The incidence of syphilis, a sexually transmitted disease caused by the Treponema pallidum subsp. pallidum (TPA), has been surging globally despite effective antibiotic therapy. A new strategy for syphilis control is the development of a multi-component syphilis vaccine with global efficacy, which requires the identification of surface-exposed candidate vaccinogens and the determination of their antigenic diversity within circulating TPA strains. To improve the quality of sequences from repetitive and paralogous regions of the TPA genome, we have developed a sequencing scheme that allows amplification and long-read sequencing of 25 targets encoding TPA proteins including 15 outer membrane proteins. We tested this approach on a set of 21 clinical TPA strains, mostly of European origin preselected by MLST typing. A total of 462 (88%) of 525 amplicons were sequenced. Of 58 new alleles identified in comparison to the SS14 and Nichols TPA reference strains, the majority encoded new protein sequences (n = 55; 94.8%). The 55 variant protein sequences were encoded by 99 individual TPA loci, where single amino acid replacements occurred most frequently (n = 50), followed by replacements of two to three amino acids (n = 35) and differences comprising four or more residues (n = 14); the latter included six intra-strain recombination events. Most differences were localized to predicted surface-exposed regions, consistent with adaptive evolution of bacterial determinants that function at the host-pathogen interface. Clinical strains having the same allelic profiles from different localities differed in several loci, suggesting that geographical origin significantly contributes to genetic diversity of circulating strains.IMPORTANCEOur findings underscore the importance of analyzing TPA clinical samples isolated from diverse geographical regions in order to understand TPA OMP variability.

• Keywords
• MinION sequencing, OMPeome, Treponema pallidum, genetic epidemiology, long-read sequencing, outer membrane proteins, syphilis,
• MeSH
• Alleles MeSH
• DNA, Bacterial genetics   MeSH
• Genetic Variation * MeSH
• Humans MeSH
• Multilocus Sequence Typing MeSH
• Bacterial Outer Membrane Proteins * genetics   MeSH
• Sequence Analysis, DNA MeSH
• Syphilis * microbiology   MeSH
• Treponema pallidum * genetics   classification   isolation & purification   MeSH
• Treponema MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Bacterial MeSH
• Bacterial Outer Membrane Proteins * MeSH

Previous in vitro long-term cultivation studies of Treponema pallidum subsp. pallidum (T. pallidum) strains have indicated potential differences in the length of generation times among individual strains. In the present study, we have determined in vitro growth rates of seven T. pallidum strains including three from the Nichols-like cluster (DAL-1, Madras, and Haiti B) and four from the SS14-like cluster (Mexico A, SS14, Grady, and Philadelphia 1). Despite the observed considerable variability, the results of standard subcultures identified growth differences between the two clusters during the long-term cultivation. Furthermore, in vitro monocultures with defined inoculum revealed differences among individual strains. During three week-long binary co-cultivations of seven strains (n = 21), different growth rates were confirmed for individual strains (p < 0.001) using PCR amplicon sequencing of genomic regions differentiating treponemal-pairs. The order of strains by decreasing growth rate in vitro was DAL-1, Madras, Mexico A, Haiti B, SS14, Grady, and Philadelphia 1. While the generation time of strain DAL-1 was 32.97 h, the slowest strain, Philadelphia 1, had generation time 43.5 h. These experiments revealed significant physiological differences between the T. pallidum strains, which may also be involved in the variable presentation of syphilis symptoms observed in previous decades.

• MeSH
• Humans MeSH
• Syphilis * microbiology   MeSH
• Treponema pallidum * growth & development   genetics   classification   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Knowledge of Treponema pallidum subspecies pallidum (TPA) outer membrane protein (OMP) sequence variability is essential for understanding spirochete proliferation within endemic populations as well as the design of a globally effective syphilis vaccine. Our group has identified extracellular loops (ECLs) of TPA BamA (TP0326) and members of the FadL family (TP0548, TP0856, TP0858, TP0859, and TP0865) as potential components of a multivalent vaccine cocktail. As part of a consortium to explore TPA strain diversity, we mapped the variability of BamA and FadL orthologs in 186 TPA strains from Malawi, China, and Colombia onto predicted 3D structures. The 186 genomes fell into eight subclades (five Nichols- and three SS14-lineage) with substantial geographic restriction. Single nucleotide variants accounted for the large majority of proteoforms, with variability notably higher within the Nichols-lineage strains. Most mutations were in regions of the proteins predicted to be extracellular and harboring B cell epitopes. We observed a striking difference in the degree of variability between the six OMPs, suggesting that these proteins are following divergent evolutionary paths. Concatenation of OMP sequences recapitulated the phylogenetic structure of the TPA strains, effectively segregating within clades and largely clustering by subclades. Finally, we noted that BamA and FadL candidate ECL vaccinogens, previously shown to elicit antibodies that kill treponemes during in vitro cultivation, are well conserved. Taken as a whole, our study establishes a structural-phylogenetic approach for analyzing the forces shaping the host-pathogen interface in syphilis within endemic populations while informing the selection of vaccine targets.IMPORTANCESyphilis remains a major global health concern, reinforcing the need for a safe and effective vaccine. Understanding the variability of TPA OMPs is essential for tracking pathogen evolution and informing vaccine design. Here, we analyzed the variability of six TPA OMPs in 186 strains from Malawi, China, and Colombia, identifying protein-specific evolutionary patterns. Most mutations were localized in extracellular regions and, notably, appeared to correlate with the phylogenetic structure of TPA. Despite OMP heterogeneity, several candidate vaccinogens remained highly conserved, reinforcing their potential as globally effective vaccine targets. Our study establishes a structural-phylogenetic framework for dissecting the forces shaping the host-spirochete interface within endemic populations and provides a foundation for designing a globally effective syphilis vaccine.

• Keywords
• outer membrane proteins, protein variability, syphilis, vaccines, whole-genome sequencing,
• MeSH
• Bacterial Vaccines * genetics   immunology   MeSH
• Phylogeny MeSH
• Genetic Variation * MeSH
• Humans MeSH
• Evolution, Molecular * MeSH
• Bacterial Outer Membrane Proteins * genetics   immunology   chemistry   MeSH
• Syphilis microbiology   prevention & control   MeSH
• Treponema pallidum * genetics   immunology   classification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• China MeSH
• Colombia MeSH
• Malawi MeSH
• Names of Substances
• Bacterial Vaccines * MeSH
• Bacterial Outer Membrane Proteins * MeSH

BACKGROUND: For many years, syphilis treatment was considered straightforward due to the universal susceptibility of Treponema pallidum subsp. pallidum (TPA) to penicillin antibiotics. METHODS: Penicillin-binding protein genes from a ceftriaxone treatment failure T. pallidum isolate were assessed, and the introduction of identified mutations into two laboratory strains via natural competence was aimed for, followed by in vitro analysis of antibiotic susceptibility of the recombinants. RESULTS: TPA from the ceftriaxone treatment failure case contained A1873G and G2122A mutations in the TP0705 gene. Introduction of the A1873G mutation into laboratory strains DAL-1 and SS14 resulted in partial resistance to ceftriaxone and penicillin G in vitro. Furthermore, in silico analyses revealed that the majority of contemporary TPA SS14-like strains harbors this mutation and are thus partially resistant to ceftriaxone and penicillin G. CONCLUSIONS: This finding indicates that TPA strains accumulate mutations that increase their resistance to β-lactam antibiotics. Alternative approaches for controlling syphilis will be needed, including the development of the syphilis vaccine.

Penicillin antibiotics have been used to treat syphilis since the 1950s. Resistance to antibiotics is a growing concern. We investigated cases where antibiotics had failed to treat infection and found two mutations in a specific gene that could be responsible. Introduction of one of these mutations into two laboratory T. pallidum strains (the bacteria that cause syphilis) resulted in partial resistance to both ceftriaxone and penicillin antibiotics. Moreover, analysis of existing data revealed the presence of this mutation in numerous circulating T. pallidum strains, suggesting widespread partial resistance may already exist and increasing concerns about the future emergence of fully resistant syphilis strains.

• Publication type
• Journal Article MeSH

BACKGROUND: Treponema pallidum subspecies pertenue (TPE) is the causative agent of human and nonhuman primate (NHP) yaws infection. The discovery of yaws bacterium in wild populations of NHPs opened the question of transmission mechanisms within NHPs, and this work aims to take a closer look at the transmission of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Our study determined eleven whole TPE genomes from NHP isolates collected from three national parks in Tanzania: Lake Manyara National Park (NP), Serengeti NP, and Ruaha NP. The bacteria were isolated from four species of NHPs: Chlorocebus pygerythrus (vervet monkey), Cercopithecus mitis (blue monkey), Papio anubis (olive baboon), and Papio cynocephalus (yellow baboon). Combined with previously generated genomes of TPE originating from NHPs in Tanzania (n = 11), 22 whole-genome TPE sequences have now been analyzed. Out of 231 possible combinations of genome-to-genome comparisons, five revealed an unexpectedly high degree of genetic similarity in samples collected from different NHP species, consistent with inter-species transmission of TPE among NHPs. We estimated a substitution rate of TPE of NHP origin, ranging between 1.77 × 10-7 and 3.43 × 10-7 per genomic site per year. CONCLUSIONS/SIGNIFICANCE: The model estimations predicted that the inter-species transmission happened recently, within decades, roughly in an order of magnitude shorter time compared to time needed for the natural diversification of all tested TPE of Tanzanian NHP origin. Moreover, the geographical separation of the sampling sites (NPs) does not preclude TPE transmission between and within NHP species.

• MeSH
• Yaws * transmission   microbiology   veterinary   epidemiology   MeSH
• Phylogeny MeSH
• Genome, Bacterial * MeSH
• Primates * microbiology   MeSH
• Whole Genome Sequencing MeSH
• Treponema pallidum * genetics   isolation & purification   classification   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Tanzania epidemiology   MeSH

The incidence of syphilis has risen worldwide in the last decade in spite of being an easily treated infection. The causative agent of this sexually transmitted disease is the bacterium Treponema pallidum subspecies pallidum (TPA), very closely related to subsp. pertenue (TPE) and endemicum (TEN), responsible for the human treponematoses yaws and bejel, respectively. Although much focus has been placed on the question of the spatial and temporary origins of TPA, the processes driving the evolution and epidemiological spread of TPA since its divergence from TPE and TEN are not well understood. Here, we investigate the effects of recombination and selection as forces of genetic diversity and differentiation acting during the evolution of T. pallidum subspecies. Using a custom-tailored procedure, named phylogenetic incongruence method, with 75 complete genome sequences, we found strong evidence for recombination among the T. pallidum subspecies, involving 12 genes and 21 events. In most cases, only one recombination event per gene was detected and all but one event corresponded to intersubspecies transfers, from TPE/TEN to TPA. We found a clear signal of natural selection acting on the recombinant genes, which is more intense in their recombinant regions. The phylogenetic location of the recombination events detected and the functional role of the genes with signals of positive selection suggest that these evolutionary processes had a key role in the evolution and recent expansion of the syphilis bacteria and significant implications for the selection of vaccine candidates and the design of a broadly protective syphilis vaccine.

• Keywords
• genome analysis, phylogenetic congruence, recombination, selection, treponematoses,
• MeSH
• Yaws * microbiology   MeSH
• Phylogeny MeSH
• Treponemal Infections * microbiology   MeSH
• Humans MeSH
• Syphilis * epidemiology   microbiology   MeSH
• Treponema pallidum genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

In our most recent study, we found that in Tanzania infection with Treponema pallidum (TP) subsp. pertenue (TPE) is present in four different monkey species. In order to gain information on the diversity and epidemiological spread of the infection in Tanzanian nonhuman primates (NHP), we identified two suitable candidate genes for multi-locus sequence typing (MLST). We demonstrate the functionality of the MLST system in invasively and non-invasively collected samples. While we were not able to demonstrate frequent interspecies transmission of TPE in Tanzanian monkeys, our results show a clustering of TPE strains according to geography and not host species, which is suggestive for rare transmission events between different NHP species. In addition to the geographic stability, we describe the relative temporal stability of the strains infecting NHPs and identified multi-strain infection. Differences between TPE strains of NHP and human origin are highlighted. Our results show that antibiotic resistance does not occur in Tanzanian TPE strains of NHP origin.

• MeSH
• Chlorocebus aethiops microbiology   MeSH
• Cercopithecus microbiology   MeSH
• Species Specificity MeSH
• Feces microbiology   MeSH
• Phylogeny MeSH
• Genetic Variation MeSH
• Genetic Association Studies MeSH
• Gorilla gorilla microbiology   MeSH
• Host Specificity * MeSH
• Treponemal Infections epidemiology   microbiology   transmission   veterinary   MeSH
• Polymorphism, Single Nucleotide MeSH
• Multilocus Sequence Typing MeSH
• Ape Diseases epidemiology   microbiology   transmission   MeSH
• Monkey Diseases epidemiology   microbiology   transmission   MeSH
• Papio anubis microbiology   MeSH
• Papio cynocephalus microbiology   MeSH
• Treponema classification   genetics   isolation & purification   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Congo epidemiology   MeSH
• Tanzania epidemiology   MeSH

Syphilis, caused by Treponema pallidum subsp. pallidum (TPA), remains an important public health problem with an increasing worldwide prevalence. Despite recent advances in in vitro cultivation, genetic variability of this pathogen during infection is poorly understood. Here, we present contemporary and geographically diverse complete treponemal genome sequences isolated directly from patients using a methyl-directed enrichment prior to sequencing. This approach reveals that approximately 50% of the genetic diversity found in TPA is driven by inter- and/or intra-strain recombination events, particularly in strains belonging to one of the defined genetic groups of syphilis treponemes: Nichols-like strains. Recombinant loci were found to encode putative outer-membrane proteins and the recombination variability was almost exclusively found in regions predicted to be at the host-pathogen interface. Genetic recombination has been considered to be a rare event in treponemes, yet our study unexpectedly showed that it occurs at a significant level and may have important impacts in the biology of this pathogen, especially as these events occur primarily in the outer membrane proteins. This study reveals the existence of strains with different repertoires of surface-exposed antigens circulating in the current human population, which should be taken into account during syphilis vaccine development.

• Keywords
• Treponema pallidum subsp. pallidum, culture-independent bacterial enrichment, direct whole genome sequencing, recombination-driven diversity, syphilis,
• Publication type
• Journal Article MeSH

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.

• MeSH
• Genes, Bacterial * MeSH
• Adaptation, Biological * MeSH
• Adult MeSH
• Genomics MeSH
• Genotype * MeSH
• Humans MeSH
• Young Adult MeSH
• Selection, Genetic MeSH
• Syphilis microbiology   pathology   MeSH
• Treponema pallidum classification   genetics   isolation & purification   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Young Adult MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease with worldwide prevalence. Several different molecular typing schemes are currently available for this pathogen. To enable population biology studies of the syphilis agent and for epidemiological surveillance at the global scale, a harmonized typing tool needs to be introduced. Recently, we published a new multi-locus sequence typing (MLST) with the potential to significantly enhance the epidemiological data in several aspects (e.g., distinguishing genetically different clades of syphilis, subtyping inside these clades, and finally, distinguishing different subspecies of non-cultivable pathogenic treponemes). In this short report, we introduce the PubMLST database for treponemal DNA data storage and for assignments of allelic profiles and sequencing types. Moreover, we have summarized epidemiological data of all treponemal strains (n = 358) with available DNA sequences in typing loci and found several association between genetic groups and characteristics of patients. This study proposes the establishment of a single MLST of T. p. pallidum and encourages researchers and public health communities to use this PubMLST database as a universal tool for molecular typing studies of the syphilis pathogen.

• Keywords
• Molecular typing, PubMLST, Treponema pallidum subsp. pallidum,
• Publication type
• Journal Article MeSH