J. Mol. Microbiol. Biotechnol. (2001) 3(1): 37-62.

JMMB
Review
Biology
of T. pallidum
37

Biology of Treponema pallidum: Correlation of

Functional Activities With Genome Sequence Data
Steven J. Norris1,2 *, David L. Cox3,
and George M. Weinstock2
1

Department of Pathology and Laboratory Medicine,

University of Texas-Houston Medical School, P.O. Box
20708, Houston, Texas 77225, USA
2
Department of Microbiology and Molecular Genetics,
University of Texas-Houston Medical School, Houston,
Texas 77225, USA
3
Division of AIDS, STD and TB Laboratory Research,
Centers for Disease Control and Prevention, Atlanta, GA
30333, USA

Abstract
Aspects of the biology of T. pallidum subsp. pallidum,
the agent of syphilis, are examined in the context of a
century of experimental studies and the recently
determined genome sequence. T. pallidum and a group
of closely related pathogenic spirochetes have evolved
to become highly invasive, persistent pathogens with
little toxigenic activity and an inability to survive
outside the mammalian host. Analysis of the genome
sequence confirms morphologic studies indicating the
lack of lipopolysaccharide and lipid biosynthesis
mechanisms, as well as a paucity of outer membrane
protein candidates. The metabolic capabilities and
adaptability of T. pallidum are minimal, and this relative
deficiency is reflected by the absence of many
pathways, including the tricarboxylic acid cycle,
components of oxidative phosphorylation, and most
biosynthetic pathways. Although multiplication of T.
pallidum has been obtained in a tissue culture system,
continuous in vitro culture has not been achieved. The
balance of oxygen utilization and toxicity is key to the
survival and growth of T. pallidum, and the genome
sequence reveals a similarity to lactic acid bacteria
that may be useful in understanding this relationship.
The identification of relatively few genes potentially
involved in pathogenesis reflects our lack of
understanding of invasive pathogens relative to
toxigenic organisms. The genome sequence will
provide useful raw data for additional functional
studies on the structure, metabolism, and
pathogenesis of this enigmatic organism.
Introduction
Syphilis was deemed one of the three great plagues of the
early 20th century (along with cancer and tuberculosis)
because of its high prevalence and difficulty in treatment.
*For correspondence. Email Steven.J.Norris@uth.tmc.edu;
Tel. (713) 500-5338; Fax. (713) 500-0730.

2001 Horizon Scientific Press

The demonstration of experimental transmission of syphilis

to chimpanzees (Metchnikoff and Roux, 1906) was followed
soon thereafter by the identification of a pale, spiral-shaped
organism called Spirochaeta pallida by Schaudinn and
Hoffman (Schaudinn and Hoffman, 1905). With the
development of experimental models and identification of
the infectious agent, hopes were high that research on this
enigmatic organism and the scourge of syphilis would
proceed with the same rapidity as with the other pathogens
discovered and characterized during this golden era of
microbiology.
The organism, since renamed Treponema pallidum
subsp. pallidum, did not cooperate. Despite intensive efforts
by some of the best minds in science, this seemingly simple
bacterium yielded information begrudgingly and in small
increments. Ninety-five years after its discovery, T. pallidum
still has not been cultured continuously in vitro, although
progress has been made in the achievement of in vitro
multiplication. Investigators have had to rely on inoculation
of rabbits and other animals both for the propagation of T.
pallidum for study and as models of human infection. The
inability to produce large quantities of the spirochete in a
pure state, let alone to perform colony isolation, mutational
analysis, and other standard bacteriologic procedures, has
hampered progress greatly.
Prior to the 1970s, less was known about T. pallidum
than about virtually any other pathogenic bacterium. The
application of electron microscopy, protein gel
electrophoresis, biochemical techniques, molecular biology
procedures, and genome sequencing have led to dramatic
increases in our understanding of the morphology, in vivo
and in vitro growth characteristics and requirements,
metabolism, and antimicrobial susceptibility of T. pallidum.
The complete sequence of T. pallidum subsp. pallidum
(Nichols) genome was determined in 1998 (Fraser et al.,
1998), shortly after the complete genome sequence of the
Borrelia burgdorferi B31 (one of a group of closely related
Lyme disease spirochetes) was reported in 1997 (Fraser
et al., 1997). As with other genome sequences, the T.
pallidum sequence provided a complete parts list for the
organism, revealing not only what the genetic capabilities
of the spirochete are, but also what is missing. However,
gene function can only be predicted from sequence data
and must be verified experimentally. This review represents
a compendium of the experimental data available on the
biology of T. pallidum and its correlation with what is
predicted from the genome sequence.
Various aspects of the biology of T. pallidum have been
reviewed previously, and in many cases the older
references provide a more complete representation. Turner
and Hollander (Turner and Hollander, 1957) and Willcox
and Guthe (Willcox and Guthe, 1966) provided excellent
overviews of T. pallidum physiology and animal infectivity.
Two books edited by Johnson (Johnson, 1976) and Schell
and Musher (Schell and Musher, 1983) and a review by

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38 Norris et al.

et al., 1997). Several short reviews have been published

on aspects of the T. pallidum genome sequence (Norris et
al., 1998; Weinstock et al., 1998; Radolf et al., 1999; Norris
and Weinstock, 2000; Weinstock et al., 2000; Weinstock
et al., 2000), as well as a detailed comparison of the
predicted gene products of T. pallidum and B. burgdorferi
(Subramanian et al., 2000).
Pathobiology of T. pallidum

Figure 1. The invasive nature of T. pallidum , as demonstrated by

transmission electron microscopy of rabbit testicular tissue following
inoculation with T. pallidum subsp. pallidum Nichols. A longitudinal section
of a treponeme traveling between host cells is present in the lower part of
the micrograph. Several cross-sections of a spirochete going through a
host cell are visible in the upper portion of the photomicrograph.

Sell and Norris (Sell and Norris, 1983) also provide general
information. Summaries of the molecular and antigenic
properties of T. pallidum were published by Strugnell et al.
(Strugnell et al., 1990) and Norris et al. (Norris and Group,
1993). The molecular architecture of the outer membrane
of T. pallidum has been an area of controversy in recent
years, and has been reviewed by Blanco et al. (Blanco et
al., 1997) and Radolf (Radolf, 1994; Radolf, 1995). A small
but informative book published by the U.S. Public Health
Service (United States Public Health Service, 1967) in 1964
still provides the best overview of clinical aspects of
syphilitic infection. The immunology of T. pallidum infection
will not be addressed here, but has been reviewed
elsewhere (Pavia et al., 1978; Schell and Musher, 1983;
Sell and Norris, 1983; Norris, 1988; Radolf, 1994; Blanco

Syphilis is a multistage disease characterized by localized,

disseminated, and chronic forms of infection (United States
Public Health Service, 1967; Musher, 1987; Larsen et al.,
1999). Inoculation occurs through direct contact of skin or
mucous membranes with infectious lesions during sexual
activity. In the primary stage, T. pallidum multiplies locally
at the site of inoculation, resulting in the characteristic
primary lesion (chancre) within days to weeks of exposure.
Dissemination occurs during the primary stage, resulting
in systemic infection. Approximately 30% of patients
develop the multiple skin lesions and protean
manifestations characteristic of secondary syphilis. Primary
and secondary lesions contain large numbers of viable T.
pallidum and as such are the principal source of
transmission. Latency is characterized by serologic
reactivity in the absence of other detectable manifestations,
and may last the lifetime of the individual. Roughly onethird of untreated syphilis patients develop tertiary syphilis,
consisting of gummatous syphilis, cardiovascular syphilis,
or neurosyphilis. T. pallidum can also be transmitted
transplacentally from mother to fetus, resulting in congenital
syphilis. Congenital infection may result in stillbirth, early
fulminant infection during infancy, or chronic disease with
bone, tooth, corneal, and neural manifestations.
Bacterial pathogens can be characterized by their
invasive and toxigenic properties. T. pallidum is a highly
invasive organism with limited toxigenic capabilities. The
invasive properties of the spirochete are demonstrated in
Figure 1. Dissemination and persistent colonization of
tissues results from the organisms characteristic corkscrew motility, hematologic spread, adherence and
penetration of epithelial cell layers and other host barriers,
and ability to evade the immune response. Most of the
pathology appears to be due to the inflammatory reaction
to infection, which damages the surrounding tissue (Sell
and Norris, 1983; Norris, 1988). With few exceptions, our

Table 1. Characteristics of Pathogenic Treponema

Agent

Disease

Distribution

Climate

Pathogenicity in Humans

Pathogenicity in Animals

Treponema pallidum Venereal syphilis

subsp. pallidum

Worldwide

Temperate

Treponema pallidum Endemic syphilis

subsp. endemicum
(bejel)

North Africa;
Near East;
South Europe
Central Africa;
South America;
Indonesia
Central and
South America
Not applicable

Desert and
Temperate

Highly invasive. Local and systemic

infection, with early, latent, late and
congenital forms
Moderately invasive. Early, latent,
and late stages

Rabbits - highly susceptible to skin and testicular

infection. Hamsters - lymph node infection, but no
skin lesions. Guinea pigs - relatively resistant
Rabbits, hamsters, and guinea pigs susceptible to
infection.

Tropical and
Desert

Moderately invasive. Skin and bone

lesions. Congenital disease rare.

Chronic skin lesions in hamsters. Rabbits and

guinea pigs also susceptible.

Tropical

Not invasive. Local dermal lesions,

no systemic involvement.
Not infectious to humans

Not infectious to laboratory animals.

Treponema pallidum Yaws

subsp. pertenue
Treponema carateum Pinta
Treponema
paraluiscuniculi
a

Venereal
spirochetosis of
rabbits

Not applicable

Compiled from references (Turner and Hollander, 1957; Willcox and Guthe, 1966; Sell and Norris, 1983).

Genital lesions in rabbits

Biology of T. pallidum 39

understanding of invasive mechanisms are extremely

limited relative to the extensive knowledge of the action of
bacterial toxins on host cells and tissues. Also, proteins
involved in invasion ( e.g. InlA and ActA of Listeria
monocytogenes) tend not to be conserved across a broad
range of bacterial species, as is often the case with bacterial
toxins (such as the RTX family). Some T. pallidum gene
products predicted from the genome sequence have
sequence similarity to known bacterial virulence factors
(Weinstock et al., 1998); however, as with other aspects
of its biology, functional studies will be necessary to provide
a clearer picture of the organisms pathogenic mechanisms.
Classification of T. pallidum
Properties of the pathogenic Treponema species and
subspecies are listed in Table 1. The Treponema causing
venereal syphilis, endemic syphilis, and yaws were
reclassified in 1984 (Smibert, 1984) to reflect new DNADNA hybridization information (reviewed by Fieldsteel,
1983). The pathogenic Treponema are virtually
indistinguishable in terms of DNA homology, morphology,
protein content and physiology. None has been cultivated
continuously in vitro. No genetic information is available
for Treponema carateum (which can not be cultured even
by animal inoculation), whereas Treponema
paraluiscuniculi causes venereal spirochetosis in rabbits
but does not cause disease in humans (Graves and
Downes, 1981). Therefore these infectious agents have
retained their status as separate species. Until the recent
advent of comparisons based on PCR and other methods
(see below), the only reliable criterion for differentiating
these organisms was their pattern of pathogenesis in
humans and in experimentally infected animals.
Miao, Fieldsteel and coworkers used DNA-DNA
hybridization to determine the degree of homology between
syphilis isolates (exemplified by the Nichols strain of T.
pallidum subsp. pallidum) and other spirochetes (Fieldsteel,
1983; Stanton et al. , 1991). Based on DNA-DNA
hybridization and estimates of G + C content, all syphilis,
yaws and endemic syphilis isolates were closely related
(>95% homology). On the other hand, DNA from these
organisms exhibited <5% cross-hybridization with DNA
from easily cultivable treponemes, including Treponema
phagedenis, Treponema refringens, and Treponema
(now Brachyspira) hyodysenteriae; their respective G + C
contents were also quite different. The 16s ribosomal RNA
sequence of T. pallidum exhibits significant identity with
rRNA sequences of other Treponema species, Spirochaeta
zuelzerae, and Spirochaeta stenostrepta (Paster et al.,
1991); in addition, Treponema species and certain
Spirochaeta species both contain cytoplasmic filaments,
an unusual intracytoplasmic structure not found in other
spirochetes (see below). These findings suggest that a
close evolutionary grouping of Treponema and some
Spirochaeta species may exist, and may justify
reclassification of some organisms.
Sequence analysis, restriction fragment length
polymorphism (RFLP), and monoclonal antibody reactivity
have been examined as means for distinguishing the
pathogenic treponemes. In comparing T. pallidum subsp.
pallidum and T. pallidum subsp. pertenue gene sequences

for the 190 kDa 4D antigen, Noordhoek et al. (Noordhoek

et al., 1990) found that only a single nucleotide out of 936
was different; even this single base pair difference was
not consistent among isolates of each subspecies. In
addition, monoclonal antibodies against T. pallidum subsp.
pallidum Nichols antigens were equally reactive with T.
pallidum subsp. pertenue proteins (Noordhoek et al., 1990),
providing further evidence that these subspecies are very
closely related.
More recently, Centurion-Lara et al. (Centurion-Lara
et al., 1998) described the occurrence of sequence and
RFLP differences in the regions flanking tpp15 (TP0171),
encoding a prominent 15 kDa lipoprotein. RFLP analysis
could be used to distinguish T. pallidum subsp. pallidum
strains from yaws and endemic syphilis isolates as well as
from T. paraluiscuniculi. Cameron et al. (Cameron et al.,
1999) described the consistent occurrence of a single
nucleotide difference between T. pallidum subsp. pallidum
isolates and subsp. pertenue and endemicum isolates in
the 579 bp gene encoding glycerophosphodiester
phosphodiesterase (gpd; TP0257). In contrast, 6 nucleotide
differences were found in gpd of T. paraluiscuniculi, 5 of
which were silent substitutions. This result verifies that T.
paraluiscuniculi has diverged to some extent from the
human treponemal pathogens, and also indicates the
strong selective pressure for the conservation of this
enzyme involved in phospholipid metabolism. The surfaceexposed protein candidate Tp92 exhibits a similar pattern
of conservation within 7 T. pallidum subsp. pallidum
isolates, with progressively greater heterogeneity observed
in subsp. pertenue and T. paraluiscuniculi isolates
(Cameron et al., 2000).
The T. pallidum repeat (tpr) genes (Fraser et al., 1998;
Stamm et al., 1998; Centurion-Lara et al., 1999) appear to
represent the regions of greatest genetic divergence in an
otherwise highly conserved genome, exhibiting both
interstrain and intrastrain differences in venereal syphilis
isolates. By sequence analysis of PCR products, tprD
exhibits at least two alleles within T. pallidum subsp.
pallidum (Centurion-Lara et al., 2000), and tprK has been
shown to have multiple alleles even within individual syphilis
isolates (Centurion-Lara et al., 2000). Whole genome PCR
analysis has revealed additional strain differences at tpr
loci (E. Sodergren, unpublished data).
A molecular subtyping system for T. pallidum subsp.
pallidum recently described by Pillay et al . (1998)
demonstrates the existence of genetic heterogeneity in a
large number of strains and patient specimens. This system
utilizes a combination of size heterogeneity within the arp
(acidic repeat protein) gene and RFLP patterns of three
members of the tpr gene family (tpr E, G and J). The arp
gene contains a series of 60 bp repeats; these repeats
code for a highly acidic amino acid sequence. Thus far, 15
arp repeat sizes (4-16, 18-20, 21, 24) and 13 tpr gene RFLP
types (designated a to m) have been identified (Pillay et
al., 1998; Pillay et al., personal communication; Sutton et
al., submitted). For example, T. pallidum subsp. pallidum
(Nichols) falls into group 14d; it has 14 repeats in the arp
gene and displays RFLP pattern d for the 3 tpr genes. By
combining the tpr and arp typing methods 43 different
subtypes have been observed among 229 specimens
(genital ulcers or blood) from the United States,

40 Norris et al.

Madagascar, and South Africa. The 14d subtype was most

common and was found in 58 (25.3%) of the 229
specimens. Furthermore, both of these genes or sets of
genes are present in T. pallidum susp. pertenue and
endemicum. The limited number of samples of these two
subspecies examined indicated lower heterogeneity than
in subsp. pallidum especially in the arp gene. The
heterogeneity in these genes suggests that additional
divergence may be present in other genes.
Genome Analysis
In 1991, Walker et al. (Walker et al., 1991) used pulsed
field electrophoresis techniques to characterize the genome
of T. pallidum. The genome was shown to consist of a
single, circular chromosome approximately 1,000,000 base
pairs (one megabase) in size, much smaller than the
previous estimate of 13.7 megabases based on DNA-DNA
hybridization kinetics (Fieldsteel, 1983). T. pallidum thus
was shown to possess one of the smallest genomes within
the prokaryotes (similar to Mycoplasma and Borrelia
species) (Krawiec and Riley, 1990).
The genome sequence of T. pallidum subsp. pallidum
(Nichols) was determined in 1998 (Fraser et al., 1998) using
now well-established random, shot-gun cloning and
sequence assembly techniques. The genome consists of
a single circular chromosome of 1,138,006 base pairs (+
possible undetected sequencing errors). The G + C content
is 52.8%, much higher than the 39% value obtained with
the B. burgdorferi genome (Fraser et al., 1997). A total of
1041 open reading frames (ORFs) were identified,
accounting for 92.9% of the total genomic DNA. Based on
protein similarity searches, predicted biological roles could
be assigned to 55% of the ORFs; 17% matched
hypothetical proteins of unknown function in other
organisms, and the remainder (28%) did not have
significant similarity to other known sequences. This ratio
is similar to those obtained with other bacterial genomes.
Surprisingly, only 476 (46%) of the predicted protein
products had orthologs in B. burgdorferi, and most of these
(76%) were proteins with a predicted biological function
(e.g. housekeeping functions). Thus relatively few (~5%)
of the T. pallidum genes appear to be spirochete-specific,
i.e. common to spirochetes but not found in other bacteria.
This result indicates significant divergence among the
Spirochaetacae. Other aspects of the T. pallidum genome
will be considered in the remaining sections of this review.
Morphology
The spirochetes, members of the order Spirochaetales,
are characterized by a morphologically unique, helical or
wave-like cell body (Figure 2). When imaged in two
dimensions by darkfield microscopy or transmission
electron microscopy, the helical cells of T. pallidum have a
regular, wave-like appearance (Hovind-Hougen, 1983). The
cells of T. pallidum range from 5 to 15 m in length, as
measured along the axis of the helix, and are typically 0.16
to 0.20 m in diameter. The sinusoidal profile of the
organism has a wavelength and amplitude of approximately
1.1 m and 0.4 m, respectively.
The cross-sectional ultrastructure of T. pallidum

Figure 2. Morphology of T. pallidum subsp. pallidum Nichols by electron

microscopy. Panel (a) is a scanning electron micrograph, (b) is a preparation
of T. pallidum negatively stained with ammonium molybdylate, and (c-f)
show ultrathin sections. Note the overall helical shape (panel a) and the
insertion (I) of several periplasmic flagella (PF) or endoflagella at each end
of the cells (panel b). In ultrathin sections, the outer membrane (OM),
cytoplasmic membrane (CM), cytoplasmic filaments (CF) and PFs are
evident. Bars = 1 m (panel a) and 0.1 m (panels b-f). Reprinted with
permission from (Larsen et al., 1999).

superficially resembles that of gram-negative bacteria in

the concentric arrangement of the outer membrane (OM),
periplasmic space, peptidoglycan layer, inner (cytoplasmic)
membrane (CM) and protoplasmic cylinder (Figure 2). This
morphologic similarity is deceptive, however, since it is now
clear that the composition of the T. pallidum OM differs
markedly from that of gram-negative bacteria (see Outer
Membrane below). The OM delimits the outer cellular
surface of the organism, and the CM surrounds the
protoplasmic cylinder. Unlike gram-negative bacteria, the
organelles of motility of T. pallidum lie entirely within the
periplasmic space, between the CM and the OM. These
organelles, termed periplasmic flagella or endoflagella,
insert in groups of two to four subterminally at either end
of the cell cylinder of T. pallidum; the insertion points are
arranged in a row parallel to the long axis of the helix. The
periplasmic flagella wrap around the helical protoplasmic

Biology of T. pallidum 41

cylinder and interdigitate near the center of the organism.

The presence of an electron dense layer closely apposed
to the CM has been identified by thin-section electron
microscopy (Hovind-Hougen, 1983), although this layer,
which by inference corresponds to the peptidoglycan cell
wall, is not always clearly visible. Biochemical evidence
indicates that T. pallidum contains typical spirochetal
peptidoglycan (Radolf et al., 1989). The presence of the
OM, CM, and periplasmic flagella, like helicity, is
characteristic of all spirochetes; differences in other
morphological parameters (including cell diameter and
length and the number and length of periplasmic flagella)
exist among various members of this diverse group of
bacteria.
Outer Membrane
The OM forms the cellular surface in most intimate contact
with the host and is also a structure with which many
components important for bacterial virulence are
associated. As a result, there has been great interest in
characterizing the OM of T. pallidum. A perspective has
arisen in the field of treponemal research, acquired from
work performed in several laboratories over a number of
years, that the surface of T. pallidum exhibits a puzzling
lack of antigenicity. The kinetics of in vitro killing of T.
pallidum by specific antibody and complement are
prolonged. A minimum of 4 hours incubation with high titer
antiserum and active complement are necessary before
any killing of treponemes occurs; typically 16 h are
necessary to achieve 100% killing (Nelson and Mayer,
1949; Miller et al., 1963; Bishop and Miller, 1976). This
observation is paralleled by the finding that the surface of
T. pallidum seems to require perturbation by chemical or
immunological means before the binding of antibody to
the organism can be detected by standard
immunocytochemical techniques (Metzger and J., 1964;
Houvind-Hougen et al., 1979; Penn, 1981; Fehniger et al.,
1986; Radolf et al., 1986).
Because T. pallidum is propagated in the rabbit testis,
the possibility has been explored that the relative antigenic
inertness of the organism may result from uniform binding
of host molecules to the treponemal surface. It has been
reported that treponemes harvested from testes have
fibronectin, albumin, and other host molecules associated
with their surface (Fitzgerald et al., 1975; Fitzgerald and
Johnson, 1979; Peterson et al., 1983). These studies have
stimulated speculation that T. pallidum utilizes a form of
antigen masking as a mechanism of evading the host
immune response, as has been hypothesized for the
schistosomes (Bloom, 1979).
Ultrastructural analyses indicate that T. pallidum may
possess a simple and elegant property that limits the
antigenicity of its surface. Freeze-fracture studies have
shown that OM of the organism is populated by a
remarkably low concentration of transmembrane proteins,
approximately 100-fold fewer than in other spirochetes and
in gram-negative bacteria (Radolf et al., 1989; Walker et
al., 1989; Walker et al., 1991). Both the concave (outer
leaflet of the lipid bilayer) and convex (inner leaflet of the
lipid bilayer) OM fracture faces of T. pallidum contain few,
sparsely distributed intramembranous particles, indicating
the presence of few integral transmembrane proteins

(Radolf et al., 1989; Walker et al., 1989). In contrast, the

intramembranous particles of the OM concave fracture face
of E. coli form a two-dimensional array that essentially
covers the entire surface of the membrane leaflet (compare
10,000 particles/m2 for E.coli to 90 particles/m2 for T.
pallidum) (Lugtenberg and Van Alphen, 1983; Radolf et
al., 1989; Walker et al., 1989). In a modified immobilization
assay it was shown that although antibody binds to the
surface of T. pallidum within one hour in the absence of
active complement, a potentiation incubation period of 8 h
is required before the addition of active complement will
mediate killing of the organism (Blanco et al., 1990).
Freeze-fracture of organisms incubated for 16 h in heatinactivated immune rabbit serum revealed that the
transmembrane proteins of the OM, which are normally
widely separated, had formed aggregates (Blanco et al.,
1990). The combined results of the modified immobilization
assay and freeze-fracture of treponemes incubated for 16
h in immune rabbit serum indicate that cross-linking of the
OM proteins by antibody is the rate-limiting step in antibodydependent, complement-mediated in vitro killing of T.
pallidum. The slow kinetics of this step are consistent with
the model that proposes that the OM of T. pallidum is
populated by a low concentration of proteins.

Outer Membrane Purification

The purification of T. pallidum outer membranes and
identification of associated proteins has been problematic
((Radolf et al., 1995; Blanco et al., 1997). The OM of T.
pallidum does not contain lipopolysaccharide (LPS) (Hardy
and Levin, 1983; Johnson, 1983; Bailey et al., 1985; Penn
et al., 1985; Radolf and Norgard, 1988), a molecule found
in great abundance in the outer leaflet of the OM of gramnegative bacteria; these studies are confirmed by the lack
of genes encoding LPS biosynthesis enzymes in the
genome (Fraser et al., 1998). LPS imparts a higher density
to the OM of gram-negative bacteria, allowing separation
of the OM from the CM in sucrose density gradients. The
lack of LPS in the OM of T. pallidum probably explains the
difficulty in applying this simple technique to separation of
the treponemal CM and OM (Norris and Group, 1993).
Moreover, gold standard markers for the outer membrane
(such as the Omp proteins of E. coli) are lacking, and
detection of outer membrane proteins in cellular fractions
is predictably difficult due to their relative paucity.
Blanco et al. (Blanco et al., 1994) used a combination
of Ficoll purification of T. pallidum, octadecyl rhodamine B
chloride labeling of intact organisms, and selective release
of OM vesicles with 0.05 M citrate buffer, pH 3.2, to purify
T. pallidum and T. vincentii OMs. Ficoll purification removed
contaminating host proteins, whereas incorporation of the
hydrophobic rhodamine compound into the surface of intact
treponemes permitted monitoring of OM purification. Blanco
et al. (Blanco et al., 1994) found that the acidic citrate buffer
released vesicles that lacked cytoplasmic membrane
markers (such as the 47 kDa lipoprotein), but contained
several polypeptides deemed treponemal rare outer
membrane proteins or Tromps.
Two of the proteins identified by this means, Tromp1
and Tromp2, have been characterized. Tromp1, also known
as TroA (Hardham et al., 1997), is a 30.4 kDa protein with
a hydrophobic leader sequence; there is contradictory

42 Norris et al.

evidence regarding whether this leader is cleaved or serves

as a hydrophobic anchor (Akins et al., 1997; Blanco et al.,
1999). Native Tromp1 purified by nondenaturing isoelectric
focusing has porin activity as determined by conductance
measurements in a lipid bilayer model (Blanco et al., 1996),
leading to the identification of Tromp1 as an integral OM
protein. However, other evidence is consistent with Tromp1
being a periplasmic binding protein for an ATP-binding
cassette (ABC) transport system. Tromp1/TroA has
sequence similarity to other known periplasmic binding
proteins and is encoded by the first gene in an operon with
genes for an ATP-binding protein (TroB), two hydrophobic
membrane proteins (TroC and TroD), and a manganesedependent regulatory protein (TroR) resembling the
Corynebacterium diphtheriae regulator DtxR (Hardham et
al., 1997; Posey et al., 1999). The last gene in the operon
encodes phosphoglycerate mutase, a glycolytic pathway
enzyme that in many organisms requires magnesium as a
cofactor. Posey et al. (Posey et al., 1999) demonstrated
that TroR binds to the tro operator in a Mn++-dependent
fashion, and like DtxR acts as a cation-dependent repressor
protein.
The three dimensional structure of a recombinant form
of TroA has been determined (Deka et al., 1999; Lee et al.,
1999). It exhibits a structure nearly identical to those of
periplasmic binding proteins, and contains a captive Zn++
ion in the crystal structure. Interestingly, mutation of the
troA homolog scaA in the Streptococcus gordonii scaCBA
operon results in poor growth in low [Mn ++]; this operon is
also inhibited by high concentrations of Zn++ (Kolenbrander
et al., 1998). Thus there is convincing evidence that TroA
is a periplasmic protein involved in the capture of Mn++ or
other metal cations for transport across the cytoplasmic
membrane. However, recent data (Zhang et al., 1999)
suggest that the recombinant protein exists in a different
conformation than its native treponemal counterpart, and
that trimer formation and porin activity can be recovered
by denaturation and renaturation of recombinant Tromp1/
TroA. Thus the structural location and function of Tromp1/
TroA remain controversial.
Tromp2, characterized by Champion et al. (Champion
et al., 1997) has a predicted signal peptidase I leader
sequence that if cleaved would yield a mature protein of
24.8 kDa. The structure is predicted to have nine
transmembrane beta-pleated sheet segments, consistent
with an integral membrane protein. Unlike Tromp1,
membrane conductance experiments did not provide clear
evidence that Tromp2 has porin activity. The native and
recombinant forms partition to the detergent phase in Triton
X-114 partitioning experiments, and recombinant Tromp2
was associated with the outer membrane and binds antiTromp2 antibodies on the surface of E. coli. Therefore
evidence to date indicates that Tromp2 is a surfaceexposed integral membrane protein. It should be noted
that Tromp2 has sequence similarity (26% identity, 40%
similarity) with 131 amino acids of FlaA, the T. pallidum
endoflagellar sheath protein (see below).
Radolf et al. (Radolf et al., 1995) utilized an alternative
approach to OM purification, combining plasmolysis with
isopycnic sucrose density gradient sedimentation.
Treatment of T. pallidum with a 20% sucrose solution
followed by adjustment to 45% sucrose dissociated the

OM from the remainder of the cell, permitting separation

on a bottom-loaded, 5% to 50% sucrose gradient. Under
these conditions, OM fragments sedimented at a density
of 1.05 g/cm 3 as compared to 1.14 g/cm 3 for the
protoplasmic cylinders. Analysis of the lipid content of the
resulting OM fraction indicated that the majority of the
cardiolipin was associated with the inner membrane (CM).
However, some cardiolipin was present in the OM fraction,
which may explain the observed opsonic activity of antiVDRL antigen antibodies (Baker-Zander et al., 1993).
Analysis of the protein content of these OM preparations
revealed the presence of orthologs of thioredoxin (Tlp),
peptidyl prolyl cis - trans isomerase (PpiB), and
glycerophosphodiester phosphodiesterase (GlpQ), as well
as the following previously described proteins: treponemal
membrane protein A (TmpA), basic membrane protein
(Bmp), Tromp1/TroA, T. pallidum lysine rich repeat protein
(TpLRR), and 17 kDa lipoprotein Tpp17 (Shevchenko et
al., 1997). Many of these proteins had previously been
shown to have a periplasmic location in T. pallidum or other
organisms. The authors conclude that some of these outer
membrane candidates may represent periplasmic or CM
contaminants. Without a definitive measure of OM
localization, distinguishing contaminants from true OM
constituents is a vexing problem.
Other approaches to identifying OM proteins have
included surface labeling of intact organisms (reviewed by
(Norris and Group, 1993; Radolf, 1995)) and construction
of phoA fusions to identify exported proteins (Blanco et al.,
1991; Hardham, 1995; Stamm et al., 1998). The first
method has proven to be insufficient due to the fragility of
the OM and exposure of subsurface components during
processing. The second identifies any protein that is
transported across the CM, and thus is also not specific
for OM proteins. Surface immunofluorescence using T.
pallidum embedded in agarose microbeads (Cox et al.,
1995) has been used to verify the subsurface location of
many proteins, but thus far has not resulted in the detection
of surface-localized proteins. Although surface exposed
proteins (Osps A-C) in the OM of B. burgdorferi (Cox et al.,
1996) have been labeled using this microbead technology,
it is still unclear if the assay is sensitive enough to detect
the much less abundant (as revealed by freeze-fracture
EM ) integral protein(s) of the T. pallidum OM. Finally,
opsonic activity of T. pallidum-specific and monospecific
antisera and the protective effects of immunization have
been employed as means of identifying surface-exposed
proteins, including GlpQ (Stebeck et al., 1997; Cameron
et al., 1998), TrpK (Centurion-Lara et al., 1999), and Tp92
(Cameron et al., 2000). However, Shevchenko et al.
(Shevchenko et al., 1999) provided evidence that GlpQ
was entirely subsurface in location, and were unable to
reproduce the opsonic or protective activities of this protein.

The tpr Gene Family

The tpr gene family was discovered by four independent
means: TnPhoA mutagenesis (Stamm et al., 1998),
genome sequence analysis (Fraser et al. , 1998),
subtraction hybridization and differential immunologic
screening (Centurion-Lara et al., 1999). This family of 12
paralogous genes (tprA through tprL) is found as individual
genes or gene clusters throughout the genome, and some

Biology of T. pallidum 43

loci are accompanied by additional paralogous gene

families (tpr-associated genes, such as taaA through taaD).
The predicted Tpr proteins can be subdivided into 3 families
and vary in size from 55.5 kDa to 81.5 kDa. The sequence
identity of overlapping regions of the predicted Tpr products
ranges from 29.2 to 100 percent. In addition, they are similar
to the major sheath protein (Msp) of T. denticola (36.7 to
42.8 percent identity). Msp is a 58 kDa protein that is
thought to form a regular hexagonal array on the surface
of T. denticola (Haapasalo et al., 1992; Fenno et al., 1996),
although its surface localization and arrangement have
been questioned (Caimano et al., 1999).
Thus far, the detection of Tpr protein expression in T.
pallidum has not been reported, and preliminary studies
indicate that they either are expressed in low quantities or
are relatively non-reactive in standard western blot
techniques (S. J. N., unpublished). Centurion-Lara et al.
(1999) reported that rabbit antiserum raised against a
recombinant form of the TprK variable domain enhanced
the phagocytosis of T. pallidum by rabbit peritoneal
macrophages; this result suggests that TprK or crossreactive proteins are expressed on the surface of T.
pallidum and thus serve as opsonic targets. Moreover,
rabbits immunized with recombinant forms of TprK and
TprF exhibited decreased lesion severity, indicating that
anti-Tpr immune responses provide some degree of
protection against syphilitic infection (Centurion-Lara et al.,
1999; Weinstock et al. , 2000). Based on reverse
transcription PCR, each of the tpr genes appear to be
expressed in the Nichols strain (RT-PCR) (J. M. Hardham,
M. P. McLeod, E. Sodergren, J. K. Howell, G. M. Weinstock,
and S. J. Norris, unpublished data), but semi-quantitative
RT-PCR suggests that tprK and tprH transcripts are
expressed at higher levels than those of other tpr genes
(Centurion-Lara et al., 1999). Although heterogeneity in
tpr genes exists both between and within T. pallidum strains
and subspecies (see Classification of T. pallidum), to date
there is no evidence for rapid recombination that could give
rise to antigenic variation.

Periplasmic Flagella and Motility

The gross architecture of the periplasmic flagellum of T.
pallidum is similar to that of other bacterial flagella, in that
three structural components, the basal body, the hook, and
the filament, have been identified (Hovind-Hougen, 1972;
Hovind-Hougen, 1983). The genes corresponding to well
conserved basal body and hook proteins are present in
large operons in the T. pallidum genome sequence (Fraser
et al. , 1998), and mutation of these genes in other
spirochetes results in loss of motility (Charon et al., 1992;
Limberger et al., 1994; Li et al., 1996; Ruby et al., 1997;
Limberger et al., 1999). The T. pallidum flagellar filament
has been the subject of intense study. Upon release from
the cell under appropriate conditions, the filament retains
a helical shape (Hovind-Hougen, 1972; Hovind-Hougen,
1983). The filament is composed of an inner core (10 nm
diameter), and an outer sheath (core + outer sheath, 17
nm diameter) (Hovind-Hougen, 1983).
The polypeptides comprising the core and sheath
structures have been characterized in great detail. The
periplasmic flagellar filament of T. pallidum is composed
of four major flagellin subunits, in contrast to the single

flagellin found in most bacteria (Penn et al., 1985;

Cockayne et al., 1987; Blanco et al., 1988; Norris et al.,
1988). The flagellins are subdivided into two distinct
classes, as originally defined by two-dimensional
electrophoresis, Western blotting and N-terminal amino
acid sequence analysis (Norris et al., 1988). The class A
flagellin FlaA is a 37-kDa polypeptide that forms the sheath
of the periplasmic flagellum, while the core of the filament
is composed of three class B flagellin molecules with
relative molecular masses (Mrs) of 34,500 (FlaB1), 33,000
(FlaB2) and 30,000 (FlaB3) (Penn et al., 1985; Cockayne
et al., 1987; Blanco et al., 1988; Norris et al., 1988). The
genes that encode the flagellins of T. pallidum have been
cloned and sequenced (Isaacs et al., 1989; Pallesen and
Hindersson, 1989; Champion et al., 1990; Isaacs and
Radolf, 1990). The class B flagellin genes share significant
sequence homology with one another and with the
ubiquitous flagellin sequence found in both gram-negative
and gram-positive bacteria (Norris et al., 1988; Pallesen
and Hindersson, 1989; Champion et al., 1990). The genes
flaB1 and flaB3 were found to be separated by 277 base
pairs; these genes may be transcribed as polycistronic
message from a single promoter located upstream of flaB1
(Champion et al., 1990).
The importance of the periplasmic flagella derives from
their function in cell locomotion and their highly antigenic
nature. Motility is an important virulence factor for T.
pallidum, promoting tissue invasion and dissemination. The
role of the periplasmic flagella in motility has been well
established in the cultivatable spirochetes (Canale-Parola,
1978; Bromley and Charon, 1979; Paster and CanaleParola, 1980; Charon et al., 1984; Goldstein and Charon,
1988; Kimsey and Spielman, 1990), although the
mechanism is not entirely understood. The corkscrew
motion characteristic of spirochetes allows them to translate
through media of relatively high viscosity (Canale-Parola,
1978; Kimsey and Spielman, 1990). Like other spirochetes,
treponemes spin about their longitudinal axis as the result
of rotation of the endoflagella (Canale-Parola, 1978;
Bromley and Charon, 1979; Paster and Canale-Parola,
1980; Charon et al., 1984; Goldstein and Charon, 1988;
Kimsey and Spielman, 1990). In nonviscous media, this
motion leads to vigorous rotation but no translational
movement. The real character of treponemal motility is
observed in tissue or highly viscous medium, where the
organisms can swim at a rate of up to 19 micrometers per
second (Kimsey and Spielman, 1990; Charon et al., 1992;
Ruby and Charon, 1998). T. pallidum can rapidly reverse
direction in response to obstacles and, because of its
flexibility, change its course through bending. Observing
T. pallidum in a bit of tissue under a darkfield microscope
provides a convincing demonstration of its invasive
capabilities.
Periplasmic flagella stimulate a strong, early antibody
response that persists throughout infection (Hanff et al.,
1982; Pedersen et al., 1982; Pedersen et al., 1982; Hanff
et al., 1983). The activity of antiserum to the periplasmic
flagella in the standard immobilization assay and the lack
of activity of this antiserum in the modified immobilization
assay suggest that the periplasmic flagella may be
important second-hit targets in the humoral immune
response (Blanco et al., 1990). The presence of pathogen-

44 Norris et al.

specific epitopes (Norris and Group, 1993) in the central

region of FlaB proteins may enable the development of
more specific serodiagnostic tests for syphilis, similar to
recombinant flagellar antigens examined for serodiagnosis
of Lyme disease (Gomes-Solecki et al., 2000).

Peptidoglycan
Biochemical characterization of the residual sacculus left
after exhaustive Triton X-114 extraction of T. pallidum
demonstrated the presence of muramic acid, a signature
amino sugar of peptidoglycan (Radolf et al. , 1989).
Ornithine, which commonly constitutes the sole diamino
acid found in spirochetal peptidoglycan, was also identified,
whereas diaminopimelic acid was not present (Radolf et
al., 1989). These findings are consistent with those
obtained in studies of peptidoglycan from other spirochetes
and confirm the presence of a peptidoglycan layer as
inferred from thin-section electron micrographs (HovindHougen, 1983).
Cytoplasmic Membrane
The CM of T. pallidum, as revealed by freeze-fracture
studies, is ultrastructurally similar to the CM of typical gramnegative bacteria (Lugtenberg and Van Alphen, 1983;
Radolf et al., 1989; Walker et al., 1989). The inner leaflet
of the CM of E. coli and other gram-negative bacteria
typically contains a high concentration of intramembranous
particles. The corresponding CM fracture face of T. pallidum
is also densely populated with intramembranous particles,
in sharp contrast to either fracture face of the OM of the
organism (Radolf et al., 1989; Walker et al., 1989).
T. pallidum has been shown to contain a set of
lipoproteins that are both abundant and highly
immunogenic (Chamberlain et al., 1989; Chamberlain et
al., 1989; Schouls et al., 1989; Purcell et al., 1990; Swancutt
et al., 1990; Schouls et al., 1991; Yelton et al., 1991; Norris
and Group, 1993). The lipoproteins were originally identified
as molecules that partition into the detergent phase
following solubilization of T. pallidum with the non-ionic
detergent Triton X-114, a characteristic of hydrophobic
membrane proteins (Chamberlain et al., 1989). Many of
these lipoproteins, including Tpp47, Tpp17, Tpp15, and
perhaps GlpQ (Shevchenko et al., 1999) appear to be
predominantly associated with the periplasmic leaflet of
the CM, based on the nonreactivity of antisera against these
proteins in intact organisms and the occurrence of reactivity
upon treatment of T. pallidum with concentrations of Triton
X-100 that disrupt the outer membrane (Cox et al., 1995).
In addition, antiserum to the Triton-X114 soluble proteins
(principally lipoproteins) did not kill T. pallidum in the
modified immobilization assay; under identical conditions,
immune rabbit serum has treponemicidal activity (Blanco
et al., 1990).
Penicillin-binding proteins, which are involved in the
transpeptidation reactions of peptidoglycan synthesis, are
commonly found in the CM of gram-negative bacteria
(Waxman and Strominger, 1983). Penicillin-binding proteins
have also been identified as constituents of T. pallidum
(Cunningham et al., 1987; Radolf et al., 1989). After
extraction of T. pallidum with detergents under conditions
that solubilize the OM but leave the CM morphologically
intact, the penicillin-binding proteins remain with the

protoplasmic cylinders (Cunningham et al., 1987; Radolf

et al., 1989). The hydrophobic nature of the penicillinbinding proteins is indicated by their retention in the
detergent phase after complete membrane solubilization
and phase-partitioning with Triton X-114 (Radolf et al.,
1989). Weigel et al. (Weigel et al., 1994) demonstrated
that the major 47 kDa lipoprotein of T. pallidum (Tp47) is a
penicillin-binding protein with carboxypeptidase activity,
despite having no close orthologs. This result suggests
that other prominent CM-associated lipoproteins of T.
pallidum (such as 44.5, 17, and 15 kDa proteins) may also
be involved in a novel peptidoglycan biosynthesis pathway.

Cytoplasmic Filaments
An unusual morphological feature of T. pallidum, and most
other members of the genus Treponema and some
Spirochaeta species, is the presence of cytoplasmic
filaments that lie just beneath the CM and in direct
apposition to the periplasmic flagella. Although these
structures have been called cytoplasmic tubules (Bermudes
et al., 1994), electron microscopy of several cultivatable
treponemes (Eipert and Black, 1979; Masuda and Kawata,
1989) and T. pallidum (You et al., 1996) indicates that they
are fibrillar in structure. The filaments originate close to
the basal bodies of the periplasmic flagella and in some
cases appear to attach to them (Hovind-Hougen and BirchAndersen, 1971; You et al., 1996); however, more recent
studies with wild type and flagella-deficient mutants of T.
phagedenis (Izard et al., 1999) indicate that flagellar
components do not appear to be associated with the
cytoplasmic filaments and are not required for their
assembly. It is not known whether the filaments extend
the entire length of the cell or end near the middle, as is
the case for the flagella. The filaments have a ribbon-like
appearance with a rectangular cross-section of ~7.0 to 7.5
nm by 1 nm. Several cytoplasmic filaments aggregate along
their flat surface to form a tightly packed array that is visible
in electron micrographs of cross-sections of treponemes.
The major subunit of the cytoplasmic filaments from T.
phagedenis and other treponemes appears to be a
conserved polypeptide with an Mr of 82,000 to 83,000
(Masuda and Kawata, 1989). The gene encoding the major
cytoplasmic filament protein CfpA of T. pallidum subsp.
pallidum was identified b y N-terminal sequence analysis
of the cytoplasmic filament protein of T. phagedenis and
the corresponding protein of T. pallidum (You et al., 1996).
Overproduction of CfpA in E. coli results in formation of
short, irregular bundles suggestive of partial self-assembly
(You et al., 1996). Targeted mutagenesis of cfpA in
Treponema denticola results in altered motility of the
spirochetes, indicating that the cytoplasmic filaments are
involved in some way with motility; there are also indications
that chromosomal segregation and cell division are affected
(J. Izard and R. J. Limberger, unpublished data). These
structures are not found in most spirochetes, and as such
are not globally required for spirochetal motility and cell
division processes.
Other Cytoplasmic Constituents
Ribosomes are present as electron dense bodies in the
cytoplasm of lead citrate- and uranyl acetate- stained thin
sections of T. pallidum (Hovind-Hougen, 1983). The

Biology of T. pallidum 45

genome of T. pallidum contains two copies of ribosomal

RNA genes in the typical bacterial 16S-23S-5S
arrangement (Fraser et al., 1998); in contrast, the B.
burgdorferi genome contains two 23S-5S gene operons
and a separate, single copy of the 16S rRNA gene (Fraser
et al., 1997). The T. pallidum genome contains a full
complement of genes encoding ribosomal proteins
arranged in large and small operons as well as isolated
genes.
Other cytoplasmic structures, such as mesosomes,
vacuoles, and nuclear regions have been reported based
on examination of thin sections (Hovind-Hougen, 1983).
These structures have not been studied in detail and are
subject to the caveat that they are difficult to distinguish
from preparation-induced artifacts.
Culture of T. pallidum

In Vivo Propagation
At present, none of the strains or subspecies of T. pallidum
can be grown continuously in vitro. Therefore, nearly all
studies of T. pallidum have utilized organisms propagated
in vivo by infection of laboratory animals. The Nichols strain
of T. pallidum subsp. pallidum, which was originally isolated
in 1912 from the cerebrospinal fluid of a neurosyphilis
patient (Nichols and Hough, 1913) and has retained its
virulence for humans (Magnuson et al., 1956; Fitzgerald
et al., 1976), is most commonly used strain of T. pallidum.
Shortly after the description of T. pallidum by Schaudinn
and Hoffman (Schaudinn and Hoffman, 1905), several
investigators found that venereal syphilis isolates could be
propagated easily in rabbits. The primary and latent stages
in the rabbit closely resemble the human disease, but
secondary and tertiary manifestations are not observed in
rabbits. Guinea pigs and hamsters are less susceptible to
T. pallidum subsp. pallidum infection and lesion
development. However, these animals have proven to be
useful models of infection with endemic syphilis
(subspecies endemicum) and yaws (subspecies pertenue)
isolates. Primates can be infected with T. pallidum subsp.
pallidum, as originally described by Metchnikoff and Roux
(Metchnikoff and Roux, 1906); however, the variability of
manifestations (Turner and Hollander, 1957) and high cost
of these animals render such studies impractical. Mice can
be infected T. pallidum (Magnuson et al., 1949; Klein et
al., 1980) but are not effective models of human disease
because of the minimal and short-lived pathology observed.
Animal models and procedures for experimental
infection with T. pallidum strains are described in detail
elsewhere. A definitive reference for animal models of
treponemal infections is Biology of the Treponematoses,
published by Turner and Hollander in 1957 (Turner and
Hollander, 1957), and is highly recommended to anyone
interested in the study of these organisms. The methods
involved in the detection and quantitation of T. pallidum by
darkfield microscopy and the inoculation of animals have
been described by Miller (Miller, 1971).
Based on the injection of successive dilutions of T.
pallidum subsp. pallidum (Nichols strain) suspensions,
Magnuson et al. (Magnuson and Eagle, 1948) concluded
that a single organism is sufficient to cause infection of
rabbits. The intradermal and intratesticular routes of

inoculation appeared to be equally sensitive indicators of

infectivity; our own studies are consistent with this
observation (S.J. Norris, unpublished). In the Sing Sing
Prison study published in 1956 (Magnuson et al., 1956),
intradermal inoculation of human volunteers with the
Nichols strain of T. pallidum also indicated a high degree
of susceptibility. Prisoners with varied histories of syphilis
exposure were inoculated with T. pallidum, observed for
lesion development, and subsequently treated with a
curative dose of penicillin. The median infectious dose of
57 organisms in volunteers with no previous history of
syphilis, whereas the median infectious dose in rabbits
infected with the same inoculum was 23 organisms
(Magnuson et al., 1956).
The in vivo multiplication rate of T. pallidum has been
estimated by sequential quantitation of T. pallidum in
infected rabbit tissue (Magnuson and Eagle, 1948) and by
correlating infectious dose with the time of lesion
development (Cumberland and Turner, 1949). By either
method, the division rate is extremely slow, with an
estimated doubling time of 30 to 33 hours (Magnuson and
Eagle, 1948; Cumberland and Turner, 1949). These results
correspond well with in vitro multiplication rates, as
described below.

In Vitro Survival and Multiplication

Attempts to culture T. pallidum in vitro were stimulated by
its discovery as the causative agent of syphilis in 1905.
Within the next few years, there were several reports of
successful cultivation, but subsequent attempts at
confirmation yielded negative results (reviewed by Kast
and Kolmer, 1929 and Turner and Hollander, 1957). In
some instances, nonpathogenic spirochetes were cultured
from syphilitic tissue and identified as avirulent strains of
T. pallidum, but these isolates were subsequently shown
to represent separate species of saprophytic organisms
(e.g. Treponema phagedenis Reiter and Treponema
denticola Noguchi).
In 1948, Nelson and coworkers (Nelson, 1948; Nelson
and Steinman, 1948; Nelson and Mayer, 1949) were the
first investigators to systematically apply quantitative
techniques to the study of factors affecting the retention of
motility and virulence by T. pallidum subsp. pallidum. They
found that bovine serum (or tissue extract), bovine serum
albumin, sodium pyruvate, carbon dioxide, three sulfhydryl
compounds (sodium thioglycollate, cysteine, and
glutathione), and incubation at 30C were beneficial to the
survival of T. pallidum in vitro. Incubation under these
conditions maintained treponemal motility and some degree
of virulence for 6 to 8 days. The survival provided by
Nelsons medium permitted the development of the T.
pallidum immobilization (TPI) test, one of the first tests for
detecting anti-T. pallidum antibody in patients sera (Nelson
and Diesendruck, 1951). Using a modification of Nelsons
medium, incubation under air, and a paraffin overlay
containing an oil-soluble antioxidant, Weber (Weber, 1960)
was able to maintain treponemal motility for up to 18 days.
Kimm et al. (Kimm et al., 1960; Kimm et al., 1962) reported
that inclusion of glucose and magnesium resulted in a slight
extension of survival, whereas Graves et al. (1975) stressed
the importance of a low redox potential.
Two important revelations occurred in the mid-1970s.

46 Norris et al.

Figure 3. Multiplication and motility of Treponema pallidum subsp. pallidum

(Nichols) in the Fieldsteel et al. (Fieldsteel et al., 1981) culture system as
modified by Cox et al. (Cox et al., 1990). In this representative experiment,
the number of T. pallidum increased ~100 fold over the 17 day period of
incubation, and motility (a measure of treponemal viability) was maintained
at a high level.

First, T. pallidum was found to utilize oxygen in its

metabolism (Cox and Barber, 1974; Baseman et al., 1976;
Cox, 1983) and to survive for longer periods in a
microaerobic environment as compared to anaerobiosis
(reviewed by Jenkin and Sandok, 1983). Subsequent
studies confirmed that T. pallidum is a microaerophile and
not an obligate anaerobe as originally thought. Second, it
was found (or rather rediscovered) that T. pallidum readily
adhered to the surface of mammalian cells in culture, and
that the presence of tissue culture cells prolonged the
survival of the bacterium (Steinhardt, 1913; Kast and
Kolmer, 1943; Fitzgerald, 1983). These findings are
discussed in greater detail below in the context of
physiology and host cell interactions; however, they provide
an important backdrop for the discussion of the
multiplication of T. pallidum in the Fieldsteel culture system.

In Vitro Multiplication of T. pallidum: the Fieldsteel et

al. Culture System
In 1981, Fieldsteel, Cox, and. Moeckli (Fieldsteel et al.,
1981) obtained consistent and significant multiplication of
T. pallidum in a tissue culture system. Previous studies in
Fieldsteels laboratory (Fieldsteel et al., 1979) had utilized
mammalian cell cultures in Leighton tubes incubated
vertically to provide an oxygen gradient. They found that
T. pallidum adhered to the mammalian cells and appeared
to multiply in a region of the gradient corresponding to low
to moderate oxygen levels. Adaptation of these conditions
to standard monolayer cultures resulted in the consistent
observation of treponemal multiplication (Fieldsteel et al.,
1981), as confirmed in other laboratories (Norris, 1982;
Levy, 1984).
A key feature of the Fieldsteel et al. culture system is
its reproducibility. Although in vitro culture of T. pallidum

Figure 4. Scanning electron micrographs of T. pallidum on the surface of

SF1Ep cottontail rabbit epithelial cells after 9 days (A) and 12 days (B) of in
vitro culture. These clusters may represent microcolonies resulting from
the division of a single organism. Bars = 1 m. Reprinted with permission
from (Fieldsteel et al., 1981).

had been reported previously, this was the first system in

which significant increases were obtained consistently.
Several hundred experiments with significant multiplication
have been carried out by the laboratories involved in this
research (Fieldsteel et al., 1981; Fieldsteel et al., 1982;
Norris, 1982; Cox et al., 1984; Levy, 1984; Konishi et al.,
1986; Norris and Edmondson, 1986; Norris and
Edmondson, 1987; Norris and Edmondson, 1988; Riley
and Cox, 1988; Cox et al., 1990). Following a lag period of
36 to 48 hours, T. pallidum multiplies with a doubling time
of 35 to 40 hours (Figure 3), consistent with its slow
replication rate in vivo (Magnuson and Eagle, 1948;
Cumberland and Turner, 1949). Viability, as measured by
both motility and virulence, remain high during the first 12

Biology of T. pallidum 47

Table 2. Key Components of the Fieldsteel et al. (Fieldsteel et al., 1981) Culture System
1.
2.
3.
4.

Tissue Culture
Medium
Atmosphere
Temperature

Sf1Ep cottontail rabbit epithelial cells

A modified form of Eagles MEM containing 20% heat-inactivated fetal bovine serum, 0.66 mM dithiothreitol, and testicular extract
Microaerobic (1% to 3% O2), 5% CO2, balance N2
33C to 35C

Cox et al. (Cox et al., 1990; Cox, 1994) Modifications

1.

Tissue Culture

2.

Medium

3.
4.
5.

Atmosphere
Temperature
Results of
Modifications

Sf1Ep cottontail rabbit epithelial cells or

RAB9 rabbit fibroblast cells
Static monolayer or microcarrier bead (107) cultures
T. pallidum Culture Medium (TpCM) - modified BRMM with reduced amounts of essential (50%) and nonessential (25%) amino
acids and vitamins (25%). Addition of oxygen radical scavengers superoxide dismutase (25 U/ml), catalase (10 U/ml), CoCl2
(21 nM), cocarboxlyase (4.3 nM), histidine (0.23 mM), mannitol (0.55 mM). Heat inactivation of testis extract.
3 to 4 percent O2, 5% C02, balance N2
34C to 35C
Two-fold increase in total growth; treponemes remain highly motile for 17 to19 days; cytopathic effects observed in mammalian
cell cultures with >100 T. pallidum per cell; limited success with serial passage (see Figure 6).

days of culture. The yield of T. pallidum per standard 25

cm2 culture is limited to 2 x 108 to 5 x 108 regardless of the
inoculum size (within the range of 1 x 106 to 1 x 108). This
ceiling is most likely due to the depletion of one or more
growth-limiting components by the treponemes, or to the
accumulation of toxic products. Multiplication typically
ceases after 10 to 12 days of in vitro culture. Continuous
culture has not as yet been achieved, although promising
subculture results have been obtained (see Serial Passage
of T. pallidum below).
In vitro multiplication of T. pallidum requires the
combination of several key elements, as originally
described by Fieldsteel et al. (Fieldsteel et al., 1981) and
modified by Cox et al. (Cox et al., 1990; Cox, 1994) (Table
2). Treponemal growth in this system only occurs in the
presence of mammalian cells; Sf1Ep cottontail rabbit
epithelial cells and RAB-9 rabbit fibroblasts are particularly
effective. T. pallidum extracted from infected rabbit tissue
is inoculated into glass or plastic tissue culture flasks
containing tissue culture cells and a modified tissue culture
medium. The treponemes adhere to the mammalian cells
and appear to multiply in microcolonies on the cell surface
(Figure 4).
Serum, glucose, and dithiothreitol (an antioxidant) are
the most critical medium components. Only certain lots of
fetal bovine serum or calf serum support T. pallidum survival
and growth; 20% is the optimal concentration (Fieldsteel
et al., 1981; Fieldsteel et al., 1982; Norris and Edmondson,
1987). Human serum will also support growth (Norris and
Edmondson, 1986). The required component(s) present
in serum are associated with the protein fraction (Norris
and Edmondson, 1986) and are thought to be lipids bound
to serum proteins, which are also required by other
spirochetes, including easily cultivable Treponema ,
Borrelia, and Leptospira (Smibert, 1973; Ellinghausen,
1976; Smibert, 1976; Van Horn and Smibert, 1983).
Another important component of the Fieldsteel et al.
culture system is a 1:30 dilution of testis extract (TEx). As
originally described (Fieldsteel et al., 1981), TEx was
prepared by centrifugation or filtration of the infected testis
extract used for culture inoculation. Without this component,
growth is 30% to 50% less than in cultures with TEx. The
extract can also be obtained from uninfected rat, hamster,
or rabbit testes (Fieldsteel et al., 1982). Heat inactivation

of TEx (56C for 30 min.) improves treponemal

multiplication by 50 to 75 percent (D. L. Cox, unpublished),
presumably by inactivating complement or other
components extracted from the tissue. A 1:60 dilution of
extracts of rabbit brain or flexor muscle can substitute for
TEx in the culture system (D. L. Cox, unpublished). Both
of these tissues contain high concentrations of three potent
antioxidants, carnosine, homocarnosine, and anserine
(Kohen et al., 1988). However, the exact nature or activities
of the stimulatory components present in tissue extracts
have not as yet been determined.
It is now well established that T. pallidum is a
microaerophilic organism. The oxygen concentration range
that is best for survival and multiplication is 1.5 to 5 percent
(Figure 5) (Cover et al., 1982; Fieldsteel et al., 1982; Cox
et al., 1990), although higher concentrations can be used
if additional antioxidants are present (Cox et al., 1990).
Temperature is also critical, with optimal growth occurring
at 33C to 35C (Fieldsteel et al., 1982).
Cox et al. (Cox et al., 1990) improved the average
culture yield by approximately two-fold by several
modifications of the incubation conditions, including the
addition of oxygen radical scavengers (Table 2).
Incremental increases in growth and survival resulting from
refinements of the culture system could lead to continuous
culture, just as multiplication in primary cultures was
achieved by the additive effects of several key factors.
Extended survival and growth of venereal syphilis
strains other than the Nichols strain have also been
obtained (Cox et al., 1984; Kohen et al., 1988), although
less multiplication was observed. Little or no multiplication
was obtained with yaws and endemic syphilis isolates
under the standard Fieldsteel et al. conditions (Cox et al.,
1984).
The restricted growth obtained in vitro as compared
to in infected tissue may reflect differences in the tissue
culture environment and the in vivo milieu (Norris and
Edmondson, 1987). Tissue conditions are relatively
homeostatic, whereas the in vitro environment changes
significantly and may eventually produce toxic conditions
such as low pH or high redox potential. Depletion of
essential nutrients that are supplied continuously in vivo
would result in starvation, limiting the number of
replications. However, simple medium replacement

48 Norris et al.

suspension cultures. 2) Beads carrying other cell types

can be added to the suspensions to more closely simulate
tissue conditions. 3) Culture conditions (i.e. pH, glucose,
other nutrients, etc.) can be more easily monitored and
adjusted in suspension flasks than in conventional tissue
culture flasks by using a chemostatic system. 4) They can
be readily adapted to large scale production of treponemes.
In the second modification, T. pallidum cultivation has
been adapted to 6- and 24-well microtiter plates (D. L. Cox,
unpublished). For 24 well plates, the optimal volume of
TpCM medium is 1.25 to 1.5 ml; for 6 well plates, 5 ml is
used. The optimal Sf1Ep cell concentration is 4 x 104 for
24 well plates and 2.5 x 105 for 6 well plates. Microtiter
plate cultures are ideal for experiments exploring many
variables (such as antimicrobial susceptibility testing).
Figure 5. Influence of atmospheric oxygen levels on the in vitro multiplication
of T. pallidum in BRMM medium using the Fieldsteel et al. culture system
(closed circles). Addition of the oxygen radical scavengers cobalt chloride,
cocarboxylase, mannitol, histidine, superoxide dismutase, and catalase in
the medium TpCM permitted treponemal growth at higher oxygen
concentrations (open circles). Reprinted with permission from Cox et al.,
(Cox et al., 1990).

prolongs multiplication and survival only slightly (S. J.

Norris, unpublished). The tissue cells may produce
nutrients or detoxifying compounds or activities ( e.g.
oxygen radical scavengers), thereby providing the required
microenvironment for growth. Irreversible changes (such
as DNA breakage (Steiner et al., 1984)) must gradually
accumulate so that the organisms eventually lose their
ability to replicate (i.e. their replicative potential), even under
permissive conditions as exemplified by reinoculation into
rabbits. Creation of a tissue-like environment (extremely
high cell density and homeostatic conditions) using capillary
perfusion cultures or tissue explant techniques (Steinhardt,
1913) may provide the conditions required for continuous
multiplication, but such cultures are technically difficult to
maintain (S.J.N., unpublished).
Alternate Methods for Cultivation
Two additional modifications of the Fieldsteel et al .
cultivation system were developed. First, Riley and Cox
(Riley and Cox, 1988) described a suspension culture
system using Sf1Ep cells grown on microcarrier beads.
Suspension cultures present several advantages over
conventional static monolayer cultures. 1) Nondisruptive
passage of infected cells can be accomplished easily with

Serial Passage of T. pallidum In Vitro

The primary goals of T. pallidum cultivation studies are 1)
continuous in vitro passage, 2) eventual replacement of
the rabbit with in vitro cultures as the source of organisms
for research and diagnostic reagents, and 3) elucidation
of the physiologic requirements of T. pallidum and their
relationship to the pathogenesis of syphilis. It is unlikely
that culture of T. pallidum will ever be a practical approach
to the routine laboratory diagnosis of syphilis.
T. pallidum suspensions for passage from one culture
to another can be prepared from culture supernatant, from
bacteria dissociated from the mammalian cell monolayers
by mechanical disruption or by treatment with trypsin or
EDTA (Norris and Edmondson, 1987). Alternatively,
microcarrier bead cultures with adherant treponemes can
be transferred intact to fresh cultures (Riley and Cox, 1988).
Initial attempts at subculture using the Fieldsteel et al.
system resulted in limited multiplication (Norris and
Edmondson, 1987; D.L. Cox, unpublished). Culture yields
decreased as the age of the primary culture increased; in
a study by Norris and Edmondson (Norris and Edmondson,
1987), increases of 18.1, 5.7, 3.0, and 0.75 fold were
obtained following subculture on days 3, 6, 9, and 12,
respectively. As a result, the maximal combined growth
obtained in primary and secondary cultures was only threefold greater than in primary cultures alone (Norris and
Edmondson, 1987). This outcome indicates that T. pallidum
a progressive loss of replicative potential under the
conditions of the standard Fieldsteel et al. system.
More promising results were obtained using the Cox
et al. modifications (Table 2). A series of 12 experiments

Table 3. Summary of Physiology and Growth Requirements

1.
2.
3.

4.
5.
6.

Oxygen utilization and toxicity. T. pallidum is microaerophilic. It requires oxygen for energy production, but is extremely sensitive to reactive oxygen species.
Energy production. T. pallidum possesses complete glycolysis and hexose monophosphate shunt pathways, but lacks Krebs (tricarboxylic acid) cycle and oxidative phosphorylation
pathways.
Nutritional requirements.
a. Carbon source - D-glucose, maltose, and mannose are metabolized by and support the growth of T. pallidum, but other sugars apparently can not be utilized.
b. Amino acids and vitamins. T. pallidum lacks pathways for biosynthesis of most amino acids and vitamins. It is able to utilize and incorporate exogenously supplied
amino acids.
c. Serum. Treponemal survival and growth is dependent upon components present in the protein fraction of serum. Serum apparently provides fatty acids and other activities.
Temperature. T. pallidum survival and growth is highly temperature dependent both in vivo and in vitro. The optimal temperature range is 33C to 35C.
Host cell interactions. T. pallidum readily adheres to mammalian cells. Host cells are required for multiplication of T. pallidum in the Fieldsteel et al. culture system, but the reason
for this dependence is not known.
Other activities.
a. Motility. The motility of T. pallidum promotes dissemination.
b. Toxicity. T. pallidum does not appear to produce any potent toxins, but may exert cytopathic effects on mammalian cells when present at extremely high concentrations.
c. Capsule. Although it has been postulated that T. pallidum produces a capsule, the mucoid material present in T. pallidum-infected tissue is predominantly host-derived. The
genome lacks recognizable capsule biosynthesis genes.

Biology of T. pallidum 49

were performed in which T. pallidum was passaged 2 or 3

times in vitro, using either monolayer cultures or suspension
cultures as the source of inoculum (D. L. Cox, unpublished).
Significant enhancement of multiplication was obtained in
four experiments, as depicted in Figure 6. The first two
subculture studies were performed with standard
monolayer cultures of Sf1Ep cells and resulted in
cumulative increases of 136- and 445-fold in the number
of T. pallidum, as compared to 26- and 61-fold increases
in primary cultures without subculture. The third experiment
used microcarrier suspension cultures of Sf1Ep cells.
Microcarrier beads with attached Sf1Ep cells and
treponemes were passed after one week of culture, when
a 21-fold increase had already occurred. During the second
passage, the treponemes multiplied 81-fold over an
additional 12 days, producing a cumulative increase of
nearly 1700-fold. The fourth successful attempt was in static
monolayer cultures of RAB-9 cells (a rabbit skin fibroblast
cell line, ATCC CRL 1414). Three successive passages
were performed; secondary cultures were established after
7 days of in vitro culture, and these were subcultured at 18
days. When the secondary cultures were transferred to

fresh cultures, the resulting passage grew only 5.2-fold,

but >95% of the treponemes were still motile after a total
of 28 days of in vitro culture. The overall increase obtained
was 2057-fold, equivalent to 11 successive cell divisions.
It must be emphasized that these results have not been
obtained consistently in every experiment. It is not clearly
understood why enhanced growth was obtained in some
experiments and not in others. Given the fastidious nature
of T. pallidum and our limited knowledge of its growth
requirements, the sporadic success of serial passage is
not unexpected. Further definition and optimization of T.
pallidum growth requirements should improve culture
stability and reproducibility and hence may lead to
continuous in vitro passage.
Physiology
A summary of our current knowledge of the physiology and
growth requirements of T. pallidum is given in Table 4. A
more extensive discussion of each of these topics is
provided below.

Figure 6. Serial passage of T. pallidum subsp. pallidum in tissue culture. Results represent 4 experiments in which significant enhancement of treponemal
growth was observed in secondary and tertiary cultures; little or no enhancement of growth relative to the primary cultures was obtained in 8 other experiments.
Cell cultures consisted of standard monolayers of Sf1Ep cells (Experiments 1 and 2), microcarrier bead suspension cultures of Sf1Ep cells (Experiment 3),
or monolayers of RAB-9 cells (Experiment 4). In these experiments, the maximum cumulative fold increase was 136, 445, 1,691, and 2,057, respectively (D.
L. Cox, unpublished data).

50 Norris et al.

The Microaerophilic Nature of T. pallidum

Although long thought to be an obligate anaerobe, it is
now clear that T. pallidum is a microaerophilic organism.
Its microaerophilic nature results from the balance between
dependence upon oxygen for energy production on one
hand and extreme susceptibility to the toxic effects of
oxygen radicals on the other. In 1974, Cox and Barber
(Cox and Barber, 1974) reported that suspensions of T.
pallidum extracted from rabbit tissue consumed oxygen at
a rate similar to that of aerobic bacteria. This finding was
initially viewed with skepticism, but several other lines of
evidence now show that the organism requires oxygen for
energy production. First, it utilizes oxygen as a terminal
electron acceptor (Lysko and Cox, 1977; Lysko and Cox,
1978; Barbieri and Cox, 1981) (see Energy Production
below). Second, anabolic activity, as measured by
incorporation of radioactive precursors in protein, RNA, and
DNA, is maximal in the presence of low to moderate
concentrations of oxygen (Baseman et al., 1976; Nichols
and Baseman, 1978; Sandok and Jenkin, 1978; Baseman
et al., 1979; Norris et al., 1980; Cover et al., 1982). Third,
the survival of T. pallidum is prolonged by the presence of
1.5 to 5 percent O2, both in cell-free systems (Norris et al.,
1978; Jenkin and Sandok, 1983) and in the presence of
tissue culture cells (Fitzgerald et al., 1977; Sandok et al.,
1978; Fieldsteel et al., 1979). Lastly, multiplication of T.
pallidum in the Fieldsteel et al. system is dependent upon
the presence of oxygen (Fieldsteel et al., 1982; Cox et al.,
1990). As shown in Figure 5, growth in BRMM medium is
maximal at 3% atmospheric 02 and drops off dramatically
as oxygen levels are either increased or decreased.
Despite this dependence, T. pallidum is extremely
susceptible to oxygen toxicity and dies within ~4 hours of
exposure to atmospheric levels of O2 (21%) if reducing
agents are absent from the medium (Norris et al., 1978;
Cover et al., 1982). This sensitivity is apparently due to
the organisms lack of superoxide dismutase, catalase, and
other oxygen radical scavengers, as confirmed by the
genome sequence (Fraser et al., 1998). Superoxide
dismutase, which catalyzes the conversion of superoxide
anions to hydrogen peroxide and water, has not been
detected in T. pallidum preparations (Austin et al., 1981;
Steiner et al., 1984). Austin et al. (Austin et al., 1981) found
that catalase activity was present in cell free extracts of T.
pallidum. However, Steiner et al. (Steiner et al., 1984)
showed that the catalase present was a contaminant from
the rabbit tissue. It had electrophoretic mobility identical to
that of rabbit catalase, and its activity was neutralized by
goat anti-rabbit catalase antiserum. Hence T. pallidum
apparently lacks two enzyme activities that are commonly
found in aerobic organisms, but absent in obligate
anaerobes. A deficiency in protective mechanisms against
toxic products of oxygen most likely accounts for oxygen
sensitivity.
Recently two independent groups (Jovanovic et al.,
2000; Lombard et al., 2000) reported the presence of a 26
kDa iron-containing protein in T. pallidum (TP0823) with
superoxide reductase activity. Like superoxide dismutase,
superoxide reductase catalyzes the conversion of
superoxide anion to peroxide, but requires the presence
of a reducing cofactor such as NAD(P)H. The treponemal
enzyme has very similar characteristics to neelaredoxin,

which has typically been found in hyperthermophilic and

sulfate reducing bacteria. Thus, its discovery in T. pallidum
may indicate that the syphilis spirochete copes with
oxidative stress by a primitive mechanism not found in other
pathogenic organisms. As described in greater detail in
the section on metabolism, the genome of T. pallidum
encodes homologs (TP0509 and TP0921, respectively) to
the Salmonella typhimurium alkyl hydroperoxide reductase
enzyme system proteins, AhpC and AhpF, which uses
NAD(P)H to reduce peroxide to water (Jovanovic et al.,
2000). This homolog may provide the mechanism for
peroxide detoxification, but this possibility has yet to be
confirmed.
The mechanisms of oxygen-dependent killing are not
known. However, DNA damage manifested as singlestranded breaks in the genomic DNA has been noted
following exposure of T. pallidum to hydrogen peroxide
(Steiner et al., 1984). This damage was repaired very slowly
and inefficiently in comparison to E. coli a repair proficient
organism (Steiner et al., 1984). A factor that could contribute
to the poor repair of DNA damage due to oxygen toxicity
may be the high proportion of cysteine (2.4%) in the
predicted amino acid sequence of the important DNA repair
enzyme DNA polymerase I (PolA) (Rodes et al., 2000).
This level of cysteine is the higher than that of in any PolA
sequenced to date and is 7.5 times higher than that found
in E. coli (0.32%). The abnormally high cysteine
composition could increase the susceptibility of treponemal
polA to inactivation by oxygen and its byproducts (Rodes
et al., 2000).
As with anaerobes, oxygen toxicity against T. pallidum
can be neutralized by addition of reducing agents such as
sulfhydryl compounds. Contrary to popular belief, reducing
agents do not remove molecular oxygen from the medium;
rather, they react with reactive oxygen intermediates (e.g.
hydrogen peroxide, superoxide anion, and hydroxyl
radical), preventing their reaction with cell components.
Dithiothreitol appears to be the most effective in prolonging
survival, although cysteine, glutathione, and sodium
metabisulfite have also been shown to have protective
activity (Nelson, 1948; Nelson and Steinman, 1948; Weber,
1960; Fitzgerald et al., 1977; Norris et al., 1978; Sandok
et al. , 1978; Chalmers and Taylor-Robinson, 1979;
Fieldsteel et al., 1979; Fitzgerald et al., 1980; Fitzgerald,
1983; Cox et al., 1990). Other oxygen radical scavengers,
including cobalt chloride, cocarboxylase, mannitol, and
histidine, also increase survival (Steiner et al., 1983) and
replication of T. pallidum in the Fieldsteel et al. system (Cox
et al., 1990). Addition of the enzymes superoxide dismutase
and catalase resulted in a 38% increase in yield in this
system, although the efficacy of catalase may be limited
by its rapid inactivation (Cox et al., 1990). Overall, Cox et
al. (Cox et al., 1990) were able to increase average growth
from 35-fold to 70-fold by using a medium that contains all
of the above antioxidants and enzymes. Peroxidases may
also have protective activity (Steiner et al., 1983).
Protection of T. pallidum from reactive oxygen
intermediates is critical in attempts to achieve continuous
in vitro multiplication. Mammalian cells may promote the
survival and growth of T. pallidum (both in vivo and in vitro)
by providing this activity (Steiner et al., 1983).

Biology of T. pallidum 51

Temperature Dependence
The survival and growth of the T. pallidum subspecies
exhibit marked temperature dependence, both in vivo and
in vitro. During human syphilitic infection, spirochetes can
be found in any area of the body, in some cases causing
fulminant infections in internal regions; the most obvious
example is congenital syphilis, where organisms can be
found in great numbers in the fetus and placenta. Therefore
T. pallidum subsp. pallidum is capable of survival and
growth at 37C. However, multiplication appears to be
favored in low temperature areas of the body, most notably
the skin. During secondary syphilis, the disseminated
infection seems to be most severe (as well as most obvious)
in the skin, although internal organs are also affected. Yaws
and pinta lesions tend to be located on the distal
extremities, which may also be related to temperature
effects (Hollander, 1981). It has been theorized that the
geographic distribution of the nonvenereal treponematoses
(yaws and pinta in tropical regions, endemic syphilis in arid
and temperate regions) is influenced by differential effects
of climate on the transmission, growth and pathogenesis
of the causative organisms (Hackett, 1963). A more
extreme view (not widely held) is that all human treponemal
infections are caused by the same organism, and variation
in manifestations is due to climate and mode of
transmission (Hollander, 1981).
The temperature effect is dramatic in experimental
infection of rabbits, as reviewed by Turner and Hollander
(Turner and Hollander, 1957). Because of the higher body
temperature, fulminant T. pallidum multiplication and lesion
formation occurs only in exposed areas of the skin and in
the testes (Hollander and Turner, 1954). Infection and lesion
development are optimal at cool room temperatures of 16
to 20C. There is also a lower temperature limit in vivo, as
demonstrated in a dramatic experiment by Hollander and
Turner (Hollander and Turner, 1954). Rabbits inoculated
intradermally on the ear did not develop lesions. If, however,
a unilateral cervical sympathectomy was performed
(causing vasodilation of the ipsolateral face and ear),
lesions developed in the warm, vasodilated ear (mean
temperature = 29C to 34C) but not the cold ear (22C to
27C). From these studies, the authors concluded that the
optimal tissue temperature range for T. pallidum infection
is between 30 and 35C.
Very similar results have been obtained in vitro, where
the optimal range is 33 to 35C. Survival is extended
progressively as temperatures are reduced from 40 to
20C (Weber, 1960), and motility and virulence can be
preserved for at least a week at 4C (Turner and Hollander,
1957; J. N. Miller, unpublished). However, the organisms
are also metabolically inactive at the lower temperatures.
Fieldsteel et al. (Fieldsteel et al., 1982) showed that optimal
multiplication of T. pallidum occurred at temperatures of
33 to 35C. Similarly, Baseman and Hayes (Baseman and
Hayes, 1974) found that incorporation of [3H]-labeled amino
acids into protein was maximal at 32 to 36C. T. pallidum
rapidly loses its motility and anabolic potential at
temperatures > 38C, indicating the presence of yet another
physiologic limit. Suspensions can be rendered nonviable
by incubation at 45C for 30 minutes.
The upper temperature limit may be due in part to the
lack of a heat shock response in T. pallidum (Norris, 1991;

Stamm et al., 1991). In most organisms, exposure to high

temperatures results in increased expression of proteins
of the heat shock regulon, including GroEL and DnaK
(Neidhardt and van Bogelin, 1987). These chaperonins are
involved in protein folding and apparently help to counteract
the detrimental effects of high temperatures. No increase
in the expression of these proteins has been observed in
T. pallidum incubated at elevated temperatures. This
unusual deficiency may represent an evolutionary loss of
adaptibility.
Metabolism
Much of the experimental evidence regarding the catabolic
activities and energy production of T. pallidum is based on
studies conducted during the 1970s in the laboratories of
C. D. Cox and J. B. Baseman (reviewed by (Cox, 1983)).
Glucose apparently serves as the principal carbon and
energy source of T. pallidum, although pyruvate can also
be metabolized. Of 22 carbohydrates tested, only glucose
and pyruvate were degraded to CO 2 (Nichols and
Baseman, 1975; Schiller and Cox, 1977). D-glucose, its
disaccharide maltose, and mannose are capable of
supporting multiplication of T. pallidum in the Fieldsteel et
al. system, whereas a number of other hexoses, pentoses,

Figure 7. Assessment of carbohydrate utilization by in vitro culture. T.

pallidum subsp. pallidum Nichols was incubated in the Sf1Ep cell culture
system in the presence of 2.5 mg/ml of the carbohydrates shown, and the
fold increase in numbers of T. pallidum per culture was assessed on days
7, 10, and 12 of culture. Substantial multiplication of T. pallidum occurred
only in the presence of D-glucose, maltose, and mannose. The data
represent the combined results of 6 experiments (S. J. Norris and N. J.
Farley, unpublished data).

52 Norris et al.

and pyruvate are ineffective in this regard (Figure 7; S. J.

Norris and N. J. Farley, unpublished). The spirochete is
incapable of metabolizing fatty acids via -oxidation
(Schiller and Cox, 1977), further restricting the substrates
available for energy production.
T. pallidum has an active glycolytic pathway, but genes
encoding the enzymes involved in the Krebs tricarboxylic
acid cycle are completely absent from the genome (Fraser
et al., 1998). Schiller and Cox (Schiller and Cox, 1977)
showed that cell-free extracts contain the enzymatic
activities associated with the Embden-Meyerhoff-Parnas
and the hexose monophosphate shunt pathways. Although
two of the eight enzyme activities of the Krebs cycle were
detected in T. pallidum extracts by Schiller et al. (Schiller
and Cox, 1977), genes encoding these well-conserved
enzymes could not be identified in the T. pallidum genome
sequence (Fraser et al., 1998). Therefore it is likely that
the activities detected resulted from contamination with
rabbit testicular tissue. The final products of glucose
catabolism are acetate, lactate, and CO2, with the relative
quantities of acetate and lactate produced being dependent
upon oxygen concentration (Nichols and Baseman, 1975;
Barbieri and Cox, 1979)
Oxygen Utilization and Toxicity
Lysko et al. (Lysko and Cox, 1978) reported the presence
of an active terminal electron transport system in T.
pallidum, with oxygen serving as the final electron acceptor.
Flavoproteins and cytochromes b, c, and o were detected
spectrophotometrically in these studies. Again, these
activities were apparently due to contaminating rabbit
constituents, inasmuch as genes encoding highly
conserved electron transport proteins could not be identified
in the T. pallidum genome. Enzymes responsible for
synthesis of quinones and other electron transport
compounds are also absent. If a terminal electron transport
system exists in T. pallidum, it must consist of gene products
with no apparent homology to known electron transport
constituents.
The only gene product in the T. pallidum genome that
clearly utilizes O2 as a substrate is NADH oxidase (Fraser
et al., 1998), which converts NADH and O2 to NAD+ and
water. NADH oxidase activity is apparently associated with
the inner membrane in crude lysates of T. pallidum (Norris
and Group, 1993). The homologous enzyme was shown
recently to have a protective effect against oxygen toxicity
in B. hyodysenteriae (Stanton et al., 1999).
Overall, T. pallidum appears to be most similar to lactic
acid bacteria in terms of its relationship with oxygen. Lactic
acid bacteria, which include Lactobacillus, Streptococcus,
and some Bacillus species, are facultatively or obligately
anaerobic bacteria. Like T. pallidum, they lack a functional
tricarboxylic cycle, cytochromes and other heme proteins,
and other evidence of an electron transport chain resulting
in oxidative phosphorylation. In lactic acid bacteria, ATP
production is primarily dependent upon the glycolytic
pathway and is therefore largely independent of the
presence of O2. NADH oxidase (Nox) appears to play two
roles in these organisms: 1) oxidation of excess NADH;
and 2) removal of molecular O 2 and the potential for
reactive oxygen intermediate (ROI) production. NADH is
produced by the glycolytic pathway, and an NADH/NAD+

imbalance would result if the excess NADH were not

dissipated. Nox fulfills this role in the absence of an electron
transport system. O2 by itself is relatively non-reactive and
non-toxic, and only becomes toxic when converted to
reactive oxygen intermediates (ROIs) such as H2O2,
superoxide anion, and hydroxyl radical. Because lactic acid
bacteria lack catalase and (in some cases) superoxide
dismutase, it is necessary to limit ROI exposure by other
means. One of the best-studied systems is that of
Amphibacillus xylanus, which utilizes Nox coupled with alkyl
hydroperoxide reductase (AhpC) (Niimura et al., 1993;
Niimura et al., 1995; Niimura and Massey, 1996). In this
case, Nox actually converts O2 to H2O2, which is then
scavenged through a coupled reaction with AhpC. A similar
system exists in S. typhimurium, and A. xylanus Nox and
S. typhimurium AhpC components can complement one
another to catalyze the 4-electron reduction of oxygen to
water (Niimura et al., 1995). In Streptococcus mutans, the
system is more complex, with the coexistence of two NADH
oxidase isoforms, Nox-1 and Nox-2, along with AhpC. Nox1 produces H2O2 and is coupled with AhpC to convert the
final product to water (Poole et al., 2000). Nox-2 catalyzes
the conversion of NADH and O2 into H2O without an H2O2
intermediate. In S. mutans, the combination of Nox-1 and
AhpC appears to play an important role in reducing oxygen
toxicity, whereas Nox-2 is primarily involved in maintaining
an appropriate NADH/NAD+ red-ox state (Higuchi et al.,
1999). The putative NADH oxidase of T. pallidum (TP0921)
appears to be Nox-2 like, in that it is highly homologous
to S. mutans Nox-2 (59% identity/75% similarity) and only
weakly similar to Nox-1 (24%/41%). The T. pallidum
genome also encodes a highly conserved AhpC ortholog
(TP0509), but potential Nox-1 candidates are more similar
to thioredoxin reductase. Further functional studies will be
required to define the roles of the Nox and AhpC orthologs
of T. pallidum in oxygen scavenging or utilization pathways.
Anabolic Activity
T. pallidum is fully capable of DNA, RNA, and protein
synthesis as measured by incorporation of radiolabeled
precursors (Baseman and Hayes, 1974; Baseman et al.,
1976; Baseman and Hayes, 1977; Nichols and Baseman,
1978; Sandok et al., 1978; Baseman et al., 1979; Norris et
al., 1980). Amino acids, adenine, and uracil are readily
incorporated; nucleosides are not incorporated as
efficiently, probably because of reduced uptake.
(Barbieri et al., 1981) further demonstrated that carbon
from glucose was incorporated into lipids, nucleic acids,
and proteins. In lipids, this incorporation was restricted to
phospholipids and glycolipids and was not found in free
fatty acids and cardiolipin. These results indicate that fatty
acids are not synthesized de novo, consistent with the
observation that fatty acids are acquired from exogenous
sources and incorporated into lipids in an unmodified form
(Nichols and Baseman, 1975; Schiller and Cox, 1977). The
distribution of labeled carbon atoms was limited to the
ribose and deoxyribose portions in nucleic acids and was
primarily associated with the amino acid aspartate in
proteins. These findings are consistent with the absence
of recognizable pathways for the synthesis of nucleic acid
bases and most amino acids, based on the genome
sequence (Fraser et al., 1998). However, some studies

Biology of T. pallidum 53

show that T. pallidum is capable of at least limited

multiplication in medium lacking amino acids and vitamins
(see Nutritional Requirements below). In addition,
Gherardini et al. (Gherardini et al., 1990) showed that T.
pallidum DNA contained genes capable of complementing
E. coli proC mutants defective in proline biosynthesis,
indicating that T. pallidum is capable of synthesizing at least
some amino acids. The gene complementation approach
should be extremely valuable as a surrogate genetic
system for further defining the metabolic capabilities of T.
pallidum based on the genome sequence.
Nutritional Requirements
Assessment of the nutritional requirements of T. pallidum
has been complicated by the inability to obtain multiplication
in pure culture. In addition, most T. pallidum survival media
are extremely complex and include undefined components
(such as serum or tissue extract). Although the medium
developed by Nelson (Nelson, 1948) is relatively simple,
survival is poor unless the infected tissue is extracted for
prolonged periods (~1 hour) permitting the diffusion of
tissue components into the medium. The availability of the
Fieldsteel et al. system (Fieldsteel et al., 1981) permits
the analysis of factors affecting multiplication, although the
presence of tissue culture cells complicates interpretation.
As indicated above, there appears to be an absolute
requirement for D-glucose as a carbon and energy source
(Nichols and Baseman, 1975; Schiller and Cox, 1977). As
shown in Figure 7, D-glucose, the glucose disaccharide
maltose, and mannose were the only sugar compounds
that were capable of supporting the in vitro multiplication
of T. pallidum (S. J. Norris and N. J. Farley, unpublished
data). To further delineate the growth requirements, the
amino acid and vitamin components were omitted from
the culture medium (BRMM) with the expectation that
treponemal multiplication would not be supported. Instead,
the spirochetes in this deficient medium (BRMM-D)
multiplied as well as, and in some cases better than,
cultures in the complete medium. Treponemal survival and
multiplication were observed in BRMM-D even if dialyzed
serum was used in place of whole fetal bovine serum.
Therefore T. pallidum was capable of surviving and
multiplying in a medium containing glucose, salts,
dithiothreitol and dialyzed serum. It is of course possible
(or likely) that the tissue culture cells are secreting nutrients
required by the treponemes. Surprisingly, the Sf1Ep rabbit
epithelial cells used in the cultures also survived well under
these conditions, probably a reflection of the slow growth
rate and low metabolic activity of these cells.
It has long been recognized that serum is an essential
medium component for T. pallidum survival. Removal of
serum (and tissue extract, which also contains serum
components) results in loss of motility within minutes. Rice
and Nelson (Rice and Nelson, 1951) identified a crystalline
compound derived from serum ultrafiltrate that replaced
the serum requirement; the compound was identified as a
non-reducing hydrocarbon with a formula of C15H26O10,
but was not characterized further. Only certain commercial
lots of fetal bovine serum are effective in supporting the
growth of T. pallidum. Many lots appear to contain toxic
components, which may relate to the method of
preparation. Serum lots may also vary in the amounts of

beneficial components. D. L. Cox and coworkers

(unpublished data) examined 70 lots of serum from
HyClone Laboratories, which routinely analyzes each lot
of serum for more than 200 biochemical components. By
analyzing the amount of treponemal multiplication relative
to serum composition, the concentrations of seven
compounds were found to correlate with improved
treponemal growth: prostaglandin F1, erythrose,
arachidonic acid, cytosine, malic acid, methionine, and
estrone. Compounds that correlated with decreased
treponemal multiplication were hemoglobin, 17--estradiol,
methionine sulfoxide, and free fatty acids (D. L. Cox,
unpublished data). Further studies in which these
compounds are added in varied amounts are needed to
verify that they have stimulatory or inhibitory activities.
Norris and Edmondson (1986) found that serum
component(s) required for in vitro multiplication of T.
pallidum were not soluble small molecular weight
compounds, but were instead associated with the protein
fraction. Dialyzed serum or a serum protein fraction
precipitated with polyethylene glycol supported the growth
of T. pallidum in the Fieldsteel et al. culture system, whereas
serum ultrafiltrate (containing small molecular weight
compounds) did not support either growth or retention of
motility. It is likely that the required component(s) include
lipids carried by albumin and other serum proteins. As
indicated previously, T. pallidum apparently is incapable of
synthesizing long chain fatty acids, and incorporates them
in lipids and lipoproteins in an unmodified form (Nichols
and Baseman, 1975; Schiller and Cox, 1977). All easily
cultivable parasitic spirochetes, including Leptospira,
Borrelia, and nonpathogenic Treponema species, require
fatty acids complexed with albumin or other serum proteins
(Smibert, 1973; Ellinghausen, 1976; Smibert, 1976; Van
Horn and Smibert, 1983). Studies with T. denticola and T.
vincentii by Van Horn and Smibert (1983) indicated that
proteins present in the -globulin fraction of serum may
serve as the principal source of lipids. Fatty acids may be
released by lipases produced by the treponemes and then
bound by albumin, which serves a detoxifying function.
Alderete and Baseman (Alderete and Baseman, 1989)
showed that human plasma lipoproteins are bound by T.
pallidum suspensions, and that this binding is enhanced
by the presence of dextran sulfate. It is possible that T.
pallidum may acquire lipids by direct binding to high-density
or low-density lipoproteins.
As described above, T. pallidum has many metabolic
similarities to lactic acid bacteria. Although iron is required
by most organisms, lactic acid bacteria are unusual in that
they lack iron acquisition mechanisms, have few if any
proteins that require iron as a cofactor, and possesses Mnrequiring enzymes that fulfill functions usually provided by
heme-containing proteins (Archibald, 1986). Posey and
Gherardini (2000) demonstrated recently that B. burgdorferi
grew normally in the presence of iron chelators, and that
the genome of B. burgdorferi has few detectable sequences
predicted to encode iron metalloproteins. In addition, B.
burgdorferi does not accumulate detectable levels of iron,
but accumulates manganese, calcium, and magnesium and
requires these divalent cations for growth (Posey and
Gherardini, 2000). T. pallidum also lacks genes encoding
iron metalloproteins such as cytochromes, superoxide

54 Norris et al.

Figure 8. In vitro system for assessing the susceptibility of T. pallidum subsp.

pallidum (Norris and Edmondson, 1988), as demonstrated with the
cephalosporin ceftriaxone. The Nichols strain of T. pallidum was incubated
in the Fieldsteel et al. culture system in presence of varied concentrations
of ceftriaxone. After 7 days of incubation, the number of T. pallidum per
culture was determined and compared with the original inoculum. Each
curve represents a separate experiment, and each data point is the mean
of three separate cultures. The minimal inhibitory concentration (MIC) is
defined as the lowest concentration that completely inhibits multiplication
(culture yield < inoculum); in this instance, the average MIC for ceftriaxone
was 0.0007 micrograms/ml. This system provides a means of determining
both the bacteriostatic and bactericidal activities of compounds against T.
pallidum.

dismutase, catalase, and succinate dehydrogenase. In

addition, it possesses an ABC transport operon that is tightly
regulated by Mn concentrations (Posey et al., 1999). It is
possible that T. pallidum, like lactic acid bacteria and B.
burgdorferi, has developed an iron-independent lifestyle.
However, Alderete et al. (Alderete et al., 1988) found that
lactoferrin and transferrin bound to the surface of T.
pallidum, and the spirochete was particularly efficient at
acquiring radiolabeled Fe++ from transferrin. This binding
activity could potentially be involved in the acquisition of
iron during in vivo infection.
Interactions with Mammalian Cells
The association of T. pallidum with mammalian cells is
important in terms of understanding both the physiology of
the bacterium and the complex host-parasite interactions
that occur during infection. The key aspects of this
interaction have been reviewed previously (Fitzgerald,
1983; Baseman et al., 1986) and are considered below.
Adherence
T. pallidum readily and tenaciously attaches to the surface
of mammalian cells in culture (Fitzgerald et al., 1975;
Fitzgerald et al., 1977; Fitzgerald et al., 1977; Hayes et
al., 1977; Fitzgerald et al., 1982; Oakes et al., 1982; Quist
et al., 1983; Wong et al., 1983; Baseman et al., 1986;
Thomas et al., 1989). Literally hundreds of treponemes
can associate with each tissue culture cell within minutes.
Although adherence has been said to occur through a tiplike organelle (Hayes et al., 1977; Baseman et al., 1986),
binding of T. pallidum to tissue culture cells can occur at
any location on the spirochete (Konishi et al., 1986). The
predominant occurrence of treponemes attached by their
end is due to the motility of the organism, which tends to

draw the attachment point to the end opposite of the

direction of translational motion; when the treponeme
reverses its direction of rotation, the attachment point shifts
along the length of the organism to the other end. The
same phenomenon is observed with spirochetes attached
to antibody-coated polystyrene beads (Charon et al., 1984).
Therefore it is likely that the receptors for attachment are
evenly distributed over the surface of the bacterium, rather
than localized on the ends. In transmission electron
microscopy studies, Konishi et al. (Konishi et al., 1986)
noted the presence of an electron dense layer at the
suspected site of attachment which may represent a fusion
point between the treponemal OM and the host cells
plasma membrane.
Adherence seems to be a common property among
pathogenic spirochetes, including B. burgdorferi .
Attachment requires structural integrity and viability of both
the spirochete and the mammalian cell. Although some
variation in the extent of surface attachment has been
observed, T. pallidum will adhere to virtually any nucleated
cell type, including fibroblasts, epithelial cells, endothelial
cells, muscle fibers, and neurons; it does not adhere to
erythrocytes.
It has been postulated that fibronectin plays a major
role in this adherence (Hardy and Levin, 1983; Fitzgerald
et al., 1984; Fitzgerald and Repesh, 1985; Steiner and Sell,
1985; Thomas et al., 1985; Thomas et al., 1985; Thomas
et al., 1985; Baseman et al., 1986; Baughn, 1986; Baughn,
1987). Fibronectin is a host extracellular matrix protein
involved in cell-cell adherence in tissue. This dimeric
glycoprotein has several functional domains, including
those that bind to heparin, gelatin, and cell surface
receptors. In a model developed by Baseman and
coworkers (Baseman et al., 1986), the cell-binding domains
of fibronectin binds to both the treponeme and mammalian
cell surface, essentially crosslinking the two cells. Several
lines of evidence support this hypothesis. 1) T. pallidum
adheres to fibronectin-coated surfaces (Hardy and Levin,
1983; Fitzgerald et al., 1984). 2) Anti-fibronectin antibody
inhibits attachment; in particular, monoclonal antibodies
against the cell-binding domain decrease adherence
(Thomas et al., 1985). 3) heptapeptides containing the
specific cell-binding domain sequence Arg-Gly-Asp-Ser
blocked adherence of T. pallidum to fibronectin, whereas
peptides in which one of these amino acids is changed
are not inhibitory (Thomas et al., 1985).
The fact that attachment can be blocked to some extent
by addition of anti-T. pallidum antiserum (Fitzgerald et al.,
1984; Thomas et al., 1985) indicates that specific receptors
on the surface of T. pallidum may be involved in cell binding.
There is disagreement in the literature regarding the
number and affinity of fibronectin receptors on T. pallidum
(Steiner and Sell, 1985; Thomas et al., 1985; Baughn, 1986;
Baughn, 1987; Cox et al., 1992), but most estimates
indicate the presence of ~3,000 to 5,000 receptors per cell
and an affinity (Ka) of ~2 x 107 M-1 (Baughn, 1987).
Using a surface-specific radioimmunoassay, Cox et
al. (Cox et al., 1992) provided evidence that the amount of
fibronectin bound to the OM of intact T. pallidum is much
less than that indicated by previous studies. Significant
binding of either 125I-labeled fibronectin or antibodies
directed against fibronectin was not detected with this

Biology of T. pallidum 55

assay, although fibronectin readily associated with

organisms treated with detergent to remove the OM. Thus
estimates of fibronectin binding is highly dependent on the
condition of the treponemes. The picture is complicated
further by the variable presence of host ground substance
(Ziegler et al., 1976; Fitzgerald et al., 1979; Strugnell et
al., 1984; Fitzgerald et al., 1985; van der Sluis et al., 1985;
Strugnell et al., 1986; van der Sluis et al., 1987) and other
host proteins (Lugtenberg and Van Alphen, 1983) on the
surface of freshly extracted T. pallidum, which could also
increase or decrease fibronectin binding.
Baseman and Hayes (Nelson and Mayer, 1949)
identified three T. pallidum proteins as putative cell
adhesion receptors (adhesins). Detergent solubilized,
radiolabeled T. pallidum proteins were incubated with
formalin-fixed human epithelial cells; the cell monolayer
was washed and then assessed for the presence of bound
treponemal proteins. Three polypeptides, called P1 (Mr =
89,500), P2 (37,000), and P3 (32,000) were found to be
associated with the cells. These same proteins are capable
of binding fibronectin (Peterson et al., 1983), and are
thought to share a common domain involved in fibronectin
binding and cell adhesion (Thomas et al. , 1985).
Unfortunately, there is little evidence that these proteins
are surface-associated; in fact, P1 appears to be same
protein as CfpA, the 83 kDa subunit of the cytoplasmic
filaments, an internal structure (Norris and Group, 1993;
You et al., 1996). The genes encoding P1 and P2 were
cloned into E. coli (Peterson et al., 1987), but further
characterization of these proteins has not been reported.
Host Cell Association and T. pallidum Survival and
Multiplication
As mentioned previously, the presence of mammalian cells
promotes the survival and multiplication of T. pallidum in
vitro (Steinhardt, 1913; Kast and Kolmer, 1943; Perry, 1948;
Fitzgerald et al., 1975; Fieldsteel et al., 1977; Fitzgerald et
al., 1977; Sandok et al., 1978; Fieldsteel et al., 1981;
Fitzgerald, 1983). Sf1Ep, a slow-growing cottontail rabbit
epithelial cell culture, is particularly effective in this regard
(Fieldsteel et al., 1977). Mammalian cells may produce a
tissue-like microenvironment by providing nutrients,
protective activities, or an anchoring site. Close association
is required; separation of the treponemes from the
mammalian cells by a permeable filter prevents their
growth. In addition, treponemal survival and multiplication
appears to depend on host cell protein synthesis, because
2.5 g/ml cycloheximide (which inhibits eukaryotic but not
prokaryotic protein synthesis) decreases T. pallidum
multiplication (D. L. Cox, unpublished). Further analysis is
required to examine the nature of host cell dependence
and to determine whether medium factor(s) can be
identified which can supplant this requirement and hence
permit growth of T. pallidum in a host cell-free environment.
Tissue Invasion and Dissemination
T. pallidums motility coupled with its ability to bind to host
cells provides a powerful mechanism for local and systemic
dissemination. Most of the dissemination of treponemes
from the initial nidus of infection is thought to be
hematogenous. Infectious T. pallidum adheres to isolated
capillaries (Quist et al., 1983) and endothelial cells (Thomas

et al., 1989). T. pallidum also readily penetrates through

the intercellular junctions of endothelial cell monolayers,
as shown in a model system described by Thomas et al.
(Thomas et al., 1988). Human or rabbit aortic endothelial
cells cultured on polycarbonate filters form continuous tight
junctions, as demonstrated by transendothelial electrical
resistance measurements (Thomas et al., 1988). Viable T.
pallidum rapidly penetrate through the intercellular
junctions, so that treponemes placed on the top side of
the monolayer can be found on the other side within 2
hours. Heat-killed T. pallidum or the nonpathogenic
treponeme T. phagedenis do not cross the endothelial
barrier in significant numbers. Riviere et al. (Riviere et al.,
1989) took this model one step further by measuring the
migration of T. pallidum through the mouse abdominal wall.
Segments of the abdominal wall (consisting of the serosa,
connective tissue and muscle layers) were placed in a
culture chamber, forming a watertight barrier between the
two sides. After 24 hours, up to 6 x 107 of the 5 x108
organisms placed in the chamber had migrated across the
abdominal wall, whereas few, if any, T. phagedenis or heatkilled T. pallidum had crossed the barrier. Migration
occurred only from the epithelial side to the connective
tissue side, not vice versa. The highly invasive nature of
this pathogen is impressively demonstrated by this system.
This invasive activity may also be important in the
establishment of latent infection. Latency represents a
steady state between active infection and eradication of
the pathogen by the immune system. Because of its
invasiveness, T. pallidum may penetrate to areas that are
inaccessible to the humoral and cellular arms of the immune
response and hence avoid elimination (Medici, 1972). Sell
et al. (Sell et al., 1985) examined the mechanisms of
immunity to syphilis by reinfecting immune rabbits
intradermally with large numbers of viable T. pallidum. Most
of the organisms were eliminated with little inflammatory
response. Several days following infection, T. pallidum
could still be found in certain protective niches (such as
nerves, arrector pili muscles, hair follicles, and dense
collagen bundles), thereby evading the immune response.
During natural infection, small numbers of T. pallidum could
persist in similar protective niches, leading to long-term
latent infection with the possible development of late
manifestations.
Apparently viable treponemes can be found within host
cells, either during active infection in vivo or in tissue
cultures (Sykes and Miller, 1971; Lauderdale and Goldman,
1972; Sykes et al., 1974). Intracellular localization could
potentially be involved in latent infection.
Cytotoxicity
One possible mechanism in the pathogenesis of syphilis
is the creation of toxic products by T. pallidum. Fitzgerald
and coworkers (Fitzgerald et al., 1982; Oakes et al., 1982;
Quist et al., 1983) have described cell disruption and other
cytopathic effects in cultures of fibroblasts, epithelial cells,
isolated capillaries, muscle cells, and neurons following
incubation with extremely high concentrations of T. pallidum
(>9 x 107/ml). Lower concentrations of T. pallidum or cellfree supernatants have little, if any, toxic effect on
mammalian cells.
Cytopathic effects have also been observed during in

56 Norris et al.

vitro culture of T. pallidum (D. L. Cox, unpublished).

Enhanced treponemal multiplication and viability was
obtained with the Cox et al. modification (Cox et al., 1990),
resulting in increased numbers of T. pallidum per host cell.
Under these conditions, Sf1Ep and RAB-9 cells exhibited
cytopathic effects after 10 to 12 days of culture, and the
severity increased during the next 3 to 5 days. Titration
studies determined that the effects occurred within 48 hours
after there were >100 treponemes per cell; uninfected
cultures did not show these effects. The cellular swelling,
detachment, and disintegration observed were similar to
the effects previously reported by Fitzgerald and coworkers
(Fitzgerald et al., 1982; Oakes et al., 1982; Quist et al.,
1983). The pH and glucose concentration of the medium
were not abnormally low (pH 6.7-7.0; glucose 0.7 to 1.0
mg/ml). The mitochondria of heavily infected cells exhibited
greater fluorescence in the presence of the fluorescent dye
rhodamine 123 than did lightly infected or non-infected
cells, indicating a higher membrane potential and metabolic
activity (D. L. Cox, unpublished). This phenomenon
commonly occurs in cells that have been damaged and
may correspond to the activation of cellular repair
mechanisms (Goldstein and Korczack, 1981; Johnson et
al., 1981; DeBiosio et al., 1987).
It remains to be determined whether this type of
cytotoxicity is active in vivo. Also, it is unclear if the toxicity
observed is specific in nature (i.e. involving certain toxins
or virulence factors) or is nonspecific. High concentrations
of any bacterium may bring about changes in culture
conditions leading to mammalian cell death; such
nonspecific cytotoxic effects are commonly seen in
bacterially contaminated tissue cultures. Most tissue
necrosis that occurs during syphilitic infection seems to be
due to vessel occlusion or rupture in heavily infected tissue
(such as chancres), or to tissue displacement caused by
space-filling inflammatory reactions (as in gummas).
Weinstock and coworkers (Weinstock et al., 1998;
Norris and Weinstock, 2000; Weinstock et al., 2000;
Weinstock et al., 2000) identified five T. pallidum genes
encoding potential cytotoxins, based on amino acid
similarities. These have been termed tlyC (TP0649), hlyIII
(TP1037), hlyA (TP0026), hlyB (TP0027), and hlyC
(TP0936). The identification of the homologous proteins in
other organisms as cytolysins is tenuous, in that in most
cases the purified protein has not been demonstrated to
have hemolytic or cytolytic activity. Therefore it is necessary
to express and characterize the activities of the T. pallidum
proteins. As is often the case with heterologous bacterial
proteins, expression these T. pallidum proteins in E. coli
has been problematic, requiring the examination of several
different vectors (Weinstock et al., 2000). The reactivity of
the recombinant proteins with sera from syphilis patients
was varied, with TlyC exhibiting the most consistent
reactivity (Weinstock et al., 2000). The cytolytic activity of
these products is under investigation.
Mucoid Material
It has long been noted that a mucoid material accumulates
in T. pallidum lesions, particularly during experimental
infection of rabbits (Turner and Hollander, 1957;
Christiansen, 1963; Ziegler et al., 1976; Fitzgerald and
Johnson, 1979a; Fitzgerald et al., 1979; Strugnell et al.,

1984; Fitzgerald et al., 1985; van der Sluis et al., 1985;

Strugnell et al., 1986; van der Sluis et al., 1987). Coupled
with the knowledge that freshly extracted treponemes are
resistant to antibody binding and killing, it has been
postulated that this material is a capsule or slime layer
produced by the bacterium, and that this layer protects T.
pallidum from the immune response. Studies by Strugnell
and coworkers (Strugnell et al., 1984; Strugnell et al., 1986)
demonstrated that most of this material is actually
proteoglycans synthesized by the host. Using slices of
infected testicular tissue incubated in vitro, they found that
incorporation of [ 35S]-sulfate into the mucoid material was
blocked by the eukaryotic inhibitor cycloheximide but not
by prokaryotic inhibitor erythromycin (Strugnell et al., 1986).
[ 3 H]-labeled N-acetyl glucosamine is incorporated into high
molecular-weight material by T. pallidum, but this material
has not been characterized (Strugnell et al., 1984). The
genome of T. pallidum lacks the complex operons
associated with capsular synthesis found in other
organisms (Fraser et al., 1998). The host tissue apparently
produces excessive amounts of extracellular matrix
components in response to T. pallidum infection, particularly
when the infected animals are treated with corticosteroids
(Turner and Hollander, 1954).
Fitzgerald and coworkers (Fitzgerald and Johnson,
1979b; Fitzgerald and Gannon, 1983; Fitzgerald and
Repesh, 1987) have found that hyaluronidase activity is
associated with T. pallidum extracted from rabbit tissue,
although it is unclear whether the activity is of bacterial or
host origin. It was hypothesized that hyaluronidase
promotes the dissemination of T. pallidum during the course
of infection (Fitzgerald and Repesh, 1987). Gene(s)
responsible for this activity have not been identified.
Antimicrobial Susceptibility
One of the most clinically important properties of any
infectious agent is its susceptibility to antimicrobial agents.
In vivo therapy is still the gold standard in assessing the
efficacy of antimicrobials against T. pallidum (Lukehart et
al., 1984; Lukehart and Baker-Zander, 1987; Lukehart et
al., 1990), but in vitro assays provide a means of screening
compounds and defining effective concentrations. In the
past, loss of motility or virulence of T. pallidum or inhibition
of growth of nonpathogenic Treponema species have been
used to evaluate in vitro susceptibility (Rein, 1976).
Two additional methods have been developed. The
first utilizes the incorporation of [ 35S]-methionine to assess
the inhibitory effects of agents on macromolecular synthesis
by T. pallidum (Stapleton et al., 1985; Stamm et al., 1988).
This approach is particularly useful for assaying the activity
of protein synthesis inhibitors and was used to demonstrate
the resistance of a venereal syphilis isolate to erythromycin
(Stamm et al., 1988). This isolate, Street Strain 14, has
since been shown to contain a single point mutation in
both copies of 23S rRNA, which may account for this
strains resistance to macrolides (Stamm and Bergen,
2000).
The other procedure (Norris and Edmondson, 1988)
involves the addition of varied concentrations of an
antimicrobial compound to T. pallidum cultured by the
Fieldsteel et al. procedure (Fieldsteel et al., 1981). After 7

Biology of T. pallidum 57

days of incubation, the number of T. pallidum per culture is

determined. This technique is a very sensitive measure of
growth inhibition, and can reproducibly measure activity
within a two-fold dilution (Figure 8). The minimal inhibitory
concentration (MIC) is calculated as the concentration that
completely inhibits T. pallidum multiplication (culture yield
at 7 days < inoculum). The minimal bactericidal
concentration (MBC) is estimated by inoculation of the T.
pallidum cultures into rabbits after the 7-day incubation
period; the lowest concentration that destroys infectivity is
considered to be the MBC. This system has been used to
assess the in vitro activity of cephalosporins, quinolones,
and other compounds (Table 4) (Norris and Edmondson,
1988; S.J. Norris and N.J. Farley, unpublished data). The
cephalosporins have low MICs in this assay, in agreement
with the high efficacy of -lactam antibiotics against T.
pallidum. In contrast, the quinolone compounds tested
generally have a low activity against T. pallidum, as is also
indicated by in vivo studies in rabbits (S. J. Norris,
unpublished). This insensitivity is consistent with the
predicted similarity of T. pallidum GyrA and GyrB to those
of relatively quinolone-resistant organisms (Huang, 1996;
Stamm et al., 1997; Fraser et al., 1998).
Summary

T. pallidum is an unusual organism in a number of respects.

In addition to its spirochetal morphology, its outer
membrane contains no lipopolysaccharide and relatively
few intramembranous proteins; as such, it may evade the
immune response by presenting an antigenically inert
surface. It is also one of the few important human
pathogens that has not been cultured continuously in vitro.
This hindrance is most likely related to a combination of
factors including extreme nutritional requirements due to
deficiencies in biosynthetic capabilities, the narrow
equilibrium between oxygen dependence and toxicity, and

Table 4. Minimal inhibitory concentrations (MICs) and minimal bactericidal

concentrations (MBCs) of cephalosporins and quinolones against T. pallidum
subsp. pallidum (Nichols) in a tissue culture system (Norris and Edmondson,
1988); S. J. Norris and N. J. Farley, unpublished).
Agent
Cephalosporins
Ceftriaxone
Cefetamet
Cefteram
Ceftazidime
Quinolones
Clinafloxacin
Ciprofloxacin
Difloxacin
Norfloxacin
Pefloxacin
Temafloxacin
Other agents
Penicillin G
Tetracycline
Erythromycin
Spectinomycina
a

MIC (g/ml)

MBC (g/ml)

0.0007
0.04
0.004
0.007

0.002
ND
ND
ND

1
5
10
6
8
8

2
4
ND
ND
ND
ND

0.0005
0.2
0.005
0.5

0.0025
0.5
0.005
0.5

Lack of efficacy in vivo due to poor tissue penetration (Rice et al., 1988).

an undetermined dependence on mammalian cells that

may involve one or both of these factors. In keeping with
its invasive nature, the genome of T. pallidum yielded a
relatively short list of potential virulence factors. However,
further characterization of the tpr gene family, putative
hemolysins, and other virulence factor candidates may
reveal the importance of these proteins in T. pallidum
infection. The genome sequence provides raw data that
will greatly enhance the examination of each of these areas.
For example, gene products with signal peptidase I leader
sequences would be expected to include outer membrane
proteins, the apparent relationship between T. pallidum and
lactic acid bacteria identifies Nox/AhpF, AhpC, and
neelredoxin as being potential key factors in oxygen
metabolism, and mining of the genome may yield as yet
unidentified adhesins and other factors important in
dissemination. The real benefit of the genome sequence
lies not in sequence gazing, but in facilitating the application
of functional genomic approaches to understanding the
biology of T. pallidum.
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