Whole genome sequence of the Treponema pallidum subsp. endemicum strain Iraq B: A subpopulation of bejel treponemes contains full-length tprF and tprG genes similar to those present in T. p. subsp. pertenue strains.

Treponema pallidum subsp. endemicum (TEN) is the causative agent of endemic syphilis (bejel). Until now, only a single TEN strain, Bosnia A, has been completely sequenced. The only other laboratory TEN strain available, Iraq B, was isolated in Iraq in 1951 by researchers from the US Centers for Disease Control and Prevention. In this study, the complete genome of the Iraq B strain was amplified as overlapping PCR products and sequenced using the pooled segment genome sequencing method and Illumina sequencing. Total average genome sequencing coverage reached 3469×, with a total genome size of 1,137,653 bp. Compared to the genome sequence of Bosnia A, a set of 37 single nucleotide differences, 4 indels, 2 differences in the number of tandem repetitions, and 18 differences in the length of homopolymeric regions were found in the Iraq B genome. Moreover, the tprF and tprG genes that were previously found deleted in the genome of the TEN Bosnia A strain (spanning 2.3 kb in length) were present in a subpopulation of TEN Iraq B and Bosnia A microbes, and their sequence was highly similar to those found in T. p. subsp. pertenue strains, which cause the disease yaws. The genome sequence of TEN Iraq B revealed close genetic relatedness between both available bejel-causing laboratory strains (i.e., Iraq B and Bosnia A) and also genetic variability within the bejel treponemes comparable to that found within yaws- or syphilis-causing strains. In addition, genetic relatedness to TPE strains was demonstrated by the sequence of the tprF and tprG genes found in subpopulations of both TEN Iraq B and Bosnia A. The loss of the tprF and tprG genes in most TEN microbes suggest that TEN genomes have been evolving via the loss of genomic regions, a phenomenon previously found among the treponemes causing both syphilis and rabbit syphilis.

First report of hare treponematosis seroprevalence of European brown hares (Lepus europaeus) in the Czech Republic: seroprevalence negatively correlates with altitude of sampling areas.

BACKGROUND: The aim of this study was to quantify the seroprevalence of hare treponematosis in European brown hare (Lepus europaeus) populations in the Czech Republic and to test for an association between treponematosis prevalence and the altitude of the areas in which hares were sampled. We tested 289 serum samples of brown hares collected between 2015 and 2017. The sampling areas included 12 districts (73 villages) distributed throughout the Czech Republic. Serum samples were tested for the presence of antibodies against the causative agent of hare treponematosis (Treponema paraluisleporidarum ecovar Lepus, TPeL) using two serological tests for human syphilis that cross-react with TPeL: the Treponema pallidum hemagglutination assay (TPHA) and the fluorescent treponemal antibody absorption (FTA-ABS) test. To account for the imperfect diagnostic sensitivity and specificity of each test, apparent prevalence estimates of TPeL were converted to true prevalence estimates using the Rogan Gladen estimator. The correlation between TPeL true seroprevalence and altitude of sampling areas was analyzed using Pearson's correlation coefficient at three levels of spatial resolution: (1) four groups, each composed of two merged districts, with ≥20 samples collected, differing in their altitude median (206, 348, 495, and 522 m above sea level); (2) separately tested eight districts, where ≥20 samples were collected per district; and (3) 27 groups composed of villages of the same altitude level distributed across the whole dataset. RESULTS: One hundred and seven of the 289 samples were seropositive to both tests, the FTA-ABS test was positive for an additional 47 samples. Seropositive samples were found in all 12 districts. True seroprevalence of TPeL in the sampled hares was 52% (95% confidence interval 46 to 58%). A statistically significant negative correlation between TPeL seroprevalence and altitude was identified at the district level (Pearson's r = - 0.722, p = 0.043). CONCLUSIONS: Between 2015 and 2017 hare treponematosis was present at a relatively high prevalence in brown hares in all 12 districts in the Czech Republic where sampling was carried out. The seroprevalence of TPeL in brown hares was negatively correlated with the altitude of the areas in which hares were sampled.

Identification of positively selected genes in human pathogenic treponemes: Syphilis-, yaws-, and bejel-causing strains differ in sets of genes showing adaptive evolution.

BACKGROUND: Pathogenic treponemes related to Treponema pallidum are both human (causing syphilis, yaws, bejel) and animal pathogens (infections of primates, venereal spirochetosis in rabbits). A set of 11 treponemal genome sequences including those of five Treponema pallidum ssp. pallidum (TPA) strains (Nichols, DAL-1, Mexico A, SS14, Chicago), four T. p. ssp. pertenue (TPE) strains (CDC-2, Gauthier, Samoa D, Fribourg-Blanc), one T. p. ssp. endemicum (TEN) strain (Bosnia A) and one strain (Cuniculi A) of Treponema paraluisleporidarum ecovar Cuniculus (TPeC) were tested for the presence of positively selected genes. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1068 orthologous genes annotated in all 11 genomes were tested for the presence of positively selected genes using both site and branch-site models with CODEML (PAML package). Subsequent analyses with sequences obtained from 62 treponemal draft genomes were used for the identification of positively selected amino acid positions. Synthetic biotinylated peptides were designed to cover positively selected protein regions and these peptides were tested for reactivity with the patient's syphilis sera. Altogether, 22 positively selected genes were identified in the TP genomes and TPA sets of positively selected genes differed from TPE genes. While genetic variability among TPA strains was predominantly present in a number of genetic loci, genetic variability within TPE and TEN strains was distributed more equally along the chromosome. Several syphilitic sera were shown to react with some peptides derived from the protein sequences evolving under positive selection. CONCLUSIONS/SIGNIFICANCE: The syphilis-, yaws-, and bejel-causing strains differed relative to sets of positively selected genes. Most of the positively selected chromosomal loci were identified among the TPA treponemes. The local accumulation of genetic variability suggests that the diversification of TPA strains took place predominantly in a limited number of genomic regions compared to the more dispersed genetic diversity differentiating TPE and TEN strains. The identification of positively selected sites in tpr genes and genes encoding outer membrane proteins suggests their role during infection of human and animal hosts. The driving force for adaptive evolution at these loci thus appears to be the host immune response as supported by observed reactivity of syphilitic sera with some peptides derived from protein sequences showing adaptive evolution.

Complete genome sequences of two strains of Treponema pallidum subsp. pertenue from Indonesia: Modular structure of several treponemal genes.

BACKGROUND: Treponema pallidum subsp. pertenue (TPE) is the causative agent of yaws, a multistage disease endemic in tropical regions in Africa, Asia, Oceania, and South America. To date, seven TPE strains have been completely sequenced and analyzed including five TPE strains of human origin (CDC-2, CDC 2575, Gauthier, Ghana-051, and Samoa D) and two TPE strains isolated from the baboons (Fribourg-Blanc and LMNP-1). This study revealed the complete genome sequences of two TPE strains, Kampung Dalan K363 and Sei Geringging K403, isolated in 1990 from villages in the Pariaman region of Sumatra, Indonesia and compared these genome sequences with other known TPE genomes. METHODOLOGY/PRINCIPAL FINDINGS: The genomes were determined using the pooled segment genome sequencing method combined with the Illumina sequencing platform resulting in an average coverage depth of 1,021x and 644x for the TPE Kampung Dalan K363 and TPE Sei Geringging K403 genomes, respectively. Both Indonesian TPE strains were genetically related to each other and were more distantly related to other, previously characterized TPE strains. The modular character of several genes, including TP0136 and TP0858 gene orthologs, was identified by analysis of the corresponding sequences. To systematically detect genes potentially having a modular genetic structure, we performed a whole genome analysis-of-occurrence of direct or inverted repeats of 17 or more nucleotides in length. Besides in tpr genes, a frequent presence of repeats was found in the genetic regions spanning TP0126-TP0136, TP0856-TP0858, and TP0896 genes. CONCLUSIONS/SIGNIFICANCE: Comparisons of genome sequences of TPE Kampung Dalan K363 and Sei Geringging K403 with other TPE strains revealed a modular structure of several genomic loci including the TP0136, TP0856, and TP0858 genes. Diversification of TPE genomes appears to be facilitated by intra-strain genome recombination events.

Sequencing of Treponema pallidum subsp. pallidum from isolate UZ1974 using Anti-Treponemal Antibodies Enrichment: First complete whole genome sequence obtained directly from human clinical material.

Treponema pallidum subsp. pallidum (TPA) is the infectious agent of syphilis, a disease that infects more than 5 million people annually. Since TPA is an uncultivable bacterium, most of the information on TPA genetics comes from genome sequencing and molecular typing studies. This study presents the first complete TPA genome (without sequencing gaps) of clinical isolate (UZ1974), which was obtained directly from clinical material, without multiplication in rabbits. Whole genome sequencing was performed using a newly developed Anti-Treponemal Antibody Enrichment technique combined with previously reported Pooled Segment Genome Sequencing. We identified the UW074B genome, isolated from a sample previously propagated in rabbits, to be the closest relative of the UZ1974 genome and calculated the TPA mutation rate as 2.8 x 10(-10) per site per generation.

Molecular characterization of Treponema pallidum subsp. pallidum in Switzerland and France with a new multilocus sequence typing scheme.

Syphilis is an important public health problem and an increasing incidence has been noted in recent years. Characterization of strain diversity through molecular data plays a critical role in the epidemiological understanding of this re-emergence. We here propose a new high-resolution multilocus sequence typing (MLST) scheme for Treponema pallidum subsp. pallidum (TPA). We analyzed 30 complete and draft TPA genomes obtained directly from clinical samples or from rabbit propagated strains to identify suitable typing loci and tested the new scheme on 120 clinical samples collected in Switzerland and France. Our analyses yielded three loci with high discriminatory power: TP0136, TP0548, and TP0705. Together with analysis of the 23S rRNA gene mutations for macrolide resistance, we propose these loci as MLST for TPA. Among clinical samples, 23 allelic profiles as well as a high percentage (80% samples) of macrolide resistance were revealed. The new MLST has higher discriminatory power compared to previous typing schemes, enabling distinction of TPA from other treponemal bacteria, distinction between the two main TPA clades (Nichols and SS14), and differentiation of strains within these clades.

Gene target selection for loop-mediated isothermal amplification for rapid discrimination of Treponema pallidum subspecies.

We show proof of concept for gene targets (polA, tprL, and TP_0619) that can be used in loop-mediated isothermal amplification (LAMP) assays to rapidly differentiate infection with any of the three Treponema pallidum subspecies (pallidum (TPA), pertenue (TPE), and endemicum (TEN)) and which are known to infect humans and nonhuman primates (NHPs). Four TPA, six human, and two NHP TPE strains, as well as two human TEN strains were used to establish and validate the LAMP assays. All three LAMP assays were highly specific for the target DNA. Amplification was rapid (5-15 min) and within a range of 10E+6 to 10E+2 of target DNA molecules. Performance in NHP clinical samples was similar to the one seen in human TPE strains. The newly designed LAMP assays provide proof of concept for a diagnostic tool that enhances yaws clinical diagnosis. It is highly specific for the target DNA and does not require expensive laboratory equipment. Test results can potentially be interpreted with the naked eye, which makes it suitable for the use in remote clinical settings.

Genetics of human and animal uncultivable treponemal pathogens.

Treponema pallidum is an uncultivable bacterium and the causative agent of syphilis (subsp. pallidum [TPA]), human yaws (subsp. pertenue [TPE]), and bejel (subsp. endemicum). Several species of nonhuman primates in Africa are infected by treponemes genetically undistinguishable from known human TPE strains. Besides Treponema pallidum, the equally uncultivable Treponema carateum causes pinta in humans. In lagomorphs, Treponema paraluisleporidarum ecovar Cuniculus and ecovar Lepus are the causative agents of rabbit and hare syphilis, respectively. All uncultivable pathogenic treponemes harbor a relatively small chromosome (1.1334-1.1405 Mbp) and show gene synteny with minimal genetic differences (>98% identity at the DNA level) between subspecies and species. While uncultivable pathogenic treponemes contain a highly conserved core genome, there are a number of highly variable and/or recombinant chromosomal loci. This is also reflected in the occurrence of intrastrain heterogeneity (genetic diversity within an infecting bacterial population). Molecular differences at several different chromosomal loci identified among TPA strains or isolates have been used for molecular typing and the epidemiological characterization of syphilis isolates. This review summarizes genome structure of uncultivable pathogenic treponemes including genetically variable regions.

Reanalysis of Chinese Treponema pallidum samples: all Chinese samples cluster with SS14-like group of syphilis-causing treponemes.

OBJECTIVE: Treponema pallidum subsp. pallidum (TPA) is the causative agent of syphilis. Genetic analyses of TPA reference strains and human clinical isolates have revealed two genetically distinct groups of syphilis-causing treponemes, called Nichols-like and SS14-like groups. So far, no genetic intermediates, i.e. strains containing a mixed pattern of Nichols-like and SS14-like genomic sequences, have been identified. Recently, Sun et al. (Oncotarget 2016. https://doi.org/10.18632/oncotarget.10154 ) described a new "phylogenetic group" (called Lineage 2) among Chinese TPA strains. This lineage exhibited a "mosaic genomic structure" of Nichols-like and SS14-like lineages. RESULTS: We reanalyzed the primary sequencing data (Project Number PRJNA305961) from the Sun et al. publication with respect to the molecular basis of Lineage 2. While Sun et al. based the analysis on several selected genomic single nucleotide variants (SNVs) and a subset of highly variable but phylogenetically poorly informative genes, which may confound the phylogenetic analysis, our reanalysis primarily focused on a complete set of whole genomic SNVs. Based on our reanalysis, only two separate TPA clusters were identified: one consisted of Nichols-like TPA strains, the other was formed by the SS14-like TPA strains, including all Chinese strains.

Complete genome sequences of two strains of Treponema pallidum subsp. pertenue from Ghana, Africa: Identical genome sequences in samples isolated more than 7 years apart.

BACKGROUND: Treponema pallidum subsp. pertenue (TPE) is the causative agent of yaws, a multi-stage disease, endemic in tropical regions of Africa, Asia, Oceania, and South America. To date, four TPE strains have been completely sequenced including three TPE strains of human origin (Samoa D, CDC-2, and Gauthier) and one TPE strain (Fribourg-Blanc) isolated from a baboon. All TPE strains are highly similar to T. pallidum subsp. pallidum (TPA) strains. The mutation rate in syphilis and related treponemes has not been experimentally determined yet. METHODOLOGY/PRINCIPAL FINDINGS: Complete genomes of two TPE strains, CDC 2575 and Ghana-051, that infected patients in Ghana and were isolated in 1980 and 1988, respectively, were sequenced and analyzed. Both strains had identical consensus genome nucleotide sequences raising the question whether TPE CDC 2575 and Ghana-051 represent two different strains. Several lines of evidence support the fact that both strains represent independent samples including regions showing intrastrain heterogeneity (13 and 5 intrastrain heterogeneous sites in TPE Ghana-051 and TPE CDC 2575, respectively). Four of these heterogeneous sites were found in both genomes but the frequency of alternative alleles differed. The identical consensus genome sequences were used to estimate the upper limit of the yaws treponeme evolution rate, which was 4.1 x 10-10 nucleotide changes per site per generation. CONCLUSIONS/SIGNIFICANCE: The estimated upper limit for the mutation rate of TPE was slightly lower than the mutation rate of E. coli, which was determined during a long-term experiment. Given the known diversity between TPA and TPE genomes and the assumption that both TPA and TPE have a similar mutation rate, the most recent common ancestor of syphilis and yaws treponemes appears to be more than ten thousand years old and likely even older.

Molecular Typing of Syphilis-Causing Strains Among Human Immunodeficiency Virus-Positive Patients in Antwerp, Belgium.

Centers for Disease Control and Prevention and sequencing-based treponeme typing was used to analyze 72 blood samples, collected from human immunodeficiency virus and syphilis co-infected patients during 2014 to 2015 in Antwerp, Belgium. Twenty-nine (40.3%) isolates were polymerase chain reaction positive for Treponema pallidum, and all tested were macrolide-resistant. Four genotypes were identified by sequencing-based typing including two new genotypes, U4NR8 and SU9R8, whereas enhanced Centers for Disease Control and Prevention typing revealed 7 subtypes.

Human Treponema pallidum 11q/j isolate belongs to subsp. endemicum but contains two loci with a sequence in TP0548 and TP0488 similar to subsp. pertenue and subsp. pallidum, respectively.

BACKGROUND: Treponema pallidum subsp. endemicum (TEN) is the causative agent of endemic syphilis (bejel). An unusual human TEN 11q/j isolate was obtained from a syphilis-like primary genital lesion from a patient that returned to France from Pakistan. METHODOLOGY/PRINCIPAL FINDINGS: The TEN 11q/j isolate was characterized using nested PCR followed by Sanger sequencing and/or direct Illumina sequencing. Altogether, 44 chromosomal regions were analyzed. Overall, the 11q/j isolate clustered with TEN strains Bosnia A and Iraq B as expected from previous TEN classification of the 11q/j isolate. However, the 11q/j sequence in a 505 bp-long region at the TP0488 locus was similar to Treponema pallidum subsp. pallidum (TPA) strains, but not to TEN Bosnia A and Iraq B sequences, suggesting a recombination event at this locus. Similarly, the 11q/j sequence in a 613 bp-long region at the TP0548 locus was similar to Treponema pallidum subsp. pertenue (TPE) strains, but not to TEN sequences. CONCLUSIONS/SIGNIFICANCE: A detailed analysis of two recombinant loci found in the 11q/j clinical isolate revealed that the recombination event occurred just once, in the TP0488, with the donor sequence originating from a TPA strain. Since TEN Bosnia A and Iraq B were found to contain TPA-like sequences at the TP0548 locus, the recombination at TP0548 took place in a treponeme that was an ancestor to both TEN Bosnia A and Iraq B. The sequence of 11q/j isolate in TP0548 represents an ancestral TEN sequence that is similar to yaws-causing treponemes. In addition to the importance of the 11q/j isolate for reconstruction of the TEN phylogeny, this case emphasizes the possible role of TEN strains in development of syphilis-like lesions.

Origin of modern syphilis and emergence of a pandemic Treponema pallidum cluster.

The abrupt onslaught of the syphilis pandemic that started in the late fifteenth century established this devastating infectious disease as one of the most feared in human history1. Surprisingly, despite the availability of effective antibiotic treatment since the mid-twentieth century, this bacterial infection, which is caused by Treponema pallidum subsp. pallidum (TPA), has been re-emerging globally in the last few decades with an estimated 10.6 million cases in 2008 (ref. 2). Although resistance to penicillin has not yet been identified, an increasing number of strains fail to respond to the second-line antibiotic azithromycin3. Little is known about the genetic patterns in current infections or the evolutionary origins of the disease due to the low quantities of treponemal DNA in clinical samples and difficulties in cultivating the pathogen4. Here, we used DNA capture and whole-genome sequencing to successfully interrogate genome-wide variation from syphilis patient specimens, combined with laboratory samples of TPA and two other subspecies. Phylogenetic comparisons based on the sequenced genomes indicate that the TPA strains examined share a common ancestor after the fifteenth century, within the early modern era. Moreover, most contemporary strains are azithromycin-resistant and are members of a globally dominant cluster, named here as SS14-Ω. The cluster diversified from a common ancestor in the mid-twentieth century subsequent to the discovery of antibiotics. Its recent phylogenetic divergence and global presence point to the emergence of a pandemic strain cluster.

Characterizing the Syphilis-Causing Treponema pallidum ssp. pallidum Proteome Using Complementary Mass Spectrometry.

BACKGROUND: The spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection. METHODOLOGY/PRINCIPAL FINDINGS: To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance. CONCLUSIONS: This is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism.

A Retrospective Study on Genetic Heterogeneity within Treponema Strains: Subpopulations Are Genetically Distinct in a Limited Number of Positions.

BACKGROUND: Pathogenic uncultivable treponemes comprise human and animal pathogens including agents of syphilis, yaws, bejel, pinta, and venereal spirochetosis in rabbits and hares. A set of 10 treponemal genome sequences including those of 4 Treponema pallidum ssp. pallidum (TPA) strains (Nichols, DAL-1, Mexico A, SS14), 4 T. p. ssp. pertenue (TPE) strains (CDC-2, Gauthier, Samoa D, Fribourg-Blanc), 1 T. p. ssp. endemicum (TEN) strain (Bosnia A) and one strain (Cuniculi A) of Treponema paraluisleporidarum ecovar Cuniculus (TPLC) were examined with respect to the presence of nucleotide intrastrain heterogeneous sites. METHODOLOGY/PRINCIPAL FINDINGS: The number of identified intrastrain heterogeneous sites in individual genomes ranged between 0 and 7. Altogether, 23 intrastrain heterogeneous sites (in 17 genes) were found in 5 out of 10 investigated treponemal genomes including TPA strains Nichols (n = 5), DAL-1 (n = 4), and SS14 (n = 7), TPE strain Samoa D (n = 1), and TEN strain Bosnia A (n = 5). Although only one heterogeneous site was identified among 4 tested TPE strains, 16 such sites were identified among 4 TPA strains. Heterogeneous sites were mostly strain-specific and were identified in four tpr genes (tprC, GI, I, K), in genes involved in bacterial motility and chemotaxis (fliI, cheC-fliY), in genes involved in cell structure (murC), translation (prfA), general and DNA metabolism (putative SAM dependent methyltransferase, topA), and in seven hypothetical genes. CONCLUSIONS/SIGNIFICANCE: Heterogeneous sites likely represent both the selection of adaptive changes during infection of the host as well as an ongoing diversifying evolutionary process.

Whole genome sequence of the Treponema pallidum subsp. endemicum strain Bosnia A: the genome is related to yaws treponemes but contains few loci similar to syphilis treponemes.

BACKGROUND: T. pallidum subsp. endemicum (TEN) is the causative agent of bejel (also known as endemic syphilis). Clinical symptoms of syphilis and bejel are overlapping and the epidemiological context is important for correct diagnosis of both diseases. In contrast to syphilis, caused by T. pallidum subsp. pallidum (TPA), TEN infections are usually spread by direct contact or contaminated utensils rather than by sexual contact. Bejel is most often seen in western Africa and in the Middle East. The strain Bosnia A was isolated in 1950 in Bosnia, southern Europe. METHODOLOGY/PRINCIPAL FINDINGS: The complete genome of the Bosnia A strain was amplified and sequenced using the pooled segment genome sequencing (PSGS) method and a combination of three next-generation sequencing techniques (SOLiD, Roche 454, and Illumina). Using this approach, a total combined average genome coverage of 513× was achieved. The size of the Bosnia A genome was found to be 1,137,653 bp, i.e. 1.6-2.8 kbp shorter than any previously published genomes of uncultivable pathogenic treponemes. Conserved gene synteny was found in the Bosnia A genome compared to other sequenced syphilis and yaws treponemes. The TEN Bosnia A genome was distinct but very similar to the genome of yaws-causing T. pallidum subsp. pertenue (TPE) strains. Interestingly, the TEN Bosnia A genome was found to contain several sequences, which so far, have been uniquely identified only in syphilis treponemes. CONCLUSIONS/SIGNIFICANCE: The genome of TEN Bosnia A contains several sequences thought to be unique to TPA strains; these sequences very likely represent remnants of recombination events during the evolution of TEN treponemes. This finding emphasizes a possible role of repeated horizontal gene transfer between treponemal subspecies in shaping the Bosnia A genome.