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The human-infective Treponema pallidum subsp. pallidum, pertenue, and endemicum are the causative agents of syphilis, yaws, and bejel, respectively. Two highly related treponemes that are naturally infectious for animals have also been identified; these are the rabbit-infective species T. paraluiscuniculi and the Simian isolate obtained from skin lesions of a monkey (12). Although the human-infective pathogenic treponemes cause clinically distinct diseases, differentiation between T. pallidum subsp. pallidum and the other T. pallidum subspecies on the genetic level has only recently been described (6).

Syphilis remains a public health concern worldwide, with an estimated 3.5 million cases occurring annually (16). Successful control of syphilis depends on the development of an effective vaccine that demonstrates cross-protection between T. pallidum subsp. pallidum strains. To date, complete protection has been demonstrated only in experimental animals using impractical immunization protocols involving gamma-irradiated treponemes (15). Partial protection has been achieved in experimental animals by immunizing with treponemes that were antiformin treated (21) or aged at 4°C (14), as well as with several recombinant or native T. pallidum subsp. pallidum proteins, including protein 4D (1), purified endoflagella (8), and TmpB (23). Recent investigations conducted in our laboratory have identified three additional recombinant antigens that provide significant protection against experimental syphilis infections; these are TprK (5), Tp92 (3), and glycerophosphodiester phosphodiesterase (Gpd) (2, 20). 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.

In this study, we further extend our investigations into the suitability of Gpd as a potential vaccine candidate by determining the degree of Gpd sequence conservation among pathogenic treponemes.

Bacterial species. The Gpd coding sequence was PCR amplified from genomic DNAs isolated from a variety of treponemal strains. All strains were propagated in New Zealand White rabbits as previously described (13). T. pallidum subsp. pallidum Nichols was originally sent to the University of Washington by James N. Miller (University of California, Los Angeles) in 1979, and T. pallidum subsp. pertenue Gauthier was supplied by Peter Perine (Centers for Disease Control and Prevention, Atlanta, Ga.) in 1981. T. pallidum subsp. pallidum Bal-3, Bal-7, and Bal 73-1; T. paraluiscuniculi Cuniculi A; T. pallidum subsp. pertenue Haiti B; T. pallidum subsp. endemicum Iraq B; and the Simian isolate were supplied by Paul Hardy (Johns Hopkins University, Baltimore, Md.). T. pallidum subsp. pallidum Sea 81-3 and Sea 83-1 were isolated by Sheila A. Lukehart from the cerebrospinal fluid of untreated syphilis patients.

PCR amplifications. To obtain the entire gpd open reading frame, primers were designed from the 5' (5'-TGCACGGTGACGATCTGTGC-3') and 3' (5'-GGTACCAGGCGACACTGAAC-3') noncoding regions flanking the gpd gene (11). These primers are located 48 bp upstream and 51 bp downstream, respectively, of the gpd open reading frame. PCR amplification of the gpd gene was performed by using a 100-µl reaction mixture containing 200 µM deoxynucleoside triphosphates, each primer at 0.25 µM, 1× Taq polymerase buffer (50 mM Tris-HCl [pH 9.0] at 20°C, 1.5 mM MgCl₂, 20 mM NH₄SO₄), and 1 µl of genomic DNA containing 5,000 to 10,000 treponeme equivalents for each strain. The PCR conditions were 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 each reaction, a hot-start PCR (9) was performed by adding 2.5 U of Taq polymerase after the initial denaturation step. Following the PCR, the amplification products were cloned into the pGEM-T vector (Promega, Madison, Wis.) and each insert was sequenced in its entirety in both directions. To reduce the possibility of PCR- or sequencing-induced errors, two clones derived from independent PCR amplifications were sequenced for each strain.

Sequence analysis. Double-stranded plasmid DNA was extracted by using the Qiagen Plasmid Mini Kit (Qiagen, Chatsworth, Calif.), and both strands of insert DNA were sequenced by using the Applied Biosystems dye terminator sequencing kit (PE Applied Biosystems, Foster City, Calif.) and the ABI 373A DNA sequencer in accordance with the manufacturer's instructions. In all cases, both universal sequencing primers and internal primers designed from the insert sequence were used. Nucleotide sequences were translated and analyzed by using the Sequencher Version 3.1RC4 sequence analysis software (Gene Codes Corporation, Ann Arbor, Mich.). Alignment of protein and DNA sequences was performed by using the Clustal W general-purpose multiple-alignment program (22).

RFLP analysis. RFLP analysis was performed on the gpd open reading frame amplified from each treponeme strain. One microgram of each of the amplified templates was digested with PleI (New England Biolabs, Beverly, Mass.) for 4 h at 37°C prior to electrophoresis on a 1.5% NuSieve (FMC BioProducts, Rockland, Maine) agarose gel.

View larger version (47K): [in this window] [in a new window]   FIG. 1.   RFLP analysis of the gpd amplicons from various pathogenic treponeme strains. The gpd open reading frame was amplified from each of the specified strains, digested with PleI, and subjected to agarose gel electrophoresis and ethidium bromide staining. The left lane shows the 100-bp DNA ladder (New England Biolabs). Shown also is the undigested gpd amplicon from the Nichols strain. The sizes, in base pairs, of the DNA fragments generated by PleI digestion of the gpd amplicons from the various strains are shown on the right.

Nucleotide sequence accession numbers. The nucleotide sequences of the gpd genes from the Nichols, Bal-3, Bal-7, Bal 73-1, Sea 81-3, Sea 83-1, Mexico A, Haiti B, Gauthier, Iraq B, Simian, and Cuniculi A strains have been assigned GenBank accession no. AF004286 and AF127415 to AF127425, respectively.