protein has shown that both 31-kDa proteins identified by twodimensional
blot analysis of outer membrane are Tromp1. Analysis of the deduced amino
acid sequence showed an N-terminal hydrophobic region consistent with a
signal peptide. Two potential leader peptidase I cleavage sites are noted,
including threonine-histidine-alanine at residues 30 through 32 and
alanine-alanine-alanine at residues 38 through 40. Identification of the
cleavage site by N-terminal amino acid sequence of the native protein has
not been possible because of the limited amount of native protein
recoverable. However, other data discussed later in this review suggest
that alanine-alanine-alanine is the correct cleavage site. The deduced
Tromp1 amino acid sequence also supports the concept that Tromp1 topology
is in accord with the structural paradigms of other gram-negative outer
membrane proteins. Beta-moment analysis, which shows amphipathic sequence
regions, has predicted that Tromp1 has 14 membrane-spanning amphipathic
betasheet segments typical of gram-negative outer membrane proteins
(29,30).

The lethality for Escherichia coli transformants harboring the intact
tromp1 structural gene, which was found by immunoblot analysis to be
expressing Tromp1, is similar to that observed for many recombinant
gram-negative porin proteins expressed in E. coli (39). The tromp1 gene has
recently been found to be part of a putative transport operon that includes
an adenosine triphosphate binding protein and a hydrophobic membrane
protein (Hardham et al., Gordon Research Conference, 1996). To determine if
Tromp1 was a porin, native Tromp1 was purified from Triton X100 solubilized
T. pallidum by nondenaturing isoelectric focusing. The demonstration of
porin activity, by the black lipid bilayer assay, has confirmed that Tromp1
is a membrane-spanning outer membrane protein of T. pallidum, the first
such protein to be identified for this organism. Native Tromp1 showed two
distinct conductance distributions about means of 0.15 and 0.7 nS. The
larger of these channels is similar in mean conductance to the 0.76 nS
activity observed by using purified outer membrane. The smaller 0.15 nS
conductance channel had not been detected previously, and while two
channels of different sizes have been reported for other porins (40), this
may simply be the result of partial damage during isolation.

Because most gram-negative bacterial porins exist in the outer membrane in
either a sodium dodecyl sulfate (SDS) unstable or stable trimer
conformation (29), it was theorized that Tromp1 would also be organized in
an oligomeric form in the outer membrane of T. pallidum. Antiserum against
a Tromp1 fusion protein has in fact identified two higher molecular mass
forms of native Tromp1 on immunoblots of T. pallidum with sizes of
approximately 55- and 80-kDa. A 98-kDa size is further found to be the only
detectable form of native Tromp1 when T. pallidum is solubilized at room
temperature in a low concentration (0.02%) of SDS. These findings would
appear to be consistent with the idea that native Tromp1 exists in the T.
pallidum outer membrane in an SDS-unstable trimer conformation, a property
that would be common to several other gram-negative outer membrane porins
(29).

Expression of a Porin Active Recombinant Tromp1 (rTromp1)

Porin proteins of gram-negative pathogens not only function as portals for
nutrient acquisition across the outer membrane but also play a role in
pathogenesis by acting as adhesins (32,33) and targets for bactericidal
antibody (34-37). The membrane-spanning conformation of membrane porins is
critical for their biologic function. Many surface-exposed epitopes on
porins shown to be targets for bactericidal antibodies are conformational
(34,36). Thus, correct outer membrane protein conformation should be a key
consideration in the study of porins as they relate to bacterial virulence
and host immunity. The previous demonstration of immune serum antibody
mediated aggregation of TROMPs, as viewed by freeze-fracture electron
microscopy (20), suggested that Tromp1 could be a key surface exposed
target for antibody that mediates killing (20,41) or opsonization (42,43)
of T. pallidum. Because of these possibilities, studies have been initiated
to express and isolate a porin form of recombinant Tromp1 (rTromp1) with
native conformation and in amounts necessary to conduct functional studies.

The controlled nonlethal expression in E. coli of exported rTromp1 was
accomplished by inserting the tromp1 gene, including the region encoding
its native signal peptide, into a relatively low copy number expression
plasmid that has an inducible T7 promoter (44). Uninduced basal levels of
T7 RNA polymerase resulted in the stable expression, export, and outer
membrane localization of rTromp1. Expression detected in whole cell lysates
showed several forms of rTromp1, ranging in molecular mass from 31- to
35-kDa. These different sizes of rTromp1 may represent different
conformations of the protein as observed for other recombinant outer
membrane proteins expressed in E. coli. Only the 35-kDa form of rTromp1 is
detected in outer membranes isolated from E. coli. One explanation for the
higher molecular mass outer membrane form is its possible association with
lipopolysaccharides, known to form a complex with gram-negative outer
membrane porin proteins (45). Such an interaction would certainly be
noteworthy because the T. pallidum outer membrane does not contain
lipopolysaccharides (24).

The demonstration of porin activity by using rTromp1 isolated from E. coli
outer membranes has further confirmed the membrane-spanning conformation of
Tromp1. Single channel conductance measurements of rTromp1 in the black
lipid bilayer assay showed two distinct distributions of activity about 0.4
and 0.8 nS. The 0.8 nS conductance is clearly similar to the 0.7 nS
conductance determined for native Tromp1 (38) and the 0.76 nS conductance
determined for purified outer membrane, indicating that the poreforming
conformation of a portion of rTromp1 is similar if not identical to that of
the native protein. Although it is not known why rTromp1 also showed a 0.4
nS conductance measurement, one possibility is an altered conformation of
the protein after the isolation process.

A further question regarding the outer membrane localization of porin
active rTromp1 has been whether surface exposed antigenic sites are
present. Whole mount immunoelectron microscopy has demonstrated the surface
binding of immune rabbit serum antibody on E. coli expressing rTromp1 (44).
Antibody raised against a soluble fusion protein form of rTromp1, which was
found by immunoblot analysis to be 100-fold more sensitive in detecting
Tromp1 than the immune rabbit serum antibody, did not show surface binding
of antibody on E. coli expressing rTromp1. This soluble fusion protein form
of rTromp1 does not have porin activity when tested in the black lipid
bilayer assay. Thus, a reasonable explanation for the immunoelectron
microscopy observations is that the bound immune rabbit serum antibodies
recognize conformational surface epitopes on rTromp1, a possibility which
may also extend to native Tromp1 on the surface of T. pallidum.

The importance of porin protein conformation to biologic function has been
further demonstrated in studies using rTromp1. The primary amino acid
sequence of Tromp1 suggests two possible signal peptide processing sites
within the first 40 residues at threonine-histidine-alanine and at
alanine-alanine-alanine. Signal peptide fusion constructs at these two
possible cleavage sites using the OmpT signal peptide of E. coli result in
both forms of rTromp1 being exported and targeted to the E. coli outer
membrane. Only the OmpT signal fused at the alanine-alanine-alanine
position resulted in an exported product that has a molecular mass closest
to that of native Tromp1. Moreover, while the outer membrane form of
rTromp1 processed at threonine-histidine-alanine showed porin activity, the
average channel conductance was 3.2 nS, which is considerably larger than
the 0.70.8 nS channels observed for native and recombinant Tromp1 exported
with its native signal peptide. The greater lethality and limited recovery
of the OmpTTromp1 fusion construct processed at alanine-alanine-alanine has
precluded at this time the ability to test this outer membrane form for
porin activity. These findings suggest that alanine-alanine-alanine is the
likely processing site in the Tromp1 sequence for leader peptidase I. More
importantly, these findings have indicated that subtle changes in the
length of the primary amino acid sequence of Tromp1 can have significant
effects upon porin activity and are likely the result of an altered
conformation of the protein. For this reason and as described previously,
proper Tromp1 conformation is an important consideration for future studies
addressing its role in syphilis pathogenesis.

T. Pallidum Rare Outer Membrane Protein 2 (Tromp2)

In the search for additional TROMP species, we have recently focused on the
28-kDa protein identified in purified T. pallidum outer membrane
preparations (23). As in the protein for Tromp1, internal amino acid
sequences from the native 28-kDa protein were used to clone the encoding
structural gene, now designated tromp2 (46). Analysis of the deduced amino
acid sequence showed a 24-residue N-terminal hydrophobic region consistent
with a signal peptide and terminating in a typical leader peptidase I
cleavage site of leucine-alanine-alanine. As for Tromp1, the deduced amino
acid sequence for Tromp2 is also in accord with the structural paradigms of
other gram-negative outer membrane proteins. Beta-moment analysis has
predicted that Tromp2 has 9 membrane-spanning amphipathic betasheet
segments.

Recombinant expression and export of Tromp2 (rTromp2) in E. coli has
recently been accomplished by using the entire tromp2 structural gene in a
relatively low copy number plasmid that has an inducible T7 promoter (46).
In contrast to Tromp1, Tromp2 expression was not found to be lethal to E.
coli, even under maximum inducing conditions. Immunoblot analysis using
antiserum generated to rTromp2 has shown that virtually all of the
recombinant protein produced is targeted to the E. coli outer membrane.
When tested in the black lipid bilayer assay, rTromp2 isolated from E. coli
outer membranes showed only occasional channel formation. The total number
of porin insertional events was extremely low when compared with the amount
of rTromp2 tested. This is in contrast to both native and recombinant
Tromp1 where numerous insertional porin events were readily observed. Thus,
whether Tromp2 truly has porin function is not clear at this time.

As for rTromp1, a question regarding the outer membrane localization of
rTromp2 was the existence of surface exposed antigenic sites. The use of
whole mount immunoelectron microscopy demonstrated the surface binding of
immune rabbit serum antibody on E. coli expressing rTromp2 (46). By
comparison, antibody raised against a denatured form of rTromp2, which by
immunoblot analysis was found to be more sensitive in detecting rTromp2
than immune rabbit serum, did not show surface binding on E. coli
expressing rTromp2. This finding is again similar to that described above
for rTromp1 and again suggests that the bound immune rabbit serum
antibodies recognize conformational-surface epitopes on rTromp2, a
possibility which may extend to native Tromp2 on T. pallidum.

T. Pallidum Rare Outer Membrane Protein 3 (Tromp3)

Of the three outer membrane protein species identified (28-, 31-, and
65-kDa), the 65-kDa protein, tentatively termed Tromp3, is present in the
least amount on the basis of 2-dimensional SDS-PAGE gold-stained
immunoblots of purified outer membrane (23). In addition, the presence of
the 65-kDa protein from one outer membrane preparation to the next has been
inconsistent, suggesting that this protein may be differentially expressed.
The extremely small amount of Tromp3 has at this time precluded amino acid
sequencing and, therefore, the cloning of the gene that encodes this
protein.

The Potential Role of TROMPs in Acquired Immunity Against Syphilis

Freeze-fracture electron microscopy has demonstrated the ability of serum
from immune syphilitic rabbits to aggregate TROMPs (20). These studies have
been extended by using serum from infected rabbits with varying degrees of
challenge immunity and have shown that TROMP aggregation correlates
directly with the development of challenge immunity (Lewinski et al.,
unpub. obs., 1994). These findings are in accord with past studies that
have demonstrated a significant to complete level of protection of animals
after passive immunization with serum from immune donors (47). In a recent
study, we have used the small amount of attainable T. pallidum outer
membrane to immunize mice. We have found that serum from the immunized mice
possesses complement-dependent treponemicidal activity (100% killing of T.
pallidum) that is 30 times greater than that of serum from immune
syphilitic rabbits (Blanco et al., unpub. obs.). Immunoblot analysis using
this serum has shown that only the outer membrane associated proteins are
detected. We have also found that absorption of this serum to remove
antibodies to the T. pallidum lipoproteins does not diminish the titer of
the 100% killing activity. Such levels of serum treponemicidal activity
have heretofore never been generated by immunization of mice or rabbits
with either native or recombinant T. pallidum antigens. These observations
are consistent with the idea that TROMPs represent the key surface exposed
targets for treponemicidal antibody and perhaps a protective host immune
response.

Our recent ability to express in sufficient amounts recombinant Tromp1 and
Tromp2 provides an opportunity to address directly the ability of these T.
pallidum outer membrane proteins to elicit protective immunity in
experimental animals. However, the correct outer membrane conformation of
the recombinant TROMPs may prove a key factor in whether protective
immunity results, as is the case for several bacterial porins. We hope that
TROMP immunization will generate a humoral and/or cellular response capable
of efficiently killing T. pallidum and provide a significant level of
acquired resistance.

Acknowledgments

We thank Dr. Eldon M. Walker for the electron microscopy presented in this
review and Dr. Cheryl I. Champion for her extensive contributions to the
studies involving Tromp1 and Tromp2.

This paper is dedicated to Dr. Mary C. Pangborn, the discoverer of
cardiolipin at the Division of Laboratories and Research, New York State
Department of Health, for her 90th birthday, and to Dr. Thomas B. Turner,
the pioneer of treponemal research conducted at the Johns Hopkins School of
Medicine, for his 100th birthday.

Dr. Blanco is a member of the Department of Microbiology and Immunology,
UCLA School of Medicine, Los Angeles. He has been involved over the past 16
years in syphilis research focusing on structure-function relationships and
the immunobiology of surface and subsurface proteins.

Address for correspondence: David R. Blanco, Dept. Microbiology and
Immunology, CHS 43-239, UCLA School of Medicine, 10833 LeConte Ave., Los
Angeles, CA 90095; fax:310-206-3865;e-mail:
dblanco@microimmun.medsch.ucla.edu.

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Emerging Infectious Diseases
National Center for Infectious Diseases
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