Abstract
The tprK gene of Treponema pallidum subsp. pallidum, the causative agent of venereal syphilis, belongs to a 12-member gene family and encodes a protein with a predicted cleavable signal sequence and predicted transmembrane domains. Except for the Nichols type strain, all rabbit-propagated isolates of T. pallidum examined thus far are comprised of mixed populations of organisms with heterogeneous tprK sequences. We show that tprK sequences in treponemes obtained directly from syphilis patients are also heterogeneous. Clustering analysis demonstrates that primary chancre tprK sequences are more likely to cluster within a sample than among samples and that tighter clustering is seen within chancre samples than within rabbit-propagated isolates. Closer analysis of tprK sequences from a rabbit-propagated isolate reveals that individual variable regions have different levels of diversity, suggesting that variable regions may have different intrinsic rates of sequence change or may be under different levels of selection. Most variable regions show increased sequence diversity upon passage. We speculate that the diversification of tprK during infection allows organisms to evade the host immune response, contributing to reinfection and persistent infection.
The spirochete Treponema pallidum subsp. pallidum causes multistage disease and establishes persistent infection in the absence of antibiotic therapy. Although robust humoral and cellular immune responses are able to clear most treponemes from primary and secondary syphilis lesions, some treponemes survive to establish lifelong persistent infection. The mechanism by which these organisms evade the immune response to persist for decades is not known.
Like gram-negative bacteria, T. pallidum has an inner and an outer membrane, but freeze-fracture electron microscopy has shown that, compared to Escherichia coli, the outer membrane of T. pallidum is relatively devoid of integral membrane proteins (19, 25). The extreme fragility of T. pallidum to physical manipulation has thus far impeded attempts to ascertain the identity of these rare outer membrane proteins (5). The tpr family of genes, some of which have homology to the major surface protein genes of Treponema denticola, has been identified in T. pallidum (3, 7, 21). Cleavable signal sequence and transmembrane domains are predicted for several Tprs (3), making them candidates for outer membrane exposure. Examination of sequences from various clinical isolates demonstrates that one of the Tprs, TprK, is heterogeneous in seven discrete variable (V) regions that are separated by conserved sequences (4, 20). Differences in the V regions are due to base pair changes, as well as insertions and deletions that preserve the coding frame of tprK. Hazlett et al. (8) showed that the tprK translational start is downstream of the start site predicted in the T. pallidum genome sequence (7), which supports the presence of a cleavable signal sequence upstream of the TprK V regions. There is a single tprK locus in the genome of the laboratory strain Nichols and Street strain 14 (20), and our laboratory has been able to isolate populations of T. pallidum that are clonal in tprK (unpublished results). This suggests that heterogeneous T. pallidum isolates are comprised of populations of organisms that each carry a single, unique tprK sequence.
A model has been proposed for a segmented gene conversion-like mechanism of variation of tprK (A. Centurion-Lara, R. E. LaFond, K. Hevner, C. Godornes, B. J. Molini, and S. A. Lukehart, unpublished data). Each tprK V region is flanked by a pair of unique terminal direct repeats, and internal repeat sequences are also found within each V region. Whole or partial fragments of tprK V region sequences, including terminal and/or internal repeat sequences, are found in donor regions adjacent to the tprD gene. In this model, DNA segments are copied from donor sites and inserted into the tprK expression locus V regions, creating new sequences in the insertion site. Donor cassettes can introduce single- or multiple-base-pair changes, insertions, or deletions found among tprK V regions. This model allows for the generation of the wide array of tprK variants found in T. pallidum isolates.
We demonstrate here that tprK diversity is present during syphilis infection in individuals and accumulates upon rabbit passage of T. pallidum. Clustering analysis shows that tprK sequences from rabbit-propagated isolates are more closely related within than among isolates. Sequences derived directly from syphilis patients are diverse but cluster more tightly within infected individuals than do rabbit-propagated isolates. Individual V regions from a rabbit-propagated isolate display differing levels of heterogeneity, and we show that a low-heterogeneity V region accumulates diversity upon serial passage.
MATERIALS AND METHODS
Collection and isolation of T. pallidum samples.
Swabs were obtained from the primary chancres of three patients with early syphilis (MD504, MD530, and MD55) at the Sexually Transmitted Disease Clinic at Johns Hopkins University, Baltimore, Md. T. pallidum was detected by dark-field microscopy in the chancres of all three patients, and MD504 and MD530 had reactive serum Reactive Plasma Reagin tests for syphilis. The present study was approved by the Human Subjects Divisions of Johns Hopkins University and the University of Washington. Swabs were stored in 500 μl of 1× lysis buffer (10 mM Tris, pH 8; 0.1 M EDTA, pH 8; 0.5% sodium dodecyl sulfate). T. pallidum Sea 81-4 was isolated by rabbit inoculation of material collected from the primary chancre of a patient attending the Seattle-King County STD Clinic at Harborview Medical Center, and the isolate was propagated by serial intratesticular passage through New Zealand White rabbits (R&R Rabbitry, Kenwood, Wash.) as previously described (10). T. pallidum Sea 81-3, Sea 83-1, and Sea 84-2 were isolated by rabbit inoculation of the cerebrospinal fluid from three patients with neurosyphilis (11), and serially passaged in New Zealand White rabbits 4, 6, and 10 times, respectively. The respective committees at the University of Washington approved all human subject and animal protocols. Treponemes were harvested from infected rabbit testes and enumerated by dark-field microscopy. For DNA sequencing, bacteria were pelleted at 12,000 × g for 30 min and resuspended in 1 ml of 1× lysis buffer.
Treponemal DNA extraction and PCR amplification of tprK.
DNA was extracted from bacterial pellets and swab samples by using the QIAamp DNA Mini kit (Qiagen, Inc., Valencia, Calif.), taking careful precautions to avoid DNA cross-contamination between isolates. For Sea 81-4, DNA was extracted from a known number of treponemes, and an aliquot of DNA equivalent to 100 organisms was used for amplification of tprK. DNA was amplified in this manner from serial passages of T. pallidum Sea 81-4: 4 (low-passage), 12 (medium-passage), or 21 (high-passage) rounds after isolation from the patient. DNA was also amplified from rabbit-passaged T. pallidum isolates Sea 81-3 (passaged 4 times), Sea 83-1 (passaged 6 times), and Sea 84-2 (passaged 10 times). The primers used for amplification of DNA from isolates encompass the seven V regions of tprK (sense, 5′-ATATTGAAGGCTATGCGGAGCTG; antisense, 5′-TACCCCACACTCGTAATACCC). Sea 81-4 DNA was amplified with TaqDNA polymerase (Promega Corp., Madison, Wis.) by using PCR conditions as described elsewhere (4), except that the annealing temperature was 60°C and the number of cycles was 45. PCR amplification of the three cerebrospinal fluid isolates was performed with 1 μM concentrations of the above primers in a 100-μl reaction containing a 200 μM concentration of deoxynucleoside triphosphates (Promega), 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 0.1 mg of bovine serum albumin/ml, 2.5 U of PfuTurbo Hotstart DNA polymerase (Stratagene, La Jolla, Calif.), and 5 μl of purified DNA. Cycling conditions were as described elsewhere (4) except that the annealing temperature was 60°C. Primers used for amplifying primary chancre DNA encompass the open reading frame of tprK (sense, 5′-GTGGTGTATCAGCGGGTAGG; antisense, 5′-GACATGCCCCTACGAATTG). PCR conditions were as described above for Sea 81-4.
Clustering analysis of tprK sequences.
tprK PCR products were cloned and sequenced as described elsewhere (4). Sequences were deposited to GenBank under accession numbers (AY346012 to AY346089). DNA and translated amino acid sequences of tprK containing all seven V regions (corresponding to bp 182 to 1380 of the T. pallidum genome tprK sequence) were aligned by using CLUSTAL W (European Molecular Biology Laboratory, European Bioinformatics Institute [http://www.ebi.ac.uk/clustalw/]) (24), and alignments were adjusted manually to correct misalignments or gaps introduced by the program. DNA sequence alignments were subjected to neighbor-joining and parsimony analysis by using PHYLIP (Phylogeny Inference Package, version 3.57c; Department of Genetics, University of Washington [http://evolution.genetics.washington.edu/phylip.html]). The trees were subjected to 100 random bootstrap resamplings, and bootstrap confidence levels were determined; values of >70 are considered to be significant (6).
RESULTS
Comparative analysis of diverse tprK sequences from patient samples and rabbit-propagated isolates.
In the present study, we sought to analyze tprK sequences from treponemes obtained directly from the primary chancres of three patients, and compare them to tprK sequences from three rabbit-propagated isolates. The tprK gene was either amplified directly from swabs of primary chancres of three patients with early syphilis—MD504, MD530, and MD55—or was amplified from the harvested treponemes of the rabbit-propagated isolates Sea 81-3, Sea 83-1, and Sea 84-2. The amplicons were then cloned into plasmids and sequenced. Sequencing of 14 plasmid clones from MD504 and 9 plasmid clones from MD55 yielded eight and seven different tprK sequences, respectively. Five different sequences were found in the 13 plasmid clones analyzed from MD530. Sequences that are heterogeneous in at least one of the seven V regions in tprK within each sample are considered different alleles; each sequence is identified by a lowercase letter, and amino acid alignments of the V regions of the unique sequences from each patient sample are shown in 1A (constant regions are 99.7% identical at the DNA level and are therefore not shown). The N- and C-terminal ends of the V regions in Fig. 1 correspond to the terminal base pair repeats described above. Some V regions are shared within the plasmid clones sequenced from an individual patient, and several are shared between two patients. For example, the V1 sequence GIASDGGAIKH is found in all MD504 plasmid clones, as well as in plasmid clone “a” of MD55, and all but one of the MD55 and MD530 clones share the same V5 sequence. In addition, the V2 sequence in MD504 plasmid clone “h” is found in five MD530 clones, and the V7 sequence YGGTNKKNDAAPATKWKAEYCGYYE is shared by three plasmid clones from MD504 and four plasmid clones from MD55. In contrast to sequences from patient lesions, each plasmid clone from rabbit-propagated isolates yielded a different tprK sequence. As in patient-derived samples, heterogeneity in tprK is found only in the V regions (Fig. 1B). Although there are many examples of V region sequences that are shared within an isolate (i.e., the V1 sequence GIASDGGAIKH in Sea 81-3), only the V4 sequence TDVGRKKDGAQGTVGAD is shared between the Sea 81-3 and Sea 84-2 isolates.
FIG. 1.
Amino acid alignments of the variable (V) regions of tprK from directly sequenced patient samples (A) and rabbit-propagated isolates (B). Shaded areas indicate sequence identity; dashes indicate gaps in the alignment. The translational start shown here was identified by Hazlett et al. (8) as different from that in the genome sequence. Conserved regions are identical to those previously published (4, 20); the number of conserved amino acids not shown is indicated.
To further explore tprK heterogeneity, we determined the number of different sequences for each of the V regions (Table 1). With the exception of V4, there are more different V region sequences in rabbit-propagated isolates than in the patient-derived samples. The rabbit-propagated isolates have a considerably wider range of sequence length in V3 and V6, although V7 has a wider range of length in patient-derived treponemes.
TABLE 1.
Analysis of TprK V regions from patient samples and rabbit-propagated isolates
| V region | No. of different V region sequences in patient sample(s): |
Length of V region (aa)a in patient samples | No. of different V region sequences in rabbit-propagated isolate(s): |
Length of V region (aa) in rabbit-propagated isolates | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| MD504 (n = 8) | MD530 (n = 5) | MD55 (n = 7) | Combined (n = 20) | Sea 81-3 (n = 7) | Sea 83-1 (n = 8) | Sea 84-2 (n = 5) | Combined (n = 20) | |||
| V1 | 1 | 1 | 2 | 3 | 11-15 | 2 | 4 | 1 | 7 | 11-14 |
| V2 | 3 | 1 | 2 | 5 | 20-22 | 6 | 3 | 2 | 11 | 20-22 |
| V3 | 1 | 2 | 3 | 6 | 19-23 | 3 | 4 | 3 | 10 | 15-27 |
| V4 | 3 | 2 | 1 | 6 | 17-20 | 2 | 2 | 2 | 5 | 17-20 |
| V5 | 3 | 1 | 2 | 5 | 28 | 6 | 5 | 4 | 15 | 28-30 |
| V6 | 4 | 4 | 3 | 11 | 15-21 | 7 | 5 | 5 | 17 | 13-27 |
| V7 | 4 | 3 | 2 | 8 | 24-44 | 2 | 5 | 5 | 12 | 23-27 |
aa, amino acid(s).
Clustering analysis of tprK.
In the alignments in Fig. 1, some V regions do not show heterogeneity within a patient sample or isolate. To determine the relatedness within and among tprK sequences derived from different sources, we performed clustering analysis by using the neighbor-joining method. The tprK sequences from MD504, MD530, and MD55 fall into three tight clusters that correspond to the individual patient samples (Fig. 2A). Bootstrap analysis shows that these identical cluster groups occur in all 100 resampled trees. This indicates that tprK sequences are more related within treponemes from an individual patient than among patients.
FIG. 2.
Clustering analysis of tprK DNA sequences obtained directly from primary chancres of patients MD504, MD530, and MD55 (A) and from isolates that were propagated 4 (Sea 81-3), 6 (Sea 83-1), or 10 (Sea 84-2) times in rabbits (B). Cluster groups are made up of unique sequences from an individual patient or isolate, and are enclosed by a solid oval. Lowercase letters represent the unique sequences in each sample and, in panel A, numbers in parentheses indicate the number of sequenced plasmid clones that contain that sequence. In panel B, each plasmid clone contains a unique sequence. Trees were subject to 100 random bootstrap resamplings, and bootstrap numbers for major branches are shown. The marker bar represents a 1% difference in nucleotide sequences; within a given sample, there is greater genetic distance among rabbit-propagated sequences than among primary chancre sequences.
To test the hypothesis that heterogeneity in tprK accumulates during rabbit passage, we compared the tprK sequences obtained directly from patient specimens to those from three rabbit-propagated isolates. Clustering analysis of seven, eight, and five different tprK sequences, respectively, from T. pallidum Sea 81-3, Sea 83-1, and Sea 84-2 are shown in Fig. 2B. As in the primary chancre samples, the sequences generally cluster by isolate, but the genetic distance among the sequences within a rabbit-propagated isolate is greater than seen in the chancre samples. Two of the isolates have sequences clustering at the end of major branches; only the branch containing sequences from Sea 81-3, which was passaged the fewest number of times, has a bootstrap number higher than the minimum confidence level of 70. The Sea 84-2 isolate, which underwent the highest number of rabbit passages, does not cluster at the end of a single major branch. This analysis shows that tprK sequences from rabbit-propagated isolates are more diverse than those from directly sequenced patient samples, suggesting that propagation of isolates gives organisms the opportunity to accumulate diversity in tprK.
Clustering analysis by using the parsimony method for the trees in Fig. 2 yields bootstrap numbers (data not shown) similar to those resulting from the neighbor-joining method. Because the differences in tprK are due to insertions and deletions of nucleotide sequences of variable lengths in addition to apparent base pair changes, we also performed neighbor-joining and parsimony analysis on sequence alignments with the gaps removed. These analyses yielded results similar to those shown in Fig. 2 (data not shown).
Accumulation of diversity in T. pallidum Sea 81-4 tprK.
Our analyses of tprK have revealed that individual V regions have different degrees of diversity within and among T. pallidum isolates (4). To investigate this phenomenon more closely, we examined the unique sequences for each V region in the rabbit-propagated Sea 81-4 isolate. To investigate the possibility that a given V region sequence might change with repeated passage, we sequenced tprK from Sea 81-4 organisms harvested after 4 (low-passage), 12 (medium-passage), or 21 (high-passage) rounds of propagation in rabbits.
For each passage level, genomic DNA equivalent to that from 100 organisms was amplified and sequenced. We analyzed 10 tprK sequences from the low-passage harvest and 14 sequences from each of the medium- and high-passage harvests. Each of the 38 tprK sequences from Sea 81-4 is unique across the seven V regions, pointing to the high level of sequence heterogeneity found in this gene; however, some individual V region sequences are shared among different tprK sequences. Among 38 Sea 81-4 clones, V1 and V4 are the least heterogeneous with three and nine different sequences, respectively (Fig. 3). V6 and V7 with 24 and 20 different sequences, respectively, have the highest level of heterogeneity in the Sea 81-4 isolate.
FIG. 3.
Cumulative number of new V region sequences found in 38 tprK sequences from T. pallidum Sea 81-4 after low (▨), medium (▩), and high (▥) passage.
Figure 3 also illustrates that the accumulation of diversity differs among V regions. For example, two different V1 sequences are found at low passage, one new sequence is seen at medium passage, and no new sequences are found at high passage. In contrast, there are nine different V6 sequences at low passage, five new sequences are seen at medium passage, and ten additional sequences are found at high passage. Further analysis reveals that V regions with less diversity (V1, V2, V4, and V5) each have a common sequence found at all three passage levels. In contrast, no common sequence is found at all passage levels for the most diverse V regions (V3, V6, and V7). Accumulation of diversity is most clearly illustrated by detailed analysis of the founder tprK V4 sequence in Sea 81-4, TDVGHKKNGANGDIGAD (Table 2). This sequence is also seen, though in decreasing frequency, in the medium- and high-passage organisms. In the medium-passage organisms three new V4 sequences were identified, and five additional V4 sequences were identified in high-passage organisms. The amino acid changes predicted by these sequence changes are shown in Table 2.
TABLE 2.
Predicted TprK V4 amino acid sequences from T. pallidum Sea 81-4
| Predicted amino acid sequencea | No. (%) of sequences |
||
|---|---|---|---|
| Low passage (n = 10) | Medium passage (n = 14) | High passage (n = 14) | |
| TDVGHKKNGANGDIGAD | 10 (100) | 8 (57) | 6 (43) |
| TDVGHKKNGAQGTVGAD | 4 (29) | 1 (7) | |
| TDVGHKRNGANGDIGAD | 1 (7) | 0 (0) | |
| TDVGHKKNGADGDIGAD | 1 (7) | 0 (0) | |
| TDVGRKKDGAQGTVGAD | 2 (14) | ||
| TDVGRKKDGANGDIGAD | 1 (7) | ||
| TDVGHKKENAANGDIGAD | 1 (7) | ||
| TDVGHKKENAAPDDIGAD | 1 (7) | ||
| TDVGHKKENAANVNGTVGAD | 2 (14) | ||
Amino acid changes in medium-passage sequences are single underlined; amino acid changes in high-passage sequences are double underlined.
DISCUSSION
Outer surface molecules of many bacterial pathogens undergo antigenic variation, allowing organisms to evade the host immune system and establish chronic infection. To produce a vast repertoire of serotypes, gene conversion occurs in msp2 and msp3 of the rickettsial pathogen Anaplasma marginale (2, 15) and in the vsp and vlp genes of Borrelia hermsii, the relapsing fever Borrelia spirochete (18, 22). The vlsE gene of the Lyme disease agent Borrelia burgdorferi also undergoes antigenic variation to produce a high level of diversity (23, 26, 27), and changes in the VlsE variable regions have been shown to alter VlsE antigenicity during infection (14). Similar to known mechanisms of antigenic variation in Anaplasma and Borrelia, our working model for antigenic variation of tprK is gene conversion from common donor sites.
Studies in our laboratories (16a, 17) have shown that antibodies to TprK arising from infection with the Nichols strain and other T. pallidum isolates are specifically targeted to the V regions, whereas T cells recognize epitopes in the conserved regions. Furthermore, immunization with recombinant Nichols TprK is partially protective against Nichols challenge in repeated studies (3, 16) but provides less protection against infectious challenge with T. pallidum strains expressing heterologous TprKs (16a). The major mode of clearance from early syphilis lesions is phagocytosis of opsonized treponemes (1, 9), and the subset of treponemes that remains at the site of infection after clearance is resistant to phagocytosis in vitro (12). Antiserum to TprK has been shown to be opsonic for T. pallidum (3), suggesting that TprK is a target of immune clearance. We hypothesize that TprK heterogeneity may contribute to the ability of T. pallidum to evade the host immune response.
In the present study, we show that V1 is the least heterogeneous of the seven V regions in Sea 81-4. Previous studies in our laboratories demonstrated that, during infection with the Nichols strain and with non-Nichols isolates, antibodies are induced against all V regions except V1 (16a, 17). Taken together, these results suggest that V1 is under less immune pressure than the more heterogeneous V regions. V1 may also be less diverse than other V regions because it is involved in some necessary function for TprK. Nichols strain TprK transmembrane topology (3) and Kyte-Doolittle and TMpred tests of multiple TprK sequences from the Sea 81-4 isolate (data not shown) show that V1 is located in a hydrophilic region of the protein; nonetheless, V1 may be topologically hidden or otherwise inaccessible to B cells, thus restricting exposure of V1 to the immune system.
Further evidence that V regions have different levels of diversification is provided by our analysis of tprK sequences from the T. pallidum isolate Sea 81-4 after low, medium, and high passages. Sequences from V regions with low to moderate heterogeneity, such as V4, were maintained at all passage levels, whereas no identical sequences from V regions with high heterogeneity were found at all three passage levels. Because treponemes were harvested at the peak of infection and before the initiation of significant immune clearance, this analysis suggests that V regions have different intrinsic rates of diversification.
We found that tprK sequences in primary chancres are less diverse than those in rabbit-propagated isolates. Early studies on the transmission of T. pallidum showed that intradermal injection of as few as 10 organisms may lead to infection in humans (13). Syphilis transmission occurs primarily during early infection when numbers of treponemes are high; however, the replication rate of T. pallidum is quite slow, and the average time to produce a primary chancre in the exposed individual is 2 to 6 weeks, suggesting that the infectious dose is low. We propose that a very low number of treponemes is transmitted in natural infection and that treponemes in early chancres (at the time of transmission) have not yet been subjected to significant immune selection. This is reflected in the clustering analysis showing that tprK sequences are more related within infected individuals than among individuals. This result suggests that treponemes in individuals are derived from a limited number of transmitted organisms with related tprK sequences.
The Sea 81-3, Sea 83-1, and Sea 84-2 isolates were generated by infecting rabbits with cerebrospinal fluid from syphilis-infected patients and are likely to have arisen from very low numbers of treponemes, just as in the directly sequenced chancre samples. After rabbit passage, however, treponemes within each rabbit-propagated isolate are more diverse than those within treponemes from primary chancres, as indicated by the greater genetic distance between sequences. In addition, sequences from Sea 81-3, which was passaged 4 times in rabbits, cluster more tightly than sequences from Sea 84-2, which was passaged 10 times. In contrast to transmission of low numbers of organisms in nature, T. pallidum is propagated in the laboratory by passaging 106 to 108 organisms to naive rabbits. Harvesting occurs at the peak of infection and before significant immune clearance begins; therefore, as in natural infection, there is not yet any obvious effect of immune selection. Because the number of organisms passaged in laboratory propagation of T. pallidum is likely to be much higher than in human transmission, rabbit propagation may afford a greater opportunity for accumulation of diversity in tprK than does natural infection.
Our laboratories have generated strains of T. pallidum that carry a single tprK sequence, and we are placing these strains under immune pressure by allowing immune clearance in vivo before passaging. In addition to investigating generation of tprK upon passage of clonal strains, we will also directly determine whether tprK varies in a single infection by examining sequences during early and late infection. These experiments will test whether certain V regions undergo stronger immune selection than others and will provide further evidence for tprK diversification as a mechanism of antigenic variation. The expression of new TprK proteins may contribute to the ability of T. pallidum to persist in the face of a robust immune response and to reinfect previously exposed individuals.
Acknowledgments
This work was supported by NIH grants AI34616 and AI42143 (to S.A.L.), AI43456 (to W.C.V.V.), and AI45724 (to A.M.R.). R.E.L. was supported by NIH training grant AI07140.
We thank Timothy Rose and John Mittler for helpful discussions and Heidi Pecoraro for manuscript preparation.
REFERENCES
- 1.Baker-Zander, S., and S. Sell. 1980. A histopathologic and immunologic study of the course of syphilis in the experimentally infected rabbit: demonstration of long-lasting cellular immunity. Am. J. Pathol. 101:387-413. [PMC free article] [PubMed] [Google Scholar]
- 2.Brayton, K. A., G. H. Palmer, A. Lundgren, J. Yi, and A. F. Barbet. 2002. Antigenic variation of Anaplasma marginale msp2 occurs by combinatorial gene conversion. Mol. Microbiol. 43:1151-1159. [DOI] [PubMed] [Google Scholar]
- 3.Centurion-Lara, A., C. Castro, L. Barrett, C. Cameron, M. Mostowfi, W. C. Van Voorhis, and S. A. Lukehart. 1999. Treponema pallidum major sheath protein homologue TprK is a target of opsonic antibody and the protective immune response. J. Exp. Med. 189:647-656. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Centurion-Lara, A., C. Godornes, C. Castro, W. C. Van Voorhis, and S. A. Lukehart. 2000. The tprK gene is heterogeneous among Treponema pallidum strains and has multiple alleles. Infect. Immun. 68:824-831. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Cox, D. L., P. Chang, A. W. McDowall, and J. D. Radolf. 1992. The outer membrane, not a coat of host proteins, limits antigenicity of virulent Treponema pallidum. Infect. Immun. 60:1076-1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783-791. [DOI] [PubMed] [Google Scholar]
- 7.Fraser, C. M., S. J. Norris, G. M. Weinstock, O. White, G. G. Sutton, R. Dodson, M. Gwinn, E. K. Hickey, R. Clayton, K. A. Ketchum, E. Sodergren, J. M. Hardham, M. P. McLeod, S. Salzberg, J. Peterson, H. Khalak, D. Richardson, J. K. Howell, M. Chidambaram, T. Utterback, L. McDonald, P. Artiach, C. Bowman, M. D. Cotton, J. C. Venter, et al. 1998. Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science 281:375-388. [DOI] [PubMed] [Google Scholar]
- 8.Hazlett, K. R., T. J. Sellati, T. T. Nguyen, D. L. Cox, M. L. Clawson, M. J. Caimano, and J. D. Radolf. 2001. The TprK protein of Treponema pallidum is periplasmic and is not a target of opsonic antibody or protective immunity. J. Exp. Med. 193:1015-1026. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Lukehart, S. A., S. A. Baker-Zander, R. M. Lloyd, and S. Sell. 1980. Characterization of lymphocyte responsiveness in early experimental syphilis. II. Nature of cellular infiltration and Treponema pallidum distribution in testicular lesions. J. Immunol. 124:461-467. [PubMed] [Google Scholar]
- 10.Lukehart, S. A., S. A. Baker-Zander, and S. Sell. 1980. Characterization of lymphocyte responsiveness in early experimental syphilis. I. In vitro response to mitogens and Treponema pallidum antigens. J. Immunol. 124:454-460. [PubMed] [Google Scholar]
- 11.Lukehart, S. A., E. W. Hook III, S. A. Baker-Zander, A. C. Collier, C. W. Critchlow, and H. H. Handsfield. 1988. Invasion of the central nervous system by Treponema pallidum: implications for diagnosis and treatment. Ann. Intern. Med. 109:855-862. [DOI] [PubMed] [Google Scholar]
- 12.Lukehart, S. A., J. M. Shaffer, and S. A. Baker-Zander. 1992. A subpopulation of Treponema pallidum is resistant to phagocytosis: possible mechanism of persistence. J. Infect. Dis. 166:1449-1453. [DOI] [PubMed] [Google Scholar]
- 13.Magnuson, H. J., E. W. Thomas, S. Olansky, B. I. Kaplan, L. DeMello, J. C. Cutler. 1956. Inoculation syphilis in human volunteers. Medicine 35:33-82. [DOI] [PubMed] [Google Scholar]
- 14.McDowell, J. V., S. Y. Sung, L. T. Hu, and R. T. Marconi. 2002. Evidence that the variable regions of the central domain of VlsE are antigenic during infection with Lyme disease spirochetes. Infect. Immun. 70:4196-4203. [DOI] [PMC free article] [PubMed]
- 15.Meeus, P. F., K. A. Brayton, G. H. Palmer, and A. F. Barbet. 2003. Conservation of a gene conversion mechanism in two distantly related paralogues of Anaplasma marginale. Mol. Microbiol. 47:633-643. [DOI] [PubMed] [Google Scholar]
- 16.Morgan, C. A., S. A. Lukehart, and W. C. Van Voorhis. 2002. Immunization with the N-terminal portion of Treponema pallidum repeat protein K attenuates syphilitic lesion development in the rabbit model. Infect. Immun. 70:6811-6816. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16a.Morgan, C. A., S. A. Lukehart, and W. C. Van Voorhis. 2003. Protection against syphilis correlates with specificity of antibodies to the variable regions of Treponema pallidum repeat protein K. Infect. Immun. 71:5605-5612. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Morgan, C. A., B. J. Molini, S. A. Lukehart, and W. C. Van Voorhis. 2002. Segregation of B and T cell epitopes of Treponema pallidum repeat protein K to variable and conserved regions during experimental syphilis infection. J. Immunol. 169:952-957. [DOI] [PubMed] [Google Scholar]
- 18.Plasterk, R. H., M. I. Simon, and A. G. Barbour. 1985. Transposition of structural genes to an expression sequence on a linear plasmid causes antigenic variation in the bacterium Borrelia hermsii. Nature 318:257-263. [DOI] [PubMed] [Google Scholar]
- 19.Radolf, J. D., M. V. Norgard, and W. W. Schulz. 1989. Outer membrane ultrastructure explains the limited antigenicity of virulent Treponema pallidum. Proc. Natl. Acad. Sci. USA 86:2051-2055. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Stamm, L. V., and H. L. Bergen. 2000. The sequence-variable, single-copy tprK gene of Treponema pallidum Nichols strain UNC and Street strain 14 encodes heterogeneous TprK proteins. Infect. Immun. 68:6482-6486. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Stamm, L. V., S. R. Greene, H. L. Bergen, J. M. Hardham, and N. Y. Barnes. 1998. Identification and sequence analysis of Treponema pallidum tprJ, a member of a polymorphic multigene family. FEMS Microbiol. Lett. 169:155-163. [DOI] [PubMed] [Google Scholar]
- 22.Stoenner, H. G., T. Dodd, and C. Larsen. 1982. Antigenic variation of Borrelia hermsii. J. Exp. Med. 156:1297-1311. [DOI] [PMC free article] [PubMed]
- 23.Sung, S.-Y., J. V. McDowell, and R. T. Marconi. 2001. Evidence for the contribution of point mutations to vlsE variation and for apparent constraints on the net accumulation of sequence changes in vlsE during infection with Lyme disease spirochetes. J. Bacteriol. 183:5855-5861. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Walker, E. M., G. A. Zampighi, D. R. Blanco, J. N. Miller, and M. A. Lovett. 1989. Demonstration of rare protein in the outer membrane of Treponema pallidum subsp. pallidum by freeze-fracture analysis. J. Bacteriol. 171:5005-5011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Zhang, J. R., J. M. Hardham, A. G. Barbour, and S. J. Norris. 1997. Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes. Cell 89:275-285. [DOI] [PubMed] [Google Scholar]
- 27.Zhang, J. R., and S. J. Norris. 1998. Genetic variation of the Borrelia burgdorferi gene vlsE involves cassette-specific, segmental gene conversion. Infect. Immun. 66:3698-3704. [DOI] [PMC free article] [PubMed]