COMMENT
displace the existing chaperone,
replacing the chaperone–adhesin
interaction with a subunit–adhesin
interaction.
Much information about secre-
tion in Gram-negative bacteria has
emerged over the past few years,
but an enormous amount of infor-
mation is yet to be uncovered. We
are excited by the potential of the
chaperone/usher system for fur-
thering our understanding of se-
cretion and look forward to com-
paring and contrasting this system
with the other assembly/secretion
systems. We believe that continued
exploration will reveal general
Treponema pallidum: doing a
remarkable job with what it’s got
Justin D. Radolf, Bret Steiner and Dmitriy Shevchenko
D
iscovered almost a century
ago, Treponema pallidum
subsp. pallidum, the syphilis-
causing spirochete, continues to be
an enigma. Well into the molecular
era, there is still much debate con-
cerning its composition and ultra-
structure and the nature of the im-
mune responses it evokes within the
mammalian host1. The T. pallidum
genome was sequenced2 in the expec-
tation that the resulting treasure-
trove of information would provide
the tools to resolve these controver-
sies and, more importantly, expe-
dite the search for a safe and effec-
tive vaccine, which has long eluded
syphilis researchers. Although it is
too early to tell, of course, whether
this impressive technological feat
will yield the promised scientific
breakthroughs, at the very least it has
reinforced the notion that T. pal-
lidum plays by a very different set of
rules compared with other bacterial
pathogens. The salient features of the
genome are summarized in Box 1.
A solution to the cultivation
problem?
The primary problem confronting
contemporary syphilis research is
0966-842X/99/$ - see front matter © 1999 Elsevier Science. All rights reserved.
TRENDS IN
concepts applicable to all of the
assembly/secretion systems.
E.T. Saulino, D.G. Thanassi and
S.J. Hultgren
Dept of Molecular Microbiology,
Washington University School of
Medicine,
Box 8230, 660 Euclid Avenue,
St Louis, MO 63110-1093, USA
References
1 Saulino, E.T. et al. (1998) EMBO J. 17,
2177–2185
2 Thanassi, D.G. et al. (1998) Proc. Natl.
Acad. Sci. U. S. A. 95, 3146–3151
3 Bitter, W. et al. (1998) Mol. Microbiol.
the inability to cultivate T. pallidum
on artificial medium. T. pallidum
has long been known to lack nu-
merous biosynthetic and catabolic
capabilities3. The genomic sequence
has further refined this picture
of it as a prokaryotic, metabolic
‘basket case’ and clarified pre-
genomic metabolic studies that
relied on spirochete preparations
contaminated with rabbit testicu-
lar material3. T. pallidum can uti-
lize carbohydrates as its sole energy
source and lacks the tricarboxylic
acid cycle and oxidative phospho-
rylation pathways. In addition, it
is unable to synthesize fatty acids,
enzyme cofactors and most amino
acids. It is an axiom of microbial
pathogenesis that the struggle for
J.D. Radolf* is in the Depts of Internal
Medicine and Microbiology; and
D. Shevchenko is in the Dept of Internal
Medicine, University of Texas
Southwestern Medical Center, 5323 Harry
Hines Blvd, Dallas, TX 75235-9113, USA;
B. Steiner is in the Centers for Disease
Control and Prevention, 1600 Clifton
Road NE, Atlanta, GA 30333, USA.
*tel: 11 214 648 6896,
fax: 11 214 648 5476,
e-mail: jradol@mednet.swmed.edu
PII: S0966-842X(98)01422-X
MICROBIOLOGY 7 VOL. 7 NO. 1 JANUARY 1999
27, 209–220
4 Linderoth, N., Simon, M. and Russel, M.
(1997) Science 278, 1635–1637
5 Koomey, M. (1995) Trends Microbiol. 3,
409–411
6 Kubori, T. et al. (1998) Science 280,
602–605
7 Ginocchio, C.C. et al. (1994) Cell 76,
717–724
8 Jacob-Dubuisson, F., Striker, R. and
Hultgren, S.J. (1994) J. Biol. Chem. 269,
12447–12455
9 Karlyshev, A.V. et al. (1992) FEBS Lett.
297, 77–80
10 Labigne-Roussel, A.F. et al. (1984)
Infect. Immun. 46, 251–259
11 Bullitt, E. et al. (1996) Proc. Natl. Acad.
Sci. U. S. A. 93, 12890–12895
iron acquisition is a defining feature
of the host–pathogen relationship
and that iron, which is highly se-
questered within the host, is essen-
tial for bacterial metabolism4. Re-
markably, T. pallidum appears to
forego the need for iron acquisition
mechanisms because it lacks many
of the enzymes and proteins that use
iron or heme cofactors. Taken as a
whole, the diverse metabolic de-
ficiencies documented by the gen-
omic sequence support the idea
that the treponeme’s refractoriness
to in vitro cultivation results from
the absence of one or more vital
nutrients from past media formu-
lations. Unfortunately, discovering
the missing ingredient(s) is no sim-
ple matter given the number of possi-
bilities raised by the sequence and
the fact that virtually every conceiv-
able nutritional supplement has
already been investigated without
success5. Additional clues to solving
the cultivation problem might
emerge from comparisons of the
T. pallidum genome with those
of two other minimalist, yet culti-
vatable, bacterial pathogens: Myco-
plasma genitalium6 and the fellow
spirochete, Borrelia burgdorferi7.
COMMENT

Box 1. The Treponema pallidum genome at a glancea

General features
• Circular chromosome of 1.138 Mb, 1041 predicted open reading frames (ORFs); lacks plasmids.
• Predicted biological roles for 577 ORFs (55%); 287 ORFs (28%) are novel genes.

Transcription and translation

• Genes for a, b and b9 core RNA polymerase subunits.
• Five sigma factors (s24, s28, s43, s54 and s70); lacks s38 (rpoS) and s32 (heat shock).
• 44 tRNA synthetase genes; lacks glutaminyl-tRNA synthetase.
• Two ribosomal RNA operons with typical (16S-tRNA-23S-5S) eubacterial organization.

DNA replication, repair, recombination and restriction/modification

• a, b, e, g and t subunits of DNA polymerase III.
• Types I and II topoisomerases; lacks type IV topoisomerase.
• DNA adenine methyltransferase.
• Major pathways of uvr excision repair and mutL/mutS mismatch repair.
• recF recombination pathway; lacks sbcB, recB, recC and recD.
• No recognizable systems for DNA restriction/modification.

Regulatory functions
• Two response-regulator two-component systems.
• Several putative transcriptional repressors.
• Potential phosphorylation-based regulatory system involving orthologs of PtsI, Hpr, PtsK and PtsN.

Metabolic pathways, transporters and other cellular processes

• Lacks pathways for synthesis of fatty acids, nucleotides, enzyme cofactors and most amino acids.
• Uses ribonucleotide diphosphate reductase to reduce ribonucleosides to deoxyribonucleotides.
• Contains all of the enzymes for glycolysis; lacks genes for the tricarboxylic acid cycle and oxidative phosphorylation pathways.
• Lacks genes to use amino acids and fatty acids as energy sources.
• Encodes genes for 18 distinct ATP-binding cassette (ABC) transporters with specificities for amino acids, carbohydrates,
cations and thiamine.
• Lacks a phosphoenolpyruvate:phosphotransferase system.
• Lacks a recognizable system for phosphate uptake.
• Lacks genes for superoxide dismutase, catalase or peroxidase.

Motility and chemotaxis

• 36 genes encoding proteins involved in flagellar structure and function.
• Two copies of the flagellar motor switch protein, FliG.
• 13 chemotaxis proteins with putative specificity for amino acids or carbohydrates.

Cell envelope
• Complete pathway for synthesis of peptidoglycan.
• Complete machinery for protein export and lipid modification of proteins.
• 22 putative lipoproteins.
• 12 paralogs (Tpr proteins) with sequence relatedness to the major sheath protein (Msp) of Treponema denticola (three are
full-length proteins with amino-terminal export signals).
• Orthologs for OmpH of Yersinia enterocolitica and Omp85 of Neisseria gonorrhoeae.
• Lacks recognizable protein secretory machinery.
• Lacks a pathway for lipopolysaccharide biosynthesis.

Virulence determinants
• Five putative hemolysins/cytotoxins.

a
Adapted from Ref. 2.

The genome and the quest for
outer membrane proteins
The outer membrane of T. pallidum
differs from those of Gram-nega-
tive bacteria in that it lacks lipo-
polysaccharide (endotoxin) and con-
tains a much higher lipid : protein
ratio8. The paucity of proteins in the
T. pallidum outer membrane9,10,
coupled with the ostensibly poor
reactivity of these proteins with the
anti-treponemal antibodies in syph-
ilitic serum11, has frustrated efforts
to characterize these entities via
conventional molecular methods,
including outer membrane iso
lation1,12. The genomic sequence has
opened up new avenues in the quest
for rare outer membrane proteins1.
T. pallidum does not contain or-
thologs for highly conserved outer
membrane proteins of enteric
Gram-negative bacteria, such as
porins, further underscoring the
compositional differences between
treponemal and Gram-negative bac-
terial outer membranes. However,
it does contain orthologs for the
lesser known OmpH of Yersinia en-
tercolitica and Omp85 of Neisseria

TRENDS IN MICROBIOLOGY 8 VOL. 7 NO. 1 JANUARY 1999


gonorrhoeae. Undoubtedly, the big-
gest surprise from the sequence is
that T. pallidum also contains a
family of 12 paralogs [designated
Tpr (T. pallidum repeat) proteins]
with sequence relatedness to the
major sheath protein (Msp) of the
cultivatable oral spirochete Tre-
ponema denticola13. The number
and sequence variability of these
proteins, compared with the single-
copy Msp of T. denticola, have in-
vited speculation that they comprise
a system for antigenic variation that
contributes to the relapsing nature
of syphilis. However, this sugges-
tion should be treated with caution.
Examination of the Tpr sequences
reveals that all but four lack poten-
tially cleavable amino-terminal sig-
nal peptides and that one of these
four sequences is likely to be trun-
cated by a frameshift (Fig. 1).
Although one could postulate a
series of recombinational events
analogous to those occurring in
other systems for antigenic vari-
ation, studies thus far indicate that
the tpr genes are highly stable on
repeated passage14. Nevertheless,
the fact that only a minority of the
Tpr proteins is likely to be surface
exposed should simplify efforts
to evaluate their effectiveness as
inducers of protective immunity.
Syphilis pathogenesis – the
unsolved riddle
Since the recognition of venereal
syphilis as a distinct clinical entity
in the late 15th century, physicians
have marveled at, and been baffled
by, its protean manifestations15. Un-
raveling the genetic basis for the re-
markable invasiveness and complex
tissue tropisms of this bacterium15
is the ultimate objective of patho-
genesis-related research. Perhaps
the greatest disappointment of the
genomic sequence is how little in-
sight it provides into the parasitic
strategies used by this spirochete.
Although T. pallidum does con-
tain open reading frames (ORFs)
encoding putative hemolysins/
cytotoxins of uncertain pathogenic
significance, it does not contain
orthologs for any well-known
virulence factors. Also lacking are
the components of a secretory
apparatus, which would be needed
to deliver virulence determinants
TRENDS IN MICROBIOLOGY
COMMENT
Trends in Microbiology
Msp 58.3 (56.2) kDa
TprA 28.7 (25.6)/66.8 (63.7)∗ kDa
TprB 71.1 kDa
TprH 76.1 kDa
TprK 55.5 kDa
TprL 56.5 kDa
TprC 64.7 kDa
TprD 64.7 kDa
TprF 39.3/43.7∗ kDa
TprI 66.5 kDa
TprE 81.5 (79.3) kDa
TprG 81.3 (79.0) kDa
TprJ 81.4 (79.1) kDa
Fig. 1. Export signals of the family of Treponema pallidum repeat (Tpr) proteins. The 12 pro-
teins are arranged into sequence-related subgroups. The major sheath protein (Msp) of
Treponema denticola is shown for comparison. The black boxes at the amino-termini of TprA,
TprE, TprG and TprJ represent potential signal peptides. Unfilled boxes in TprA and TprF indi-
cate the portions of the respective open reading frames downstream from the frameshifts.
Predicted molecular masses are shown at the right of each protein. The molecular masses
shown in parentheses for TprA, TprE, TprG and TprJ are those predicted for the putative mature
(i.e. processed) proteins. The asterisks indicate the molecular masses predicted for the TprA
and TprF proteins without frameshifts.
into the host environment. Thus, (Schell, R.F. and Musher, D.M., eds),
the sequence confronts us with the pp. 57–70, Marcel Dekker
realization that the enigma of 4 Payne, S.M. (1993) Trends Microbiol. 1,
T. pallidum is actually twofold. It 66–69
not only points to our need for 5 Jenkin, H.M. and Sandok, P.L. (1983) in
new physiological paradigms to Pathogenesis and Immunology of
explain how this unorthodox bac- Treponemal Infection (Schell, R.F. and
Musher, D.M., eds), pp. 71–98, Marcel
terium copes with the demands of
Dekker
life within a hostile host milieu but 6 Fraser, C.M. et al. (1995) Science 270,
it also points to the enormous gaps 397–403
in our understanding of the dis- 7 Fraser, C.M. et al. (1997) Nature 390,
tinctive, yet highly successful, 580–586
brand of human parasitism em- 8 Radolf, J.D. et al. (1995) Infect. Immun.
ployed by this bacterium. Many of 63, 4244–4252
the solutions to these biological 9 Radolf, J.D., Norgard, M.V. and Schulz,
conundrums are likely to reside in W.W. (1989) Proc. Natl. Acad. Sci.
the .40% of the genome that con- U. S. A. 86, 2051–2055
sists of functionally uncharacter- 10 Walker, E.M. et al. (1989) J. Bacteriol.
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perimental strategies to decipher 11 Cox, D.L. et al. (1995) Mol. Microbiol.
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12 Shevchenko, D.V. et al. (1997) Infect.
into a coherent picture will be the
Immun. 67, 4179–4189
new frontier for syphilis research. 13 Fenno, J.C. et al. (1997) J. Bacteriol.
179, 1082–1089
14 Pillay, A. et al. (1998) Sex. Transm. Dis.
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1 Radolf, J.D. (1995) Mol. Microbiol. 16, 25, 408–414
1067–1073 15 Tramont, E.C. (1994) in Principles and
2 Fraser, C.M. et al. (1998) Science 281, Practice of Infectious Diseases
375–388 (Mandell, G.L., Bennett, J.E. and
3 Cox, C.D. (1983) in Pathogenesis and Dolin, R., eds), pp. 2117–2133,
Immunology of Treponemal Infection Churchill Livingstone
9 VOL. 7 NO. 1 JANUARY 1999