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Opsonic Potential, Protective Capacity, and Sequence Conservation of the Treponema pallidum subspecies pallidum Tp92

  1. Caroline E. Cameron,
  2. Sheila A. Lukehart,
  3. Christa Castro,
  4. Barbara Molini,
  5. Charmie Godornes and
  6. Wesley C. Van Voorhis
  1. Department of Medicine, University of Washington, Seattle
  1. Reprints or correspondence: Dr. Wesley C. Van Voorhis, Dept. of Medicine, University of Washington, 1959 Pacific Ave. NE, Box 357185, Seattle, WA 98195 (wesley{at}u.washington.edu).
 
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Abstract

By means of a differential screening technique, a 92-kDa antigen, designated Tp92, was identified from Treponema pallidum subspecies pallidum. This protein is similar in sequence to the protective surface antigens D15 from Haemophilus influenzae and Oma87 from Pasteurella multocida. Amino acid sequence analyses revealed a cleavable N-terminal signal sequence and predicted the outer membrane location for Tp92. In support of this, antiserum raised against recombinant Tp92 promotes opsonization and phagocytosis of T. pallidum by rabbit macrophages, and anti-Tp92 reactivity is absent from washed treponemal preparations presumed to be lacking outer membranes. The Tp92 amino acid sequence is 95.5%–100% conserved among 11 strains representing 4 pathogenic treponemes, and immunization with recombinant Tp92 partially protected rabbits from subsequent T. pallidum challenge. These results demonstrate that Tp92 is an invariant, immunoprotective antigen that may be present on the surface of T. pallidum and may represent a potential vaccine candidate for syphilis.

Infectious syphilis, caused by the spirochete bacterium Treponema pallidum subspecies pallidum, remains a public health concern worldwide. In 1995, the World Health Organization's global estimate of syphilis prevalence was 12 million new cases, the majority of which occurred in developing nations [1]. A rapid increase in the incidence of syphilis has been observed in parts of the former Soviet Union, with 50-fold more cases reported in 1996 than in 1990 [2]. Syphilis also is prevalent in the southern region of the United States, where focal syphilis epidemics occur periodically [3, 4]. In the absence of appropriate antibiotic treatment, T. pallidum subsp. pallidum establishes a lifelong chronic infection that may progress to the debilitating and potentially fatal tertiary disease in about one-third of infected persons. Apart from the serious nature of the disease itself, a number of studies suggest that syphilis may increase the risk of acquisition and transmission of human immunodeficiency virus (HIV) [5–8]. In addition, syphilis may be more difficult to eradicate from HIV-infected persons, thus increasing the direct morbidity and mortality associated with treponemal infections [9–11].

The failure of public health measures to control syphilis worldwide and the increased potential for HIV transmission emphasize the urgent need for a syphilis vaccine. To date, complete protection against syphilis has been demonstrated only in experimental animals, with impractical immunization protocols involving gamma-irradiated treponemes [12]. Partial protection has been achieved in experimental animals by immunizing them with antiformin-treated treponemes [13], 4°C “aged” treponemes [14], or several recombinant or native T. pallidum subsp. pallidum proteins, including protein 4D [15], purified endoflagella [16], and TmpB [17]. Recent investigations conducted in our laboratory have identified 2 additional recombinant antigens that provide significant protection against experimental syphilis; these are Tpr K, one of the members of the recently described T. pallidum repeat (Tpr) family of molecules [18–21], and the enzyme glycerophosphodiester phosphodiesterase (Gpd) [22, 23]. Antigens that confer complete protection against infection have yet to be discovered, and successful vaccination regimens against syphilis may involve concurrent vaccination with promising immunoprotective antigens as part of a vaccine cocktail.

The primary clearance mechanism responsible for removal of T. pallidum from syphilitic chancres is believed to be antibody-mediated treponemal opsonization and subsequent phagocytosis and killing by macrophages. In support of this, antibody has been demonstrated to be required for phagocytosis of treponemes by macrophages in vitro [24] and for macrophage-mediated killing of T. pallidum [25]. In addition, the systemic appearance of opsonic antibody has been shown to immediately precede bacterial clearance in the experimental rabbit model [26]. These observations demonstrate the importance of identifying the target antigens of opsonic antibody, because they will be central to T. pallidum elimination from primary syphilis lesions and therefore may be immunoprotective in nature.

The T. pallidum target antigens of opsonic antibody are presumed to reside on the treponemal surface, thus allowing opsonic antibodies to bind to intact T. pallidum and phagocytosis to proceed. Therefore, the identification of these target antigens will simultaneously allow identification of T. pallidum outer membrane proteins. The definitive description of such proteins has been technically difficult due to the fragile nature of the T. pallidum outer membrane, the low density of outer membrane-spanning proteins [27, 28], and the presence of a cell surface—masking slime layer of host and/or bacterial origin [29]. To date, the most compelling candidates for proteins exposed on the T. pallidum surface are some of the Tpr family of molecules [18–21]. These are the encoded protein products of a family of 12 related genes that share sequence similarity with the major surface protein of T. denticola [30]. One member of this gene family, tprK, encodes a protein that is a target of opsonic antibodies, thus suggesting that this protein is exposed at the surface of the treponeme [18]. Other putative T. pallidum surface proteins that have been described include Gpd [22, 23] and T. pallidum rare outer membrane proteins (Tromps) 1 [31] and 2 [32], although controversy exists as to whether Gpd [33] and Tromp1 [34] actually reside on the outer leaflet of the treponeme outer membrane, and the cellular location of Tromp2 has not been independently confirmed. The recent release of the T. pallidum genome sequence [20] has facilitated identification of open-reading frames (ORFs) encoding additional putative outer membrane proteins; however, confirmatory biologic experiments need to be performed to ensure that these proteins truly reside on the bacterial surface.

To directly identify putative T. pallidum outer membrane proteins that are the targets of opsonic antibody, we made use of the fact that rabbit macrophages phagocytize T. pallidum in vitro, using antiserum from T. pallidum—infected rabbits as a source of opsonizing antibody (opsonic rabbit serum; ORS) [24]. In contrast, antiserum from rabbits immunized with heat-killed T. pallidum fails to opsonize T. pallidum (nonopsonic rabbit serum; NORS; S. A. Lukehart, unpublished data). This observed differential reactivity was exploited to identify opsonic antibody target antigens by immunologically screening a T. pallidum λ genomic expression library in duplicate with either ORS or NORS. This differential immunologic screen was used to successfully identify 2 T. pallidum ORS-reactive antigens, Gpd [22, 23] and Tpr K [18], which partially protect rabbits from subsequent T. pallidum challenge and are putative outer membrane proteins. Here we report the identification of a third ORS-reactive antigen selected by the differential expression library screen. This molecule, designated the T. pallidum Tp92 (T. pallidum antigen, 92 kDa), is a sequence-invariant putative outer membrane protein that is the target of opsonic antibodies and induces a protective immune response.

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Materials and Methods

Bacterial strains

All treponemal strains were propagated in New Zealand White rabbits as described elsewhere [35]. T. pallidum subsp. pallidum, Nichols strain, was originally sent to the University of Washington by J. N. Miller (University of California, Los Angeles) in 1979, and T. pallidum subsp. pertenue, Gauthier strain, was supplied by P. Perine (Centers for Disease Control, Atlanta) in 1981. T. pallidum subsp. pallidum (Bal-2, Bal-3, Bal-7, and Bal 73-1 strains), T. paraluiscuniculi (Cuniculi A strain), T. pallidum subsp. pertenue (Haiti B strain), and the simian isolate were supplied by P. Hardy and E. Nell (Johns Hopkins University, Baltimore). T. pallidum subsp. pallidum, Sea 81-3 and Sea 83-1 strains, were isolated by S. A. Lukehart from the cerebrospinal fluid of patients with untreated syphilis. Escherichia coli XL-1 Blue, SolR, and BL21 (DE3) pLysS strains were obtained from Stratagene (La Jolla, CA).

Expression library screening

The T. pallidum subsp. pallidum tp92 gene from the Nichols strain was identified by differentially screening a T. pallidum genomic expression library, as described elsewhere [22]. Briefly, the library was differentially screened with a T. pallidum—specific immune rabbit serum depleted of activity against the major known treponemal antigens but still retaining its opsonic capacity (ORS) and with a nonopsonic antiserum prepared by using heat-killed (63°C for 1 h) T. pallidum (NORS) [22].

Polymerase chain reaction (PCR) amplification of tp92 from pathogenic treponemes

The Tp92 coding sequence was PCR-amplified from genomic DNA isolated from 11 strains of pathogenic treponemes. To obtain the entire ORF, primers were designed from the 5′ and 3′ noncoding regions flanking the tp92 gene (primers 1 and 2, respectively, table 1). These primers are located 55 bp upstream and 49 bp downstream, respectively, of the tp92 ORF. PCR amplification of tp92 was performed as described elsewhere [36]. The PCR reaction conditions were as follows: 30 cycles of 1 min of denaturation at 94°C, 1 min of annealing at 60°C, and 2 min of extension at 74°C for the Bal 73-1, Bal-2, Bal-3, Bal-7, Sea 81-3, Sea 83-1, Haiti B, Nichols, and simian templates; 35 cycles of 1 min of denaturation at 94°C, 1 min of annealing at 55°C, and 2 min and 30 s of extension at 74°C for the Gauthier template; and 35 cycles of 1 min of denaturation at 94°C, 1 min of annealing at 60°C, and 2 min and 30 s of extension at 74°C for the Cuniculi A template.

Figure 1
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Figure 1

Comparison of the predicted serine-rich sequence of Tp92 from various pathogenic treponemes. Amino acid residues are numbered according to their order in Tp92 sequences from Treponema pallidum subsp. pallidum strains. *, Sequence identities; ·, sequence similarities; —, sequence deletions. Boxed regions represent sequences missing from the Tp92 sequence of the corresponding pathogenic treponeme compared with that from T. pallidum subsp. pallidum strains. The double-boxed region in T. paraluiscuniculi represents the missing serine-rich sequence that has been replaced by an alternative stretch of amino acids, compared with the Tp92 sequences from T. pallidum subsp. pallidum strains.

Figure 2
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Figure 2

Overexpression of recombinant Treponema pallidum Tp92 and analysis of anti-Tp92 immunoreactivity. A, Coomassie blue-stained SDS-PAGE analysis of lysates of isopropyl β-d-thiogalactopyranoside-induced (lane 1) and uninduced (lane 2) Escherichia coli BL21 (DE3) pLysS expressing the intact open-reading frame tp92-pRSETc construct and nickel chromatography-purified recombinant Tp92 (lane 3). B, Immunoblot analysis of anti-Tp92 (lane 1) and preimmune (lane 2) sera on nickel-purified recombinant Tp92. Coomassie blue-stained and immunoreactive protein bands of molecular mass <70 kDa present in A (lane 3) and B (lane 1), respectively, presumably represent breakdown products of the 70-kDa recombinant protein. Lanes contain 5 μg of total bacterial lysate or 2 μg of purified recombinant protein; molecular mass standards (kDa) are at left. Arrow, recombinant T. pallidum Tp92.

Figure 3
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Figure 3

Investigation of expression of recombinant Tp92. A, Schematic diagram of sections of the tp92 open-reading frame (ORF), represented by each of the cloned tp92 gene fragments. For each Tp92 protein the expected molecular mass is given in kilodaltons, and the hexahistidine tag and serine-rich sequence are indicated by “H” and “S,” respectively, in lightly shaded boxes. B, Coomassie blue-stained SDS-PAGE analysis of expressed Tp92 fragments. Each lane contains 10 nmol of the following recombinant proteins: lane 1, SA85-1.1-III control hexahistidine recombinant protein used for adsorption of anti-Tp92 serum; lane 2, intact ORF recombinant Tp92; lane 3, Tp92 fragment 1; lane 4, Tp92 fragment 2; lane 5, Tp92 fragment 3; lane 6, Tp92 fragment 4; lane 7, Tp92 fragment 5; and lane 8, Tp92 fragment 6. C, Immunoblot analysis showing anti-Tp92 immunoreactivity against 10 nmol of each expressed Tp92 fragment; lane assignment is the same as in B. Immunoreactivity was detected only against intact ORF recombinant Tp92 and Tp92 fragments 1 and 6. This suggests that portions of the tp92 ORF covered by fragments 2–5 were either not expressed or expressed at a very minimal level in intact ORF recombinant Tp92. For panels B and C, Coomassie blue—stained and immunoreactive protein bands of molecular mass less than the predicted mass of the expressed recombinant molecule presumably represent breakdown products of that recombinant molecule. Molecular mass standards (kDa) are at left.

Figure 4
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Figure 4

Immunoblot analysis of anti-Tp92 immunoreactivity on Treponema pallidum lysates. Antiserum to recombinant T. pallidum Tp92 was used to probe a lysate of unwashed T. pallidum (lane 1) and lysates of T. pallidum washed 1 (lane 2) and 3 (lane 3) times. Lanes 4 and 5 show the level of reactivity of control preimmune serum and anti-rabbit IgG secondary antibody alone, respectively, on unwashed T. pallidum lysate. Each lane contains 1.4 × 107 treponemes; molecular mass standards are at left (kDa). Arrow, T. pallidum Tp92. The strongly immunoreactive band at ∼55 kDa represents the heavy chain of rabbit antibody molecules that are contaminating the T. pallidum lysate preparations and are detected by anti-rabbit IgG secondary antibody.

Figure 5
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Figure 5

Opsonic potential of recombinant Treponema pallidum Tp92. Shown are the percentage of rabbit peritoneal macrophages phagocytosing T. pallidum (Tp) after 4-h incubation with 1 : 100 dilution of either immune rabbit serum (IRS), normal rabbit serum (NRS), or anti⪯T. pallidum Tp92 polyclonal antiserum. Two separate experiments were performed to test for opsonization, with a total of 6 replicate assays per condition. Error bars show SEs; P compares immune rabbit serum or anti-Tp92 with normal rabbit serum (Student's t test).

Figure 6
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Figure 6

Immunoprotective capacity of recombinant Treponema pallidum Tp92 in protection experiment I. Shown are representative rabbits from the control unimmunized group (A) and the recombinant Tp92-immunized group (B). The control unimmunized rabbit developed typical red, raised, highly indurated lesions that progressed to ulceration. The Tp92-immunized rabbit developed atypical pale, flat, slightly indurated, nonulcerative lesions. Black ink spots are adjacent to the intradermal challenge sites. Photographs were taken on day 25 after challenge.

Table 1
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Table 1

Primers specific for Treponema pallidum tp92.

Overexpression studies

The ORF encoding Tp92 was PCR-amplified from T. pallidum subsp. pallidum (Nichols strain) genomic DNA with primers designed from the 5′ (primer 3, table 1) and 3′ (primer 4, table 1) ends of the Tp92-coding region. This DNA sequence extends from bp 74 to bp 2514 of the 2514-bp tp92 ORF, and the recombinant protein expressed from this construct is hereafter referred to as intact ORF recombinant Tp92. To ensure optimal expression of the recombinant molecule within E. coli, the DNA sequence encoding the N-terminal 25 amino acids, which includes the predicted signal sequence, was excluded from the primer design and, thus, from the resulting expressed recombinant molecule. Fragments of the tp92 ORF were PCR-amplified with the following primer sets (table 1): fragment 1, bp 74–1776, primers 5 (5′) and 6 (3′); fragment 2, bp 1778–2514, primers 7 (5′) and 8 (3′); fragment 3, bp 1778–2292, primers 9 (5′) and 10 (3′); fragment 4, bp 2243–2514, primers 11 (5′) and 12 (3′); fragment 5, bp 2351–2514, primers 13 (5′) and 14 (3′); and fragment 6, bp 74–2292, primers 15 (5′) and 16 (3′). PCR amplification for each primer set was performed as described elsewhere [36], with 30 cycles of 1 min of denaturation at 94°C, 1 min of annealing at 60°C, and 2 min of extension at 74°C. After PCR, the amplification products were digested with BamHI and EcoRI, ligated to a similarly digested pRSETc T7 expression vector (Invitrogen, Carlsbad, CA) [37], and transformed first into E. coli XL-1 Blue and then into the E. coli expression strain BL21 (DE3) pLysS. We verified the reading frame and sequence of the expression constructs by DNA sequencing, using the T7 promoter primer (Pharmacia, Piscataway, NJ) and internal primers designed from the tp92 nucleotide sequence (primers 17–21, table 1). Expression of the Tp92 constructs was performed by using 500 mL of LB broth seeded with 50 mL of E. coli (OD at 600 nm of 0.6) transformed with each of the tp92-pRSETc constructs. Recombinant expression was induced by 0.4 mM isopropyl β-D-thiogalactopyranoside during log-phase growth. Bacteria were harvested by centrifugation and lysed in the presence of protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin A; all from Sigma, St. Louis). Inclusion bodies containing each of the recombinant proteins were isolated and solubilized in 6 M urea, and the recombinant 6-histidine—containing proteins were purified by nickel chromatography [37]. An irrelevant hexahistidine-tagged recombinant protein, SA85-1.1-III from Trypanosoma cruzi, was also expressed to use as a negative control and has been described elsewhere [38]. Protein quantitation of each of the recombinant proteins was performed with the BCA Protein Assay kit (Pierce, Rockford, IL).

DNA sequencing

Double-stranded plasmid DNA was extracted with the Plasmid Mini kit (Qiagen, Chatsworth, CA), and both strands of insert DNA were sequenced with a dye terminator sequencing kit (Applied Biosystems, Foster City, CA) and a DNA sequencer (ABI 373A; Applied Biosystems) in accordance with the manufacturer's instructions.

DNA and protein sequence analyses

Nucleotide sequences were translated and analyzed with Sequencher, version 3.1RC4, sequence analysis software (Gene Codes, Ann Arbor, MI). Database searches were performed by means of the basic local alignment search tool (BLAST) algorithm [39] and the blastn, blastx, or blastp program. The published T. pallidum genome [20] was used to obtain the complete tp92 ORF and the corresponding noncoding flanking regions. Protein and DNA sequences were aligned with the Clustal W general-purpose multiple-alignment program [40]. The percentages of positional identity and similarity between sequences were calculated from the number of identical or similar residues, respectively, between aligned sequences; insertions and deletions were not scored. For the predicted amino acid sequence of Tp92 and the homologue of this molecule from other organisms, the molecular mass was calculated with the Compute pI/MW Tool. For the T. pallidum Tp92, transmembrane topology analysis was performed with the TMpred program, and signal sequence and cellular location predictions were made with the PSORT program.

Antisera

Immune rabbit serum was collected from rabbits infected with T. pallidum for >90 days. Anti-Tp92 polyclonal serum was raised in New Zealand White rabbits by immunizing 5 times with 125 μg each of the purified intact ORF recombinant Tp92 emulsified in the Ribi adjuvant system: monophosphoryl lipid A + trehalose dicorynomycolate + cell wall skeleton (Sigma). Intradermal, subcutaneous, intramuscular, and intraperitoneal immunizations were administered at 3-week intervals, as outlined by the Ribi adjuvant system, and antiserum was collected at the time of administration of the final immunization. To remove any immunoreactivity directed against the 6-histidine tag present at the N-terminus of all the recombinant proteins, an immunoaffinity column was prepared by coupling the control SA85-1.1-III recombinant protein to cyanogen bromide—activated sepharose 4B (Sigma) by means of conventional techniques [41]. IgG was purified from the anti-Tp92 polyclonal serum (ImmunoPure [A] IgG Purification Kit; Pierce) and was subjected to 2 sequential passages over the SA85-1.1-III—sepharose 4B column to adsorb out all antibodies directed against the histidine tag from the anti-Tp92 IgG preparation [41]. Elution fractions (500 μL each) were collected, and protein was quantified by the BCA assay.

Opsonization assay

Immune rabbit serum, normal rabbit serum, and anti-Tp92 polyclonal serum were tested in 2 separate experiments, with a total number of 6 replicate assays per serum, for their ability to opsonize T. pallidum by means of a standard phagocytosis assay, as described elsewhere [42]. All antisera were used at a 1 : 100 dilution and were incubated for 4 h with rabbit peritoneal macrophages and viable, unwashed T. pallidum before determination of the percentage of macrophages phagocytosing treponemes. Statistical analysis was performed by 2-tailed Student's t test.

PAGE and immunoblot analyses

SDS-PAGE and immunoblotting were performed as described elsewhere [43], except that samples were blotted to Immobilon-polyvinylidene fluoride membrane (Millipore, Bedford, MA). All immunoblots were blocked with 5% milk powder in Tris-buffered saline with 0.1% Tween 20, all primary and secondary antibodies were diluted in 5% milk powder in Tris-buffered saline, and all immunoblots were developed with BCIP (5-bromo-4-chloro-3-indolyl phosphate)— Nitro Blue Tetrazolium color substrate detection (Promega, Madison, WI). High-range molecular weight markers (Rainbow; Amersham, Cleveland) were used as standards. Heterologous expression of the intact ORF recombinant Tp92 and the recombinant Tp92 fragments was monitored by SDS-PAGE analysis of 5 μg of total bacterial lysate or 2 μg of purified recombinant protein and subsequent staining with Coomassie blue R-250. Ten-nanomole fractions of the purified recombinant Tp92 fragments were electrophoresed, followed by staining with Coomassie blue R-250, to allow equimolar comparison of the expressed recombinant Tp92 fragments. The level of immunoreactivity of either immune rabbit serum or anti-Tp92 polyclonal serum on purified intact ORF recombinant Tp92 was assayed by electrophoresis and blotting of 2 μg of purified recombinant protein and probing with a 1 : 200 dilution of immune rabbit serum or anti-Tp92 polyclonal serum, respectively, followed by a 1 : 3000 dilution of alkaline phosphatase—labeled goat anti-rabbit IgG (Fc; Promega). The level of immunoreactivity of anti-Tp92 polyclonal serum on the recombinant Tp92 fragments was analyzed by electrophoresis and blotting of 10 nmol of either the intact ORF recombinant Tp92, the Tp92 peptide fragments, or the control SA85-1.1-III recombinant protein, followed by probing with the SA85-1.1-III-adsorbed anti-Tp92 purified IgG at a concentration of 2.25 μg/mL and a 1 : 3000 dilution of alkaline phosphatase—labeled goat anti-rabbit IgG (Fc). for analysis of the level of immunoreactivity of anti-Tp92 serum on washed and unwashed treponemes, T. pallidum was extracted from infected testes as described elsewhere [24] and either immediately resuspended in SDS-PAGE sample buffer (unwashed preparation) or washed 1 time or 3 times with 10 mM Tris-HCl, pH 7.5, by centrifugation (15,000 g) before resuspension of the treponemes in sample buffer. Approximately 1.4 × 107 T. pallidum were electrophoresed for each sample (unwashed, washed 1 time, washed 3 times), blotted, and probed with a 1 : 200 dilution of anti-Tp92 polyclonal rabbit serum followed by a 1 : 3000 dilution of alkaline phosphatase-labeled goat anti-rabbit IgG (Fc).

Protection experiments

New Zealand White rabbits were immunized (intramuscularly, subcutaneously, intraperitoneally, and intradermally) at 3-week intervals with Ribi adjuvant and 125 μg of purified intact ORF recombinant Tp92 in 3 independent protection experiments. In protection experiments I and III, 1 and 3 rabbits, respectively, were immunized a total of 5 times, and in protection experiment II, 3 rabbits were immunized a total of 3 times. At 1–4 weeks after administration of the final immunization in each protection experiment (experiment I, 4 days; experiment II, 13 days; experiment III, 25 days), the immunized rabbits and a total of 4 unimmunized control rabbits (corresponding to a single control rabbit in protection experiments I and II and 2 control rabbits in protection experiment III) were subjected to intradermal challenge at each of 8 sites on their shaved backs with 105 T. pallidum subsp. pallidum (Nichols strain) per site. The rabbits were examined daily to monitor the development, morphologic appearance, and progression of lesions appearing at the challenge sites. Lesion development was designated for each individual rabbit as “typical” if lesions were red, raised, and indurated and generally progressed to ulceration and as “atypical” if lesions were pale, flat, only slightly indurated, and generally nonulcerative. Before lesion ulceration on the control animals (days 18, 19, and 24 after challenge for protection experiments I, II, and III, respectively), lesion aspirates were collected from challenge sites on all animals and examined by darkfield microscopy for viable treponemes. After challenge, rabbits were assayed for serologic evidence of infection at monthly intervals by VDRL and fluorescent treponemal antibody absorption tests. Statistical analyses were performed by analysis of variance with repeated measures.

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Results

Identification of T. pallidum subsp. pallidum tp92

A λ T. pallidum subsp. pallidum genomic expression library was constructed and screened with a T. pallidum—specific opsonic antiserum preparation [22]. To aid in distinguishing plaques specifically reacting with opsonic antibodies from background immunoreactive plaques, duplicate plaque lifts were differentially screened with a T. pallidum—specific nonopsonic antiserum. Plaques exhibiting consistent immunoreactivity with the opsonic antiserum but no immunoreactivity with the nonopsonic antiserum on the primary and secondary screens were selected for further study and subjected to tertiary screening to obtain well-isolated plaques.

In vivo excision of 1 immunoreactive plaque produced a pBluescript phagemid containing an insert of ∼3.0 kb, as shown by restriction digest analysis (data not shown). Nucleotide sequence analysis of the insert revealed a 2439-bp ORF encoding a predicted 812—amino acid translated product that was fused in-frame with the λ ZAP II vector. Comparison of the insert sequence with an early version (July 1997; before gene annotation) of the released T. pallidum genome sequence (http://mmg.uth.tmc.edu/treponema/tpall.html) identified 75 bp present in the genome sequence that were missing from the 5′ end of the insert sequence of the immunoreactive clone. This DNA sequence was downstream from a putative ribosome-binding site (GGAGAA) and thus was presumed to correspond to the 5′ end of the ORF and to encode the N-terminal 25 amino acids of the translated protein product. PSORT analysis (http://psort.nibb.ac.jp:8800) of the complete 837-residue translated protein predicts a 21—amino acid cleavable N-terminal signal sequence and an 84.6% likelihood that this putative protein is located in the T. pallidum outer membrane. The mature translated protein, lacking the 21-residue signal sequence, has a predicted molecular mass of 92,040 Da. This translated protein was thus designated Tp92. The nucleotide sequence of tp92 from the Nichols strain of T. pallidum is available from EMBL/GenBank/DDBJ (accession no. AF042789).

Subsequent release of the completed T. pallidum genome identified a putative ORF corresponding to tp92 between bp 344,276 and 346,837 of the genome (ORF TP0326; GenBank, AE001212) [20]. This 2562-bp ORF predicts a slightly larger translated protein than that predicted by our tp92 ORF, encoding an extra 16 amino acids at the N-terminus. This discrepancy arises because of the assignment of an alternative initiator methionine for the genome's TP0326 ORF. The genome sequence assigns the methionine start codon for TP0326 at bp 344,276, whereas our methionine start codon corresponds to bp 344,324 within TP0326. PSORT analysis on the genome's 853-residue translated protein product of ORF TP0326 predicts a protein with no N-terminal signal sequence that is located in the bacterial inner membrane. Conversely, our 837-residue translated protein product contains a cleavable N-terminal signal sequence and is predicted to be located in the bacterial outer membrane. Two lines of evidence suggest that our prediction of the start codon for the tp92 ORF is correct. First, upstream of the methionine codon at bp 344,276 there is no discernible ribosome-binding site, in contrast to the putative ribosomebinding site upstream of the methionine codon at bp 344,324. Second, the predicted molecular mass of our 837-residue T. pallidum Tp92 more closely matches the masses predicted or observed for most of the Tp92 homologues found in other bacterial organisms (see below).

Sequence analyses

As shown in table 2, sequence database analysis using the blastp algorithm [39] revealed that the T. pallidum Tp92 shares the highest degree of sequence similarity with a putative outer membrane protein identified by genome sequencing of the related spirochete, Borrelia burgdorferi (28.1% identical, 44.7% similar) [44]. The T. pallidum Tp92 also shares about equal levels of sequence similarity with high-molecular-weight outer membrane proteins identified from a large variety of bacterial species (18.6%–22.1% identical, 35.1%–40.9% similar). The observed sequence similarity within this group of bacterial proteins is evenly distributed throughout the coding sequence of Tp92, with the exception of a serine-rich region at the C-terminal end of the translated protein (S3-A-S11-R-T2-S2) that is unique to the T. pallidum Tp92. The presence of transmembrane segments within Tp92 was analyzed with the TMPred program (http://ulrec3.unil.ch/software/TMPRED), resulting in the prediction of 3 transmembrane helices (data not shown). In this putative model, the C-terminal serine-rich stretch of Tp92 is predicted to be located within an external loop on the outer face of the outer membrane.

Table 2
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Table 2

Sequence comparison of Treponema pallidum Tp92 and putative outer membrane proteins from a variety of bacterial species.

Sequence conservation of Tp92 among pathogenic treponemes

To assess the degree of sequence conservation of Tp92 among pathogenic treponemes, the tp92 ORF was PCR-amplified and subsequently sequenced from 6 additional T. pallidum subsp. pallidum strains, 2 T. pallidum subsp. pertenue strains (causative agent of the disease yaws), 1 T. paraluiscuniculi strain (causes venereal syphilis in rabbits), and the simian isolate (obtained from skin lesions of a monkey) [48]. The overall percentage of sequence conservation for each of these strains, compared with the T. pallidum subsp. pallidum Nichols strain, is summarized in table 3. The amino acid sequence of Tp92 is highly conserved, with a range of 95.5%–100% identity and 96.8%–100% similarity shared between the Nichols strain Tp92 sequence and that of the various other pathogenic treponeme strains. However, several of the amino acid sequence changes that do exist are of particular interest. First, the finding that the Haiti B strain, which is reportedly a T. pallidum subsp. pertenue strain, shows sequence identity with the pallidum subspecies and not with the nonsyphilis strains supports the proposal by Centurion-Lara et al. [49] that this strain was misidentified and should be classified as a T. pallidum subsp. pallidum strain. Similar sequence analysis on the gpd sequence from the Haiti B strain [36] further supports its identification as a T. pallidum subsp. pallidum strain. Second, comparison of the entire Tp92 amino acid sequences from the various pathogenic treponemes shows 8 shared amino acid alterations in the Bal-2 and Sea 81-3 strains, thus suggesting a common origin for these 2 strains. Similarly, comparison of the Tp92 amino acid sequences from the Gauthier and simian strains shows 5 shared amino acid alterations in these 2 strains, also suggesting that these 2 strains originated from a common strain source. Third, a distinctive sequence deletion pattern is present in the Tp92 sequences from non—T. pallidum subsp. pallidum strains. As illustrated in figure 1, the tp92 genes of the Gauthier and simian strains have bp 2336–2350 deleted, which corresponds to deletion of aa 780–784 that comprise the end of the Tp92 signature serine-rich region. The tp92 gene sequence of the Cuniculi A strain possesses an additional complexity, in that bp 2293–2352, which encode the characteristic serine stretch comprising aa 765–784, are deleted. This DNA sequence is replaced with 30 bp that encode an alternative 10 amino acids that, although serine rich, represent a minimal serine content compared with that of the same stretch of amino acids in the other treponeme strains. All DNA sequence deletions are in-frame and do not introduce premature termination codons into the tp92 ORF. The nucleotide sequences of the tp92 genes from Bal 73-1, Bal-2, Bal-3, Bal 7, Sea 81-3, Sea 83-1, Haiti B, Gauthier, simian, and Cuniculi A strains are available from EMBL/GenBank/DDBJ (AF152007—AF152016, respectively).

Table 3
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Table 3

Summary of Tp92 sequence conservation among pathogenic treponemes.

Overexpression of the T. pallidum Tp92

Heterologous expression of the tp92 ORF in E. coli BL21 (DE3) pLysS by means of the isopropyl β-d-thiogalactopyranoside-inducible pRSETc T7 expression system resulted in production of a recombinant molecule with a molecular mass of ∼70 kDa (figure 2A, lane 1). Expression of the 70-kDa recombinant protein was significantly decreased in E. coli lysates in which protein expression from the pRSETc T7 promoter had not been induced by isopropyl β-d-thiogalactopyranoside addition (lane 2). Nickel resin chromatography of E. coli expressing the tp92-pRSETc construct allowed purification of the histidine-tagged recombinant molecule away from contaminating E. coli proteins (lane 3). The 70-kDa protein reacted with rabbit sera from T. pallidum—infected rabbits but not sera from uninfected rabbits (data not shown). This demonstrates that the 70-kDa recombinant protein is a T. pallidum antigen that is recognized during infection. In addition to the recombinant 70-kDa molecule, peptides of a smaller molecular mass were present in the nickel-purified preparation and presumably represent breakdown products of the 70-kDa recombinant Tp92.

The recombinant T. pallidum Tp92 was used to generate polyclonal antiserum, and subsequent immunoblot analysis showed an immunoreactive 70-kDa protein in both the nickel-purified recombinant protein preparation (figure 2B, lane 1) and lysates of E. coli expressing the tp92-pRSETc construct (data not shown). No corresponding immunoreactive protein was observed when either control preimmune serum on the nickelpurified recombinant protein preparation (lane 2) or the anti-Tp92 serum on preparations of E. coli expressing the pRSETc vector alone (data not shown) were tested.

The 70-kDa molecular mass of the recombinant protein is unexpectedly lower than the 97-kDa molecular mass predicted for the histidine-tagged recombinant molecule (92 kDa for the T. pallidum Tp92 plus 5 kDa extra for the N-terminal hexa-histidine tag). This low molecular mass is not the result of truncated PCR amplification of the tp92 ORF, because sequencing of the tp92-pRSETc construct verified that the entire 2439-bp insert encoding the 812-residue translated protein product was present without mutations. Instead, several alternative explanations may exist for the lower-than-expected molecular mass of the recombinant Tp92. First, the Tp92 amino acid sequence contains a high proportion of hydrophobic residues and thus may have a tendency to bind more SDS and have a higher electrophoretic mobility. This phenomenon has also been observed with several other hydrophobic proteins, including the lac permease from E. coli [50] and ISG100 from Trypanosoma brucei [51]. Second, despite the addition of a protease inhibitor cocktail to the recombinant bacterial pellet before lysis, the 70-kDa molecular mass of the recombinant Tp92 may be the result of proteolytic cleavage. In this case, the protein bands of a lower molecular mass would represent proteolytic fragments cleaved from the recombinant Tp92. Lastly, evidence suggests that foreign genes containing a stretch of consecutive serine codons may be poorly expressed in E. coli. Although the exact mechanism of inefficient expression is not understood, the level of protein expression has been found to be inversely proportional to the number of serine codons present [52].

To investigate which portion of the Tp92 molecule had been expressed in the intact ORF recombinant protein, 6 peptide fragments representing the entire amino acid sequence of Tp92 were expressed in E. coli. Figure 3A delineates the sections of the tp92 ORF covered by each of the cloned tp92 gene fragments. SDS-PAGE and subsequent Coomassie blue staining of the nickel-purified recombinant Tp92 fragments verified that peptide fragments of the expected molecular mass had been expressed (figure 3B). To determine which regions of the Tp92 amino acid sequence had in fact been expressed in the intact ORF recombinant Tp92, the level of immunoreactivity of antiserum generated against the intact ORF recombinant Tp92 on these fragments was investigated by immunoblot analysis. As shown in figure 3C, the anti-Tp92 serum was reactive against the intact ORF recombinant Tp92 (lane 2), as well as Tp92 fragments 1 (lane 3) and 6 (lane 8). No detectable reactivity was observed against Tp92 fragments 2–5 (lanes 4–7, respectively). These results suggest that the majority of protein product expressed from the tp92-pRSETc construct consists of only the first ∼567 amino acids out of the expected 812 amino acid residues predicted for the intact ORF recombinant Tp92, thus resulting in a recombinant molecule of ∼70 kDa. Although a small amount of the full-length 97-kDa recombinant Tp92 may have been produced in these expression studies, the results of figure 3C suggest that this amount is minimal and below the detection limit of our immunoassay.

Characterization of anti-Tp92 immunoreactivity on T. pallidum lysates

The level of reactivity of the anti-Tp92 polyclonal antiserum on lysates of washed and unwashed T. pallidum preparations was investigated by immunoblot analysis. As shown in figure 4, an immunoreactive band corresponding to the 92-kDa T. pallidum Tp92 was present in lysates of unwashed treponemes extracted directly from infected rabbit testes (lane 1). In contrast, no immunoreactive 92-kDa bands were observed in equal quantities of lysates prepared from T. pallidum washed 1 time (lane 2) and 3 times (lane 3) after extraction from rabbit testes or in lysates of unwashed treponemes with control preimmune serum (lane 4). Previous investigations have demonstrated that the fragile outer membrane is partially removed during washing of T. pallidum by centrifugation [53], and thus the above results suggest loss of Tp92 with the treponeme outer membrane during washing.

Opsonic potential of the T. pallidum Tp92

The anti-Tp92 serum was also investigated for its ability to opsonize T. pallidum in 2 separate experiments (triplicate wells per experiment) by means of a standard phagocytosis assay. As shown in figure 5, the anti-Tp92 polyclonal antiserum was significantly opsonic for the Nichols strain of T. pallidum, compared with normal rabbit serum (P = .0089). The level of opsonic activity observed for anti-Tp92 approximated that observed with serum collected from rabbits chronically infected with T. pallidum (immune vs. normal rabbit serum; P<.0001).

Immunoprotective capacity of T. pallidum Tp92

The protection afforded by immunization with the T. pallidum Tp92 was tested in the rabbit syphilis model. In 3 independent protection experiments, 7 rabbits were immunized either 3 or 5 times each with the purified intact ORF recombinant Tp92 (70-kDa protein) emulsified in Ribi adjuvant. The degree of anti-Tp92 immunoreactivity present in serum drawn from each rabbit 1 week after the final immunization was determined by immunoblot analysis (data not shown). The rabbits that received 5 Tp92 immunizations in protection experiments I and III demonstrated about equal levels of strong, specific immunoreactivity against the recombinant Tp92. As expected, the rabbits in protection experiment II, which received only 3 Tp92 immunizations, demonstrated a lower level of Tp92-specific immunoreactivity, with 1 rabbit demonstrating no detectable reactivity.

After administration of the final immunization in each protection experiment, rabbits were subjected to intradermal challenge at 8 independent sites with 105 T. pallidum per site. Four control rabbits received no prior immunization but underwent the same intradermal challenge. Table 4 summarizes the postchallenge analyses performed on the rabbits to determine the degree of protection provided by immunization with the T. pallidum recombinant Tp92. As shown in table 4, the control animals developed typical red, raised, and highly indurated lesions, the majority of which progressed to ulceration. In contrast, the rabbits immunized with the T. pallidum recombinant Tp92 before challenge all demonstrated alteration of lesion development. The degree of protection varied among the immunized rabbits, with the highest levels of protection observed for those rabbits exhibiting strong anti-Tp92 immunoreactivity in immunoblot analysis (rabbits from protection experiments I and III). Significant attenuation of lesion development was observed in these rabbits, with atypical pale, flat, slightly indurated, and for the most part, nonulcerative lesions appearing at the sites of challenge. In these protection experiments, the number of lesions progressing to ulceration and the number of darkfield-positive lesions in the Tp92-immunized rabbits were significantly smaller than those of lesions appearing in the control animals. All animals were shown to be infected with T. pallidum, as demonstrated by seroconversion of the rabbits in each of the protection experiments (data not shown).

Table 4
View larger version:
Table 4

Summary of postchallenge analyses of control unimmunized rabbits and recombinant Treponema pallidum Tp92—immunized rabbits.

Figure 6 shows lesions on day 25 after T. pallidum challenge in representative rabbits from the control unimmunized group (figure 6A) and the Tp92-immunized group (figure 6B). The control rabbit has developed ulcerative lesions, whereas the Tp92-immunized rabbit has cleared its lesions without ulceration, and only flat, lightly pigmented patches remain. Parallel experiments revealed that immunization with the unrelated, nontreponemal recombinant molecule SA85-1.1-III in Ribi adjuvant provided no protection (data not shown), thus demonstrating that neither the adjuvant nor the N-terminal histidine tag contributed to the protection observed in the Tp92-immunized rabbits.

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Discussion

This report describes the identification and characterization of a 92-kDa T. pallidum protein that shares sequence similarity with outer membrane proteins from a wide range of bacterial species, including the related spirochete B. burgdorferi and 2 sexually transmitted disease-causing bacterial species, Neisseria gonorrhoeae and Chlamydia trachomatis. Although the majority of these proteins have been identified through genome sequencing of the bacterial species in which they are found, and thus are hypothetical, 6 have been independently isolated by means of molecular biological or protein immunochemical approaches. These include an unknown protein from E. coli (GenBank, P39170), OMP1 from Brucella abortus (GenBank, U51683), Omp85 proteins from N. meningitidis and N. gonorrhoeae [47], Oma87 from Pasteurella multocida [46], and D15 from Haemophilus influenzae [45]. Characterization of the latter 4 proteins confirms that they are present on the bacterial surface [46, 47, 54], and passive immunization with antiserum against Oma87 and D15 has been shown in animal models to be protective against P. multocida and H. influenzae challenge, respectively [46, 54–56]. Results reported here suggest that Tp92 is a similar protective outer membrane antigen of T. pallidum.

Evidence for the surface location of Tp92 in T. pallidum comes from the observation that antibodies directed against Tp92 have significant opsonic activity for living T. pallidum, thus demonstrating that this protein is accessible on the surface of intact treponemes. Indirect evidence for the presence of Tp92 in T. pallidum outer membranes was obtained by immunoblot analysis with the anti-Tp92 serum on T. pallidum lysate preparations. A loss of immunoreactivity was observed in lysates prepared from treponemes whose outer membranes had been partially removed by washing before lysis, compared with lysates prepared from unwashed treponemes in which the fragile outer membrane and its constituent proteins remain intact before lysis. Analysis of the amino acid sequence of Tp92 also provides supporting evidence for the presence of Tp92 on the bacterial surface. The first 21 amino acid residues at the N-terminus of Tp92 predict a cleavable signal sequence that is characteristic of proteins translocated across the bacterial inner membrane [57]. In addition, the C-terminus of Tp92 contains hydrophobic residues at positions 1, 5, and 7 from the C-terminus, which loosely conforms to the consensus hydrophobicity pattern predicted for bacterial outer membrane proteins of hydrophobic residues at positions 1, 3, 5, 7, and 9 from the C-terminus [58]. Furthermore, PSORT analysis predicts an 84.6% probability that Tp92 resides in the T. pallidum outer membrane. These combined results suggest that Tp92 is associated with the T. pallidum outer membrane, and additional biochemical studies are currently under way to investigate the true cellular location of this molecule.

PCR amplification and subsequent sequence analysis of the tp92 ORF from 11 strains representing a total of 4 pathogenic treponemes revealed minimal amino acid sequence divergence between the various strains. Similarly, the D15 antigen is conserved among H. influenzae strains and thus also represents an invariant antigen [56]. Of the divergence that does occur in the Tp92 sequence, the majority is found in non—T. pallidum subsp. pallidum strains and lies within a serine-rich sequence that is unique to Tp92. The C-terminal end of this serine stretch is deleted in the Tp92 sequences from both the simian isolate and the T. pallidum subsp. pertenue Gauthier strain. This sequence is not deleted in the Tp92 sequence from the Haiti B strain, providing further evidence that its classification as a T. pallidum subsp. pertenue strain is incorrect [49]. It is also interesting to note that the entire C-terminal serine-rich sequence has been deleted from the Tp92 sequence of the rabbit pathogen T. paraluiscuniculi, Cuniculi A strain, the only different treponeme species represented in this analysis.

The potential significance of the serine-rich sequence present in Tp92 becomes apparent when one considers that similar serine-rich sequence stretches are observed in cell surface proteins from a variety of pathogenic organisms. These include ISG100 from T. brucei [51], the 39K protein of fowlpox virus [59], SREHP from Entamoeba histolytica [60], the H(+)-ATPase [61], SMS1 gene product [62], TIP1-related family of proteins [63], gp115 [64] and α-agglutinin [65] from Saccharomyces cerevisiae, and SERA from Plasmodium falciparum [66]. Although no function has yet been established for the majority of these serine-rich proteins, the latter 2 proteins have been shown to mediate attachment to cells. Specifically, the interaction of α-agglutinin with another S. cerevisiae surface protein, α-agglutinin, mediates adhesion between yeast cells during the mating process [65], and the P. falciparum SERA binds to phospholipids of red blood cell membranes and in this way is suggested to facilitate parasite invasion into host cells [66].

Similarly, as a putative outer membrane protein, the T. pallidum Tp92 may constitute another such attachment ligand. Numerous studies have shown that T. pallidum attaches to host cells [67–76], although the T. pallidum proteins mediating such attachment have not yet been identified. In this scenario, the stretch of serine residues present in the C-terminal end of the Tp92 sequence, which have been predicted to reside within an external loop on the outer face of the outer membrane, could act as potential sites for hydrogen bonding to carbohydrates present on the surface of host cells, as has been suggested for the interaction of α-agglutinin with carbohydrates of the yeast cell wall [65]. Additionally, the serine residues present in Tp92 may function as potential sites for O-glycosylation, as seen in the yeast protein gp115 [64]. Although glycosylation is far less common in prokaryotes than in eukaryotes, a recent report has shown that the flagellar core proteins of T. pallidum are glycosylated [77], thus establishing the possibility of additional glycosylated treponemal proteins. A proposed function of O-glycosylation is to induce proteins to adopt stiff and extended conformations, due to steric interactions that occur between the carbohydrate residues and the peptide core [78]. If the T. pallidum Tp92 is indeed present on the bacterial surface, such a “rodlike” conformation may enable the protein to extend through the slime layer covering the bacterial outer membrane, thus exposing the functional domain of Tp92 on the cell surface and allowing access to host cells and/or cellular substances.

The immunoprotective potential of the T. pallidum Tp92 was also investigated in this study for several reasons. First, antisera raised against the analogous proteins Oma87 and D15 from P. multocida and H. influenzae, respectively, have been shown to induce protection in animal models [46, 54–56]. Second, the invariant nature of Tp92 among various T. pallidum subsp. pallidum strains makes it an attractive candidate for design of a universal subunit vaccine against syphilis. Finally, the identification of Tp92 as a target of opsonic antibodies, through both the differential immunologic expression library screen and the phagocytosis assays, combined with the central role that opsonization and phagocytosis play in bacterial clearance, suggests this antigen may have immunoprotective capability. Indeed, immunization of rabbits with the T. pallidum Tp92 resulted in partial protection from subsequent T. pallidum challenge, with alteration of lesion development at the sites of challenge compared with that seen in unimmunized control rabbits. Not surprisingly, the level of protection achieved strongly corresponded to the immune response generated in the immunized rabbits. Rabbits who received the greatest number of Tp92 immunizations, and thus exhibited the highest level of Tp92-specific immunoreactivity, demonstrated significant protection on challenge. This degree of protection is promising, considering that the ∼245 amino acid residues at the C-terminus of the Tp92 sequence appear to have been expressed either at a very minimal level, or not at all, in our initial attempt at heterologous expression of the T. pallidum Tp92. Future investigations will focus on using alternative heterologous expression systems to produce the entire 816-residue mature Tp92 to determine whether immunization with this intact molecule confers additional protection against T. pallidum challenge.

In summary, the T. pallidum Tp92 represents a target of opsonic antibodies and an invariant, immunoprotective antigen that may be useful as a putative vaccine candidate for syphilis. Further studies will be performed to determine whether covaccination of Tp92 with other promising immunoprotective antigens, such as Gpd [23] and Tpr K [18], can achieve complete immunity against T. pallidum challenge.

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Acknowledgments

We thank Stacy Roberts, Lynn Barrett, Anna Dukes, Karin Haverlah, Charles Carlos, and Kindra Mikota for excellent technical assistance.

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Footnotes

  • All animals used in these experiments were cared for according to the Guide for the Care and Use of Laboratory Animals prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, and according to the guidelines established by the University of Washington.

  • Grant support: NIH (AI-34616, AI-18988, AI-42143, and AI-31448 to W.C.V.V. and S.A.L.); Natural Sciences and Engineering Research Council of Canada and the Medical Research Council of Canada (postdoctoral fellowships to C.E.C.).

  • Received October 11, 1999.
  • Revision received December 14, 1999.
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