Treponema pallidum subspecies pallidum strain was propagated by intratesticular inoculation of adult New Zealand White rabbits as described previously 17. Animal protocols described in this work were approved by the University of Connecticut Health Center Animal Care Committee under the auspices of Animal Welfare Assurance no. A3471-01. Escherichia coli strain DH5α was the recipient strain for recombinant constructs and was grown in Luria-Bertani (LB) broth with appropriate antibiotic supplementation.

The plasmids pKS/pho and pSK/pho containing the leader-less alkaline phosphatase (phoA) gene of E. coli in the multiple cloning site of pBluescript have been described previously 19. Using primers K1-5′, K2-5′, K3-5′, and K1-3′, three in-frame tprK–phoA fusions were generated by amplifying and cloning DNA containing codons 21 through 61 of the TprK open reading frame (ORF) along with various amounts of upstream DNA into the Xba1 and BamH1 sites of pKS/phoA and pSK/phoA. E. coli clones harboring sequenced PhoA constructs were plated on LB agar containing 100 μg/ml of ampicillin and 40 μg/ml of 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich).

Two polyhistidine-tagged rTprK expression constructs were generated. The first encodes the full-length TprK protein downstream of a putative signal peptidase I cleavage site (LWA^↓Q). The second encodes the central region (amino acids 36–350) of TprK as per Centurion-Lara et al. 7. Although this region was originally annotated as the “variable” domain 7, we refer to it as the “central” domain to avoid confusion with the recently described seven discrete regions of variability (V1–V7) present in different TprK alleles 89. DNAs encoding the TprK full-length protein and central domain were amplified from Nichols-Farmington T. pallidum DNA using primer pairs K-full-5′/K-full-3′ and K-cen-5′/K-cen-3′, respectively, and cloned into EcoR1/BamH1-digested pProEx-Htb (GIBCO BRL). DNA encoding the mature FlaA protein was amplified with primers FlaA-5′ and FlaA-3′ and cloned into BamH1 cut pProEx-Hta (GIBCO BRL). Recombinant TprK clones were completely sequenced in both directions to verify the identity and fidelity of the cloned PCR products; for both the full-length and central domain fusions, the deduced amino acid sequences were identical to those of T. pallidum (Nichols-Farmington) TprK (see Fig. 8). Fusion proteins were expressed by the addition of isopropylthiogalactopyranoside to 1 mM and purified on a Ni-NTA matrix (QIAGEN) according to the manufacturer's instructions for insoluble proteins. The identities of the purified rTprK full-length and central domain proteins were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry at the Keck Foundation Biotechnology Resource Laboratory at Yale University.

Rat anti-TprK antisera were generated by priming Sprague Dawley rats by intraperitoneal injection with 15 μg of full-length rTprK protein in CFA. After 1 mo, animals were boosted four times over 8 wk with a 1:1 mixture of the central domain and full-length rTprK (15 mg of each protein/boost) in IFA. The mAb 1-7H11.1G6, directed against full-length rTprK, was produced at the Hybridoma Center for Agricultural and Biological Sciences at Oklahoma State University. Rabbits were immunized with rTprK as described below; sera were collected 10 d after the final boost. Rat anti-Fla antisera were generated in Sprague-Dawley rats by intraperitoneal injection with 50 μg/rat of rFlaA protein in CFA, followed at weeks 6 and 8 by similarly administered boosts in IFA. Rat anti-Tp47 and rabbit anti-endoflagella (TpEf) antisera have been described previously 131720.

ELISAs were performed as described previously 13. The wells of microtiter plates coated with dilutions (100 ng/well to 1 pg/well) of full-length, or central domain rTprK were incubated with 1:50 or 1:1,000 dilutions of either NRS, IRS, IRS depleted of anti-TprK Ab (see below), rat, or rabbit anti-TprK antisera.

SDS-PAGE of samples followed by transfer to nitrocellulose membranes was preformed as described previously 14. Blots were incubated with 1:1,000 dilutions of rat antisera or 1:10 dilutions of hybridoma supernatant and subsequently developed by either colorimetric (FlaA and Tp47) or chemiluminescent (TprK) methods as described previously 1418.

Freshly extracted T. pallidum was solubilized overnight at 4°C in PBS containing 2% Triton X-114 (octylphenoxypolyethoxyethanol). Insoluble material was removed by centrifugation and the supernatant was phase separated as described previously 13. The resulting fractions were analyzed by immunoblot.

Proteinase K (PK) digestion was performed as described previously 1321. Freshly isolated T. pallidum were centrifuged at 20,000 g for 15 min and resuspended in PBS containing 1 mM CaCl₂ and 5 mM MgCl₂. After addition of either PBS, PK to 0.4 mg/ml, or PK plus Triton X-100 (t-octylphenoxypolyethoxyethanol) to 0.01%, spirochetes were incubated at 37°C for 30 min followed by the addition of PMSF to 1 mg/ml. Lysates of untreated, PK-treated, and Triton/PK-treated T. pallidum were analyzed by immunoblot.

Preparation of agarose-encapsulated treponemes has been described previously 1317. To discriminate between surface and subsurface exposure of TprK by individual spirochetes, encapsulated organisms were simultaneously probed with 1:30 dilutions of both rat anti-TprK and rabbit anti-TpEf antisera in the absence or presence of 0.05% Triton X-100. After a 1-h incubation and three gentle washes, the beads were incubated with 3 μg of biotinylated goat anti–rat IgG, washed, and then incubated with 3 μg of streptavidin-Alexa^® 546 conjugate (Molecular Probes) and 3 μg goat anti–rabbit conjugated to Alexa^® 488 (Molecular Probes). For each condition, two slides from each of three separate experiments were prepared, and ∼100 organisms per slide were scored for labeling with Alexa^® 546 (TprK) and Alexa^® 488 (endoflagella). Fluorescence emission overlap did not occur as the fluorescence of singly labeled organisms (either TprK or TpEf) was only observed with the appropriate filter.

With minor exception, opsonophagocytosis assays were performed as described previously 71322. In brief, rabbit peritoneal macrophages were incubated with T. pallidum (10 organisms per macrophage) in the presence of 10% heat-inactivated (56°C for 30 min) NRS, IRS, or rabbit anti-TprK antisera. After incubation with T. pallidum, macrophages were washed, fixed, and processed for immunofluorescence microscopy using HSS and FITC-conjugated goat anti–human IgG as described previously. In a separate series of experiments, opsonophagocytosis assays were performed using IRS from which anti-TprK Ab had been removed by two sequential rounds of incubation with purified full-length rTprK. In the first round, 40 μg of pelleted, insoluble TprK protein was resuspended in 1 ml of pooled IRS as a colloidal solution and incubated for 2 h followed by two 30-min centrifugations at 100,000 g to remove rTprK and bound Ab. The supernatant was further depleted by incubation with 40 μg of rTprK immobilized on a nitrocellulose membrane (Schleicher & Schuell). After centrifugation at 25,000 g, the supernatant was quantitatively recovered. Depletion of TprK immunoreactivity was confirmed by ELISA using both the full-length (see Fig. 3 B, inset) and central domain (data not shown) recombinant proteins and by immunoblot analysis (data not shown).

Rabbits were immunized over a 2-mo period with a 1:1 mixture of the TprK central domain (125 μg) and full-length protein (125 μg) per rabbit in Ribi Adjuvant (MPL + TDM + CWS; Corixa Corporation) according to the manufacturer's instructions. It should be noted that, except for the inclusion of full-length protein, this immunization protocol was identical to that of Centurion-Lara et al. 7. The protective capacity of TprK was evaluated in two separate experiments. In the first, two TprK-immunized rabbits and two serologically nonreactive control animals were challenged with 10³ virulent treponemes by intradermal injection on their shaved backs at each of six sites. In the second experiment, identical challenges were administered to four TprK-immunized and four sham-immunized animals. Animals were examined daily to monitor the development, morphologic appearance, and progression of lesions which were scored for erythema, induration, and ulceration. Lesion aspirates were obtained at designated time points for darkfield microscopy and, in experiment two, for PCR amplification and sequence analysis of the tprK gene. At the termination of experiment one (90 d after challenge), the spleens or popliteal nodes were excised for rabbit infectivity testing (RIT; reference 23). After the development of orchitis or seroconversion, recipient animals were killed and treponemes were harvested as described above.

TprK genes were amplified with primers K1-5′/Kseq-4 or TprK5′/TprK3′ (Table ) and the Expand High Fidelity PCR system (Boehringer). PCR was performed as follows: 94°C for 2 min followed by 35 cycles of 94°C for 10 s, 65°C for 10 s, 72°C for 2 min followed by a single terminal extension for 7 min at 72°C; products were TA cloned or purified using the Concert Rapid PCR purification system (Life Technologies). All DNAs were extracted with fresh reagents in a PCR-dedicated clean room. Sequencing was performed using the primers shown in Table and with an Applied Biosystems Inc. model 377 automated DNA sequencer with the BigDye cycle sequencing kit.

The likelihood ratio test (LRT), a computer algorithm which can detect molecular clocks in phylogenetic reconstructions 2425, was applied to alignments of TprK, TprJ, and the P1 protein of Haemophilis influenzae. Phylogeny trees were constructed with the maximum likelihood algorithm in PUZZLE (v4.0.2; reference 26) with and without a clock assumption, using the Dayhoff model of substitution 27, gamma-distributed rate heterogeneity, 10,000 puzzling steps, and neighbor-joining reconstruction.

As a starting point for our study, we used limiting dilution RT-PCR to assess the transcription of tprK relative to other members of the tpr paralogous gene family and the abundantly expressed flaA gene. Before performing these experiments, we determined the amplification efficiency of each primer pair with limiting dilutions of T. pallidum DNA template. The resulting efficiency values (Table ) were used to correct the densitometric analyses of the RT-PCR products which were subsequently normalized to flaA. As shown in Fig. 1, transcripts for all tpr genes were detected, albeit at significantly lower levels than for flaA (all P values <0.001). The levels of transcript for tprs C/D, E, G, H, J, and K were not significantly different (P > 0.05), whereas transcripts for tprs A, B, F, I, and L were significantly less abundant than that for tprK (P < 0.05).

In the T. pallidum genomic sequence 5, the translational start for TprK was assigned to an ATG codon (Fig. 2 A). The predicted 505 residue polypeptide does not possess an NH₂-terminal leader peptide, a prerequisite for OM localization 16, but does contain a potential transmembrane domain at amino acids 36 through 48. Based on this sequence, the PSORT algorithm assigns TprK to the cytoplasmic membrane. Centurion-Lara et al. 7, on the other hand, proposed the GTG codon 75 bases downstream of the ATG as the protein's translational start. The resulting 480 residue protein contains a potential NH₂-terminal export signal with a signal peptidase I cleavage site, consistent with either a PP or OM location. Using this revised sequence, PSORT predicts either a PP or OM location with the former heavily favored (scores of 0.927 and 0.28 for PP and OM, respectively). Scores for known PP and OM proteins, FlaA (PP 0.783, OM 0.221) and E. coli OmpF (PP 0.381, OM 0.944), support the algorithm's predictive capacity. As the GTG start would allow TprK to potentially reside in the OM and the ATG start would preclude an OM localization, we next sought to determine the correct translational start of the protein. The low abundance of the native protein precluded obtaining NH₂-terminal sequence by automated Edman degradation. As an alternative strategy, we generated chimeras in which the tprK sequence distal to the leader peptide/transmembrane domain was fused in-frame with an alkaline phosphatase reporter construct lacking a leader peptide (19; Fig. 2). The chimeras contained decreasing lengths of upstream sequence such that the shortest insert (tprK-phoA3) began downstream of the ATG start predicted by Fraser et al. 5, but upstream of the GTG start predicted by Centurion-Lara et al. 7. Moreover, by cloning these constructs in both orientations relative to the lac promoter of pBluescript, we reasoned that we also would be able to identify any autonomously functioning TprK promoter(s). To ensure that the translation of PhoA fusions could initiate only from within TprK sequences, all three constructs contained either naturally occurring or engineered stop codons in-frame with the vector-encoded LacZ α-peptide. In the SK orientation (transcription driven by the lac promoter), all of the fusions expressed PhoA activity (Fig. 2 B), strongly supporting the translational start assigned by Centurion-Lara et al. 7 and the presence of an NH₂-terminal leader peptide. In the KS orientation (transcription driven by the native promoter), fusions TprK-PhoA1 and TprK-PhoA2 expressed PhoA activity, whereas fusion TprK-PhoA3, which contains only 15 bp of sequence upstream of the GTG codon, did not. This result indicates that the 78 bp of DNA deleted from TprK-PhoA3 relative to TprK-PhoA2 contains an autonomous promoter. Indeed, consensus −35 and −10 promoter elements with a 17-bp spacer were identified within this stretch; immediately upstream from the GTG start codon is a consensus ribosomal binding site (Fig. 2 B).

Despite the evidence that TprK is periplasmic, an examination of its protective capacity appeared to be warranted in light of the previous report of TprK-induced partial protection 7. In two independent experiments involving a total of 12 rabbits, the time course for lesion development and resolution and the gross appearance of lesions were indistinguishable between 6 TprK-immunized, and 6 control animals. Typical examples of lesion development are shown in Fig. 7. TprK immunization also had no discernible effect on the presence of spirochetes within tissues. In the first experiment, spirochetes were recovered by RIT from spleen and/or lymph nodes from one of the two TprK-immunized and one of the two unimmunized rabbits. In the second experiment, lesion aspirates from two to three sites on each animal were obtained for darkfield microscopy 24 and 31 d after challenge. On day 24, seven of eight aspirates from the TprK-immunized animals were darkfield positive as opposed to eight of eight from the sham-immunized controls. All aspirates were darkfield positive on day 31.

As noted earlier, the discovery of variant tprK genes among geographically disparate T. pallidum strains and within some treponemal subpopulations has fueled speculation that host immune responses select for TprK sequence variants 89. If this were the case, then TprK “escape variants” should be overrepresented among treponemes recovered from TprK-immunized animals. To examine this issue, we PCR amplified and sequenced tprK genes from our challenge strain (Nichols-Farmington), the isolates recovered by RIT in experiment one, and the spirochetes in the lesion aspirates from experiment two. As described in Materials and Methods, extensive precautions were taken to avoid PCR artifacts which would confound this analysis. As is the case for all TprK proteins reported to date, the Nichols-Farmington TprK was not an exact match for any other polypeptide in the databases (Fig. 8 A, and not shown). However, it was most similar to the Nichols-Seattle and Nichols-Houston TprKs with 97% amino acid identity to each. The 12 amino acid differences (10 substitutions and 2 deletions) between the Farmington and Seattle orthologs are located in four of the seven recently described TprK variable regions (i.e., V1, V3, V6, and V7; Fig. 8 A; references 8 and 9). The amino acid identity of the Nichols-Farmington TprK with the remaining orthologs in the databases ranged from 87 to 91% with the differences mapping to the seven variable regions. It is noteworthy that the PSORT algorithm heavily favors a PP location for all TprK orthologs (Fig. 8 A and data not shown). Downstream of the tprK coding region, the Nichols-Farmington strain contained the same 67-bp deletion reported for the Nichols-Seattle and Bal7 strains but which is absent from all other strains examined thus far (data not shown; references 8 and 9). In contrast to the heterogeneity among the geographically distinct TprK proteins, the TprK sequences of treponemes recovered from five different TprK-immunized animals were identical to that of Nichols-Farmington (Fig. 8 B). The sole sequence variant, with two amino acid changes in region V7, was recovered from an unimmunized control rabbit (Fig. 8 B).

As the above results suggested that tprK is not subject to immunological pressure, we next used the LRT to determine whether the variation among TprK sequences is consistent with evolutionary drift. LRT, a phylogenetic tool used in molecular evolutionary biology, can determine whether variation among related sequences has occurred at a constant or at a discontinuous rate 2425. The hypothesis underlying our LRT analysis of TprK was that proteins subject to immunological pressure would show a discontinuous rate of change and therefore not display a molecular clock. To assess this hypothesis, we tested for the presence of molecular clocks in TprK, TprJ, and the H. influenzae P1 protein. TprJ is predicted by PSORT to be anchored to the cytoplasmic membrane via an uncleaved leader peptide 57; a protein sequestered in this manner should not be subject to immunological pressure. In this regard, TprJ has been shown to be stable in T. pallidum Nichols for >1 yr despite repeated passage in rabbits 30. P1 is an OM protein with surface-exposed hypervariable domains which are known to be subject to immunological selection 31. Consistent with our hypothesis, the molecular clock was rejected for the immunologically driven variation of P1 (Table ). Both TprJ and TprK displayed a molecular clock (Table ) indicating constant rates of change consistent with evolutionary drift.