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Journal of Bacteriology, April 2000, p. 2332-2335, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Multiple Alleles of Treponema pallidum
Repeat Gene D in Treponema pallidum Isolates
Arturo
Centurion-Lara,
Eileen
S.
Sun,
Lynn K.
Barrett,
Christa
Castro,
Sheila A.
Lukehart, and
Wesley C.
Van
Voorhis*
Departments of Medicine and Pathobiology,
University of Washington, Seattle, Washington 98195-7185
Received 20 September 1999/Accepted 5 January 2000
 |
ABSTRACT |
Two new tprD alleles have been identified in
Treponema pallidum: tprD2 is found in 7 of 12 T. pallidum subsp. pallidum isolates and 7 of 8 non-pallidum isolates, and tprD3 is found in
one T. pallidum subsp. pertenue isolate.
Antibodies against TprD2 are found in persons with syphilis,
demonstrating that tprD2 is expressed during infection.
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TEXT |
Syphilis, caused by Treponema
pallidum subsp. pallidum, is a chronic disease
characterized by periods of activity and latency, with clearance of
early lesions but persistence of infection. The mechanisms T. pallidum uses to persist in humans are still not known, but it is
possible that alterations in surface proteins will be involved because
of their interaction with the host and their visibility to the immune response.
A family of 12 tpr genes is contained in the T. pallidum subsp. pallidum Nichols strain genome
(7), some of which code for candidate surface-exposed
proteins (3, 7, 12, 13). The tpr genes of
T. pallidum are of interest for a variety of reasons. Their
gene products are homologous to the Msp proteins of Treponema
denticola, which have been implicated in cell attachment and porin
function (6, 11). Anti-TprK antibody has been shown to
opsonize T. pallidum Nichols strain for phagocytosis
(3); thus, TprK may be exposed at the cell
surface. Immunization with TprK is partially protective against
challenge with T. pallidum Nichols strain (3),
suggesting some tpr gene products are a focus of the
protective immune response. Finally, the variable nature of the
tpr genes suggests a role in immune evasion and persistence
if different tpr genes are sequentially expressed. The 12 tpr gene products can be categorized into three subfamilies (3). Subfamilies I (TprCDFI) and II (TprEGJ) have conserved amino- and carboxyl-terminal sequences, but variable central amino acid
sequences (3). Subfamily III Tpr proteins (TprABHKL) have scattered variable and conserved sequences throughout their length (3).
A novel tpr gene was discovered.
Multiple
tprK alleles have been found in recent isolates of T. pallidum (5), in contrast to the single tprK
allele identified in the laboratory-adapted Nichols strain
(7). Because of our interest in tpr
heterogeneity, we compared tpr gene sequences from other
T. pallidum isolates to those from the Nichols strain. For
example, genomic DNA from the T. pallidum subsp.
pallidum Mexico A isolate was used as a template for PCR
with primers A and B (Table 1), which are
complementary to conserved regions flanking the central variable
domains of tpr subfamilies I and II. The rabbit propagation,
sources of the treponeme isolates, extraction of genomic DNA, and PCR
conditions have been described previously (2-5). One of the
amplicons was homologous to subfamily I (tprCDFI) at the
conserved 5' and 3' ends, yet distinct from all of the subfamily I
genes in much of the variable region (GenBank accession no. AF187953).
This novel tpr was also found in the T. pallidum
subsp. pallidum Bal-3 isolate (GenBank accession no. AF187952).
The novel tpr gene occupies the tprD locus,
and thus was termed tprD2.
In order to localize the novel
tpr gene in the genome, inverse PCR was used to amplify a
fragment of genomic DNA containing the 5' portion of the novel
tpr gene and the 5' flanking DNA. Genomic Bal-3 DNA (100 ng)
was digested with Sau3A1 and ligated with T4 DNA ligase (New
England Biolabs, Beverly, Mass.) in a 120-µl volume, such that
circles were likely to be formed. This was used as a template in a PCR
with primers C and G (Table 1 and Fig. 1)
and yielded a 1.7-kb amplicon. Sequencing of this amplicon demonstrated
that the novel tpr gene is flanked at the 5' end by DNA with
almost complete identity to the TP0132 and TP0133 genes, which are at
the 5' flanking end of tprD in the T. pallidum
Nichols genome (Fig. 1).
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FIG. 1.
Diagram of the tprD locus. Three different
alleles of tprD (tprD, tprD2, and
tprD3) are present in the tprD locus in the
strains examined. The regions denoted as arrows and with TP or
tpr are predicted coding regions with putative start codons
at the beginning of the arrow and putative stop codons at the point of
the arrow. IGR, intergenic region. PCR products are shown as thin
lines, and the primers used to produce the products are shown as
arrowheads. The different shading patterns and lines within the
tprD, tprD2, and tprD3 alleles
demonstrate the differences between the alleles.
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|
The entire
tprD locus and flanking regions (Fig.
1, primers
H and I) were amplified from
T. pallidum subsp.
pallidum isolates
Bal-3, Mexico A, Sea 81-3, Sea 81-4, and
Bal-7 and from the Gauthier
strain of
T. pallidum subsp.
pertenue. The sequence of these amplicons
demonstrated that
a novel sequence is found in the
tprD locus
of the Bal-3,
Mexico A, Sea 81-4, and Sea 81-3 isolates (GenBank
accession no.
AF187952,
AF217539,
AF217540, and
AF217541,
respectively), and this
allele was termed
tprD2 (Fig.
1). The
sequences of the
tprD2 allele and flanking regions were identical
in all four
of these isolates. The amplicon from Bal-7 (GenBank
accession no.
AF217537) was identical to the
tprD gene found
in the
Nichols strain, and the amplicon from the Gauthier strain
(GenBank
accession no.
AF217538) was different from both
tprD and
tprD2 and was termed
tprD3.
There are four regions of heterogeneity between
tprD2 and
tprD: a 330-bp central variable region and three smaller
variable
regions to the 3' end of the open reading frames. No
significant
homology (>18-bp identity) to these four variable regions
of
tprD2 is present anywhere in the
T. pallidum
Nichols genome. These four
variable regions are also reflected by
differences in the predicted
amino acid sequences of
tprD2
and
tprD (Fig.
2). Both TprD
and
TprD2 have predicted cleavable signal sequences (at amino acid
17),
but TprD2 is highly predicted to be in the outer membrane,
while TprD
is predicted to localize in the inner membrane by PSORT
analysis
(
http://psort.nibb.ac.jp/). Outer membrane expression
of TprD2 could
increase the repertoire of variable Tpr proteins
on the surface for
antigenic variation or to change the functionality
of the protein.
TprD3 is 95% identical to TprD, with the major
region of heterogeneity
located between amino acid residues 285
and 304, but with scattered
amino acid differences found throughout
the sequence. Like TprD, TprD3
is predicted by PSORT analysis
to have a cleavable signal sequence, but
to be located in the
inner membrane. It is noteworthy that, while the
5' conserved
region (amino acids 1 to 284) of TprD2 is identical to the
genome
sequence for TprD, the same region in TprD3 has the signature
at
amino acids 234 to 245 (EQHYRKGTEDST) that
characterizes TprF
and TprI predicted from the genome sequence
(
7).
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FIG. 2.
Amino acid sequence alignments of tprD,
tprD2, and tprD3. The alignments of the predicted
amino acid sequences of the tprD, tprD2, and
tprD3 alleles are shown.
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tprD2 was identified in about half of the
Treponema isolates.
Primers C and D (Table 1) were
designed to amplify a tprD2-specific 273-bp amplicon (Fig.
1) to test for the presence of tprD2 in a variety of
Treponema isolates. We examined 12 isolates of T. pallidum subsp. pallidum, 4 isolates of T. pallidum subsp. pertenue (yaws spirochetes), 1 isolate
of T. pallidum subsp. endemicum (endemic syphilis
spirochete), the Simian isolate (primate spirochete [8]), and 2 isolates of Treponema
paraluiscuniculi (rabbit venereal spirochetes) (Table
2). tprD2 was detected in 7 of
12 T. pallidum subsp. pallidum genomes and in 7 of 8 of the non-syphilis treponeme genomes (Table 2). Primers specific
for tprA (primers E and F, Table 1) gave the predicted
315-bp amplicon with each of these isolates, demonstrating that the DNA
from each treponeme isolate was intact and amplifiable (not shown).
Rabbit DNA, a likely contaminant, did not give an amplicon with these
sets of primers. Thus, it appears that the tprD2 allele is
present in about half of the T. pallidum subsp.
pallidum isolates tested, but not the Nichols strain that
was used for the genome sequencing project (7).
Humans with syphilis make antibodies to TprD2-specific
peptide.
An amplicon encoding a 90-amino-acid peptide (amino acids
301 to 391 in Fig. 2) unique to the predicted TprD2 protein was amplified (primers C and D, Table 1 and Fig. 1), expressed as a
six-histidine fusion protein, and purified (TprD2-specific peptide) (3). This TprD2-specific peptide had no homology to any of the other predicted tpr gene products or homology to any of
the predicted open reading frames of the T. pallidum Nichols
strain genome.
To test for immunoreactivity to the TprD2-specific peptide, human sera
(obtained with informed consent as approved by the
University of
Washington Institutional Review Board for human
subjects) were
preabsorbed overnight with 5%
Escherichia coli lysate
containing an irrelevant recombinant
Trypanosoma cruzi SA85-1.1-III protein in pRSET (
9). Western blotting with 100
ng of recombinant TprD2-specific peptide, 1:100 diluted human
sera, and
1:3,000 alkaline phosphatase-conjugated goat anti-human
immunoglobulin
G (Sigma, St. Louis, Mo.) was performed as previously
described
(
1). Antibodies in sera from five of seven persons
with
secondary syphilis were reactive with the TprD2-specific
peptide, while
no anti-TprD2 activity was detected in sera from
seven uninfected
persons, two persons with primary syphilis, and
three persons with late
latent syphilis. Representative immunoblots
are shown in Fig.
3. The antibodies to TprD2-specific
peptide
that were generated during syphilis infection demonstrate that
TprD2 is expressed by
T. pallidum subsp.
pallidum
in humans. The
proportion of syphilitic sera reactive with
TprD2-specific peptide
(5 of 12 tested) is about the same proportion of
T. pallidum subsp.
pallidum isolates carrying the
tprD2 allele (7 of 12 tested).
This suggests that most
isolates containing the
tprD2 allele also
express it.
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FIG. 3.
Western blots demonstrating antibodies in sera from
persons with secondary syphilis react with the TprD2-specific peptide.
Shown are immunoblots with the 14-kDa TprD2-specific recombinant
peptide from a representative experiment. The immunoblots were reacted
with sera from two uninfected controls (U), five persons with secondary
syphilis (noted by no. 2), and two persons with late latent syphilis
(LL). Antibody reactivity to the tprD2-specific peptide is
seen with the sera from three of five persons with secondary syphilis.
Shown to the right are the positions of the molecular mass markers.
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|
We have demonstrated that 7 of 12 of the
T. pallidum subsp.
pallidum isolates and most of the non-syphilis treponemes
tested
have a novel
tprD2 allele. The one non-syphilis
treponeme that
did not contain
tprD2, the Gauthier strain of
T. pallidum subsp.
pertenue, contains another
variant of
tprD, termed
tprD3. In the
four
tprD2-containing isolates examined, the
tprD2
gene occupies
the position of the
tprD gene, as defined in
the Nichols strain;
similarly,
tprD3 occupies the same
position in the Gauthier genome.
Substitution of
tprD2 and
tprD3 for
tprD generates additional
diversity of
the
tpr genes in some strains. The differences in
the
predicted amino acid sequences of
tprD and
tprD2
are localized
to a 110-amino-acid central variable region and three
smaller
variable regions toward the carboxyl terminus of TprD2. If
these
differences are in the immunodominant or exposed regions of these
molecules, this could help to explain why immunity from heterologous
challenge is not as complete as it is with homologous challenge
(
10). Alternatively, these variable domains may provide a
functional
capacity, like cell binding, and the sequence variation may
extend
the functional capacity of individual isolates. It is notable
that TprD2, but not TprD or TprD3, is predicted to be in the outer
membrane, suggesting a different functional role for
TprD2.
| |
ACKNOWLEDGMENTS |
We thank Barbara Molini for excellent technical assistance.
This work was supported by Public Health Service grants AI34616,
AI42143, and AI31448 (Sexually Transmitted Diseases Cooperative Research Center New Investigator Award to A.C.-L.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departments of
Medicine and Pathobiology, University of Washington, Box 357185, Seattle, WA 98195-7185. Phone: (206) 543-0821. Fax: (206) 685-8681. E-mail: wesley{at}u.washington.edu.
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Journal of Bacteriology, April 2000, p. 2332-2335, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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