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Abstract
Background
Syphilis is the best known treponemal infection and the disease has captured the attention
of well-known physicians and the imaginations of writers and artists. In contrast,
the nonvenereal treponemal infections (yaws, bejel, and pinta) have been endemic in
remote regions of Africa, Southeast Asia, and South America, and are thus less well-known
in western culture. However, these endemic treponematoses were so prevalent that,
between 1952 and 1964, the World Health Organization (WHO) undertook a massive eradication
campaign in which over 300 million people in Africa, South America, Southeast Asia,
the South Pacific islands, and the Middle East were examined and ∼50 million were
treated with penicillin [1]. It is estimated that the burden of disease was reduced
by 95%. In the mid 1980s, the Fogarty International Center and WHO sponsored a series
of regional meetings to gather information about the status of the endemic treponematoses
[2]–[4]. Numerous foci of infection were identified in Africa, Asia, and South America,
and the recommendation was made to focus new efforts on control, with the goal of
elimination. The HIV epidemic intervened and the attention of the global health community
was appropriately diverted to AIDS, thus interrupting the momentum for elimination
of the treponematoses. Since that time, because of little to no surveillance in many
areas, the current magnitude of the diseases is unknown, although foci of yaws and
endemic syphilis have periodically been reported. Several countries, including Indonesia
and India, have current ongoing yaws control programs. In 2007, the WHO once again
committed itself to eliminate yaws (http://www.who.int/mediacentre/news/notes/2007/np04/en/index.html).
Traditionally, the human Treponematoses have been differentiated based upon their
mode of transmission (sexual vs. non-venereal), clinical manifestations, serious sequelae
(e.g. central nervous system [CNS] or cardiovascular involvement, congenital infection),
and experimental host range (Table 1). In the mid-1900's, a disagreement raged over
whether the etiologic agents of these infections were the same species, with mode
of transmission and clinical course defined by culture and climate, or were different
species with important biological differences [5],[6]. The agents are morphologically
identical, and all induce reactivity in the standard serological tests used for syphilis
diagnosis. Recent molecular studies have identified genetic signatures that can differentiate
the existing strains of Treponema pallidum subspecies pallidum (syphilis), subspecies
pertenue (yaws), and subspecies endemicum (bejel) [7], yet the molecular bases for
the described differences in transmission and clinical course have not been defined.
10.1371/journal.pntd.0000184.t001
Table 1
Traditional Characteristics of Treponemal Infections
Species
Disease
Mode of Transmission
Serious Sequelae
Preferred Experimental Host
T. pallidum subsp. pallidum
Syphilis
Sexual
Cardiovascular and CNS disease, gummas, congenital syphilis
Rabbit
T. pallidum subsp. pertenue
Yaws
Direct nonsexual contact
Gummas
Hamster
T. pallidum subsp. endemicum
Bejel
Direct nonsexual contact
Gummas
Hamster
T. carateum
Pinta
Direct nonsexual contact
None
Non-human primate
How definitive are these clinical and epidemiological differences? Syphilis is usually
transmitted sexually, but there are many examples in older textbooks of nonsexual
transmission via direct contact; examples include digital infection of dentists via
contact with oral lesions of syphilis patients, and infection of the nipple in wet
nurses via oral lesions in infected infants. In contrast, yaws is generally transmitted
by skin-to-skin contact in children, and infectious lesions are typically resolved
before sexual debut, thus transmission during sexual activities is not recognized.
Thus, mode of transmission appears to be defined by opportunity, rather than biology.
There is similar overlap in clinical manifestations. The lesions of yaws are typically
described as “frambesiform” (raised “berry-like” lesions compared to the ulcer or
maculopapular rash of syphilis) yet there are numerous descriptions in the literature
of less dramatic, even macular, yaws lesions in drier climates [8]. Conversely, the
condylomata lata of secondary syphilis can be raised and multilobed (“frambesiform”),
and they appear in moist body folds, a common location for the frambesiform lesions
of secondary yaws. Although gummatous destruction, particularly of bone and cartilage,
is well-recognized in syphilis, yaws, and bejel, only syphilis is said to lead to
CNS involvement and congenital infection. However, in a very thoroughly researched
review article, Roman and Roman [9] argue eloquently that there is ample evidence
for CNS, cardiovascular, and congenital infection in yaws. In that review, Blacklock
[10] is quoted as stating that the “criteria used to differentiate syphilis and yaws
were ‘nothing more than the statement of an epidemiological observation.’” As an example,
the Haiti B strain was originally identified as T. pallidum subsp. pertenue because
it was isolated from an 11-year old child with “typical generalized frambesiform yaws”
[11], in a epidemiological setting of childhood infection. Yet molecular studies in
several labs [12]–[14] have subsequently demonstrated that the Haiti B strain has
molecular signatures consistent with T. pallidum subsp. pallidum. The Madras strain
is another such example of a “yaws” isolate that appears in fact to be a pallidum
strain based on molecular data. With regard to host range differences, the Haiti B
strain was used extensively by Schell et al. [15]–[17] in their studies of yaws infection
in the hamster model. It has been stated that hamsters do not regularly develop clinical
manifestations of syphilitic disease [11],[17], yet hamsters are readily infected
by the Haiti B strain, now considered to be a pallidum strain. Thus the clinical,
epidemiological, and host range criteria used to differentiate the agents of the treponematoses
are soft.
Molecular studies have identified signature polymorphisms that serve to differentiate
the known strains of the three subspecies pallidum, pertenue, and endemicum [7]. While
these studies suggest that there are genetic differences among subspecies, the strains
used to identify these distinctive signatures were classified by the clinical and
epidemiological criteria described above and thus the molecular signatures may be
biased. As mentioned above, two strains (Haiti B and Madras) were isolated from characteristic
yaws lesions, yet they have the molecular signatures of the pallidum subspecies.
A New Study: On the Origin of the Treponematoses—A Phylogenetic Approach
Harper and colleagues [18] have examined a number of chromosomal regions in a total
of 23 strains/samples representing the three T. pallidum subspecies and have identified
regions of mutation (single nucleotide polymorphisms [SNP] or indels). Some of these
polymorphisms have been previously described and others are novel. Importantly, this
analysis included two new yaws samples from Guyana, representing the only South American
yaws samples evaluated to date. Based upon 17 regions of mutation in the established
strains, the authors constructed a phylogenetic tree of the concatenated sequences,
and propose that this tree identifies the yaws subspecies as being the oldest, with
the bejel and syphilis subspecies evolving subsequently. Unfortunately the DNA from
the two new yaws samples from Guyana was too degraded to conduct extensive sequencing,
so these samples were not included in the phylogenetic analysis. From these samples,
the authors selected a subset of seven genetic regions for analysis. Based upon the
homology of four SNPs in the Guyana samples with the group of pallidum strains, the
authors conclude that the syphilis subspecies evolved from New World yaws strains.
Strengths and Limitations of the Study
The basis of the claim for a New World origin of venereal syphilis is sequence similarity
between the Guyana yaws samples and the pallidum strains. However, the sequence similarity
is based on only four SNPs. Furthermore, three of the SNPs cause non-synonymous changes
and occur in a very short region (∼15 amino acids) of the tprI protein. This is an
extraordinarily high rate of evolutionary change in a genus that has been characterized
by very little change. Two of the SNPs (tprI 137 and 151) are shown to have evolved
two independent times according to the authors' network analysis (Fig. 3 of Harper
et al. [18]), a result that again makes little sense in a genus characterized by very
little variation. One of those SNPs (tprI 151) also differs between two pertenue strains
CDC-1 and CDC-2, which are strains that might be predicted to be identical given the
very close geographical and chronological proximity of isolation. Finally, tprI is
thought to be involved in pathogenesis [19] and thus is subject to the effects of
natural selection, which violates the assumptions of phylogenetic analysis. Clearly
the tprI locus is atypical of the treponemal genome and, thus, is not the best choice
when trying to resolve the decades-old debate concerning the origin of venereal syphilis.
Additionally, the phylogenetic and network analyses presented by Harper et al. are
contradictory in that the phylogeny supposedly supports the evolution of pallidum
from endemicum (Fig 2 of Harper et al. [18]) but the network (Fig 3 of Harper et al.
[18]) is used to infer the origin of pallidum from New World pertenue strains. Part
of the problem may be the fact that the phylogeny does not show significant structure,
contrary to the authors' claims. When the tree is redrawn to show only branches with
minimal 50% bootstrap support, the pertenue cluster disappears and all three subspecies,
plus the simian isolate, branch off the most basal branch simultaneously (see Figure
1). This means that no evolutionary order can be inferred. Furthermore, since all
strains were collected contemporaneously (at least on an evolutionary time scale),
the branch lengths should all be approximately equivalent since a phylogeny reflects
only mutational evolution (i.e. all treponemal strains should be equidistant from
their common ancestor). The fact that the pallidum strains have longer branch lengths
does not mean they evolved more recently, but instead is consistent with an argument
for increased recombination or selection along the pallidum branch, i.e. essentially
any phenomenon that violates the evolution-by-mutation-only assumption of a phylogenetic
analysis. It is also perhaps noteworthy that the pallidum strains are all from the
New World except for two strains (South Africa and Madras), whereas the endemicum
and pertenue strains are all from the Old World (with the exception of the new Guyana
strains). Thus, the reported sequence homology between the Guyana and pallidum strains
may simply reflect geographic clustering of New World vs. Old World strains. It would
be interesting, though perhaps not possible, to examine older European or Asian pallidum
strains to see whether the phylogeny is altered.
10.1371/journal.pntd.0000184.g001
Figure 1
A Phylogenetic Tree Depicting the Relationships between T. pallidum strains.
This phylogeny is identical to the phylogeny of Harper et al. (Fig. 2) except that
it has been re-estimated to show only branches with >50% bootstrap support for both
maximum likelihood and maximum parsimony analyses. As with Harper et al.'s phylogeny,
vertical distance is not meaningful, i.e. the placement of taxa along the vertical
lines is inconsequential. Pertenue, endemicum and pallidum strains are depicted in
green, blue, and red, respectively.
Caution, therefore, must be used in drawing conclusions about the evolution of “subspecies”
that may represent a biological continuum, rather than discrete agents. Certainly,
firm conclusions should not be based upon a few SNPs in two samples taken from a single
location.
Next Steps
Despite the limitations of the Harper et al. analysis (as acknowledged by the authors
in the discussion), the results reinforce another long-term question: How could the
limited divergence between Treponema species and subspecies give rise to the observed
differences in pathogenesis? Examination of ∼7 kb of sequence resulted in the identification
of only 26 nucleotide substitutions among T. pallidum strains, or one difference for
every 275 bp (99.6% identity). As indicated in the article, this figure most likely
represents a gross overestimate of the overall degree of heterogeneity, because some
of the DNA segments were selected because of their high variability. A recent report
by Strouhal et al. [20] compared the sequences of T. pallidum subsp. pallidum (Nichols)
and Treponema paraluiscuniculi (Strain Cuniculi A), the closely related spirochete
that causes venereal spirochaetosis in rabbits but is not pathogenic to humans. A
genome-wide analysis using microarray and whole genome restriction mapping indicated
that the overall sequence similarity is in the range of 98.6 to 99.3%. Most of the
differences identified are within tpr genes or neighboring genes. Based on these two
studies and prior evidence, it is possible that genes within the tpr loci are primarily
responsible for the differences in disease manifestations and host susceptibility.
T. pallidum is one of the few human bacterial pathogens that have not been cultivated
in vitro, obviating experimental approaches such as mutational analysis and complementation
to definitively identify virulence determinants. How then can our knowledge of genes
related to treponemal evolution and pathogenesis be further refined? One possible
approach is whole genome sequencing of multiple strains and comparison of the resulting
sequences. The ongoing goal of inexpensive, ‘personalized’ human genome sequences
has resulted in the development of multiple novel sequencing approaches; some of these
methods can yield a high redundancy bacterial genome sequence for ∼US$400 in reagent
costs [21]. The new methodologies tend to yield shorter sequences per template (25
to 100 bp) and to have a higher error rate than Sanger sequencing. These shortcomings
make the newer technologies more applicable to genome re-sequencing (e.g. the analysis
of the closely related pathogenic Treponema strains) and can be ameliorated in part
by combining the results of two or more sequencing technologies.
Because of the paucity of available endemicum strains and New World pertenue isolates,
new approaches may be needed to analyze archival DNA specimens. Kolman et al. [22]
were able to identify T. pallidum sequences by PCR amplification of DNA extracted
from deformed bones (saber shins) in 200-year-old skeletal remains from Easter Island.
Processes have been developed for isolation, whole genome amplification, and sequencing
of DNA from individual bacterial cells [23],[24]. These methods, perhaps coupled with
laser capture microscopy or techniques for dissecting out organisms, could be utilized
to recover and obtain sequence information from ancient bones or preserved tissue
specimens with treponemal infections.
We have developed a microfluidic device that allows the isolation and genome amplification of individual microbial cells, thereby enabling organism-level genomic analysis of complex microbial ecosystems without the need for culture. This device was used to perform a directed survey of the human subgingival crevice and to isolate bacteria having rod-like morphology. Several isolated microbes had a 16S rRNA sequence that placed them in candidate phylum TM7, which has no cultivated or sequenced members. Genome amplification from individual TM7 cells allowed us to sequence and assemble >1,000 genes, providing insight into the physiology of members of this phylum. This approach enables single-cell genetic analysis of any uncultivated minority member of a microbial community.
Genome sequencing currently requires DNA from pools of numerous nearly identical cells (clones), leaving the genome sequences of many difficult-to-culture microorganisms unattainable. We report a sequencing strategy that eliminates culturing of microorganisms by using real-time isothermal amplification to form polymerase clones (plones) from the DNA of single cells. Two Escherichia coli plones, analyzed by Affymetrix chip hybridization, demonstrate that plonal amplification is specific and the bias is randomly distributed. Whole-genome shotgun sequencing of Prochlorococcus MIT9312 plones showed 62% coverage of the genome from one plone at a sequencing depth of 3.5x, and 66% coverage from a second plone at a depth of 4.7x. Genomic regions not revealed in the initial round of sequencing are recovered by sequencing PCR amplicons derived from plonal DNA. The mutation rate in single-cell amplification is <2 x 10(5), better than that of current genome sequencing standards. Polymerase cloning should provide a critical tool for systematic characterization of genome diversity in the biosphere.
Introduction As Naples fell before the invading army of Charles the VIII in 1495, a plague broke out among the French leader's troops [1]. When the army disbanded shortly after the campaign, the troops, composed largely of mercenaries, returned to their homes and disseminated the disease across Europe [2],[3]. Today, it is generally agreed that this outbreak was the first recorded epidemic of syphilis. Although its death toll remains controversial, there is no question that the infection devastated the continent [4]. Because the epidemic followed quickly upon the return of Columbus and his men from the New World, some speculated that the disease originated in the Americas [2]. Indeed, reports surfaced that indigenous peoples of the New World suffered from a similar malady of great antiquity [5] and that symptoms of this disease had been observed in members of Columbus's crew [3]. In the twentieth century, criticisms of the Columbian hypothesis arose, with some hypothesizing that Europeans had simply not distinguished between syphilis and other diseases such as leprosy prior to 1495 [6]. It was soon recognized that different varieties of treponemal disease exist. Unlike syphilis, which is caused by the spirochete T. pallidum subspecies pallidum, the other types normally strike during childhood and are transmitted through skin-to-skin or oral contact. All are quite similar with regard to symptoms and progression [7], but endemic syphilis, or bejel, caused by subsp. endemicum, has historically affected people living in hot, arid climates and yaws, caused by subsp. pertenue, is limited to hot and humid areas. Pinta, caused by Treponema carateum, is the most distinct member of this family of diseases. Once found in Central and South America, this mild disease is characterized solely by alterations in skin color. Today, the debate over the origin of treponemal disease encompasses arguments about whether the four infections are caused by distinct but related pathogens [8] or one protean bacterium with many manifestations [9]. Paleopathologists have played a pivotal role in addressing the question surrounding the origin of syphilis. The treponemal diseases, with the exception of pinta, leave distinct marks upon the skeleton and can thus be studied in past civilizations. Paleopathological studies of populations in the pre-Columbian New World show that treponemal disease was prevalent, with cases dating back 7,000 years and increasing over time [10]. In contrast, paleopathological studies of large pre-Columbian populations in Europe and Africa have yielded no evidence of treponemal disease [11]–[13]. However, isolated cases of pre-Columbian treponemal disease from other Old World excavation sites have been reported sporadically [14]. Although these cases have often been met with criticism regarding diagnosis, dating, and epidemiological context, they have convinced some that treponemal disease did exist in the pre-Columbian Old World [15],[16]. The T. pallidum genome is small (roughly 1,000 kilobases) and was sequenced in 1998 [17]. However, comparative genetic studies of T. pallidum [18]–[23] have been rare and relatively small in scope. One reason for this is the difficulty in obtaining non-venereal strains for study. Today only five known laboratory strains of subsp. pertenue, two strains of subsp. endemicum, and no strains or samples of T. carateum survive. Furthermore, it is uncertain whether the disease pinta still exists. No cases have been reported to the World Health Organization from the former endemic countries Mexico or Colombia since 1979 [24]. Similarly, endemic syphilis was eradicated some time ago in its European focus, Bosnia [25]. In Turkey, only one infected family has been reported in the last forty years [26], and a large survey in the United Arab Emirates revealed only non-active cases of endemic syphilis in the elderly [27]. In the Old World, yaws is still reported but appears limited to a few isolated foci in the Republic of Congo and the Democratic Republic of Congo [28],[29], as well as Indonesia and Timor-Leste, where roughly 5,000 cases are reported annually [30]. In the New World, yaws appears to be constrained to an ever-constricting area of Guyana's interior [31]. Because of the paucity of samples available for experiments, most comparative studies have included very few non-venereal strains. Another limitation on comparative studies has been the small amount of variation present in the T. pallidum genome. Variability is sufficiently low that the discovery of a single nucleotide polymorphism (SNP) has warranted publication in the past [18],[20],[21]. One study has suggested that most variation between the subsp. pallidum and pertenue genomes lies within the tpr gene family [32], a family of 12 genes with sequence similarity that make up roughly 2% of the genome. A recent examination of this large gene family demonstrated more extensive variation between strains than had previous studies but also documented an unusually high frequency of intra-gene conversion events [23]. Thus, it is possible that most polymorphism in the T. pallidum genome may be concentrated in genes with limited phylogenetic informativeness. Our goal in this study was to identify variable sites in the T. pallidum genome and characterize them in as many non-venereal strains as possible, in order to test the hypothesis that syphilis emerged in humankind's recent past, from New World-derived strains of T. pallidum. Recombination can result in a phylogeny different from the true one [33],[34], and it has been shown that gene conversion is an important evolutionary mechanism in one large T. pallidum gene family [23]. For this reason, after sequencing many sites from around the genome, we performed rigorous tests for recombination, then built a phylogeny from the non-recombining SNPs and insertions/deletions (indels) identified. These results, paired with geographic analysis of strains, provide novel information on the history of T. pallidum. Methods Origin and Preparation of Isolates Twenty-two human Treponema pallidum strains, one T. pallidum strain collected from a wild baboon, and three T. paraluiscuniculi strains, which are responsible for venereal syphilis in rabbits, were used in this study (Table 1). This included all laboratory strains of subsp. pertenue (n = 5) and subsp. endemicum (n = 2). Guyana is the only known active site of yaws infection in the western hemisphere. In order to provide representation of non-venereal strains from the Americas, subsp. pertenue strains (n = 2) were obtained from indigenous children with clinical evidence of non-venereal treponemal disease during a humanitarian medical mission to protected native reserves deep within the Guyanese interior. These samples were collected from a population that has had very little contact with the outside world, both due to the remoteness of their forest location and to legal restrictions on outsider interference. Ethical approval for the sample collection protocol was obtained from the Ethics Board at Lakeridge Health Centre (Oshawa, Canada) and included obtaining informed consent from patients. Scrapings were taken from active yaws lesions and were immediately deposited in either ethanol or saline. They were kept as cool as possible, but not frozen, for the duration of the two-week medical trip. Upon return to the laboratory, they were kept frozen until DNA was extracted. Additional subsp. pertenue samples (n = 4) came from a Dutch collection of strains destroyed years ago during a freezer breakdown. Although these strains were non-viable, the organisms were kept and a small amount of each was made available for this project. Eleven syphilis strains were chosen for analysis based on geographic and chronological span. This included two strains of uncertain subspecies and origin, Haiti B and Madras, which were originally labeled as subsp. pertenue strains, but appear to be subsp. pallidum strains based on both genetic studies and clinical manifestations in a rabbit model [18], [20]–[22],[35]. 10.1371/journal.pntd.0000148.t001 Table 1 Treponema sp. analyzed in this study (N = 26). Strain Name Subspecies Place Collected Date Collected Sample Type Source Brazzaville pertenue Congo 1960 DNA only L. Schouls CDC-1 pertenue Nigeria 1980 isolate CDC CDC-2 pertenue Nigeria 1980 isolate CDC CDC-2575 pertenue Ghana 1980 DNA only L. Schouls Gauthier pertenue Nigeria 1960 isolate S. Lukehart Ghana pertenue Ghana 1988 DNA only L. Schouls Guyana 1 pertenue Guyana 2005 DNA only This Study Guyana 2 pertenue Guyana 2005 DNA only This Study Pariaman pertenue Indonesia 1988 DNA only L. Schouls Samoa D pertenue Samoa 1953 isolate CDC Samoa F pertenue Samoa 1953 isolate CDC Bosnia A endemicum Bosnia 1950 isolate CDC Iraq B endemicum Iraq 1951 isolate CDC Chicago B pallidum Chicago 1951 isolate CDC Dallas 1 pallidum Dallas 1991 isolate CDC Grady pallidum Atlanta 1998 isolate CDC Haiti B* pallidum Haiti 1951 isolate S. Lukehart R. Limberger Madras* pallidum India 1954 isolate CDC Mexico A pallidum Mexico 1953 isolate CDC Nichols pallidum Wash, D.C. 1912 isolate CDC Philadelphia 1 pallidum Philadelphia 1988 isolate CDC South Africa pallidum South Africa 1998 DNA only A. Pillay Simian Strain ? Guinea 1962 isolate S. Lukehart T. paraluiscuniculi Strain A N/A ? ? isolate CDC T. paraluiscuniculi Strain H N/A ? ? isolate S. Lukehart T. paraluiscuniculi Strain M N/A ? ? isolate S. Lukehart *: Strains were originally labeled subsp. pertenue, but various studies suggest they are, in reality, subsp. pallidum strains. DNA was obtained from Treponema organisms grown in rabbit tissue or from clinical specimens. Laboratory isolates of T. pallidum were grown in New Zealand white rabbits, consistent with guidelines set out by the Institutional Animal Care and Use Committee at the U.S. Center for Disease Control and Prevention (CDC). DNA was isolated using the QIAamp mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions for tissue or fluid preparations, depending on the type of sample. Whole genome amplification (REPLI-g Midi Kit, Qiagen) was performed on the strains for which only a limited amount of DNA was available: the four subsp. pertenue strains from the Dutch collection and a pallidum strain obtained from a South African clinical specimen. The whole genome amplification product was used as a template for subsequent polymerase chain reactions (PCRs). Unfortunately, amplification of the whole genome of the two strains collected in Guyana could not be performed, due to DNA degradation. Sequencing Twenty-one genetic regions (Table 2) were sequenced in all strains except for the two clinical samples from Guyana. These regions were scattered around the genome (Fig. 1), and were chosen based on previously demonstrated polymorphism [18], [20]–[22],[36], implication in pathogenesis, or because they harbored repetitive sequences. Because a very limited amount of considerably degraded DNA was available from the clinical specimens collected in Guyana, only seven polymorphic sites encompassing 17 SNPs could be sequenced in these strains. The sites were chosen based on 1) which appeared to be the most phylogenetically informative at the time of sequencing; and 2) which involved small molecular weight products, easily amplified from damaged DNA. They included IGR(fliG-tp0027), deoD, gpd, tp0618, tprI, cfpA, and tpF-1. 10.1371/journal.pntd.0000148.g001 Figure 1 Genome coordinates of the 21 regions targeted for sequencing. The name of the genes and/or intergenic regions (IGRs) amplified are shown in bold next to their numeric coordinates and a point denoting their location in the genome. The coordinates of the nucleotides sequenced within each gene/IGR can be found in Table 2. 10.1371/journal.pntd.0000148.t002 Table 2 The 21 genetic regions sequenced in this study. Genetic Region1 Description/Function2 Amplicon Size (Region Sequenced) 3 Accession Numbers IGR( fliG-tp0027 ) Intergenic Region 280 bp (1–207) EU101881–EU101906 cysS (tp0091) Cysteinyl-tRNA synthetase 168 bp (440–607) EU101781–EU101804 tp0106 Transporter (possible carnitine, choline, or glycine betaine transporter) 598 bp (55–652) EU102099–EU102122 tp0123 Hypothetical protein containing TPR domains 596 bp (253–809) EU102123–EU102146 234 bp IGR( tp0129-tp0130 ) Intergenic Region (158–334) EU101955–EU101978 tp0130 Hypothetical protein, basic (361–417) IGR( tp0135-tp0136 ) Intergenic Region 301 bp (24–324) EU101979–EU102002 deoD (tp0170/pfs) Purine nucleoside phosphorylase 501 bp (482–804) EU101805–EU101830 gpd ( tp0257/glpQ ) Glycerophosphodiester phosphodiesterase 331 bp(366–633) EU101831–EU101856 tp92 ( tp0326 ) Predicted outer membrane protein 1030 bp (1530–2504) EU102075–EU102098 tp0347 Hypothetical protein 340 bp (1–228) EU102147–EU102170 IGR( cheY-tp0367 ) Intergenic Region 165 bp (1–160) EU101857–EU101880 333 bp rpiA (tp0616) Ribose 5-phosphate isomerase (546–762) EU101907–EU101930 IGR(rpiA-tp0617) Intergenic Region (1–32) tp0617 Hypothetical protein, basic (193–276) tp0618 Hypothetical protein, acidic 274 bp (236–347) EU102171–EU102194 tprI ( tp0620 ) Member of the Treponema pallidum repeat family 376 bp (1–162) EU102245–EU102267 tp0668 Conserved hypothetical protein (integral membrane) 243 bp (256–498) EU102195–EU102218 223 bp tp0740 Conserved hypothetical protein (1–42) EU102003–EU102026 IGR( tp0740-tp0741 ) Intergenic Region (1–130) tp0741 Conserved hypothetical protein (562–612) cfpA ( tp0748 ) Cytoplasmic filament protein 556 bp(23–533) EU101755–EU101780 tmpB (tp0769) Predicted outer membrane protein 966 bp (57–919) EU102051-EU102074 551 bp IGR( tex-fld ) Intergenic Region (1–369) EU101931–EU101954 fld (tp0925) Flavodoxin (1–101) 150 bp tp0989 P26 related protein (1–39) EU102027–EU102050 IGR(tp0989-tp0990) Intergenic Region (1–46) tp0990 Conserved hypothetical protein (1–65) tpf-1 ( tp1038 ) Antigen Tpf-1 171 bp (81–190) EU102219–EU102244 1 Genes are identified by both commonly used names, when present, and the gene number in the T. pallidum genome. Intergenic regions (IGRs) are identified by the genes between which they fall. Where amplicons contained multiple genetic regions, they are listed on separate lines. 2 As listed on the STDgen website (http://stdgen.northwestern.edu/). 3 The size of the amplicon, in basepairs, is listed in bold. Below, the coordinates of the region sequenced within the gene is given in nucleotide positions. Primers (Table S1) were designed using the programs MacVector (Accelrys, Burlington, MA) and Primer3 [37] and the subsp. pallidum genomic DNA sequences posted on the Los Alamos National Laboratory Bioscience Division's STD Sequence Databases webpage (http://www.stdgen.northwestern.edu). PCR amplifications were performed in 50 µL reactions containing 0.5 µM primers (Invitrogen, Carlsbad, CA), 200 µM GeneAmp dNTPs (Applied Biosystems, Foster City, CA), and 2.5 U AmpliTaq Gold polymerase with Gold Buffer and 3.0 mM MgCl2 (Applied Biosystems). PCR conditions were as follows: One cycle of 94°C for 5 minutes; 35 cycles of 94°C for 30 seconds, primer annealing at the appropriate temperature for 30 seconds (Table S1), and 72°C for 1 minute and 30 seconds; followed by a final extension for seven minutes at 72°C. Standard precautions to avoid DNA contamination were employed, including the use of negative controls, aerosol resistant pipette-tips, and a three-station PCR set-up protocol. In addition, sequences that were especially important in the phylogenetic analysis were confirmed through independent amplifications. Amplicons were purified using a gel extraction kit (Qiagen, Valencia, CA) and sequenced using either an ABI 3100/3700 Automated Capillary DNA Sequencer and the Big Dye Sequencing kit (Applied Biosystems) or the CEQ 8000 Genetic Analysis System and the DTCS Quick Start Kit (Beckman-Coulter, Fullerton, CA). Sequencing was performed at the CDC, SeqWright/Fisher Sequencing Services, or Oregon Health Sciences University's Core Laboratory. Sequences were deposited in GenBank under the accession numbers listed (Table 2). Sequence Analysis Open reading frames containing polymorphisms were translated into protein sequences to examine the resulting amino acid changes. The average number of nucleotide differences between groups, as well as the average pairwise difference for each polymorphic gene, was calculated using DnaSP [38]. All nucleotide substitutions occurring in genic areas were deemed either synonymous or non-synonymous (Table S2). The amino acid substitutions resulting from non-synonymous changes were scored according to three criteria. Amino acids were categorized according to charge, polarity and volume, and Grantham's distance [39],[40]. In the first two cases, a substitution resulting in a change from one category to another was considered a radical substitution, while others were considered conservative. In the latter case, substitutions resulting in distances greater than 100, according to Grantham's index, were categorized as radical, others conservative. Substitutions considered radical in at least two of the three categories were scored as radical (Table S2). Secondary DNA structure in the polymorphic region of IGR(fliG-tp0027) was predicted using the program Mfold [41]. Phylogenetic Analysis In order to rule out recombination in the areas analyzed, each gene was tested against all of the paralogs present in the sequenced T. pallidum subsp. pallidum Nichols strain genome, using the program RDP2 [42]. This type of search would identify recombination events between the strains sequenced in this study and the paralogs present in the sequenced genome, as well as recombination between the genes in the strains sequenced. In addition, when small stretches of extremely polymorphic DNA were identified, a BLAST search was performed in order to identify possible donor regions involved in intragenomic conversion events (http://www.ncbi.nlm.nih.gov/BLAST/). Because the complete genome sequence was only available for one strain, it is possible that some recombination events, between paralogs or donor regions not present in the sequenced genome, would not be detected. For this reason, the frequency of synonymous and non-synonymous substitutions was examined in highly polymorphic stretches; regions with a high number of synonymous substitutions and multiple substitutions per codon were considered to be possible results of recombination and were excluded from the analysis. The sequence of these regions can be found in Table S3. The complete alignments, constructed as described below and encompassing all polymorphism in which within-gene recombination had been ruled out, were also analyzed using RDP2, in order to rule out large-scale genomic recombination. In order to construct phylogenetic trees incorporating all variation, an alignment of the concatenated SNPs and indels was created using ClustalX version 1.83 [43]. The order of the regions in the concatenation corresponds to their position in the genome. Modeltest [44] was used to choose the appropriate model of nucleotide substitution, Kimura's two parameter, and phylogenetic trees were built in *PAUP 4.0 [45]. Both maximum likelihood and maximum parsimony methods were employed, to glean the most information from both parsimony-uninformative traits and indels, respectively. T. paraluiscuniculi was used as an outgroup (i.e. a taxon known to lie outside of the T. pallidum grouping) with which to root the trees and determine the directionality of substitutions. In the alignment, indels were trimmed to one basepair, in order to prevent their greater length from dominating the analysis. One thousand replicates were run to obtain bootstrap support at each node, with starting trees obtained through random, step-wise addition. Tree bisection and reconnection was used for the branch-swapping algorithm. The maximum likelihood tree was chosen for display, with bootstrap support from both methods displayed at nodes. Trees in which trimmed indels were weighted 1/5 the value of substitutions were also created, in light of the faster mutation rate of repeat regions. Because many substitutions were contained in one gene, tp92, trees in which polymorphism in tp92 was not considered were built. In addition, trees built only from synonymous SNPS or non-synonymous SNPs were created using the methods described above. Results Sequence Variation Of the twenty-one genes sequenced in this study, data from T. paraluiscuniculi was obtained for all but one gene, tprI, which had previously been shown to be absent from the genome of this species [46]. The tp0618 gene could be amplified from T. paraluiscuniculi Strain A, but not from Strains H and M. It is possible that the gene is missing from the genome of the latter two strains or that the sequence in the priming regions has diverged sufficiently to prevent amplification. No areas likely to result from recombination were identified in formal tests performed in RDP2. However, polymorphic regions in two genes, tp0618 and tp92, contained small tracts of highly variable DNA with an elevated frequency of synonymous nucleotide substitutions and multiple substitutions per codon. No possible donor regions could be identified for these tracts using BLAST searches on the T. pallidum genome. However, because such a high frequency of synonymous substitutions could be explained by recombination, and it was thus not clear how many mutational events were responsible for the polymorphism observed, the polymorphism in tp0618 and four polymorphic regions from tp92 were excluded from the phylogenetic analysis. The tp92 substitutions included in the analysis can be found in polymorphism Table S2, while all substitutions can be found in polymorphism Table S3. In the remaining 7 kilobases of the T. pallidum genome examined, which were sequenced from 20 scattered regions, a total of 70 SNPs and 12 indels were identified. Most of them are described here for the first time. Twenty-six substitutions occurred between T. pallidum strains (Table 3), amounting to about one substitution per 275 basepairs. This value is likely to significantly overestimate the amount of polymorphism typical of the genome, however. A number of regions were sequenced either because of their previously demonstrated polymorphism or because they were thought likely to contain variation. This may have weighted the regions sampled towards exceptionally polymorphic areas. The region sequenced in tp92, for example, contained 7 of the 24 substitutions observed between T. pallidum strains (Table 3). Roughly two-thirds of the total substitutions observed represented fixed differences between T. pallidum and the outgroup, T. paraluiscuniculi (Table 4). Singletons were rare, accounting for only 7 of the 70 observed substitutions (Table S2). Three of these singletons were found in the Pariaman strain, which was geographically distinct from other subsp. pertenue strains, and no singletons were observed in the other whole genome amplified strains. Thus, it appears that whole genome amplification did not introduce spurious substitutions. 10.1371/journal.pntd.0000148.t003 Table 3 Summary of polymorphism found between T. pallidum strains. Genetic Region Size of Sequenced Fragment (bp) # Indels # Polymorphic Sites # Sites with NS Substitutions # Parsimony Informative Sites1 π π S: π NS All Sites S Sites NS Sites IGR( fliG-tp0027 ) 207 1 1 - 2 - - - - tp0106 598 0 1 0 0 0.00016 0.00061 0.00000 - tp0123 557 0 1 1 0 0.00017 0.00000 0.00023 0 IGR( tp0135-tp0136 ) 301 1 0 - 1 - - - - deoD 323 0 1 0 1 0.00160 0.00611 0.00000 - gpd 268 0 1 0 1 0.00193 0.00823 0.00000 - tp92 2 975 1 7 7 6 0.00194 0.00000 0.00257 0 tp0347 228 1 1 0 2 0.00082 0.00329 0.00000 - IGR( cheY-tp0367 ) 160 0 2 - 2 - - - - rpiA 217 0 1 1 0 0.00044 0.00000 0.00059 0 tp0617 84 0 3 0 3 0.01451 0.05193 0.00000 - tprI 162 0 3 3 3 0.00947 0.00000 0.12660 0 tp0668 243 1 0 0 1 0.00000 0.00000 0.00000 0 cfpA 511 0 2 1 2 0.00152 0.00464 0.00065 7.13846 tpf-1 110 0 2 1 1 0.00542 0.00323 0.00612 0.52778 1 In parsimony analysis, gaps were considered a fifth character state. 2 In the tp92 gene, residues 1531–1593, 1672–1794, 1837–2106, 2140–2334, and 2386–2502 were included in this analysis. Regions that may have undergone recombination were omitted. 10.1371/journal.pntd.0000148.t004 Table 4 Summary of divergence between T. pallidum and T. paraluiscuniculi. Type Number Intergenic INDELS 5 SNPs 13 Genic INDELS 2 SNPs 31 A few polymorphic regions analyzed here are of special interest in light of past studies and the paucity of genetic variation described to date. Polymorphism between subsp. pertenue strains occurring in the first 200 basepairs of the tprI gene, demonstrated here, had not been described in previous studies of this gene [23],[47]. In the small region of tprI sequenced here, all 3 substitutions documented in T. pallidum strains were non-synonymous and in close proximity (Table 3). Two resulted in radical amino acid substitutions (Table S2). Variation in the cfpA gene had previously been reported in a study comparing the sequence of this gene in the Nichols and Haiti B subsp. pallidum strains [36]. We observed only one of the substitutions reported in this article, a polymorphism occurring among subsp. pallidum strains at position 92 (Fig. 2). However, we discovered one fixed difference between the non-venereal subspecies and subsp. pallidum, at residue 303 (Fig. 2). Much of the polymorphism in tp92 was not analyzed in this study, because it fell within hyper-variable regions that were difficult to align and in which recombination could not be ruled out. Even so, in the regions included, 7 non-synonymous and no synonymous substitutions occurred among T. pallidum strains (Table 3). Four of these substitutions resulted in radical amino acid changes (Table S2). Finally, in IGR(fliG-tp0027) the presence of a SNP followed by a long homonucleotide repeat region (Fig. S1) was documented in subsp. pallidum, but not in the other subspecies. The net polymorphism in the former strains was predicted, on the basis of conformational stability, to form a stem-loop structure in the intergenic region between the oppositely transcribed genes fliG and tp0027. This structure, located between the predicted promoter and transcriptional start site of the operon containing genes tp0027 and tp0028 (Fig. S1), could attenuate transcript levels of these genes. Both of these genes are homologous to tlyC, which is believed to either code for a hemolysin or for a protein that regulates hemolysin production [48]. 10.1371/journal.pntd.0000148.g002 Figure 2 Comparison of 17 single nucleotide polymorphisms present in pathogenic Treponema strains, including 2 strains of T. pallidum subsp. pertenue gathered from Guyana. A phylogenetic tree (Fig. 3), constructed using maximum likelihood and parsimony methods, demonstrated that all T. pallidum strains fell within a single clade. Within this larger clade, several T. pallidum clades with bootstrap support greater than 90% were identified. Subsp. pallidum and subsp. endemicum strains formed groupings distinct from subsp. pertenue strains. In addition, subsp. pertenue strains CDC-1, CDC-2575, and Ghana formed a clade distinct from the other strains of this subspecies, including those gathered nearby. Several clades with lower bootstrap support were also identified within the pallidum and pertenue subspecies. The subsp. pertenue strains occupy a basal location on the tree, indicating an ancestral position for them in the T. pallidum family. This basal position is supported by the average number of nucleotide differences from the T. paraluiscunili clade, which was lower for the subsp. pertenue strains (50.13) then for the subsp. endemicum and pallidum clade (55.15), or for the individual subsp. endemicum (51.33) and subsp. pallidum (56.00) clades. The genetic distance between subsp. pertenue and endemicum strains was small compared to the distance between these non-venereal subspecies and subsp. pallidum. The terminal position of the subsp. pallidum clade on the tree indicates that it diverged most recently. 10.1371/journal.pntd.0000148.g003 Figure 3 A phylogenetic tree depicting the relationships between the T. pallidum subspecies. This maximum likelihood tree is based on 20 polymorphic regions in the T. pallidum genome. Bootstrap support was estimated with 1,000 replicates in order to assess confidence at branching points and are shown within circles where values are high (>90%). Bootstrap support values for both maximum likelihood and maximum parsimony trees are shown, in that order. The results obtained from this phylogenetic analysis were robust. Trees in which trimmed indels were weighted one-fifth the value of substitutions were found to be qualitatively indistinguishable from unweighted trees. And because many of the substitutions analyzed here came from the portion of tp92 sequenced, a tree built without this gene was constructed (tree not shown). The major groupings described above were observed. However, without tp92, groupings that appeared in Fig. 3 with low bootstrap support, such as the clade containing the Mexico A and subsp. pallidum strain from South Africa, were not identified. Trees built using only synonymous or non-synonymous substitutions demonstrated that while the subspecies clades could be obtained by analyzing only synonymous substitutions, non-synonymous substitutions were responsible for increased phylogenetic resolution (trees not shown). That is, identification of all within-subspecies clades was dependent on non-synonymous substitutions. Genetic analysis of the two subsp. pertenue strains collected from indigenous groups in Guyana revealed that they were the closest relatives of modern subsp. pallidum strains identified in this study. Only a subset of seven genetic regions could be analyzed in these strains: IGR(fliG-tp0027), deoD, gpd, tp0618, tprI, cfpA, and tpF-1. These regions contained 17 SNPs at which Old World non-venereal strains differed from subsp. pallidum strains (Fig. 2). At 4 of the 17 SNPs examined, the New World subsp. pertenue strains were found to be identical to subsp. pallidum strains. These 4 SNPs occurred in 2 loci on separate sides of the genome, tprI and gpd. At the remaining 13 sites, the New World pertenue strains were identical to Old World non-venereal strains. A network path was constructed, in which these SNPs were considered in their geographic context (Fig. 4). In addition, polarity of substitutions was determined using data from the previously constructed phylogeny (Fig. 3). The tree indicated that the ancestral state of the gpd gene could be found in Old World subsp. pertenue and endemicum strains. In addition, polymorphism in the tprI gene could be used to divide T. pallidum into four groups: subsp. pallidum, subsp. endemicum, and two smaller groups of Old World subsp. pertenue. The phylogeny indicated that of the two Old World subsp. pertenue groups, the more recently diverged was the CDC-1/CDC-2575/Ghana one, while the other subsp. pertenue group was ancestral. Thus, the 4 SNP sequence present in the majority of Old World subsp. pertenue strains appears to have arisen first. The network path suggests that a series of substitutions led from this first group of Old-World pertenue strains to a second group of African subsp. pertenue strains and to subsp. endemicum strains (Fig. 4). The pattern of substitutions suggest that a hypothetical intermediate strain, arising from either the group II subsp. pertenue strains or endemicum strains, once existed and was a progenitor to both New World subsp. pertenue and to all subsp. pallidum strains. This data also suggests that the New World subsp. pertenue strains belong to a group distinct from the Old World subsp. pertenue strains, occupying a phylogenetic position somewhere between Old World non-venereal strains and modern subsp. pallidum strains. 10.1371/journal.pntd.0000148.g004 Figure 4 A network path for four informative substitutions shows that New World subsp. pertenue, or yaws-causing strains, are the closest relatives of modern subsp. pallidum strains. The geographical distribution of the endemic treponemal diseases circa 1900 is shown, based on a map created by Hackett [62]. Each polymorphism pattern is linked to the sites where the strains that contain it were gathered. Arrows convey the directionality of change, determined from the previously built phylogenetic tree as described in the text. The four substitutions were located in two genes located on separate sides of the genome, tprI and gpd. The locations from which subsp. pallidum strains were gathered are listed in Table 1. Discussion In the past, a number of different hypotheses regarding the origin of T. pallidum subsp. pallidum, the causative agent of syphilis, have been put forth. Using new data collected in this study, we assess a number of these hypotheses. The phylogenetic tree created in this study sheds light on the relative order in which the T. pallidum subspecies emerged. Subsp. pertenue strains gathered from central Africa and the South Pacific occupy basal positions on the tree, indicating that they most closely resemble the ancestral pathogen in humans (Fig. 3). The early emergence of these strains in human history is also supported by their low average nucleotide differences from T. paraluiscuniculi and their similarity to the simian strain of T. pallidum, which infects wild baboons. Although the simian strain could not be distinguished from subsp. pertenue strains using the polymorphic data in this study (Fig. 3), another study focusing on the tpr genes found that it was distinct from human T. pallidum strains examined [47]. This evidence is consistent with the hypothesis that yaws is an heirloom disease in humans, one caused by a pathogen that infected our anthropoid ancestors and has evolved with our species [49]. The presence of yaws in wild populations of our closest relatives, gorillas and chimpanzees [50],[51], further supports this theory. However, a more recent cross-species transfer event between humans and non-human primates cannot be ruled out using the available genetic data. It has been shown that inoculation with the simian strain can cause a yaws-like infection in humans [52], and it is known that infection rates are high in both humans and wild baboons in yaws-endemic areas of West Africa [53],[54]. Thus, it is possible that non-human primates serve as a source of human disease, or vice versa. Therefore, in the future it would be desirable to collect T. pallidum strains from various wild, non-human primate species and to sequence them at additional loci. Such information is likely to provide important information concerning the antiquity of yaws in humans. Subsp. endemicum strains, gathered from the Middle East and the Balkans, diverged from subsp. pertenue strains at some later date, and subsp. pallidum strains diverged most recently, indicating that they emerged relatively recently in human history (Fig. 3). The topology of the tree is consistent with the long-held belief that treponemal disease is very old and has traveled with humans during their migrations, evolving from ancestral subsp. pertenue, in hot, humid regions, into subsp. endemicum as people settled in cooler and dryer areas, and finally into subsp. pallidum [9],[55]. Examination of additional variable sites in non-venereal strains from Africa and Asia may aid in pinpointing the trajectory of this pathogen family in the Old World. The study of two yaws-causing strains from the Americas provides additional clues to understanding the history of syphilis. The genetic analysis of the two subsp. pertenue strains gathered in Guyana demonstrates that they are the closest relatives of venereal syphilis-causing strains identified in this study (Fig. 4). These strains are genetically distinct from Old World subsp. pertenue strains, having diverged more recently than Old-World non-venereal strains. The geographic analysis of strains, paired with the phylogeny, suggests a three-stage model for T. pallidum's dissemination and evolution. First, T. pallidum arose in the Old World, in the form of non-venereal infection, before spreading with humans to the Middle East/Eastern Europe, in the form of endemic syphilis, and then to the Americas, in the form of New World yaws. Second, a T. pallidum strain from the Americas was introduced back into the Old World, probably as a result of the European exploration of the Americas, becoming the progenitor of modern syphilis-causing strains. Third, modern subsp. pallidum strains disseminated from Europe to the rest of the world. Descriptions of the clinical presentation of yaws in indigenous Guyanese patients [31] support the “transitional” position of South American subsp. pertenue strains, between Old World non-venereal and subsp. pallidum strains, indicated by the genetic comparison in this study. These yaws patients, who are inhabitants of Guyana's interior, typically present with a chancre (a chronic, painless ulcer with raised margins) similar to the type seen in venereal syphilis, though found in children and in extra-genital locations. This clinical presentation is quite different from the textbook “frambesiform” lesion characteristic of yaws in Africa and Asia. It is possible that this distinctive lesion results from differences in the pathogen genome, although host differences, both cultural and genetic, may also play a role. Though modern travel increases the probability that a strain present in one place may have been recently introduced from another, the isolation of the aboriginal population from which these strains were collected, as well as their unique genetic and clinical characteristics, makes such an event unlikely. It is possible that these strains possess some of the characteristics of subsp. pallidum strains while still retaining many of the features of subsp. pertenue strains, including a non-sexual transmission mode. The condition of the two samples precluded a complete comparative study involving these strains, but the distinct genetic make-up indicated by the available data makes further study of South American subsp. pertenue strains desirable, if additional samples can be gathered in the future. Although the data suggest that venereal syphilis-causing strains arose recently, from a New World progenitor, the transmission mode of the ancestral bacterium remains unclear. The closest relatives of syphilis-causing strains in this study were non-venereally transmitted. However, because of the disappearance of endemic treponemal disease from South America it was only possible to study two indigenous strains from this continent, gathered in close proximity. It is possible that a strain even more closely related to subsp. pallidum once existed in pre-Columbian South America and was transmitted venereally. Paleopathologists have assessed the age distribution of treponemal infection in pre-Columbian Native American populations, in hopes of determining the mode of transmission in these civilizations. However, because permanent bone remodeling due to treponemal infection usually occurs in tertiary-stage disease, which can take many years to develop, it is difficult to determine at which age individuals typically contracted the infection in past populations, even when finds are abundant [56]. Several possible cases of congenital syphilis, believed by many to occur only in venereal infection, have been reported from pre-Columbian America, but the diagnoses remain tentative [56]. Some researchers have attempted to determine the nature of treponemal infection in past populations through statistical comparison of specific pathologies in the skeletal record. Based on this method, they assert that the treponemal infection present in the Dominican Republic at the time of Columbus's landing was more akin to venereal syphilis than yaws or endemic syphilis [57]. However, this method remains controversial, because of the limited samples from which specific pathology rates have been determined for each disease [56]. Therefore, it is not clear whether venereal syphilis existed in the New World prior to Columbus's arrival. While it is possible that Columbus and his crew imported venereal syphilis from the New World to Europe, it is also possible that the explorers imported a non-venereal progenitor that rapidly evolved into the pathogen we know today only after it was introduced into the Old World. Indeed, analysis of the changing descriptions of venereal syphilis following its appearance in Europe have led many to believe that the pathogen did evolve rapidly after its initial introduction [16]. Given the limitations of the available data, the question of whether the progenitor of modern syphilis-causing strains was venereal or non-venereal may remain unresolved. The results of this molecular study clarify some findings of skeletal biology while obfuscating others. The virtual absence of syphilitic lesions from Pre-Columbian Old World skeletons can be explained simply in the context of this data; syphilis did not exist in these areas until the Renaissance. On the other hand, the absence of lesions typical of yaws or endemic syphilis in these areas is puzzling in light of the genetic data. If the non-venereal treponematoses arose in the Old World long before syphilis, then why isn't there more evidence of their presence? If yaws was the first form of treponemal disease, as indicated by our study, and was limited to hot, humid areas, we would expect preservation of ancient, affected remains to be poor. This may contribute to the paucity of skeletal finds. In light of the hypotheses regarding the rapid evolution of subsp. pallidum in Renaissance Europe and the lack of knowledge concerning the genetic basis for the different clinical manifestations of the treponematoses, any evidence of positive selection or functional change in the T. pallidum genome would be of great interest. A comparison of trees constructed with only synonymous or non-synonymous substitutions emphasized the role of non-synonymous substitutions in differentiating T. pallidum strains. Ten of the 14 non-synonymous substitutions observed between T. pallidum strains occurred in just 2 genes: tprI and tp92. Similarly, 6 of 9 radical amino acid substitutions observed between T. pallidum strains occurred in these 2 genes (Table S2). Given the evidence for the role of the TprI and Tp92 proteins in pathogenicity [22],[58],[59], the substitutions clustered in the regions of these genes that were sequenced may hint at positive selection. Sequencing the entirety of these genes in many strains of T. pallidum may be worthwhile, in order to better assess this possibility. Similarly, since tp0027 and tp0028 are both homologous to tlyC, a gene that either encodes a hemolysin or regulates a cryptic one in E. coli [48], a difference in transcript level between subspecies could affect pathogenesis. To this end, a regulatory function for the predicted stem-loop structure in IGR(fliG-tp0027) in subsp. pallidum could be tested. Transcript levels of tp0027 could be characterized in the different subspecies; in addition, the two IGR variants discovered in this study could be placed upstream of a reporter gene in a genetically tractable bacterium, such as E. coli, in order to directly examine the effect of the stem-loop structure on transcript levels. Our conclusions regarding the history of T. pallidum differ from those drawn in a recent comparative study of the tpr gene family [23], in which it was asserted that the times of emergence for the pathogens that cause endemic syphilis, yaws, and syphilis were similar and dated to sometime later than the emergence of modern humans but earlier than the Renaissance. We propose several reasons for why the conclusions arrived at in the two studies diverge. The strains collected from Guyana in this study played an integral role in our analysis, because they were most closely related to syphilis-causing strains and helped establish the geographic trajectory of T. pallidum evolution. In addition, in the genetic regions we examined, recombination was much less common than in the majority of the tpr genes, if it was present at all. Our data suggest that the T. pallidum genome is evolving in a largely clonal manner amenable to phylogenetic analysis, with the exception of frequent intra-gene conversion events confined within the members of the tpr gene family [23]. Finally, because T. paraluiscuniculi was extremely similar to T. pallidum at all but one of the sequences examined (tprI is missing in the former species), it was easy to create alignments and to assess the directionality of mutations in this study. The evolutionary pathway between the tpr genes of T. paraluiscuniculi and T. pallidum is more complicated and much harder to interpret. This study has some obvious limitations. The level of polymorphism found between T. pallidum strains is quite low, as suggested by previous studies. For this reason, the level of resolution in the phylogenetic tree is relatively poor. It is likely that polymorphism data from the rest of the genome will clarify the topology of this tree, including the relationship between subsp. endemicum and subsp. pallidum strains, which are grouped together in this study with low bootstrap support, and the position of subsp. pertenue strains, which occupy a basal position on the tree but group together only by default. Similarly, the close relationship between subsp. pallidum strains and New World subsp. pertenue strains described in this paper hinges on the analysis of only 4 SNPs. Three of these SNPs were found in a single gene, tprI. The close proximity of these non-synonymous SNPs within the gene, the non-linear relationships of the strains indicated by the substitutions (Fig. 4), and the evidence that the TprI protein may be involved in pathogenesis, suggest that tprI may not be evolving neutrally. For this reason, it is unlikely that these SNPs have accumulated in a clockwork manner. Instead, the information that can be gleaned from this gene is limited to the relative order in which evolutionary events occurred. The large-scale comparative genetic studies possible on the pathogens that cause diseases such as malaria and anthrax will never be possible in T. pallidum, because of the disappearance of the non-venereal treponematoses and the strains that cause them [60],[61]. The prevalence of yaws in Guyana, the last country in South America in which yaws has been documented in recent years, has decreased annually, and surveys carried out by our group in 2006 and 2007 in endemic yaws territory demonstrated no active cases of the disease. Analysis of South American strains is necessary in order to assess the relationship between subsp. pallidum and non-venereal treponemal strains. Because it is not clear whether an opportunity to examine such strains will arise again, the results presented in this paper are of special importance in the debate over the origins of treponemal disease. In conclusion, in this study we found that syphilis-causing strains evolved relatively recently in human history and that the closest relatives of subsp. pallidum were yaws-causing strains from the New World. When this genetic data is combined with extensive documentary evidence that syphilis appeared in Europe for the first time around 1495 [12] and the apparent absence of skeletal signs of syphilis in pre-Columbian Europe and North Africa, the Columbian hypothesis for syphilis's origin gains new strength. Supporting Information Figure S1 Predicted stem-loop structure observed in all Treponema pallidum subsp. pallidum strains in IGR(fliG-tp0027). All nucleotides preceding the transcription start site are shown, with the predicted polymerase binding site in red and the putative hemolysin genes tp0027 and tp0028 following the intergenic region. The homonucleotide repeat that allows this stem-loop formation is absent in subsp. endemicum and pertenue strains, and its presence may affect transcription of the genes ahead. (0.04 MB DOC) Click here for additional data file. Table S1 Primers and annealing temperatures used in this study. (0.07 MB DOC) Click here for additional data file. Table S2 Polymorphism table. (0.07 MB XLS) Click here for additional data file. Table S3 Polymorphism table including excluded regions. (0.09 MB XLS) Click here for additional data file.
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