JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2006, p. 888–891
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 44, No. 3
Reactivity of Antibodies from Syphilis Patients to a Protein Array
Representing the Treponema pallidum Proteome†
Mary Beth Brinkman,1‡ Matthew McKevitt,1‡ Melanie McLoughlin,2Carla Perez,1Jerrilyn Howell,2
George M. Weinstock,4Steven J. Norris,2,3and Timothy Palzkill1*
Department of Molecular Virology and Microbiology1and Human Genome Sequencing Center,4Baylor College of Medicine,
One Baylor Plaza, and Department of Pathology and Laboratory Medicine2and Department of Microbiology and
Molecular Genetics,3University of Texas—Houston Medical School, 6431 Fannin Street, Houston, Texas 77030
Received 1 November 2005/Returned for modification 22 December 2005/Accepted 10 January 2006
To identify antigens important in the human immune response to syphilis, the serum antibody reactivity of
syphilitic patients was examined with 908 of the 1,039 proteins in the proteome of Treponema pallidum subsp.
pallidum using a protein array enzyme-linked immunosorbent assay. Thirty-four proteins exhibited significant
reactivity when assayed with human sera from patients in the early latent stage of syphilis. A subset of antigens
identified were further scrutinized for antibody reactivity at primary, secondary, and latent disease stages, and
the results demonstrate that the humoral immune response to individual T. pallidum proteins develops at
different rates during the time course of infection.
Over the past half century, effective antibiotic treatment
programs have made syphilis relatively uncommon in the
United States, with less than 7,100 primary and secondary
cases diagnosed in 2003 (6). However, recent data indicate that
reported cases are again increasing in subsets of the popula-
tion, and periodic epidemics of syphilis have occurred for de-
cades (5). In 1995, the number of new cases of syphilis world-
wide was estimated to be 12 million per year (29).
As a syphilitic infection can produce a variable range of
symptoms in humans, laboratory tests are often required to
definitively diagnose an infection. Due to the inability to cul-
ture the organism in vitro, a need exists for the development
and optimization of T. pallidum detection in diverse clinical
specimens (16). While enzyme-linked immunosorbent assays
(ELISAs) for Treponema are commercially available, they ex-
hibit varying efficiencies at different disease stages (23). Thus,
knowledge of the presence and timing of antigenic protein
expression by T. pallidum will allow for the selection of optimal
antigen combinations for T. pallidum detection.
New cases of syphilis occur primarily in areas of poor health
care and low socioeconomic status (19), and the availability of
a vaccine would greatly aid in reducing the worldwide inci-
dence of the disease. A syphilis vaccine could reduce depen-
dence on antibiotics, prevent side effects due to antibiotic ad-
ministration, and prevent disease before it occurs. Indeed, the
Centers for Disease Control and Prevention has included the
development of a vaccine in their plan to eradicate syphilis
from the United States (13). In 1973, complete protection from
reinfection with Treponema pallidum subsp. pallidum was seen
in rabbits immunized with large numbers of gamma-irradiated
treponemes (15). However, the large numbers of organisms
and injections used render this approach impractical for hu-
man vaccine development. Several syphilitic manifestations
can be reproduced in the rabbit, and experimental infection of
rabbits has been shown to be an effective vehicle to test vaccine
candidates (4). Optimally a subunit vaccine of recombinantly
expressed proteins or peptides could be developed. Although
many vaccine candidates have been tested, thus far no antigen
has been shown to provide complete protection from subse-
quent T. pallidum infection.
Previously, we performed a systematic screen of the T. pal-
lidum proteome to identify antigenic proteins during rabbit
infections (13). In order to identify novel human antigens, we
have extended this study to a large-scale screening of the T.
pallidum proteome using sera collected from patients with
syphilis. Our results reveal many newly identified antigens that
can be further characterized for vaccine potential as well as for
clinical diagnostic purposes.
MATERIALS AND METHODS
Bacterial strains, plasmids, and media. Glutathione S-transferase (GST) fu-
sion proteins were expressed in Escherichia coli BL21(DE3) (Invitrogen, Carls-
bad, Calif.). Plasmids expressing GST-T. pallidum subsp. pallidum strain Nichols
fusion proteins were constructed using PCR amplification of each T. pallidum
gene, ligation into a donor plasmid, and Cre-loxP recombination with a GST
expression vector using Invitrogen’s Univector cloning technology as previously
described by McKevitt et al. (14). E. coli cells were cultured in Luria Bertani
(LB) or 2YT medium (16% [wt/vol] Bacto-Tryptone, 1% [wt/vol] Bacto-Yeast,
0.5% [wt/vol] NaCl).
Serum preparation. The human serum samples were previously collected in
Texas from normal human subjects and from patients diagnosed with primary,
secondary, and early latent syphilis. Sera were pooled prior to the ELISA ex-
periments as normal human sera (six sera), primary (two sera), secondary (nine
sera), and early latent (five sera). For the initial screening of reactivity, the pool
of sera from five patients with early latent syphilis was used. Human sera col-
lected from patients diagnosed with secondary syphilis were kindly provided by
Robert Baughn, VA Medical Center, Houston, TX. All human sera were col-
lected under established guidelines with prior approval by the Committee for the
Protection of Human Subjects, University of Texas Health Science Center at
* Corresponding author. Mailing address: Department of Molecular
Virology and Microbiology, Baylor College of Medicine, One Baylor
Plaza, Houston, TX 77030. Phone: (713) 798-5609. Fax: (713) 798-
7375. E-mail: email@example.com.
† Supplemental material for this article may be found at http://jcm
‡ These authors contributed equally to the work.
Absorption of anti-E. coli protein antibodies. Before use, serum samples were
incubated with E. coli cell lysate to remove nonspecific reactivity. Briefly, E. coli
BL21(DE3) was grown overnight at 37°C in LB medium. Cell pellets were
resuspended in 10 ml bacterial protein extraction reagent (B-PER) (Pierce,
Rockford, Ill.) containing 0.375 mg/ml lyzozyme and 420 ng/ml DNaseI and then
incubated on a rocking platform for 10 min at room temperature. Cell debris was
deposited by centrifugation (10 min at 16,325 ? g at 4°C), and the supernatant
was collected for use. A mixture of 10 ?l serum, 11 ml phosphate-buffered saline
(PBS) (pH 7.4) containing 1% dry milk, and 1 ml BL21(DE3) cell lysate super-
natant was mixed on a rocking platform for 2 h at room temperature just prior
Protein expression. Expression conditions were optimized and standardized as
previously described (13, 14). Briefly, E. coli BL21(DE3) hosting the plasmid
constructs containing individual T. pallidum open reading frames (ORFs) was
inoculated into 1 ml LB media containing 25 ?g/ml kanamycin, 100 ?g/ml
ampicillin, and 2% glucose in 96-well plates. Following incubation with shaking
overnight at 37°C, 100 ?l of the culture was added to 1.5 ml 2YT containing 25
?g/ml kanamycin and 100 ?g/ml ampicillin. The cultures were incubated in 2-ml
wells containing microstir bars in a 96-well format at 30°C for 5 h, followed by
addition of isopropyl-a ¯-D-thiogalactopyranoside (0.1 mM final concentration)
and incubation for an additional 5 h. Cells were then pelleted and stored at
?80°C. Previous control experiments indicated that the T. pallidum GST fusion
proteins vary in the amount of protein expression in E. coli based on reactivity of
an anti-GST antibody but that there was not a strong correlation between the
amount of anti-GST reactivity and the amount of reactivity with sera from rabbit
ELISA protocol. Each pellet was subjected to three rounds of freeze-thawing
prior to addition of 220 ?l of bacterial protein extraction reagent (B-PER)
(Pierce, Rockford, Illinois) containing 0.375 mg/ml lysozyme and 420 ng/ml
DNaseI, which was used to lyse the pellet. The resuspended pellets were stirred
vigorously with a microstir bar at room temperature for 10 min. Reacti-Bind
glutathione-coated white 96-well plates (Pierce, Rockford, Ill.) were blocked
overnight in 150 ?l PBS-Casein (Pierce, Rockford, Ill.). A volume of 110 ?l of
each cell lysate was added to the glutathione-coated plates, followed by incuba-
tion at room temperature for 2 h. The plates were washed with 210 ?l of PBS
(pH 7.4)–0.05% Tween 20 (buffer A) using an Elx50 Auto Strip washer (Bio-Tek,
Winooski, Vt.) eight times, and the wells were then blocked with 150 ?l PBS (pH
7.4) containing 5% dry milk at room temperature for 1 h. Absorbed human
serum was diluted into buffer A to a final serum dilution of 1:1,200. A volume of
110 ?l of the serum preparation was added to each well, followed by incubation
for 2 h at room temperature. The plates were then washed eight times with buffer
A. A 1:12,000 dilution of goat anti-human immunoglobulin G (IgG) and IgM
horseradish peroxidase conjugate (110 ?l; heavy plus light chain specific and
affinity purified [Pierce, Rockford, Ill.]) was added to each plate well and was
incubated for 1 h at room temperature. In a separate experiment under identical
ELISA conditions, the goat anti-human antibody was shown to detect as few as
5 ng/well of purified human IgG (Pierce, Rockford, Ill.) and IgM (Pierce, Rock-
ford, Ill.). The plates were washed eight times with buffer A before the addition
of 150 ?l of SuperSignal ELISA Pico chemiluminescent substrate (Pierce, Rock-
ford, Ill.). Light emission from each plate well was monitored 10 min after
peroxidase substrate addition with a Genios plate reader (Tecan, Durham, N.C.)
for 200 ms. The experiments were repeated three times.
Data analysis. Each plate in the serum arrays contained immobilized GST
without a fusion protein as a negative control in order to identify statistically
relevant reactive proteins arrayed on the same plate. For analysis of the data
generated from the arrays with human serum, the ratio between the chemilumi-
nescence detected from a sample well containing a T. pallidum protein fused to
GST and the chemiluminescence detected from a sample well containing only
immobilized GST protein was calculated. These experiments were conducted
three times, and the sample signal-to-background-signal ratios were averaged.
Based on the addition of the mean of the reactivity of the normal human sera
control plus four times the standard deviation (99.9% confidence interval assum-
ing a normal distribution), a value of 1.5 or greater indicates significant interac-
tions between antibodies present in the syphilitic sera and immobilized T. palli-
RESULTS AND DISCUSSION
Identification of antigenic proteins. Of the 908 T. pallidum
proteins examined for reactivity with pooled early latent hu-
man serum, 34 proteins were considered significantly antigenic
as indicated by a signal-to-background ratio of 1.5 or greater
(Fig. 1; Table S1 in the supplemental material). The 34 pro-
teins reactive with early latent human sera were also found to
be significantly reactive in a previous study that used sera from
T. pallidum-infected rabbits in a similar assay (13). This cor-
relation validates our previous results as well as the use of the
rabbit model system for antigen identification. A total of 90
proteins, 32 of those reactive with early latent human sera as
well as additional proteins known to be antigenic in previous
studies with rabbit sera, were selected for further analysis of
reactivity with human sera from different stages of disease
progression, including sera from patients with primary syphilis,
secondary syphilis, or normal human sera (Table S2 in the
supplemental material). Thirty-eight of the 90 proteins exam-
ined exhibited signal-to-background ratios of ?1.5 with sera at
one or more of the syphilitic stages (Table 1). Fourteen were
reactive with the pooled sera from each stage and thus may
represent good candidates for immunodiagnostic assays. Six-
teen of the 38 antigens we identified were previously reported
in the T. pallidum literature as antigens (Table 1), and only two
proteins, TP0974 (hypothetical protein) and TP1015 (N utili-
zation substance protein B), did not produce a detectable re-
action in our previous immunoproteome analysis using sera
from T. pallidum-infected rabbits (13).
As seen in Table 1, no significant interactions were detected
when normal human serum was incubated with the arrayed
proteins, and the most reactive syphilitic disease stage was the
early latent stage. Six of the 38 reactive proteins, TP0133 (hy-
pothetical protein), TP0136 (hypothetical protein), TP0326
(outer membrane protein), TP0398 (flagellar hook-basal body
complex protein), TP0663 (outer membrane protein, putative),
and TP0767 (translation elongation factor G), did not exhibit
reactivity with early latent syphilis sera (Table 1). An immune
response to these proteins may be specific to the early stage of
infection, thus making them good candidates for a diagnostic
test for early syphilitic infection. In our assay, 11 proteins were
reactive (ratio, ?1.5) with the early latent pool, but they were
not reactive with sera from primary or secondary syphilis pa-
FIG. 1. Identification of antigenic proteins in the T. pallidum pro-
teome using sera collected from patients in the early latent stage of
disease. The chemiluminescence ratio refers to the relative light units
resulting from the binding of serum Ig to the T. pallidum-GST fusion
protein divided by the value obtained when wells were coated with
VOL. 44, 2006ANTIGENIC COMPOSITION OF TREPONEMA PALLIDUM 889
tients. If the development of the humoral immune response
between secondary and early latent syphilis in humans coin-
cides with protective immunity, then the 11 proteins that ex-
hibited reactivity only during early latency are of great interest.
Four of the 11 proteins, including TP0163 (Mn2?/Mg2?ABC
transport, periplasmic binding protein TroA), TP0216 (heat
shock protein 70), TP0292 (conserved hypothetical protein),
and TP1038 (bacterioferrin), have been previously identified as
antigens (3, 17, 20). Five of the seven remaining novel antigens
were also identified as antigens in our previous analysis using
sera collected from rabbits (13). As in the prior study, it is
likely that some false-negative results were obtained due to
either a loss of an antigen during the preabsorption of sera
with E. coli proteins, low expression levels, lability of some of
the protein products in E. coli, or mutations introduced into
ORFs during the cloning process (13). In addition, 131 T.
pallidum ORFs were not included in this study, either because
of the inability to clone the ORF or to convert the ORF to an
expressed GST fusion clone or because of mutations in the
cloned ORF detected by DNA sequencing (13, 14). Finally, it
TABLE 1. 38 T. pallidum proteins that exhibit signal-to-background ratios of ?1.5 with sera from one or more syphilitic disease stagesa
Name and/or function
ABC transporter, periplasmic
Heat shock protein 70
Leucine-rich repeat protein
Conserved hypothetical protein
Outer membrane protein
Cationic outer membrane protein
Flagellar hook-basal body
complex protein (FliE)
Lipoprotein, 17 kDa
Conserved hypothetical protein
Carboxypeptidase, 47 kDa
Outer membrane protein,
putative (Tromp 2)
Flagellar hook protein
Translation elongation factor G
Membrane protein (TmpA)
Outer membrane protein (TmpB)
Conserved hypothetical protein
Membrane antigen, pathogen-
Rare lipoprotein A, putative
N utilization substance protein B
Basic membrane protein (tpn39b)
257 27II 41,00210.041.0
7.3 3.0 7.1
6841 II43,041 5.05 0.7
aThe chemiluminescence ratio refers to the relative light units resulting from the binding of serum Ig to the T. pallidum–GST fusion protein, divided by the value
obtained when wells were coated with GST alone. Significant reactivity is indicated by boldface.
bPrediction of type I or II signal sequence or N-terminal transmembrane helix (TMH) or cytoplasmic (CYT) location. The precictions were made using the LipoP
1.0 server (12a).
890 BRINKMAN ET AL.J. CLIN. MICROBIOL.
is possible that the number of antigens detected in the exper-
iments reported here are limited by the number of patient sera
used for detection and that further studies with additional sera
would detect additional antigens.
The genomic or “reverse vaccinology” approach to defining
antigens is a useful method for characterizing the humoral
immune response to infectious agents (22). The characteriza-
tion of antigens such as MglB-2 and TmpC may be useful in
immunodiagnosis, in that these antigens give rise to strong,
rapid antibody responses that may increase the sensitivity of
diagnosis during the early stages of infection. Furthermore,
novel vaccine candidates, including potential surface-exposed
outer membrane proteins, may be present among the many
previously undescribed antigens identified in the human anti-T.
pallidum immunoproteome. Analysis of the protective capacity
of these recombinantly expressed antigens is presently under
way in the hope of finding a combination of proteins that
protect against T. pallidum infection.
This work was supported by NIH grants AI45842 to T.P. and
AI49557 to S.J.N.
We thank Mary Mosher for technical assistance.
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