Treponema pallidum infections occur worldwide causing, among other diseases, syphilis and yaws. In particular sexually transmitted syphilis is regarded as a re-emerging infectious disease with millions of new infections annually. Here we present three historic T. pallidum genomes (two from T. pallidum ssp. pallidum and one from T. pallidum ssp. pertenue) that have been reconstructed from skeletons recovered from the Convent of Santa Isabel in Mexico City, operational between the 17 th and 19 th century. Our analyses indicate that different T. pallidum subspecies caused similar diagnostic presentations that are normally associated with syphilis in infants, and potential evidence of a congenital infection of T. pallidum ssp. pertenue, the causative agent of yaws. This first reconstruction of T. pallidum genomes from archaeological material opens the possibility of studying its evolutionary history at a resolution previously assumed to be out of reach.

Among the worldwide prevalent treponemal diseases syphilis is a global threat that is currently re-emerging. The origins of syphilis and other treponemal diseases are as yet unresolved and are subject to an intensive scholarly debate. Until now, assumptions on its origins and evolutionary history could only be drawn from osteological analyses of past cases and genetic analysis of contemporary T. pallidum genomes; contributions from ancient DNA are very rare and have so far failed to provide genome-level data. The ancient T. pallidum genomes presented here allow us, for the first time, to perform genome-wide comparative analyses and to assess a connection between osteological manifestations of past treponemal cases and specific T. pallidum species. Our study demonstrates the possibility of retrieving ancient T. pallidum genomes from archeological material and thereby establishes a new method that could greatly contribute to uncover the mystery regarding the origins of treponemal diseases.

Here, we use DNA hybridization capture methods in combination with high-throughput sequencing to retrieve historic T. pallidum DNA (T. pallidum ssp. pallidum and T. pallidum ssp. pertenue) and to successfully reconstruct three genomes using bone material dating back to Colonial Mexico with characteristic skeletal manifestations for congenital treponematosis [ 18 ]. Our study establishes the possibility of retrieving ancient T. pallidum genomes from archeological material and allows us, for the first time, to assess the genomic principle of past treponemal infections.

Ancient DNA could help to resolve this controversy by illuminating the evolutionary history of human-pathogenic treponemes. However, detection of ancient treponemal DNA is very rare. To date, only short strain-unspecific PCR fragments have been retrieved from a 200-year-old mummy [ 13 ] and from European post-Columbian neonates [ 14 ]. Furthermore, even in modern patients with advanced stage syphilis, who may display bone lesions, molecular detection of the bacterium is challenging [ 15 ] casting doubt on the possibility of a successful recovery of T. pallidum from ancient skeletons [ 16 ]. Our latest research on non-human treponemes, however, was successful in retrieving T. pallidum ssp. pertenue DNA from non-human primate bones that were several decades old [ 17 ].

Treponema pallidum subspecies cause several diseases, among which sexually transmitted syphilis, caused by T. pallidum ssp. pallidum (TPA), and yaws, caused by T. pallidum ssp. pertenue (TPE), are the best known and are prevalent worldwide. The global disease burden is high for both diseases. Syphilis is seen as re-emerging in various regions of the world including Europe, North America, China and Australia [ 1 ] with 10.6 million cases reported in 2009 [ 2 ], while for yaws more than 300,000 new cases were recorded between 2008 and 2012 [ 3 ]. The origins and evolutionary history of these pathogens remain nebulous, including the perceived sudden appearance and pandemic spread of syphilis in Europe at the end of the 15 th century. Hypotheses surrounding the origin of syphilis are subject to an extensive scholarly debate, in particular the New World origin of syphilis with worldwide dissemination starting in the 15 th century [ 4 , 5 ] in contrast to the hypothesis of multiregional origin that posits an increase in virulence followed by a pandemic spread in the 15 th century [ 6 ]. While genetic data from contemporary T. pallidum strains has been interpreted as support for the New World origin [ 7 , 8 ], there exists potential skeletal evidence in support of the multiregional origin [ 9 , 10 ], yet this remains heavily debated [ 11 , 12 ].

The presence of 61 genes previously thought to be related to virulence based on other studies was evaluated in the ancient and modern strains. This included the Tpr family, various outer membrane proteins, adhesion proteins, lipoproteins and a few other classes [ 54 – 60 ]. The ancient strains identified as syphilis harbor all of these factors except TPANIC_RS04235 a homolog of the FadL family that enables the transport of long-chain fatty acids (LCFAs) as well as other hydrophobic nutrients [ 61 ]. In general, Treponema pallidum seems to harbor altogether five FadL orthologs [ 62 ], of which the other four are present in the ancient strains. In addition, previous studies have described a deletion of the gene TPANIC_1030 in the pertenue strains [ 41 , 63 ], which is also confirmed in the ancient pertenue strain 133 ( S3 Fig and S4 Table ).

To assess the effects of these findings in the context of single-gene phylogenies, we tested different model topologies ( S2B and S2C Fig ) on 1,028 genes in a TREE-PUZZLE analysis [ 53 ]. Out of 1,028 genes, 544, 564 and 509 genes in strains 94A, 94B and 133 have at least one informative SNP in the gene alignment, of which 221, 180 and 246, respectively, do not reject any of the model trees. Among the remaining genes, 199 genes of strain 133 rejected any positioning of the strain within the syphilis clades, while for strains 94A and 94B, 265 and 322 genes, respectively, reject any position within the yaws clade. For all remaining genes, the rejected tree topologies are listed in S1 Table . We found the highest support for an ancestral placement of 94A and 94B with only seven and eight genes rejecting this positioning ( S1 Table ). For the third historic strain, 133, its positioning in the yaws clade has the highest support only rejected by one gene (proA; TPANIC_RS01715), which contains a SNP (position 376,586) that is specifically shared with Nichols ( S1 Table ).

ClonalFrameML [ 52 ] showed evidence of recombination as previously described [ 41 ]: depending on the applied model we detected between 13 (standard model) and 16 (per-branch model) predicted recombination events corresponding to the common ancestor of 94A and 94B ( S3 Table ). The combination of recombination events from both models (standard and per-branch) was analyzed by comparing these regions to SNP positions that 94A and 94B share with different clades ( S1 Table ). Most of the regions are shared with Fribourg-Blanc and thereby further support the ancestral positioning of the historic strains. We also found one recombination event (346215–347572; TPANIC_RS01590) that is congruent with previous reports on recombination in two genes (TPANIC_RS01590, TPANIC_RS02370) in the contemporary Mexico_A strain [ 46 ]. Interestingly, for TPANIC_RS02370 no recombination event was observed in the common ancestor of 94A and 94B pointing to the possibility of a recombination event that happened later in time. We identify multiple regions where the common ancestor of 94A and 94B shows putative recombination events with the ancestors of TPA (pallidum subspecies), TPE (pertenue subspecies) and TEN (endemicum subspecies) suggestive of inter-strain recombination as has been proposed earlier [ 46 – 48 ]. Furthermore, the genes TPANIC_RS04240, TPANIC_RS04770 and TPANIC_RS05095, which were previously proposed to be recombining [ 47 – 49 ], were also detected as part of potentially recombining regions in our analysis ( S3 Table ).

To study the derived SNPs that the historic strains share with different clades in more detail we also conducted a comparative SNP analysis using 2,294 SNPs among a complete set of 42 T. pallidum strains and identified the following: for 94A and 94B, 22 SNPs and 30 SNPs, respectively, support an ancestral position (Fribourg-Blanc clade), 19 and 24 SNPs, respectively, support a position in the SS14 clade, and 4 and 5 SNPs, respectively, support a position in the Nichols clade ( Fig 2C ). In general, the loci of these shared SNPs are spread across the genome and are not specific to certain regions.

To further investigate this phenomenon we performed several analyses. We constructed a Bayesian phylogenetic tree from 1,061 SNP positions after deletion of positions with missing data from a SNP alignment of the historic strains 94A and 94B with Fribourg-Blanc, Bosnia_A, Nichols, Sea81-4, Mexico_A and SS14 using BEAST2 [ 50 ]. The uncertainty in the branching pattern of 94A and 94B was visualized using Densitree [ 51 ] by overlaying the different tree topologies from the BEAST2 run ( Fig 2B ). We found the highest support for an ancestral placement in 82.16% of the tree states ( Fig 2B , blue color). Only 17.66% of the tree states ( Fig 2B , red color) support the branching of the historic strains within the SS14 clade, and 0.18% of the tree states support a trifurcation of the 94A and 94B strains, the SS14 clade (SS14, Mexico_A) and the Nichols clade (Nichols, Sea81-4).

(A) Maximum Likelihood tree with bootstrap support for 39 modern strains and the three ancient strains. The strains 94A (magenta) and 94B (orange) branch with the syphilis SS14 clade while strain 133 (brown) branches with Fribourg-Blanc and other yaws strains. The scale represents the mean number of substitutions per site according to the GTR+GAMMA. Colored bars highlight the three subspecies Treponema pallidum ssp pallidum (TPA), pertenue (TPE) and endemicum (TEN). Strains of subspecies pallidum cause syphilis, subspecies pertenue cause yaws and subspecies endemicum causes bejel. (B) Bayesian trees visualized in Densitree overlaying phylogenetic trees based on the most probable topologies. Blue colored trees represent the most probable topology followed by red colored trees. For the ancient strains 94A and 94B two conflicting topologies are visible. The bars represent the 95% highest probability density intervals of the heights of the clades. The support value given at each clade is the fraction of trees in the tree-set that contain the clade. (C) Circos plot showing the shared SNP positions with specific clades and the coverage of the three ancient strains. From outer circle to the inner circle regions of possible recombination detected by ClonalFrameML are denoted on the outermost circle (purple). ‘ORI’ refers to the origin of replication. The genome coverages of the ancient strains 94B, 94A and 133 are represented in orange magenta and brown respectively from outward to inwards. Based on the SNPs that are specifically shared with different clades, colored bars are shown for strains 94B and 94A respectively in the innermost circles. Red bars highlight the SNP positions specifically shared with Fribourg-Blanc (supporting a phylogenetic position ancestral to the two syphilis clades). The green bars highlight the SNP positions shared with the SS14 clade while the blue bars highlight the SNP positions shared with the Nichols clade.

We reconstructed a phylogeny for the three historic and 39 publicly available modern T. pallidum genomes [ 41 ] using different reconstruction methods. Our Maximum Likelihood ( Fig 2A ) and Maximum Parsimony ( S2A Fig ) trees position two of the historic strains (94A, 94B) on the branch of the modern syphilis-causing T. pallidum ssp. pallidum strains, while the third historic strain (133) falls with the modern yaws-causing T. pallidum ssp. pertenue strains. A precise placement of sample 133, however, within the yaws clade cannot be further resolved owing to its lower coverage. The strains 94A and 94B are very similar and differ only in one single position unique to 94A (237,661) in the gene TPANIC_RS01145 coding for a cobalt ABC transporter ATP-binding protein. Twenty-seven SNP positions are unique to 94A and 94B while three SNP positions are specific to sample 133 ( S2 Table ). In the Maximum Likelihood tree they branch within the SS14 clade, while they fall basal on the syphilis branch in the Maximum Parsimony tree with reduced bootstrap support. This phenomenon could be linked to DNA mosaic patterns, such as those identified previously in three T. pallidum strains [ 46 – 48 ], leading to the suggestion that T. pallidum strains may horizontally recombine [ 49 ].

To assess the authenticity of the captured T. pallidum DNA we analyzed nucleotide misincorporation patterns characteristic of ancient DNA [ 42 , 43 ]. The observed DNA damage patterns of 10% to 17% C to T damage at the terminal base of the DNA fragments support the ancient origin of the retrieved T. pallidum DNA ( Table 1 ). Human mitochondrial fragments were simultaneously enriched via a streptavidin-based bead capture [ 44 ] resulting in complete mitochondrial genomes with haplogroups found today in Central America (D1i2 for 94A & 94B and H1c+152 for 133). Consistent with the ancient pathogen, 11% to 17% DNA damage was observed at the terminal ends, thus supporting their authenticity ( Table 1 ). Contamination of the human mitochondrial DNA calculated using the program Schmutzi [ 45 ] in all three samples was estimated at less than 2% ( Table 1 ).

Long bones with lesions suggestive of treponemal infections were sampled for all five individuals; ancient DNA was extracted [ 38 ] and double stranded next generation sequencing libraries were constructed for all samples [ 39 , 40 ]. To reduce the background of environmental DNA, whole-genome array capture for T. pallidum subspecies [ 41 ] was conducted. After Illumina sequencing to a depth of 98 to 204.5 million pre-processed pair-end reads, mapping to the reference genome (NC_021490.2) and duplicate removal, between 71,524 and 128,419 unique DNA sequencing reads were retrieved for individuals 133, 94A and 94B, resulting in an average of 3.25 to 7.72-fold genome coverage with a duplication factor of 28.64 to 37.25 and 57–94% of the reference genome covered at least 3-fold ( Table 1 ).

The skeletal changes displayed by the three individuals suggest treponemal infection that is possibly congenital given their young ages [ 26 , 37 ]; however, the nature of the skeletal changes, being non-specific yet suggestive of treponemal infection emphasizes the importance of analysis through molecular paleopathological methods. Ancient DNA provides the possibility to both identify a causative infectious organism and explore its ecological and evolutionary history.

These individuals present signs of infection in the form of abnormal skeletal changes such as periosteal reactive bone formation and diaphyseal expansion of long bones ( Fig 1 ). Pathological skeletal changes were observed macroscopically and descriptively recorded as outlined by Buikstra and Ubelaker [ 25 ]. We recorded abnormal bone formation and bone destruction, status at time of death (active or healing) and location and extent of the affected bone. The resulting data was referenced to paleopathological and clinical literature [ 25 – 31 ]. The individuals displayed varied skeletal completeness and differential preservation due to taphonomic damage. The most consistent skeletal feature suggestive of infection present in the three individuals was periostitis, which included a multilayered periosteal reaction and dactylitis ( Fig 1 )[ 32 – 35 ]. Periostitis, though not specific to treponemal disease is strongly associated with treponematosis. It has been described as a “third category” of skeletal indicators of treponematosis and is present in greater frequency compared to other changes associated with treponemal disease such as cranial and dental changes [ 27 ]. Although these individuals ( S1 Fig ) do not display pathognomonic dental changes such as Hutchinson’s incisors or “mulberry” (or Founier’s) molars which are known to have a broad occurrence (33% and 27% respectively) in individuals bearing a congenital infection of syphilis, they do display abnormal periosteal bone development of the long bones and cranial bones [ 28 ]. To our knowledge, dental features associated with congenital syphilis are restricted to permanent teeth and would only be confidently visible around 6–9 months of age as only the sites of initiated ameloblast activity are visible before then [ 27 , 36 ]. Although each individual was represented by a differing number of skeletal elements, all individuals presented layered periostitis. Individual 94A, estimated to be a six month-old infant based on skeletal development [ 24 ], displays a systemic, fulminating periosteal reaction present on the axial as well as the appendicular skeleton as illustrated by the severely affected tibia in Fig 1 . Also present in this individual was dactylitis (expansive periostitis of the metacarpals), which is associated with treponematosis in the first year of life [ 27 ], among other conditions [ 26 ]. Furthermore, this individual displays abnormal reactive bone on the endocranial surface of the pars basilaris ( Fig 1 ). Individual 94B is represented by fewer skeletal elements; however, periostitis suggestive of treponemal infection is still present on the ribs and an unidentified long bone ( Fig 1 ). Based on the crown formation of the incisors and molars this individual is estimated to have died at birth +/- 2 months. Individual 133 presents bilateral periosteal reaction appearing to be active at time of death with extensive involvement of the appendicular and axial skeleton. As illustrated in Fig 1 , the right femur of this individual displays significant periosteal bone formation with some cortical resorption. This individual is represented by a nearly complete, albeit fragmentary, skeleton, with four mandibular molars, two maxillary incisors, and two canines. Dental elements from the individual were used to estimate the age at death as six months (+/- 3 months) post partum [ 21 ].

(A) The right tibia of individual 94A displays reactive periosteal bone on the anterior aspect of the diaphysis accompanied by progressive layering of the reactive bone. (B) The pars basilaris portion of the cranium of individual 94A showing pathological reactive bone in the endocranial surface, active at time of death. (C) An unidentified long bone from individual 94B displays fulminating periosteal reaction involving the whole of the diaphysis fragment. (D) The left femur of individual 133 presenting periosteal bone formation and expansion with cortical resorption characteristic of treponemal diseases. Source of the pictures: skeletal collection from Santa Isabel Convent, Mexico City, in custody of the Laboratory of Osteology, Post Graduate Studies Division, National School of Anthropology and History (ENAH), Mexico.

We screened samples from five individuals from the former Convent of Santa Isabel, a historical site located in downtown Mexico City used by nuns of the Franciscan Order from 1681 to 1861 [ 19 ]. Burials of 239 individuals (90% of which were foetuses, neonates and infants) were excavated from the remains of the convent in the 1990s, which were buried in the niches and tombs [ 19 ]. Five individuals were selected for this study based on the appearance of skeletal changes consistent with treponemal infection at the time of death. Of the five individuals, only three individuals were positive for treponemal DNA: individual 94A, 94B and 133 ( Fig 1 , S1 Fig ). We estimated the age of these three individuals using a method based on dentition, as dental development is considered a more reliable indicator of age than developmental or degenerative skeletal changes [ 20 ]. The dental aging chart established by Ubelaker [ 21 ] was developed using American indigenous populations; however, when evaluating dental aging methods with a population of known ages, the Schour and Massler atlas [ 22 ] was found to have a higher accuracy of age estimations in individuals in below the age of 5.4 years [ 23 ]. Both methods were, therefore, employed to estimate the age of individuals as recommended previously [ 20 , 21 ]. Our results reveal one perinatal individual (94B) and two infants (94A and 133) [ 21 , 24 ]. Archaeological context as well as pH—EH quantification studies suggest a burial date during the Colonial period [ 19 ]. Radiocarbon dates of the individuals 94B and 133 suggest that both individuals were buried less than 350 years ago (younger than cal AD 1654 for 94B and younger than cal AD 1669 for 133).

Discussion

The origin of syphilis and the evolutionary history of treponemal diseases are subjects of on-going debate. Ancient T. pallidum genomes have the potential to provide evidence to address many questions raised in this context. Here we provide the first historic genomes of T. pallidum subspecies. Our data is of sufficient resolution to allow for a global genomic and phylogenetic analysis. Although our historic genomes cannot directly contribute to discussions about the origin of syphilis due to their post–Columbian age, our study demonstrates the potential of successfully obtaining authentic historic T. pallidum genomes when focusing on treponemal cases in young individuals. More historic T. pallidum genomes, in particular from the pre-Columbian era, could be able to settle the debate. However, the selection of samples, retrieval of ancient T. pallidum DNA and comparative genomic analyses pose multiple challenges.

We find strong evidence that the bone lesions in two historical treponematosis cases, one in a perinate and another in an infant, were caused by T. pallidum ssp. pallidum. Furthermore, we present skeletal and genomic evidence of T. pallidum ssp. pertenue infection in an infant individual estimated to be six months (+/- 3 months) of age at time of death. This individual calls to attention the possible existence of congenital yaws, which has been debated in the literature due to the apparent inability of T. pallidum ssp. pertenue to cross the placental barrier, despite the description of potential historical examples [64, 65]. Variation in the ability for vertical transmission has been demonstrated within T. pallidum subspecies, for example the Haiti B strain of T. pallidum ssp. pallidum also seems unable to cross the placenta [66, 67] suggesting the possibility of such variation within the pertenue subspecies strains. Thus, the debate of whether T. pallidum ssp. pertenue can [64, 65], or cannot [67, 68] cross the placental barrier to cause a form of congenital treponematosis remains unresolved. The severity of the skeletal response in individual 133 together with the young age at death (6 months, +/- 3 months) supports a diagnosis of congenital yaws; however, the known pathophysiology of T. pallidum ssp. pertenue infections, namely that skeletal changes can occur in infants infected up to four months post partum [64, 65], challenges an unequivocal diagnosis of congenital yaws for this case and highlights the importance of further attention to this research question.

Although the individuals included in our study present skeletal changes consistent with but not limited to treponematosis, producing a differential diagnosis of a greater resolution was not possible due to the shared signs of yaws and syphilis and the differential preservation of the skeletons, which compromised the ability to visualize diagnostic patterning of the pathological changes. Likewise, such challenges exist in the diagnosis of clinical cases whereby evidence of the overlapping modes of transmission and clinical features of syphilis and yaws can be found in biomedical research on modern T. pallidum strains, such as T. pallidum ssp. pallidum causing yaws-like frambesiform lesions [69] or T. pallidum ssp. pertenue invading neurological and cardiovascular tissue as seen in syphilis [64, 70]. However, our genetic results allowed us to identify syphilis infection in two of the individuals (94A, 94B) whereas one individual (94B) presents a likely congenital infection of syphilis. In addition, we identified yaws in one infant individual (133). Our work demonstrates the value of molecular identification of ancient pathogens, particularly as applied to treponemal diseases where skeletal responses to the various pathogenic subspecies are often shared, challenging the development of a confident diagnosis through osteological observation [27]. Molecular analyses not only provide greater resolution to the paleopathological diagnosis of osteological changes but they also provide relevant details related to the history and evolution of pathogens we still encounter today.

In the phylogenetic comparison of our historic strains with contemporary T. pallidum strains, we detected ambiguous SNP patterns in our strains that may suggest a number of recombination events in its evolutionary past. Although T. pallidum subspecies are thought to be clonal [71], several studies on modern treponemal DNA have reported evidence for recombination events in various T. pallidum subspecies [41, 46–48]. As recombination mechanisms are active during treponemal infections, TPA strains could have integrated some genomic regions from TPE strains during a co-infection of the same host [46, 48]. The presence of yaws in sample 133 and syphilis in samples 94A and 94B indicates that pertenue and pallidum infections were both prevalent among individuals in this region, which could have opened the possibility of recombination events facilitated by co-infections with these subspecies. Further research on ancient and modern T. pallidum strains is needed to assess this aspect of their evolution, which should be borne in mind in phylogenetic assessments.

Various studies on the pathogenicity of treponemes have suggested a number of genes potentially involved in virulence, most of which encode proteins that reside at the host-pathogen interface. Regarding the presence and absence of these genes we did not detect a difference in our ancient strains compared to modern ones with the exception of the FadL homolog TPANIC_RS04235, which is absent in our ancient syphilis strains. FadL facilitates the transport of long-chain fatty acids (LCFA), which are metabolic energy sources and can also aid in pathogenesis. Uptake of high concentrations of LCFAs released by host cells would assist in suppression of inflammatory responses thereby enabling the pathogen to colonize the host more efficiently [72, 73]. However, there are four other FadL homologs present and it is unclear if they target different compounds with varying specificity [57]. Thus, it currently remains an open question if the absence of one of the FadL homologs could influence phenotype.

In conclusion, our study demonstrates that retrieval of authentic ancient T. pallidum DNA from historic tissues to the resolution of genomic reconstruction is indeed possible, despite earlier pessimism [74]. The presence of T. pallidum ssp. pertenue in old world monkeys [17, 75] and our finding that two T. pallidum subspecies likely caused similar osteological manifestations in the past may suggest a more complex evolutionary history of T. pallidum than previously assumed. Furthermore, our detection of possible recombination events in the past highlights an important, and currently underrepresented, analytical component that should be accommodated in future models of T. pallidum's history. Our results point out the necessity of ancient T. pallidum genome-level data to better understand both osteological manifestations caused by infection of the various forms and their potential for genomic recombination in the past. Ultimately, these analyses may be able to resolve the origin of syphilis and the evolutionary history of treponemal diseases in general.