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Journal of Clinical Microbiology, September 2006, p. 3377-3380, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.00784-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Differentiation of Treponema pallidum Subspecies

Arturo Centurion-Lara,1,2* Barbara J. Molini,1 Charmie Godornes,1 Eileen Sun,2,{dagger} Karin Hevner,1 Wesley C. Van Voorhis,1,2 and Sheila A. Lukehart1,2

Departments of Medicine,1 Pathobiology, University of Washington, Seattle, Washington 981952

Received 12 April 2006/ Returned for modification 12 June 2006/ Accepted 26 June 2006


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ABSTRACT
 
Treponema pallidum includes three subspecies of antigenically highly related treponemes. These organisms cause clinically distinct diseases and cannot be distinguished by any existing test. In this report, genetic signatures are identified in two tpr genes which, in combination with the previously published signature in the 5' flanking region of the tpp15 gene, can differentiate the T. pallidum subspecies, as well as a simian treponeme.


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TEXT
 
The human pathogenic treponemes include three subspecies of Treponema pallidum (i.e., subsp. pallidum [syphilis], subsp. endemicum [bejel], and subsp. pertenue [yaws]) and T. carateum (pinta). This classification is based upon distinctive host ranges and clinical manifestations of each infection and, in part, saturation reassociation kinetic studies of the causative agents of syphilis and yaws (28). The World Health Organization estimates that there are 12 million new cases of syphilis per year (17), and the total number of cases of yaws, bejel, and pinta (the endemic treponematoses) worldwide is approximately 2.5 million (38), although good surveillance data are not available (1, 2, 5, 7, 21, 24, 32, 39). These treponemes cannot be cultured continuously in vitro and may cause life-long infections in untreated individuals. Classically, T. pallidum infections are characterized by episodes of active clinical disease interrupted by periods of asymptomatic latent infection.

The natural history of primate infection with the Fribourg-Blanc treponeme is unknown. This treponeme was isolated in 1962 from a baboon in Guinea (12, 13). It affects various species of primates, and its geographical distribution overlaps with regions where yaws is endemic in Western Africa (13). In 1971, Smith et al. showed that the Fribourg-Blanc treponeme is able to experimentally infect humans and to cause active infection (34).

The T. pallidum subspecies and the Fribourg-Blanc treponeme are morphologically indistinguishable by immunofluorescence or electron microscopy (9, 14, 19, 31) and induce similar histopathological changes and cross-reactive antibodies, indicating a high level of antigenic relatedness (3, 4, 10, 12, 14, 27, 30, 33, 34, 36, 37). We have earlier reported a single-base-pair change in the 5'-flanking region of the tpp15 gene (6), which introduces an Eco47III restriction site and permits the differentiation of T. pallidum subsp. pallidum from the other subspecies by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified products. This single-nucleotide change distinguishes only syphilis from nonsyphilis treponemes but cannot differentiate subspecies pertenue from subspecies endemicum. We now report the identification of subspecies-unique genetic signatures in two members of the tpr gene family: tprC and tprI (11). RFLP analysis of tprC and tprI amplicons, in combination with the Eco47III digestion of tpp15 gene flanking region amplicons, permits the differentiation of each of the three T. pallidum subspecies and the unclassified simian isolate.

All treponemal isolates used in the present study, except for the Pariaman and Ghana 051 pertenue strains, were propagated in New Zealand White rabbits (25) with the approval of the University of Washington Institutional Animal Care and Use Committee. DNA for the Pariaman and Ghana 051 T. pallidum subsp. pertenue strains was generously provided by Leo Schouls and Gerda Noordhoek. The origin of the isolates or DNA is indicated in Table 1. No isolates of T. carateum are known to exist; therefore, we could not analyze this species. DNA from each treponemal strain was extracted as described elsewhere (6).


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  		TABLE 1. Treponema isolates

Full-length tprC and tprI open reading frames from the simian isolate and at least one strain of each T. pallidum subspecies, as well as regions of the tprC and tprI genes from 19 strains (Table 1), were amplified, cloned, and sequenced as previously described (35). PCR primers for tprC (sense, 5'-TGAACGCGCTCAACATAGAC; antisense, 5'-CCCTCATCCGAGACAAAAT) were used to generate an amplicon of 707 bp. This PCR product was then digested using the enzymes BsrDI (8 U) and BsiEI (10 U) in a 50-µl reaction volume at 65 C for 16 h as described by the manufacturer (New England Biolabs, Beverly, MA). The restriction products were then loaded onto a 1% agarose-1.5% NuSieve agarose (Cambrex, Rockland, ME) gel and visualized using ethidium bromide staining. Another set of primers (sense, 5'-AAGCACGCGTGTACATCC; antisense, 5-ATCCCTCGCCTGTAAACTGA) was used to amplify a 493-bp fragment of tprI. BsrDI digestion of 10 µl of this PCR amplicon was performed according to the manufacturer's instructions (New England Biolabs, Beverly, MA); the digestion products were also separated in 1% agarose-1.5% NuSieve agarose (Cambrex) gels.

All treponemal strains were also analyzed using the previously published method (6) for RFLP analysis of the 5' flanking region of the tpp15 gene with redesigned primers (sense, 5'-TCCGCCTTCTCGCGTTTCTCG; antisense, 5'-TGCGAGGGTAGTTGCTGCTTCG). PCR was carried out as already described (9), and amplicons were digested as previously described. Each 50-µl digestion reaction contained either 25 U of AfeI (an Eco47III isoschizomer; NEB) per reaction.

Comparative analysis of the tprI sequences in the three T. pallidum subspecies and the Fribourg-Blanc treponeme revealed a genetic signature region at positions 1740 to 1766 containing a BsrDI restriction site (NN/CATTGC) in T. pallidum subsp. endemicum (Fig. 1A). As predicted, amplicons from the two T. pallidum subsp. endemicum strains (Bosnia A and Iraq B) were cut at this unique site, yielding fragments of 334 and 159 bp, whereas the amplicons from T. pallidum subsp. pertenue and T. pallidum subsp. pallidum strains and the simian strain show a single uncut band at 493 bp (Fig. 1A and Table 2). In this analysis, the Fribourg-Blanc strain grouped with the T. pallidum subsp. pertenue strains.


Figure 1
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  		FIG. 1. (A) RFLP analysis of the 493-bp tprI amplicons using BsrDI. Restriction fragments of 334 and 159 bp are seen only in T. pallidum subsp. endemicum strains. See also Table 2. (B) Simultaneous RFLP analysis of the 707-bp tprC amplicons using BsrDI and BsrEI. A BsrDI restriction site is found in the T. pallidum subsp. endemicum (Bosnia A and Iraq B) isolates, the Fribourg-Blanc isolate, and in the T. pallidum subsp. pallidum Nichols, Chicago, and Sea 81-4 isolates, yielding fragments of 547 and 160 bp. The BsiEI restriction site (572- and 135-bp fragments) is present in the T. pallidum subsp. pallidum Sea 81-3 and Mexico A strains which lack the BsrDI restriction site and differentiates them from the T. pallidum subsp. endemicum isolates. The T. pallidum subsp. pertenue amplicons remain uncut (707 bp). See also Table 2.


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  		TABLE 2. Restriction analysis for differentiation of Treponema species and subspecies

In contrast to tprI, the tprC locus contains the signature GGTATTACTC at positions 1726 to 1735 in T. pallidum subsp. pertenue and some T. pallidum subsp. pallidum isolates (Fig. 1B). This signature differs from the corresponding regions in the tprC genes of the T. pallidum subsp. endemicum strains and the remaining T. pallidum subsp. pallidum strains, which contain the BsrDI restriction site NN/CATTGC. Downstream of this first distinguishing region, there is a second signature that contains a BsiEI restriction site (CGRY/CG) unique to the subset of syphilis treponemes that lack the BsrDI restriction site. Consequently, BsrDI distinguishes T. pallidum subsp. endemicum and some T. pallidum subsp. pallidum isolates from the T. pallidum subsp. pertenue strains, and BsiEI differentiates T. pallidum subsp. pertenue from those T. pallidum subsp. pallidum treponemes that lack the BsrDI site. As predicted by sequence analysis (Fig. 1B and Table 2), the T. pallidum subsp. pertenue (Samoa D, Samoa F, and Gauthier) amplicons remain uncut, the T. pallidum subsp. endemicum (Bosnia A and Iraq B) and some T. pallidum subsp. pallidum (Chicago, Nichols, and Sea 81-4) isolates yield two BsrDI fragments of 547 and 160 bp, while the other T. pallidum subsp. pallidum (Mexico A and Sea 81-3) isolates give two BsiEI restriction fragments of 572 and 135 bp (see Fig. 1B). The CDC-1, CDC-2, Pariaman, and Ghana 051 T. pallidum subsp. pertenue strains, as expected, remain uncut (707 bp) by BsrDI (Table 2). In contrast to the tprI results, the Fribourg-Blanc simian isolate here shows the same restriction pattern as the T. pallidum subsp. endemicum strains; thus, the combination of tprC and tprI analyses permits the differentiation of the simian isolate from the human isolates.

Because standard serological methods cannot distinguish the treponemal infections from each other, these findings are of particular importance for infected persons from areas where yaws and bejel are endemic. A long-standing controversy in the treponemal field has been the whether venereal syphilis and the endemic treponematoses are caused by the same etiological agents. Hudson (20) hypothesized that the organisms are identical and that changes in the climatic conditions result in different clinical manifestations. In contrast, the hypothesis of Hackett (18) that these organisms are genetically distinct, although very closely related, is supported by our molecular studies and by the lack of cross-immunity among subspecies (37).

The phylogenetic status of the Fribourg-Blanc simian isolate has not yet been resolved. Initial serological and cross-immunity studies suggested that it is more closely related to subspecies pertenue than to subspecies pallidum (33). Our molecular analysis suggests that it is distinct from the pertenue, endemicum, and pallidum subspecies and may in fact be representative of another group of pathogenic treponemes. The ability of this strain to cause experimental active infections in humans indicates that primates may serve as reservoirs for zoonotic treponemal infections or new emerging human pathogens.

The strains for each subspecies that were tested in this analysis include all of the non-T. pallidum subsp. pallidum isolates currently available to the authors, and these strains represent geographically distinct regions of origin and diverse isolation dates. Our findings strongly suggest that the genetic differences identified here are indeed subspecies-specific and chromosomally stable. For the endemicum subspecies, the number of strains analyzed is small. However, studies of a larger number of strains, if they become available, may confirm our findings. We have previously demonstrated that even single nucleotide polymorphisms are reliable targets for differentiation of T. pallidum subspecies (6).

In addition to its utility in the specific diagnosis of treponemal infections from biopsy or swab samples, this method may permit subspecies differentiation in ancient DNA (22) from skeletal remains for studying the migration and evolution of the treponemal subspecies during the centuries. Ongoing studies in our laboratories may lead to the development of serological methods for differentiation of the treponematoses. Although serological tests would have more practical applications, the new methods described in the present study may also prove to be very useful in diagnosis and research.


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ACKNOWLEDGMENTS
 
We are grateful to Heidi Pecoraro for manuscript preparation and to Gerda Noordhoek, Leo Schouls, Victoria Pope, James N. Miller, Paul Hardy, Ellen Nell, Peter Perine, and Robert George for providing strains or DNA.

This study was supported by NIH grants AI42143, AI34616, and AI63940.


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FOOTNOTES
 
* Corresponding author. Mailing address: Harborview Medical Center, Box 359779, 325 9th Ave, Seattle, WA 98104. Phone: (206) 341-5364. Fax: (206) 341-5363. E-mail: acentur{at}u.washington.edu. Back

{dagger} Present address: Rosetta Inpharmatics, Seattle, Wash. Back


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Journal of Clinical Microbiology, September 2006, p. 3377-3380, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.00784-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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